Spirochetes Unwound

Still no solid evidence for the Old World origin of syphilis

2012-01-17T10:10:00.000-08:00

The first recorded syphilis epidemic flared up in war-torn Naples in 1494, only two years after Columbus discovered the New World. From there syphilis spread throughout Europe. Ever since then, controversy has raged about the origin of syphilis. A popular belief is that Columbus's crew got infected in the New World and brought the spirochete back to Europe, where they transmitted the disease to others while serving as mercenaries during the first Italian War. The competing pre-Columbian hypothesis asserts that syphilis was always present in the Old World yet wasn't recognized until 1494.

Among the treponemes, Treponema pallidum subspecies pallidum, the agent of syphilis, is the only one that's sexually transmitted. Other treponemal diseases include yaws and endemic syphilis, each caused by a genetically distinct subspecies of Treponema pallidum. The exuberant immune response to T. pallidum during the tertiary stage of treponemal disease leaves marks on the bones. Historical cases of treponemal disease can therefore be identified by looking for these skeletal lesions. Diagnosing treponemal infection in this way is tricky because a number of diseases cause similar lesions. Nevertheless, bone deformities specific to treponemal diseases do exist, most famously the worm-eaten appearance of caries sicca. Unfortunately, skeletal lesions cannot be used to reliably distinguish syphilis from the other treponemal diseases, so I will be speaking of treponemal disease instead of syphilis when discussing the skeletal evidence.

The findings of treponemal lesions in pre-1492 skeletal remains from the Old World would upend the Columbian hypothesis. A number of claims of pre-Columbian treponemal lesions on Old World skeletons have appeared in the scientific literature, with some garnering widespread media attention. One example is the findings from the excavation of an English friary at Hull Magistrate's Court, which was a subject of an episode of the PBS series Secrets of the Dead. Skeletal remains from four individuals unearthed at the site had evidence of treponemal disease. The site where the bones were found was dated to 1300-1450.

The Syphilis Enigma, part 1 of 4

The Syphilis Enigma, part 2 of 4

The Syphilis Enigma, part 3 of 4

The Syphilis Enigma, part 4 of 4

Harper and colleagues recently took another look at the published claims of pre-Columbian treponemal disease in Old World skeletal remains. Their work came out last month in the Yearbook of Physical Anthropology, the supplement to the Journal of Physical Anthropology They scrutinized the data in the 54 published reports with a standardized set of criteria for diagnosing treponemal disease and radiocarbon dating the bones. There were two parts to their analysis. First, they looked at the description of the skeletal lesions (including photographs, when available) to make sure they were the type caused solely by treponemal diseases. Second, they looked at method used to date the bones. In turns out that all 54 reports were flawed in some manner. In many reports, diagnosis of treponemal disease was based on the types of skeletal lesions that could have been induced by other diseases. In others, the bones were dated indirectly by archeological methods instead of directly by radiocarbon dating the bones. Or worse, details of the dating method were sometimes omitted.

Among the 11 specimens for which the diagnosis of treponemal disease was deemed to be correct by the authors, two were dated by radiocarbon methods to the pre-Columbian years. The time of death for the two individuals were 1424-1479 for a specimen unearthed in Safed, Israel and 1426-1486 for one dug up at the Church of St. Helen-on-the-wall in York, England. At first glance, these specimens appear to undermine the Columbian hypothesis. However, radiocarbon dating assumes that the ratio of ¹⁴C ("new" carbon, which decays at a known rate) to ¹²C ("old" carbon) remains constant in the environment and by extension living things, which exchange carbon with the environment by eating and breathing. Slight deviations of the ratio may throw off the calculated date by hundreds of years. One factor that must be accounted for is the "marine reservoir effect," which could skew the isotope ratio in humans who consume seafood. The problem is that water near the surface mixes with deeper water, which reflects the higher proportion of old carbon from the material coming off the ocean floor. The carbon in the air does not mix quickly enough with the carbon in the water to equalize the carbon ratios. Therefore, marine organisms do not contain the same carbon ratio as land organisms. Humans who consume seafood will have a slightly lower proportion of new carbon than expected, making bones from seafood consumers appear older than they really are if no correction is made. When the correction is made for the marine reservoir effect, the new dates for the specimens become 1424-1953 for the Safed specimen and 1421-1669 for the St. Helen-on-the-Walls specimen. An additional three Old World specimens were dated to the pre-Columbian period by radiocarbon dating, corrected for the marine reservoir effect. However, the specimens lacked lesions specific for treponemal disease. In sum, proper dating of the skeletal remains revealed that none of the Old World skeletons bearing treponemal marks could have originated from the pre-Columbian era. There is still no convincing evidence for the Old World origin of syphilis.

So can we now reject the pre-Columbian hypothesis? The authors make an interesting comment about this.

The trope that absence of evidence is not the same as evidence of absence will be invoked by some. While it is true that a working hypothesis may be falsified at any time, at what point does the absence of skeletal evidence of pre-Columbian treponemal disease in the Old World become compelling? After all, the best proof of a theory is failure to disprove it, and we find that despite intense research interest, credible evidence disproving the Columbian Hypothesis is lacking.

The "intense research interest" includes past examination of tens of thousands of skeletons in continental Europe and North Africa and 50,000 in England alone, with none yielding convincing evidence of pre-Columbian treponemal disease. The authors do point out that skeletal remains from vast regions of sub-Saharan Africa and Asia have not been well-studied. For this reason, it may be too soon to lay the pre-Columbian hypothesis to rest.

ACKNOWLEDGEMENTS: A big thanks to Molly Zuckerman for alerting me to her group's work and for sending me the article.

References
Harper, K.N., Zuckerman, M.K., Harper, M.L., Kingston, J.D., & Armelagos, G.J. (2011). The origin and antiquity of syphilis revisited: an appraisal of Old World pre-Columbian evidence for treponemal infection American Journal of Physical Anthropology, 146 (S53), 99-133 DOI: 10.1002/ajpa.21613

von Hunnius, T.E., Roberts, C.A., Boylston, A., & Saunders, S.R. (2006). Histological identification of syphilis in pre-Columbian England American Journal of Physical Anthropology, 129 (4), 559-566 DOI: 10.1002/ajpa.20335

Related posts by other bloggers

Revisiting Syphilis in the Old World in Bones Don't Lie
The French Disease, the Italian Disease, the Christian Disease--the New World Disease? in The Loom

A tale of two more studies: topical antibiotics applied to tick bites to prevent Lyme disease

2011-10-30T17:41:00.000-07:00

Feeding Ixodes ticks harboring Borrelia burgdorferi deposit the Lyme disease spirochete in the skin of the victim. The spirochetes remain in the skin for a few days before entering the bloodstream to spread throughout the host. The delay in dissemination provides a window of opportunity to stop the infection by simply applying antibiotics to the skin where the tick was feeding. Topical application of antibiotics would allow patients to avoid experiencing side effects associated with ingesting antibiotics.

I recently posted a critique of a study by Knauer and colleagues, who tested the ability of a topical antibiotic to prevent B. burgdorferi infection in lab mice bitten by infected ticks. As I explained in the post, the antibiotic appeared to prevent infection, but the investigators had used a weakened B. burgdorferi strain to inoculate the mice. Consequently it wasn't possible to draw any conclusions about the effectiveness of their antibiotic formulation in preventing infection.

Now let's look at two more studies that tested the ability of topical antibiotics to prevent infection in the mouse model of Lyme disease. These studies were conducted properly with highly infectious B. burgdorferi strains. One study was published almost 20 years ago. The other appeared online just last month. Both studies were published in The Journal of Infectious Diseases.

In their 1993 study Shih and Spielman were able to prevent B. burgdorferi infection by applying at least 1 milligram of tetracycline starting up to two days following the tick bite. The antibiotic had to be applied twice a day for at least three consecutive days (see table below). The presence of infection four weeks after tick feeding was assessed by xenodiagnosis, which tests whether the spirochetes could be recovered by ticks placed on the skin at a location distant from the original tick bite. The investigators also found that penicillin, amoxicillin, ceftriaxone, doxycycline, and erythromycin applied for three days beginning one day after tick feeding prevented infection.

Although this study demonstrated the promise of topical antibiotics in preventing Lyme disease in those who discover a tick feeding on them, a limitation of the study was that most of the antibiotics were dissolved in DMSO, which is not approved for use on humans. The only antibiotic dissolved in a solvent suitable for humans was erythromycin, which was added into 70% ethanol.

For their 2011 study, Wormser and colleagues decided to dissolve the antibiotics in something else that would be acceptable to apply to human skin. They rubbed a 2% erythromycin ointment or 3% tetracycline gel over the tick bite 1-2 days after the infected ticks finished feeding on the mice. The antibiotics were applied twice daily for three days. Four weeks later urinary bladder and ear tissue were cultured to see whether the mice had a disseminated infection. The authors found that their antibiotic formulations failed to prevent systemic infection, although erythromycin was able to prevent a persistent infection at the tick bite site in some of the mice (see table below).

Erythromycin and tetracycline were tested in both studies. Why the stark difference in the effectiveness of the same antibiotics in the two studies? In the Discussion of their paper, Wormser and colleagues highlighted the major methodological differences between the studies:

Different antibiotic concentrations. A much higher concentration of erythromycin was applied to the tick bites in the 1993 study.
Different solvents. DMSO, the solvent used for the 1993 study, may have promoted better penetration of tetracycline into the skin than the ointment and gel formulations selected for the Wormser study.
Different placement of infected ticks. In the 1993 study the infected ticks were placed on the ear for feeding. In the Wormser study the ticks were placed on the back, where the skin may be thicker and hence more resistant to antibiotic penetration.
Different B. burgdorferi strains. Wormser and colleagues used ticks infected with the highly invasive BL206 strain to inoculate the mice, whereas Shih and Spielman used ticks infected with the less invasive JD1 strain.
Different mouse strains. The C3H mouse strain used in the Wormser study is highly susceptible to dissemination by B. burgdorferi.

For this simple treatment approach to effective, higher concentrations of the antibiotic in a penetrating solvent such as ethanol may be necessary. Different B. burgdorferi and mouse strains should also be tested in future studies.

References

Shih, C.-M., & Spielman, A. (1993). Topical prophylaxis for Lyme disease after tick bite in a rodent model Journal of Infectious Diseases, 168 (4), 1042-1045 DOI: 10.1093/infdis/168.4.1042

Wormser, G.P., Daniels, T.J., Bittker, S., Cooper, D., Wang, G., & Pavia, C.S. (2011). Failure of topical antibiotics to prevent disseminated Borrelia burgdorferi infection following a tick bite in C3H/HeJ mice Journal of Infectious Diseases DOI: 10.1093/infdis/jir382
Knauer, J., Krupka, I., Fueldner, C., Lehmann, J., & Straubinger, R.K. (2011). Evaluation of the preventive capacities of a topically applied azithromycin formulation against Lyme borreliosis in a murine model Journal of Antimicrobial Chemotherapy DOI: 10.1093/jac/dkr371

Related post

A flawed study claiming prevention of Lyme spirochete infection with topical antibiotics

Life after leptospirosis, a pilot study

2011-10-23T13:59:00.000-07:00

The signs and symptoms associated with acute human leptospirosis are well known. Less is known about how well patients manage after they begin recovering from the illness. Most who survive the illness, even those who had severe illness, appear to regain full health eventually. However careful examination may reveal subtle deficiencies in organ function.² Moreover the spectrum of health problems that could crop up later is not known. The only late complication of leptospirosis that has been well documented is uveitis, which can lead to eye pain and vision problems.³ Old case studies have also reported recurring headaches, general malaise, and even dementia, aggression, depression, and psychosis in those surviving leptospirosis,^4,5 although these observations would need to be confirmed in well-designed clinical studies.

To gain a better understanding of the long-term outcomes of leptospirosis, a large systematic study is needed to monitor the health of leptospirosis patients as they heal from the illness. Such a study was attempted by Spichler and colleagues.¹ Their pilot study targeted patients hospitalized with leptospirosis at any time from December 2008 to May 2009 in São Paulo, Brazil, where leptospirosis is endemic. There were 180 patients hospitalized with leptospirosis during this period, but for a variety of reasons only 47 of the 180 could be enrolled in the study. All 47 had been treated for at least seven days with antibiotics (penicillin or ceftriaxone) during their hospitalization, and all had their diagnosis confirmed by serologic testing. 17 of the 47 had severe leptospirosis, defined as those who had experienced jaundice, kidney failure, bleeding (hemorrhage), pulmonary (lung) involvement, or shock while hospitalized. Fortunately no patient died.

The first outpatient visit was conducted an average of 22 ± 12 days after the patients were discharged from the hospital. 23 of the 47 (49%) continued to experience one or more of the following health problems: general malaise, headache, muscle pain, dizziness, bronchitis, and abdominal pain. Three patients still had jaundice.

Only 22 of the 47 patients came back for a second visit, which took place a mean of 40 ± 21 days after they were discharged from the hospital. Two of the 22 patients continued to suffer from medical problems.

One individual was experiencing general malaise, which wasn't a problem for him before being stricken with leptospirosis. He did always have high blood pressure, which may or may not have been a contributing factor to his malaise. An ECG showed some abnormalities with his heartbeat. Leptospira is known to cause myocarditis, an inflammation of the heart muscle. This patient continued to suffer from profound general malaise when examined one year later.

The second patient started to experience panic attacks between the two clinic visits. Were the panic attacks related to his earlier bout with leptospirosis? It's difficult to conclude anything from this single patient. Although the patient had severe leptospirosis, did not suffer from any of the known neurologic features of leptospirosis during his acute illness⁶ and had not been diagnosed with any neurologic or psychiatric condition before contracting leptospirosis. Panic disorder has never been described in leptospirosis patients in the scientific literature.

Since this was a pilot study, the investigators probably lacked the resources to address the problems that cropped up during the study. As pointed out by the authors, the major limitations of the study were that few of the eligible patients were enrolled in the study and that many dropped out between visits, resulting in a potentially biased sampling of leptospirosis patients. Additionally the follow-up visits did not include any laboratory testing to detect lingering functional deficiencies in the kidney, liver, and other organs. For these reason it's impossible to make any definitive conclusions about the recovery of these subjects. Despite the preliminary nature of this pilot study, the possible outcomes of acute leptospirosis identified in this study and earlier case studies beg for a future prospective study with a larger number of individuals living in an area where leptospirosis is endemic. A longer time frame for follow up is also necessary since uveitis can first appear up to four years after recovery from leptospirosis.³

Any future study should also follow those with mild or asymptomatic Leptospira infections. The reason is that the long-term outcome of mild disease is unknown. Since those with mild or asymptomatic infections are unlikely to seek medical attention, identifying such individuals will require investigators to monitor healthy high-risk individuals by serologic testing so that the newly infected could be identified as those with increasing anti-Leptospira antibody titers.

In light of the recent discovery of chronically infected individuals in the Peruvian Amazon,⁷ it would also be prudent to test the urine of healthy individuals for Leptospira being shed from the kidney tubules so that the findings of the Peruvian study could be confirmed. The long-term effects of chronic infection, if any, could also be identified.

Where could a future study examining the long-term outcomes of Leptospira infection be conducted? Brazil and India may be best suited for this type of study. Leptospirosis is highly endemic in those countries, and multiple research teams are already investigating the epidemiology of leptospirosis in those countries.

10-30-2011, edits for clarity.

Featured paper

1. Spichler A., Athanazio D, Seguro AC, and Vinetz JM (July 2011). Outpatient follow-up of patients hospitalized for acute leptospirosis. International Journal of Infectious Diseases 15(7):e486-e490. DOI: 10.1016/j.ijid.2011.03.020

References

2. de Francesco Daher E, Zanetta DMT, and Abdulkader RCRM (September 2004). Pattern of renal function recovery after leptospirosis acute renal failure. Nephron, Clinical Practice 98(1):c8-c14. DOI: 10.1159/000079922

3. Shukla D, Rathinam SR, and Cunningham ET (Spring 2010). Leptospiral uveitis in the developing world. International Ophthalmology Clinics 50(2):113-124. DOI:

4. Shpilberg O, Shaked Y, Maier MK, Samra D, and Samra Y (April 1990). Long-term follow-up after leptospirosis. Southern Medical Journal 83(4):405-407. Link

5. Avery TL (July 27, 1983). Leptospirosis and mental illness. New Zealand Medical Journal 96(736):589 (Letter).

6. Panicker JN, Mammachan R, and Jayakumar RV (September 2001). Primary neuroleptospirosis. Postgraduate Medical Journal 77(911):589-590. DOI: 10.1136/pmj.77.911.589

7. Ganoza CA, Matthias MA, Collins-Richards D, Brouwer KC, Cunningham CB, Segura ER, Gilman RH, Gotuzzo E, and Vinetz JM (2006). Determining risk for severe leptospirosis by molecular analysis of environmental surface waters for pathogenic Leptospira. PLoS Medicine 3(8):e308. DOI: 10.1371/journal.pmed.0030308

Related post

Healthy human carriers of the spirochete Leptospira in the Peruvian Amazon

The Lyme disease spirochete feasts on tick antifreeze

2011-10-01T22:19:00.000-07:00

In the northeastern United States the Lyme disease spirochete Borrelia burgdorferi spreads from one white-footed mouse to another by hitching a ride in the tick Ixodes scapularis. Transmission between tick and mouse occurs during the tick's rare blood meals. The larval tick acquires B. burgdorferi from an infected mouse during a blood meal late in the summer, and the spirochetes take up shelter in the tick's midgut. Later the larva molts into a nymph, which then completes the transmission cycle by feeding on an uninfected mouse during the next spring or early summer.

Although blood is potentially a rich source of nutrients for both tick and spirochete, the cells lining the tick's gut rapidly engulf the nutrients, including glucose, an energy-rich sugar favored by B. burgdorferi. The spirochetes must therefore rely on other energy sources if it is to survive the many months between tick feedings. How does B. burgdorferi fuel its survival during this period?

A study in the July issue of PLoS Pathogens has shown that B. burgdorferi metabolizes the tick's antifreeze while living in its midgut.¹ Many arthropods and insects produce large amounts of antifreeze to protect themselves from freezing temperatures. The Ixodes tick's antifreeze is glycerol, the same stuff that's often added to enzymes to keep them from freezing in laboratory freezers. The amount of glycerol found in other organisms is too low to serve as antifreeze. Instead glycerol is metabolized to extract the chemical energy stored in its bonds and to make cell membrane components. I describe below how B. burgdorferi handles glycerol, but the same enzymes are found in most organisms that metabolize glycerol, including humans.

The B. burgdorferi genome encodes homologs of a glycerol transporter (GlpF), glycerol kinase (GlpK), and glycerol-3-phosphate dehydrogenase (GlpD), which are used by the bacteria to take up and metabolize glycerol (see figure below). The figure also shows that B. burgdorferi can break down glucose by the glycolytic pathway (glycolysis) to supply its carbon and energy needs.

Modified from Figure 1 of Pappas et al., 2011. The BB numbers are the gene ID numbers assigned when the B. burgdorferi genome was sequenced. The individual steps of glycolysis are not shown. Source

After the glycerol transporter brings glycerol into the cytoplasm, glycerol kinase (GlpK) quickly phosphorylates glycerol at the expense of ATP to generate glycerol-3-phosphate.

Glycerol-3-phosphate is located at a branch point in glycerol metabolism. This key metabolite can be shunted to one of two pathways. One pathway leads to assembly of lipids, and the other leads to the glycolytic pathway, which generates ATP for B. burgdorferi.

To make more lipids, additional molecules are attached to glycerol-3-phosphate by other enzymes to generate phospholipids, glycolipids, and lipoproteins. For example, one of the two major phospholipids in B. burgdorferi membranes is phosphotidylcholine (the other is phosphotidylglycerol). Note in the figure below that glycerol-3-phosphate (in black and blue) makes up the core of phosphotidylcholine. (R₁ and R₂ denote fatty acid chains.) The glycerol or glycerol-3-phosphate base also forms the core of other phospholipids, glycolipids, and lipoproteins needed to assemble the bacterial cell membrane.

ATP provides the energy to build lipids and other components of B. burgdorferi. To generate ATP, glycerol-3-phosphate is converted by glycerol-3-phophate dehydrogenase (GlpD or G3PDH) into dihydroxyacetone phosphate, which feeds into the middle of the glycolytic pathway.

Glycerol is not a great energy source. For each molecule of glycerol, one ATP is consumed to make glycerol-3-phosphate, and two molecules of ATP are made via glycolysis, netting B. burgdorferi one molecule of ATP. On the other hand, each molecule of glucose, which is plentiful in blood, nets two molecule of ATP, twice the amount of energy extracted from glycerol.

To enlarge the glycolytic pathway, click on the image above

B. burgdorferi lacks the TCA cycle enzymes and electron transport chain, which could unleash the chemical energy stored in the bonds of pyruvate, the end product of glycolysis, to generate even more ATP. Instead pyruvate is converted into the fermentation end product lactate by lactate dehydrogenase.

Modified from Figure 2c of Harper and Harris 2005

For their study the investigators knocked out the B. burgdorferi glpD gene encoding glycerol-3-phosphate dehydrogenase so that the spirochete couldn't use glycerol as an energy source to make ATP. As expected, the glpD mutant was unable to grow to a high cell density when glycerol was the major carbon and energy source in the culture medium. Nevertheless the mutant was still able to infect laboratory mice and spread throughout their bodies almost as well as the wild-type (unmutated) strain. This makes sense since energy sources other than glycerol (such as glucose) are readily available in mammals.

To see how well the glpD mutant survived in ticks, larval Ixodes scapularis ticks were allowed to feed to satiation on groups of mice infected with the mutant and wild-type strains. Similar numbers of the mutant (632 ± 343 spirochetes/tick) and wildtype (737 ± 369 spirochetes/tick) ended up in the larva (P = 0.5646). The infected larva were maintained in the lab and allowed to molt into nymphs. 7-8 weeks after larval feeding, the number of mutant spirochetes in the nymphs (254 ± 137 spirochetes/tick) was much lower than the number of wildtype (1173 ± 637 spirochetes/tick; P = 2.76 x 10^-8). This result suggests that to thrive in the tick's midgut, B. burgdorferi has to break down glycerol, the tick's antifreeze, to generate ATP.

The glpD mutation also slowed the rapid increase in spirochete numbers seen when the infected nymph starts to feed on a mouse (see below). It's unclear how much of the blood nutrients are available to B. burgdorferi early during feeding. Blood consumption by the tick is slow initially, and a membrane called a peritrophic matrix forms in the tick midgut to encase the blood. The spirochetes in the midgut may therefore rely primarily on glycerol to power its rapid multiplication even as the nymph is feeding. Within a few days a small number of spirochetes eventually break through the midgut lining and make their way to the salivary glands, where they end up as passengers in the saliva flowing into the mouse's skin.

Figure 11 from Pappas et al., 2011. Infected nymphs were placed on mice at time zero. Filled circles, wild-type B. burgdorferi; open squares, glpD mutant. Source

The impaired growth of the glpD mutant in the feeding nymph also delayed their transmission into the mice. The nymphs fed for 62 hours before the wild-type strain was transmitted to the mice, whereas 72 hours elapsed before transmission of the glpD mutant was detected.

Why does the glpD mutant survive at all in the ticks? The answer is that there are probably other energy sources available to B. burgdorferi. The authors proposed that the sugar chitobiose, a component of the tick's cuticle and peritrophic membrane, can be consumed by B. burgdorferi living in the midgut. The transporter encoded by chbC brings chitobiose into the spirochete, where it is processed by several enzymes before being fed into the glycolytic pathway. In fact the authors found that B. burgdorferi expressed larger amounts of the chbC mRNA when in the unfed nymph than it did when in the mouse host. This result would be expected if B. burgdorferi was trying to metabolize the tick's chitobiose, which is not found in the mouse.

So to sum things up, B. burgdorferi appears to use different organic carbon sources to fulfill its energy needs depending on where it's living. In the mouse host B. burgdorferi most likely breaks down glucose, a sugar rich with potential chemical energy. Since glucose isn't available in the tick, the spirochete consumes glycerol and possibly chitobiose while living in the tick's midgut.

Reference

Pappas, C.J., Iyer, R., Petzke, M.M., Caimano, M.J., Radolf, J.D., & Schwartz, I. (2011). Borrelia burgdorferi requires glycerol for maximum fitness during the tick phase of the enzootic cycle PLoS Pathogens, 7 (7) DOI: 10.1371/journal.ppat.1002102

Image sources

Unless otherwise stated in the figure legends, the chemical reactions were taken from Biochemistry (5th Edition) by Berg, Tymoczko, and Stryer.

Harper ET and Harris RA (2005). Glycolytic Pathway, from eLS. DOI: 10.1038/npg.els.0003883

Related posts

The Lyme disease spirochete has flagella but doesn't use them to penetrate the gut of the feeding tick

A flawed study claiming prevention of Lyme spirochete infection with topical antibiotics

2011-09-28T11:18:00.000-07:00

Two recent papers tested the effectiveness of topical antibiotics in preventing Borrelia burgdorferi infection in mice following a tick bite. Infection by the Lyme disease spirochete was successfully halted in the Knauer et al. study from Germany¹ but not in the Wormser et al. study conducted in New York.² However a flaw in the Knauer study may have unfairly tipped the outcome in the antbiotic's favor. (I'll save the Wormser study for another post.)

The paper by Knauer and colleagues¹ presented two trials, which differed in how the mice were inoculated with B. burgdorferi, In the first trial the spirochetes were injected into the skin, and azithromycin was applied topically one hour, three days, and five days later at the injection site. In the second trial infected ticks transmitted the spirochetes to the mice. Azithromycin was applied topically to the feeding site immediately after the ticks stopped feeding. In both trials azithromycin was dissolved in ethanol for application to the inoculation site. Disseminated infection of the mice was assessed by culturing the heart, bladder, ear, and tarsus 56 days after inoculation.

The results from the first trial reveals the problem with the study (Table 1). Among the ten mice in the placebo group (first row), which received only ethanol, only one (10%) had any culture positive organs 56 days later. The spirochetes failed to establish a persistent infection in the other nine mice, suggesting that the investigators were working with a weakened strain of B. burgdorferi. The ethanol could have had a slight killing effect (see the second trial) yet could not have accounted for the poor infection rate in the placebo group.

The table legend claims that the difference between the placebo and treatment groups was significant, but the statistics were done on the numbers in the column labeled "Infection Status." According to the text of the paper, a positive "Infection Status" refers to those animals that managed to produce antibodies against B. burgdorferi antigens. Infection status is therefore not the proper metric to assess the infectivity of B. burgdorferi. When the statistics are performed with the appropriate numbers, located in the column under "Culture," the effect of azithromycin (1/10 culture positive in control group vs. 0/10 culture positive in any treatment group) is not significant (P = 0.9 for control vs. any treatment group).

Results from the second trial are shown in Table 2. This trial included an extra control group ("No treatment") that received neither antibiotic nor ethanol. Four of the seven mice in the untreated group (57%) ended up culture positive. This is still a low infection rate compared to the rates observed in other studies, in which 90-100% of the control animals end up infected following tick inoculation of B. burgdorferi. Two of the nine mice treated with ethanol alone (placebo) were culture positive, suggesting that ethanol alone helps prevent infection (57% culture positive in "no treatment" group vs. 22% in placebo group, P = 0.152), although the experiment would need to be repeated with larger groups of animals to make a statistically convincing case.

None of the animals treated with azithromycin were culture positive. However the number of animals was again too low to conclude that antibiotic treatment was effective (2/9 culture positive in placebo group vs. 0/9, 0/8, and 0/5 in the treatment groups, P > 0.4 for comparison of each treatment group with placebo group). The authors were able to claim statistical significance by combining the two control groups (no treatment and placebo) and the treatment groups. However it is inappropriate to combine groups in this manner to attain statistical significance.

Even if the investigators had used a larger number of animals, the problem of their weakened challenge strain remains. Application of topical antibiotics may turn out to be effective in preventing Lyme disease after a tick bite, but the study presented by Knauer and colleagues was not a fair test of this treatment approach.

References

1. Knauer, J., Krupka, I., Fueldner, C., Lehmann, J., & Straubinger, R. (2011). Evaluation of the preventive capacities of a topically applied azithromycin formulation against Lyme borreliosis in a murine model Journal of Antimicrobial Chemotherapy DOI: 10.1093/jac/dkr371

2. Wormser, G., Daniels, T., Bittker, S., Cooper, D., Wang, G., & Pavia, C. (2011). Failure of topical antibiotics to prevent disseminated Borrelia burgdorferi infection following a tick bite in C3H/HeJ mice Journal of Infectious Diseases DOI: 10.1093/infdis/jir382

BAM! A rare outer membrane protein of the stealth pathogen Treponema pallidum hidden in plain sight

2011-08-29T22:45:00.000-07:00

Our immune system is capable of generating antibody against bacterial proteins located inside and outside of the bacterial cell. However only those antibodies targeting surface proteins (or other surface components) have the potential to eliminate the bacteria.

Unlike typical Gram negative bacteria, the external surface of the Treponema pallidum outer membrane is bare except for a tiny number of proteins protruding from the membrane. Most of the proteins targeted by the host antibody response are safely tucked away beneath the surface, inaccessible to the antibodies that recognize them. T. pallidum even lacks LPS, which is a major target found on typical Gram negative bacteria. The barren surface of T. pallidum is therefore one factor that may allow the "stealth pathogen" to persist in the host despite a strong antibody response.

None of the rare T. pallidum outer membrane proteins (Omps) have been identified with certainty, until now. Scientists at the University of Connecticut have finally confirmed that TP0326 is one of these rare Omps.¹ Desrosiers and colleagues showed that the protein TP0326 was digested when proteinase K was added to live T. pallidum. Proteins known to be located in the periplasm were left untouched by the protease, indicating that the fragile outer membrane remained intact while the spirochetes were being harvested for the experiment. These results indicated that TP0326 was exposed on the surface of T. pallidum.

TP0326 (also called "Tp92") was first identified as a candidate Omp over a decade ago when rabbit antibodies against the protein were shown to promote opsonophagocytosis (engulfment) of T. pallidum.² Antibodies must physically link bacteria to phagocytes for opsonophagocytosis to proceed. Opsonophagocytosis therefore occurs only when antibodies against surface-exposed proteins are present. The amino acid sequence of TP0326 also gave clues to its location. TP0326 was identified as a homolog of the Omp85 family,² a set of proteins known to reside in the outer membrane of other Gram negative bacteria. Omp85 was later renamed BamA when it was shown to be the core component of the outer membrane protein complex "BAM" that inserts newly expressed Omps into the outer membrane.³

A number of computer programs predicted that TP0326 spanned the outer membrane as a β-barrel structure, which I described in an earlier post.⁴ The gallery of E. coli transmembrane Omps displayed below shows that the loops on one side of the barrel are exposed on the surface of the bacterium. The transmembrane portion of BamA is depicted as a box because its structure has yet to be determined experimentally, but it is also likely to have a β-barrel structure.

Figure 1 from Burgess 2008. The outer membrane is colored gray. The numbers indicate the number of β strands that cross the membrane. The periplasm is located below the outer membrane.

The amino terminus of BamA consists of five repeating structures called POTRA, which are thought to guide the β strands of new transmembrane Omps into the outer membrane.³ Note that the POTRA domains protrude into the periplasmic space. The amino terminus of TP0326 is thought to possess the POTRA domains as well.

Given the prediction that TP0326 structurally resembles BamA, it wasn't too surprising that TP0326 was exposed on the surface of T. pallidum. What was surprising was how TP0326 was targeted by the immune system in syphilis patients. Among the six patients examined by Desrosiers et al., three lacked any antibody whatsover against TP0326. Although the other three syphilis patients managed to generate antibody against TP0326, the antibodies reacted weakly (1 patient) or not at all (2 patients) with the β-barrel domain, which contained the surface-exposed loops. Instead, the antibodies targeting TP0326 reacted strongly with the subsurface POTRA domains in these three patients. Assuming that the results with these six patients can be extrapolated to other syphilis patients, humans infected with T. pallidum are incapable of generating a strong antibody response against the surface-exposed loops of TP0326.

Since the exposed regions of TP0326 appear to be an Achilles heel of T. pallidum, TP0326 may have evolved to avoid generation of antibodies targeting its vulnerable segments. Experimentally infected rabbits, which are not a natural host of T. pallidum, succeeded in generating antibody against the TP0326 β-barrel domain. Unfortunately the authors didn't present any data indicating whether the surface-exposed loops were recognized by the rabbit antibodies. However, as I mentioned above, earlier work showed that infected rabbits produce opsonic antibody against TP0326.² The same paper showed that TP0326 was somewhat effective as a subunit vaccine in the rabbit model of syphilis.² Both of these observations suggest that rabbits are able to produce antibody that reacts with the surface-exposed regions of TP0326. The rabbit model could be used to figure out conclusively whether the effectiveness of TP0326 as a vaccine relies upon generation of antibodies against the surface-exposed loops of the protein. If so, an approach for developing a syphilis vaccine would be to coax the human immune system into generating antibodies that target the surface-exposed loops of TP0326.

Featured paper

1. Desrosiers DC, Anand A, Luthra A, Dunham-Ems SM, LeDoyt M, Cummings MAD, Eshghi A, Cameron CE, Cruz AR, Salazar JC, Caimano MJ, and Radolf JD (June 2011). TP0326, a Treponema pallidum β-barrel assembly machinery A (BamA) orthologue and rare outer membrane protein. Molecular Microbiology 80(6):1496-1515. DOI: 10.1111/j.1365-2958.2011.07662.x

Related papers

2. Cameron CE, Lukehart SA, Castro C, Molini, B, Godornes C, and Van Voorhis WC (April 2000). Opsonic potential, protective capacity, and sequence conservation of the Treponema pallidum subspecies pallidum Tp92. The Journal of Infectious Diseases 181:1401-1413. DOI: 10.1086/315399

3. Knowles TJ, Scott-Tucker A, Overduin M, and Henderson IR (March 2009). Membrane protein architects: the role of the BAM complex in outer membrane protein assembly. Nature Reviews Microbiology 7(3):206-214. DOI: 10.1038/nrmicro2069

4. Cox DL, Luthra A, Dunham-Ems S, Desrosiers DC, Salazar JC, Caimano MJ , and Radolf JD (December 2010). Surface immunolabeling and consensus computational framework to identify candidate rare outer membrane proteins of Treponema pallidum. Infection and Immunity 78(12):5178-5194. DOI: 10.1128/IAI.00834-10

Image source

Burgess NK, Dao TP, Stanley AM, and Fleming KG (September 26, 2008). β-barrel proteins that reside in the Escherichia coli outer membrane in vivo demonstrate varied folding behavior in vitro. Journal of Biological Chemistry 283(39):26748-26758. DOI: 10.1074/jbc.M80275420

Related posts

The quest for outer membrane proteins of the stealth pathogen Treponema pallidum: the cliffhanger episode

Does Borrelia burgdorferi cause an inadequate antibody response by altering B cell activation in the lymph node?

2011-07-04T00:24:00.000-07:00

One of the characteristic features of Lyme disease is lymphadenopathy or swollen lymph nodes. It's not too surprising when a lymph node draining a site of infection swells. However when investigators looked at the lymph node draining the inoculation site of Borrelia burgdorferi in mice, they found that the spirochete had somehow altered the course of activation of B cells producing the antibodies that targeted the spirochete.

Before discussing the findings in the paper, a review of how the antibody response evolves in the lymph node is in order. An antibody response to microbial proteins is sparked when antigen from microbes breaching the skin layer flow into the draining lymph node or are carried to the lymph node by dendritic cells. Lymph nodes are where naive B cells, upon recognition of antigen, differentiate into plasma cells, which secrete large amounts of antibody that target the invading microbe. The antibodies which bind most tightly to protein antigens are made with T cell help in germinal centers, which emerge from the rare B cells in the lymph node that produce antibody capable of recognizing the antigen. (I say "rare" here because each B cell in the lymph node produces antibodies with different antigenic specificities to ensure that any microbe that the host may possibly encounter will be recognized by antibodies displayed by at least a few B cells.) Upon binding the antigen and reception of critical signals from T cells, the B cells migrate to areas in the lymph node containing fixed networks of follicular dendritic cells (FDCs), a type of immune cell with long branched processes that extend out from the body of the cell. (FDCs differ from the dendritic cells that bring antigen to the lymph node.) The B cells then start to proliferate wildly, doubling every 6 to 8 hours (faster than B. burgdorferi!). As the B cell numbers surge, they form germinal centers, which can be identified easily by standard histological stains (see image below). The lymph node may even swell, depending on how much the B cells proliferate.

Lymph node: (1) capsule; (2) subcapsular sinus; (3) germinal centers; (4) lymphoide nodule; (5) trabeculae. Source

As the B cells multiply in the germinal center, a process called somatic hypermutation, which is promoted by signals received from T cells, causes a large number of mistakes to be made within the segment of DNA encoding the antigen binding portion of the antibody. Consequently, the antibodies displayed by some germinal center B cells are no longer able to bind to the microbial antigen whereas those made by other B cells will bind better. The FDC processes, whose surfaces are loaded with antigen, continuously probe the antibodies expressed by the newly arising B cells. Since the new B cells are programmed to die unless they express antibody able to bind the antigen displayed by the FDCs, only B cells displaying antibody that bind most tightly to the antigen will survive. This process by which B cells expressing the antibodies with the highest affinity for antigen are selected is called affinity maturation. Somatic hypermutation and affinity maturation can only occur in germinal centers. The B cells also undergo class switching, in which the class of antibody expressed by the B cells switches from IgM and IgD, which are expressed by naive B cells, to IgA, IgE, or one of the IgG subclasses. The exact switch that occurs is governed by the cytokines that the B cells are exposed to. Which cytokines are present depends on the nature of the infection. Eventually the B cells expressing high-affinity antibodies of the appropriate class differentiate into antibody-secreting plasma cells, which are released from the lymph node to circulate throughout the body and fight the infection.

So what happens in the lymph nodes during a B. burgdorferi infection? When the investigators inoculated B. burgdorferi into or underneath the skin of mice, the lymph node draining the site swelled considerably, enlarging by more than a factor of 10 by the tenth day of infection.

From Fig. 2 of Tunev et al., 2011. Arrow points to lymph node draining the inoculation site. Source

What the investigators saw when they looked at lymph node sections under the microscope was very different from the textbook description of T-cell dependent B cell activation that I gave above. First of all, live B. burgdorferi was found in the lymph node draining the site of infection in mice. This is unusual since phagocytes would normally greet and destroy any microbe that managed to find its way into the lymph node.

From Fig. 3 of Tunev et al., 2011. Day 8 of infection. The arrows point to intact extracellular B. burgdorferi in the subcapsular sinus of the lymph node, which was culture positive beginning on day 1 of infection. Source

Second, massive proliferation of B cells accounted for lymph node swelling, but the expansion of B cell numbers wasn't occurring in well-defined germinal centers. The authors also noted that T cells were not increasing in number. These observations suggested that T cells, which are required for germinal centers to form, were not fully participating in B-cell activation. The lack of germinal centers suggested that somatic hypermutation and affinity maturation were not occurring.

From their observations, the authors speculated that B. burgdorferi somehow subverted B cell activation in the lymph node so that the end result was a large number of plasma cells secreting antibodies of poor quality. By poor "quality," I assume that the authors meant that the affinity of the antibody for B. burgdorferi proteins was low and that the "wrong" subclasses of IgG antibodies were expressed. The most abundant IgG subclasses being produced in the draining lymph node at its most swollen state were IgG2b and IgG3. Whether other IgG subclasses would be more effective at clearing B. burgdorferi from the host and whether the affinities of the antibodies for B. burgdorferi proteins were poor still need to be determined experimentally. Perhaps a classic T-cell dependent B cell response involving the formation of germinal centers accompanied by somatic hypermutation, affinity maturation, and appropriate class switching would have led to production of "high" quality antibodies. If the authors are correct, they have revealed yet another means by which B. burgdorferi could persist in the host.

Featured paper

Tunev SS, Hastey CJ, Hodzic E, Feng S, Barthold SW, and Baumgarth N (May 2011). Lymphadenopathy during Lyme borreliosis is caused by spirochete migration-induced specific B cell activation. PLoS Pathogens 7(5):e1002066. DOI: 10.1371/journal.ppat.1002066

Membrane fusion between Borrelia spirochetes, a new type of bacterial interaction

2011-06-04T21:53:00.000-07:00

It's not unusual for bacteria to collide while swimming around in culture medium. When this happens, the bacteria simply bounce off each other and swim off in different directions. However scientists have discovered that the encounter between Borrelia spirochetes, the agents of Lyme disease and relapsing fever, can progress to something more intimate.¹ When they looked at Borrelia cultures under the microscope, the spirochetes that bumped remained stuck to each other side-by-side as they swam. These encounters were usually brief, lasting for less than 10 seconds, although sometimes they lasted for more than a minute before separating. If you watch the video below, you'll see two Lyme disease B. afzelii cells near the bottom left corner coming together side-by-side and then separating several seconds later. The investigators saw similar interactions in cultures of other Lyme disease spirochetes (B. burgdorferi and B. garinii) and a relapsing fever spirochete (B. hermsii).

Movie S2 from Kudryashev et al., 2011

When I first saw the video, I thought that the spirochetes were simply getting tangled up and that it took several seconds for them to get untangled. However when the investigators examined the cultures by cryoelectron tomography (a type of electron microscopy), they saw that the Borrelia cells weren't merely tangled or stuck to each other. Their outer membranes were actually fused, sometimes so extensively that both cytoplasmic cylinders ended up in a single outer membrane sheath. Panel A below shows a cross-section of fused B. garinii cells. Panel B shows a 3-dimensional rendering of the the fused spirochetes from panel A. The two cytoplasmic cylinders (bright and dark magenta) are surrounded by a single outer membrane sheath. The flagellar filaments from both cells form a single bundle and are shown in yellow.

Figure 4A and 4B from Kudryashev et al., 2011

Artifacts are minimized in specimens observed by cryoelectron tomography because the specimen does not have to be fixed with harsh chemicals. Instead, the live specimen is placed on an electron microscope grid and plunge-frozen to preserve the structure of the biological sample. Still, as the authors point out, fusion of Borrelia cells could be an artifact of preparing the spirochetes for cryoelectron tomography. The outer membrane of Borrelia cells could have fused while collecting the spirochetes by centrifugation or when blotting excess liquid from the electron microscopy grid before freezing the specimen.

Assuming that this was not a preparation artifact, what could be the role of outer membrane fusion in the biology of Borrelia? The authors present two possibilities. First, Borrelia cells can share their outer membrane contents by fusing their outer membranes together. Sharing may be advantageous to Lyme Borrelia during transmission from the tick to the victim's skin, when the spirochetes are turning on genes encoding protective proteins such as OspC. One can imagine Borrelia cells sharing its protective surface proteins with others that have yet to express them so that a larger number of spirochetes can fend off host defenses and establish an infection. Another intriguing possibility is that DNA is exchanged between the two spirochetes. Out of the 110 pairs of fused Borrelia cells observed by cryoelectron tomography, the investigators found one pair whose inner membranes were also fused, providing a conduit (at least theoretically) for transfer of DNA. Cells in culture may not remain fused long enough to transfer DNA, but Lyme disease Borrelia lie dormant in the tick midgut for months, giving Borrelia cells lying next to each other plenty of time to exchange DNA, assuming that membrane fusion and DNA transfer can even occur in this setting.

The outer membrane of spirochetes is unique among diderm (double-membrane) bacteria because of its loose association with the underlying peptidoglycan layer. For this reason the outer membrane of all spirochetes, not just those of Borrelia, may be especially prone to fusing. This raises the possibility that the outer membranes of other spirochetes such as Leptospira and Treponema could also fuse.

Note: This work has also been described in the blog Small Things Considered.

Reference

1. Kudryashev M., Cyrklaff M., Alex B., Lemgruber L, Baumeister W, Wallich R, and Frischknecht F (May 2011). Evidence of direct cell-cell fusion in Borrelia by cryogenic electron tomography. Cellular Microbiology 13(5):731-741. DOI: 10.1111/j.1462-5822.2011.01571.x

A new attenuated leptospirosis vaccine protects hamsters from lethal infection by more than one serovar of Leptospira

2011-05-01T18:00:00.000-07:00

Scientists have demonstrated that a new attenuated leptospirosis vaccine protects laboratory hamsters from being killed by Leptospira, even when the challenge and vaccine strains belong to different serovars (immune types).^1,2 This is the first leptospirosis vaccine to confer complete cross-protection against lethal infection by a serovar different from the one used for immunization.

The leptospirosis vaccines that are out on the market are still formulated with killed Leptospira or sometimes their outer membrane. These traditional vaccines are administered primarily to dogs, cattle, and pigs. Human leptospirosis vaccines are not available in most countries, even in areas where leptospirosis is endemic.

New types of leptospirosis vaccines are needed since the traditional killed vaccines are flawed. One problem is that immunity is serovar specific. For this reason a vaccine must contain all the serovars that the target population may encounter. Even when the vaccine manufacturers figure out which serovars are circulating, a new serovar may emerge, rendering the vaccine ineffective as the new serovar spreads through the susceptible population. The vaccine must then be reformulated at substantial cost.

This is exactly what happened to the leptospirosis vaccines that are given to dogs.³ The early canine vaccines, first available in the 1970s, contained the serovars Canicola and Icterohemorrhagiae. These vaccines worked fine until the late 1980s or so, when new serovars started to appear in infected dogs, even in those that had been vaccinated. Since then vaccine makers have added the serovars Grippotyphosa and Pomona to their vaccines. Nevertheless with over 200 pathogenic serovars of Leptospira lurking out there, we don't know when or which additional serovars will emerge in the future.

It would be nice to have a single leptospirosis vaccine formulation that would protect against all serovars. The protective effect of traditional vaccines is due to antibodies generated against lipopolysaccharide (LPS), whose structure differs among the serovars of Leptospira. Since immunization elicits antibodies that recognize the LPS of only the serovars included in the vaccine, vaccinated individuals remain susceptible to infection by other serovars.

To get around this problem, scientists have been testing individual Leptospira surface proteins as potential vaccines in rodent models of leptospirosis. Leptospira surface proteins tend to be antigenically conserved among the different serovars: antibodies generated against a protein from one serovar often reacts against the same protein expressed by other serovars. According to many studies the LipL32 and Lig surface lipoproteins, when delivered as recombinant proteins, naked DNA, or by microbial vectors (adenovirus and Mycobacterium bovis), apparently protected hamsters or guinea pigs from lethal infection by Leptospira. Unfortunately one of the leaders in the leptospirosis field, Ben Adler (also an author of the two featured papers), has questioned the interpretation of these studies.⁴ He points out that the challenge strains used in several studies were not sufficiently lethal, making it easier to observe a protective effect of the vaccine. Moreover some studies claimed statistically significant protective effects of the protein vaccine when in fact there was none upon Adler's reanalysis of the data. The only protein to convincingly exert a protective effect in an appropriate animal model was LigA⁵ although the ability of the LigA subunit vaccine to cross-protect against different serovars of Leptospira has yet to be tested. However there is one major problem with using LigA as a vaccine--not all Leptospira strains have the ligA gene.⁶

In the two studies described here the investigators took a step back from looking at individual proteins and developed an attenuated strain to use for immunization. The properties of the attenuated strain, designated M1352, are described in the paper authored by Murray and colleagues.² The M1352 strain was not developed by the classic approach of continuously growing and passaging the bacteria in culture until they lost their ability to cause disease. Instead the strain was one of a large collection of mutants generated by random transposon mutagenesis of L. interrogans serovar Manilae. The M1352 strain had the transposon inserted in a gene located in a large cluster of genes encoding enzymes that assemble LPS. The mutation had subtle effects on the reactivity of M1352 with various antibodies raised against leptospiral LPS, suggesting that the LPS structure itself was somehow changed in M1352 when compared with the wild-type Manilae strain.

Since LPS is a crucial surface component that interacts with the host, it was not too surprising that M1352 was not able to cause lethal infections like its wild-type Manilae parent. When they infected hamsters with the M1352 strain, the spirochetes were unable to kill the hamsters or even establish an infection in the kidneys. Despite the efficient clearance of M1352, the Leptospira lingered long enough in the hamsters to provoke an antibody response. Western blots of L. interrogans lysates revealed strong reactivity of antibodies from the M1352-infected hamsters to a number of proteins. Because the M1352 strain generated an antibody response without establishing an infection, the authors decided to test the weakened strain as a vaccine in the hamster model in a follow-up study.¹

In the second study, Srikram and colleagues¹ demonstrated that immunization of hamsters with a single dose of live M1352 was more effective than a dose of heat-killed wild-type strain in protecting hamsters from being killed by the wild-type Manilae strain. The M1352 vaccine also did a better job in preventing colonization of the kidneys by the spirochete and in minimizing lung hemorrhage than the heat-killed vaccine.

When they challenged the vaccinated hamsters with a different serovar, a Pomona strain, all the hamsters immunized with live M1352 survived whereas 60% of animals immunized with heat-killed wild-type Manilae perished. However the M1352 vaccine didn't work perfectly. Although all hamsters immunized with live M1352 survived the challenge with the Pomona strain, the kidneys from 90% of the animals were culture positive, and 90% had hemorrhaged lungs. Nevertheless this is the first time that complete protection from death was observed following challenge of vaccinated animals by a serovar unrelated to the vaccine strain. They also showed that the M1352 vaccine had to be administered alive. Heat-killed or chemically-killed M1352 vaccine failed to protect hamsters from lethal infection.

The investigators next tried to figure out which component of the M1352 strain was the protective cross-reactive antigen targeted by the hamster's immune system. They wondered whether the live M1352 and heat-killed wild-type Manilae vaccines generated antibody responses to different proteins. When they probed separate two-dimensional blots of L. interrogans membrane preparations of serovar Pomona with antibodies from hamsters immunized with M1352 and heat-killed wild-type Manilae, a number of protein spots lit up. Most proteins, including LipL32, reacted with both sets of antibodies. These proteins are unlikely to account for the cross-protection conferred by the M1352 vaccine since the presence of these antibodies in the hamsters immunized with heat-killed Manilae failed to protect the animals from being killed by the Pomona strain. On the other hand, four Pomona proteins were recognized only by hamsters receiving the attenuated vaccine:

Loa22, the only surface protein known to be essential for L. interrogans to cause lethal infections⁷
a homolog of GspG, a component of the type II secretion system
LA1939, a possible lipoprotein of unknown function
OmpL36, a surface-exposed outer membrane protein of unknown function⁸

Since GspG is not a surface component of other bacteria and nothing is known about where LA1939 is located on Leptospira, Loa22 and OspL36 are the best candidates to test as potential cross-protective vaccines.

Featured papers

1. Srikram A, Zhang K, Bartpho T, Lo M, Hoke DE, Sermswan RW, Adler B, and Murray GL (March 15, 2011). Cross-protective immunity against leptospriosis elicited by a live, attenuated lipopolysaccharide mutant. Journal of Infectious Diseases 203(6):870-879. DOI: 10.1093/infdis/jiq12

2. Murray GL, Amporn S, Henry R, Hartskeerl RA, Sermswan RW, and Adler B (November 2010). Mutations affecting Leptospira interrogans lipopolysaccharide attenuate virulence. Molecular Microbiology 78(3): 701-709. DOI: 10.1111/j.1365-2958.2010.07360.x

Helpful references

3. Guerra MA (February 15, 2009). Leptospirosis. Journal of the American Veterinary Medical Association 234(4):472-478. DOI: 10.2460/javma.234.4.472

4. Adler B and de la Pena Moctezuma (January 27, 2010). Leptospira and leptospirosis. Veterinary Microbiology 140(3-4):287-296. DOI: 10.1016/j.vetmic.2009.03.012

5. Silva, ÉF, Medeiros MA, McBride AJA, Matsunaga J, Esteves GS, Ramos JGR, Santos CS, Croda J, Homma A, Dellagostin OA, Haake DA, Reis MG, and Ko AI (August 14, 2007). The terminal portion of leptospiral immunoglobulin-like protein LigA confers protective immunity against lethal infection in the hamster model of leptospirosis. Vaccine 25(33):6277-6286. DOI: 10.1016/j.vaccine.2007.05.053

6. McBride AJA, Cerqueira GM, Suchard MA, Moreira MA, Zuerner RL, Reis MG, Haake DA, Ko AI, and Dellagostin OA (March 2009). Infection, Genetics and Evolution 9(2):196-205. DOI: 10.1016/j.meegid.2008.10.012

7. Ristow P, Bourhy P, da Cruz McBride FW, Figueira CP, Huerre M, Ave P, Girons IS, Ko AI, and Picardeau M (July 2007). The OmpA-like protein Loa22 is essential for leptospiral virulence. PLoS Pathogens3(7):e97. DOI: 10.1371/journal.ppat.0030097

8.Pinne M and Haake DA (June 2009). A comprehensive approach to identification of surface-exposed, outer membrane-spanning proteins of Leptospira interrogans. PLoS One 4(6):e6071. DOI: 10.1371/journal.pone.0006071

Dual role of TLR8 during the engulfment of Lyme disease spirochetes by human monocytes

2011-04-12T10:51:00.000-07:00

For the first time scientists have shown that Toll-like receptor 8 (TLR8), a microbial RNA sensor located inside phagocytes, detects what is primarily an extracellular pathogen, the Lyme disease spirochete Borrelia burgdorferi.¹ As one may expect, the phagocytes secreted a mixture of inflammatory cytokines in response to the spirochete. But they also expressed at least one of the type I interferons (IFNs), which until recent years were thought to be produced only in response to viral and intracellular bacterial infections.²

The phagocytes used for the study were human monocytes, which are the more easily available bloodstream form of the macrophages found in our tissues. Macrophages are designed to capture and engulf microbial pathogens invading our bodies. While sopping up the invaders, the macrophages send out warning signals in the form of cytokines and other inflammatory molecules to alert nearby cells and to get the immune system to send more immune cells to help defend the tissue under attack.

Most of the macrophage's microbial sensors belong to a family of related membrane proteins called Toll-like receptors (TLRs). TLR1, TLR2, TLR4, and TLR6 span the plasma membrane, whereas TLR3, TLR7, TLR8, and TLR9 are located in membrane structures inside the cell. Each TLR recognizes a specific component of microbes. For example, TLR4 latches onto LPS; TLR2 forms a complex with TLR1 or TLR6 to bind the lipidated amino terminus of lipoproteins; TLR7 and TLR8 recognize single-stranded RNA; and TLR9 recognizes DNA. Engagement of a TLR by a microbial component triggers a signaling cascade within the cell leading to the transcription of genes encoding inflammatory cytokines, which are then secreted. Stimulation of TLR3, TLR4, TLR7, TLR8, and TLR9 can also activate transcription of genes encoding type I IFNs. The exact response of the macrophage depends on which TLRs are engaged by the pathogen.³

In the PNAS paper by Cervantes and colleagues, the authors searched for the sensors triggered by B. burgdorferi.¹ It had been known for a decade that one of the sensors of B. burgdorferi is TLR2, which recognizes the many lipoproteins that populate the surface of the spirochete. However, TLR2 can't be the only B. burgdorferi sensor in macrophages because a more recent study showed that mouse macrophages missing its Tlr2 gene continued to produce inflammatory cytokines, albeit at lower levels, while engulfing B. burgdorferi.⁴ This same study also showed that B. burgdorferi stimulated human monocytes to transcribe genes encoding type I IFNs and a number of genes known to be induced by type I IFNs.

The investigators first examined the effects of blocking phagocytosis of B. burgdorferi by treating the monocytes with cytochalasin D, a chemical that blocks phagocytosis. They found that cytochalasin D prevented transcription of the gene encoding the type I interferon IFN-β and reduced the amount of the inflammatory cytokine TNFα secreted from the monocytes. The little bit of TNFα that continued to be produced was probably a consequence of TLR2 being stimulated by B. burgdorferi lipoproteins on the surface of the monocytes.

Since the spirochetes had to be engulfed to observe the monocyte's complete response, an intracellular sensor must participate in sensing B. burgdorferi. The investigators therefore focused their attention on the intracellular nucleic acid sensors TLR7, TLR8, and TLR9. Earlier studies by many other labs have shown that engagement of these intracellular TLRs activated production of type I IFNs, so it made sense to examine these TLRs.

To figure out which TLR functioned as the intracellular sensor of B. burgdorferi, the investigators used synthetic fragments of DNA that specifically blocked each TLR without interfering with phagocytosis. When they applied the inhibitors individually to the monocytes, they found that only the TLR8 inhibitor blocked induction of the IFN-β gene by B. burgdorferi (see right half of panel A below). A synthetic DNA fragment that does not inhibit any TLR failed to block induction of IFN-β transcription. Therefore, TLR8 was implicated as being the intracellular sensor that detects B. burgdorferi. As a control, LPS, which is sensed by TLR4 but not TLR8, continued to induce synthesis of the IFN-β transcript in the presence of the TLR8 inhibitor (left half of panel A).

B. burgdorferi was added at a 10:1 ratio (bacteria:monocytes). Following the 4-hour incubation period, IFN-β transcript levels were measured by quantitative real-time RT-PCR. ODN, a control synthetic oligodeoxynucleotide; N.S., not significant; *, P < 0.05; **, P < 0.01 (Mann-Whitney U test)

When the investigators looked at the inflammatory cytokines being produced by the TLR8-inhibited monocytes, they discovered that TLR8 had another role in the monocyte's response to B. burgdorferi. Blocking TLR8 reduced (but did not eliminate) the secretion of the inflammatory cytokines TNFα, IL-6, IL-1β, and IL-10 (see panel B below). These results indicated that TLR2 and TLR8 both had to send signals the nucleus to maximize the amount of cytokines produced during engulfment of the spirochete. The authors also found by indirect immunofluorescence microscopy that TLR2 and TLR8 were together in the phagosomal membrane surrounding the engulfed B. burgdorferi, which they saw were being destroyed.

The amount of cytokines secreted by the human monocytes was measured following the 4-hour incubation period. "Un," no spirochetes added; "Bb," B. burgdorferi added at a 10:1 ratio (bacteria:monocytes)

So here's what the authors believe is happening during the encounter of human monocytes with B. burgdorferi. The monocyte first contacts the spirochete at the surface of the plasma membrane. The lipoproteins on the surface of the spirochete activate the TLR2 sensor on the surface of the monocyte, but the low local concentration of TLR2 in the plasma membrane hampers its full signaling potential. As the monocyte engulfs the spirochete by phagocytosis, TLR8 and TLR2 are recruited to the phagosomal membrane surrounding the spirochete, which at this point is being destroyed by antimicrobial substances being dumped into the phagosome. Destruction of the spirochete releases its RNA to stimulate TLR8, while the crowding of TLR2 in the phagosomal membrane enhances the signaling stimulated by lipoproteins. TLR8 and TLR2 work together to send a signal to the nucleus to activate transcription of numerous genes, including those encoding inflammatory cytokines. Signaling from TLR8 by a separate pathway also stimulates transcription of type I interferons.

So is TLR8 relevant to Lyme disease? The authors make the reasonable assertion that TLR8 activation benefits those infected with B. burgdorferi in part by turning on the type I IFN response.

Given that type I IFNs can shape a variety of downstream inflammatory responses through positive and/or negative regulation of hundreds of additional genes involved in secondary host defenses, TLR8 activation is likely to play a critical role in clearance of the spirochete and more importantly, disease control. [emphasis mine]

Unfortunately, the research literature tells us that type I IFNs have a dark side. Although the beneficial role of type I IFNs in fighting off viral infections is well established, whether they help or hurt us during bacterial infections is not always obvious. Type I IFNs are clearly essential in combating some bacterial infections such as lethal bloodstream infections caused by group B streptococci, Streptococcus pneumoniae, and E. coli.² As for Lyme disease, type I IFNs may help kill spirochetes, but they also promote joint inflammation in infected mice.⁵ To determine whether TLR8 contributes to Lyme arthritis, scientists will need to perform B. burgdorferi infection studies with Tlr8-knockout mice.

Featured paper

1. Cervantes, J.L., Dunham-Ems, S.M., La Vake, C.J., Petzke, M.M., Sahay, B., Sellati, T.J., Radolf, J.D., & Salazar, J.C. (2011). Phagosomal signaling by Borrelia burgdorferi in human monocytes involves Toll-like receptor (TLR) 2 and TLR8 cooperativity and TLR8-mediated induction of IFN-β Proceedings of the National Academy of Sciences, 108 (9), 3683-3688 DOI: 10.1073/pnas.1013776108

Key references

2. Mancuso G, Midiri A, Biondo C, Beninati C, Zummo S, Galbo R, Tomasello F, Gambuzza M, Macrì G, Ruggeri A, Leanderson T, & Teti G (2007). Type I IFN signaling is crucial for host resistance against different species of pathogenic bacteria. Journal of Immunology, 178 (5), 3126-3133 PMID: 17312160

3. Kawai, T., & Akira, S. (2010). The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors Nature Immunology, 11 (5), 373-384 DOI: 10.1038/ni.1863

4.Salazar, J.C., Duhnam-Ems, S., La Vake, C., Cruz, A.R., Moore, M.W., Caimano, M.J., Velez-Climent, L., Shupe, J., Krueger, W., & Radolf, J.D. (2009). Activation of human monocytes by live Borrelia burgdorferi generates TLR2-dependent and -independent responses which include induction of IFN-β PLoS Pathogens, 5 (5) DOI: 10.1371/journal.ppat.1000444

5. Miller JC, Ma Y, Bian J, Sheehan KC, Zachary JF, Weis JH, Schreiber RD, & Weis JJ (2008). A critical role for type I IFN in arthritis development following Borrelia burgdorferi infection of mice. Journal of Immunology, 181 (12), 8492-8503 PMID: 19050267

Related posts

Antigen presentation in the bloodstream: How invariant NKT cells are activated by Lyme disease spirochetes

Serologic testing for syphilis: missing the point

2011-02-24T11:12:00.000-08:00

You may have seen several news sources touting the recent CDC finding that nearly one in five positive reactions with a newer syphilis test are wrong. These headlines may grab the reader's attention, but the press took the finding out of context and failed to deliver the real message that the CDC was trying to convey. Worse, the press reports may needlessly confuse and worry those who are being treated for syphilis.

Serological tests for syphilis are grouped into two categories. Nontreponemal tests such as the VDRL and RPR are based on antibody generated against the lipid cardiolipin. Presumably cardiolipin is released from damaged tissue in syphilis patients and gets incorporated into the membrane of Treponema pallidum. The reason that these tests are "nontreponemal" is that antibodies to cardiolipin accompany many other conditions. On the other hand, treponemal tests use T. pallidum proteins or even the entire spirochete as antigen to detect antibodies against the spirochete. Although the classic treponemal tests such as the FTA-ABS (fluorescent treponemal antibody-absorption) and TP-PA (Treponema pallidum particle agglutination) are still used, the newer automated EIA (enzyme immunoassay) and CIA (immunochemiluminescence) treponemal tests enable clinical laboratories to rapidly screen a large number of sera.

The traditional approach to syphilis testing is to first screen the patient's serum with a nontreponemal test. Since nontreponemal tests can give false positive reactions, reactive sera are retested with one of the treponemal tests. However, the low cost of executing the automated treponemal tests have led some high-volume clinical laboratories to reverse the order of the assays: they screen with the EIA/CIA treponemal test and confirm positive results with a nontreponemal test. The CDC report in the Morbidity and Mortality Weekly Report deals with this so-called "reverse sequence" testing.

So where did the "nearly one in five" figure come from? From 2006 to 2010, five large clinical laboratories screened 140,176 sera specimens with the reverse sequence procedure. Of the 4,834 reactive with the EIA/CIA treponemal test, 2,743 gave negative results with the nontreponemal RPR test. When the samples that gave discrepant results were tested further with one of the classic treponemal tests, 866 of the 2,743 samples were negative. Overall, among the 4,834 samples that were reactive with the newer treponemal test, 866 or 18% were nonreactive with two subsequent tests. These 866 were assumed to be false positives.

The news media pounced on the 18% figure and declared that hundreds may have been given antibiotics to treat a disease that they didn't have. But they ignored the fact that doctors don't diagnose syphilis on the basis of a single lab test. It is standard practice to perform a second test when the first comes back positive and to do even a third one if warranted. Doctors also take into account the physical exam and the sexual and medical history of the patient before making the decision to treat with antibiotics.

Here's how the CDC responded to the assertion that those among the 18% may have been falsely diagnosed and treated unnecessarily with antibiotics:

There are two problems with this assertion. First, the current report does not document whether or not treatment was provided. Second, in those cases where treatment was provided, it may have been justified based on sexual risk and findings on clinical evaluation. It is also important to note that syphilis is not diagnosed on the basis of a single blood test. Many labs routinely will do additional testing when the first test is positive, without notifying the patient. Doctors diagnose syphilis after considering at least two syphilis tests, the patient's history, the physical exam, and a review of past syphilis test results. The MMMR analysis, while important, does not allow us to conclude that the newer tests led to inaccurate syphilis diagnosis or inappropriate treatment.

So what was the message that the CDC was trying to communicate to readers of the MMMR report? Their intention was to provide guidance in the management of cases for which the reverse sequence screening is performed instead of the traditional sequence, which is still recommended by the CDC. Specifically, when conflicting results occur (positive with the treponemal test, negative with the nontreponemal test), a third test should be done with the TP-PA. (The CDC does not recommend the FTA-ABS because it is less specific and probably less sensitive.) A positive reaction with the TP-PA indicates past or present syphilis; a negative reaction indicates that syphilis is unlikely. As always, the clinical observations and medical history of the patient should also be considered in making an informed treatment decision.

Reference

Centers for Disease Control and Prevention (February 11, 2011). Discordant results from reverse sequence syphilis screening -- five laboratories, United States, 2006-2010. MMMR. Morbidity and Mortality Weekly Report 60(5):133-137. link

Another reason not to own a pet rat

2011-02-20T01:25:00.000-08:00

A pair of European doctors wrote about the trials of a 37-year-old man who came down with Weil's disease, a severe form of leptospirosis. The case report appeared in the recent issue of The Lancet Infectious Diseases.

The individual showed up in the emergency room complaining of a sudden fever, muscle pain, and a severe headache. Notable were the signs of jaundice and conjunctival suffusion (reddening of the eye), which suggested leptospirosis.

Figure from Jansen and Schneider, 2011

Shortly after he was admitted, the patient had to be moved to the intensive care unit because his kidneys were failing. Blood tests for Leptospira antibodies came back negative, most likely because it was too early in the infection for antibodies to appear.

An infectious diseases specialist noted that the patient suffered from the classic signs of Weil's disease: kidney failure, jaundice, and an enlarged spleen. Accordingly, the patient was started on intravenous penicillin, the favored treatment for severe leptospirosis. Further blood tests conducted during the second week of treatment finally revealed antibodies to Leptospira. The patient completely healed within several weeks. The only question that remained is how the patient got infected with Leptospira.

At some point the patient admitted that he owned four pet rats. Since rats can silently carry Leptospira in their kidneys, his pets were sacrificed to check their kidneys for the presence of the spirochete. All four kidneys turned out to be culture positive for Leptospira interrogans, serovar Icterohaemorrhagiae or Copenhageni (the typing test couldn't distinguish the two serovars). The patient's antibodies reacted against the same serovars, suggesting that the source of the infection was tainted urine from his pet rats.

Although cases of leptospirosis acquired from pet rats appear to be rare, anyone who wishes to own a rat should be aware that rats can be carriers of a potentially deadly spirochete. This is yet another reason why rats should be obtained from a responsible breeder.

For another case, check out this post from the Worms & Germs Blog (make sure you read the comments for the complete story).

Reference

Jansen, A. and Schneider, T. (February 2011). Weil's disease in a rat owner. The Lancet Infectious Diseases 11(2):152. DOI: 10.1016/S1473-3099(10)70106-7

The quest for outer membrane proteins of the stealth pathogen Treponema pallidum: the cliffhanger episode

2011-02-15T09:48:00.000-08:00

Syphilis patients are able to generate antibodies against the spirochete Treponema pallidum. However, if you were to mix sera from these patients with T. pallidum in a test tube, very few of the antibodies in the sera would bind to the spirochetes. The reason is that the strange outer membrane architecture of T. pallidum makes the spirochete invisible to the antibodies. Most of the proteins and lipid molecules targeted by the antibodies lie beneath the outer membrane.

The outer membrane of T. pallidum differs considerably from that of a typical Gram-negative bacterium. The most glaring difference is that T. pallidum lacks lipopolysaccharide (LPS), a favorite target of the immune response. The outer membrane is also bare of other potential surface antigens except for a very small number of transmembrane outer membrane proteins (Omps) and (possibly) surface lipoproteins. The poor surface antigencity may help the so-called "stealth pathogen" persist in the body despite a robust immune response to the infection.

The few Omps displayed on the surface of T. pallidum must be doing something really important if the spirochete is willing to risk exposing them to attack by antibodies. For this reason, scientists have been seeking the identity of these Omps to figure out what they do. These rare Omps could also be fashioned into a long-desired syphilis vaccine.

Unfortunately, the scarcity of Omps and the delicate nature of the outer membrane of T. pallidum have stymied efforts to identify Omps. The routine centrifugation and washing steps used to prepare other bacteria for analysis easily damage the outer membrane of T. pallidum, causing the loss of Omps and exposing the abundant periplasmic and inner membrane proteins. Consequently, probes used to identify exposed proteins may react with the periplasmic and inner membrane proteins, which are normally shielded by the outer membrane. Without the proper controls, this would lead one to conclude wrongly that a non-Omp that reacts with the probe (such as antibodies raised against the protein of interest) is surface exposed. In addition, when the outer membrane is purified with the intention to identifying Omps, it is hard to distinguish the tiny amounts of Omps from proteins from other bacterial compartments contaminanting the outer membrane preparation.

Despite these technical challenges, several Omp candidates have been proposed, but those proteins are either mired in controversy (TprK, for example) or await experimental confirmation of their surface exposure. To date, no protein that has been demonstrated unambiguously to be displayed on the exterior of T. pallidum.

The past decade has seen the development of several computer programs that can be used to predict whether a given gene encodes a transmembrane Omp. The algorithms differ, but all of these programs attempt to identify amino acid sequences that fold into a β-barrel, which forms the core of transmembrane Omps whose 3D structures are known. The β-barrel forms when an anti-parallel β-sheet rolls into the shape of a barrel. Most of the β-barrel is embedded in the outer membrane so that the loops connecting the β-strands stick out from the two surfaces of the membrane. The loops displayed on the external face of the outer membrane would be accessible to antibodies. The size and composition of the loops vary among different Omps, but all β-strands tend to have alternating hydrophobic amino acids with their nonpolar side chains acid protruding out from the barrel into the hydrophobic interior of the lipid bilayer. Each β-strand consists of 9-11 amino acid residues and is tilted up to 45° out of the transmembrane axis. Different β-barrels have as few as 8 and as many as 22 transmembrane β-strands.

The ribbon representation of OmpA, an 8-stranded transmembrane Omps from E. coli, is shown below as one example.

From Figure 1b of Smith et al., 2007. The N- and C-terminal β-strands are colored brown and blue, respectively. The side chains of the "aromatic girdle" are shown.

Below, OmpA is unfurled to show the topology of the protein:

From Figure 1a of Smith et al., 2007. β-strand amino acid residues are depicted as diamonds, and loop residues are depicted as circles. Alternating hydrophobic amino acid residues within the β-strand are colored red (aromatic residues of the girdle) and yellow.

Assuming that the rare transmembrane Omps of T. pallidum share the β-barrel structure, the obvious computational approach to finding these Omps would be to run all of the proteins encoded by the T. pallidum genome through one of these programs. One problem with these programs is that they will pick up a few proteins that are not truly transmembrane Omps. To minimize this problem, Justin Radolf's group, as reported in the December 2010 issue of Infection and Immunity, ran the 1038 protein-coding sequences of T. pallidum through seven different Omp-predicting programs. They found that two proteins were predicted by all seven programs to have the β-barrel structure; another four candidates were identified by six programs.

One of the proteins at the top of the list, identified by all seven programs, was TP0326, a BamA homolog encoded by the genomes of many Gram-negative bacteria. Experiments with other bacteria have shown that BamA is a member of an outer membrane protein complex that assembles other transmembrane Omps into the outer membrane, so it would make sense for T. pallidum to possess such a protein. BamA itself is thought to be a transmembrane Omp.

This wasn't the first time that a syphilis researcher has encountered TP0326. In a study published 11 years ago, before the function of BamA was known, Caroline Cameron and colleagues demonstrated that antibodies raised against TP0326 (also called "Tp92" in their paper) stimulated macrophages to engulf T. pallidum in a process called opsonophagocytosis. In addition, TP0326 was somewhat effective as a vaccine in the rabbit model of syphilis: rabbits that had been immunized with TP0326 experienced milder skin lesions than unimmunized rabbits following inoculation of T. pallidum into the skin. These observations indirectly supported the localization of TP0326 to the outer membrane since opsonophagocytosis and effective vaccination require a target that is accessible on the surface of the spirochete. However, this earlier work lacked a more direct test such as the indirect immunofluorescence assay to confirm that TP0326 was exposed on the surface.

The problem with the standard two-step indirect immunofluorescence assay is that it is not sensitive enough to detect the rare Omps of T. pallidum. Therefore, as described in the Infection and Immunity paper, Radolf's group tinkered with the assay and managed to amplify the output signal by adding a third step to the procedure. To minimize damage to the outer membrane during the centrifugation and washing steps, the spirochetes were encased in gel microdroplets, which protected the delicate outer membrane while allowing antibodies to permeate to probe the T. pallidum surface.

With the modified immunofluorescence assay, the investigators were able to detect surface proteins with syphilitic antibodies for the first time, although only in a small minority of the spirochetes in the field of view lit up with the red color (see figure below). Presumably, the other spirochetes failed to react with the antibodies because they didn't quite have enough Omp antigens being expressed on their surface (although a more interesting explanation would be that the nonreactive spirochetes had down-regulated their surface Omps). Regardless of the true explanation, these results indicated that at least some of the antibodies generated by syphilis patients were directed against surface components of T. pallidum. When the investigators treated the spirochetes with the detergent Triton X100 to intentionally damage the outer membrane, all of the spirochetes glowed, indicating that most of the antibodies targeted proteins beneath the surface of T. pallidum. As a negative control, they demonstrated that sera from healthy patients failed to react with intact spirochetes.

To keep track of how many spirochetes were damaged by the procedure, the investigators added antibody raised against the periplasmic flagella along with the patient antibodies. The flagellar antibodies would bind to the spirochetes only if the integrity of the outer membrane was compromised by handling the spirochetes. The assay was designed so that bound flagellar antibodies would glow green.

From Figure 4 of Cox et al., 2010. (A) All spirochetes, whether or not they fluoresced, could be seen with darkfield optics (DF). Spirochetes that bound to antibodies from syphilis patients (HSS) glowed red. Spirochetes with a disrupted outer membrane reacted with the flagellar antibody (anti-FlaA) and glowed green. (B) 5.8% of the spirochetes observed were undamaged and reacted with patient antibodies (glowed red but not green). Another 5.1% were damaged (glowed red and green). 89.0% of the spirochetes failed react with the patient antibodies. 100% of the spirochetes fluoresced when treated with the detergent Triton X100 before adding the antibodies.

With an improved immunofluorescence assay, the investigators were poised to test the proteins at the top of the list for surface exposure. As I was nearing the end of the paper, I was expecting the authors to describe their test of the BamA homolog TP0326 for surface exposure. Surprisingly, they ended the paper without testing any of the proteins near the top of the list.

I can only assume that the authors are planning to submit a separate manuscript in the future describing the successful detection of TP0326 or another protein near the top of the list. But the problem with ending the paper without demonstrating surface localization of even a single protein is that one can question whether even the 3-step immunofluorescence assay is sensitive enough to detect an Omp exposed on the T. pallidum surface. They did test two proteins lower down on the list that other labs believe are surface exposed (TprK and a fibronectin-binding lipoprotein) but neither protein was detected on the outer membrane surface by the modified immunofluorescence assay. So we are left with an assay that certainly has more sensitivity, but is it sensitive enough?

To be continued...(?)

Featured paper

Cox, D.L., Luthra, A., Dunham-Ems, S., Desrosiers, D.C., Salazar, J.C., Caimano, M.J.., and Radolf, J.D. (December 2010). Surface immunolabeling and consensus computational framework to identify candidate rare outer membrane proteins of Treponema pallidum. Infection and Immunity 78(12):5178-5194. DOI: 10.1128/IAI.00834-10

Other references

Radolf, J.D. (June 1995). Treponema pallidum and the quest for outer membrane proteins. Molecular Microbiology 16(6):1067-1073.

Cameron, C.E., Lukehart, S.A., Castro, C., Molini, B., Godornes, C., and Van Voorhis, W.C. (April 2000). Opsonic potential, protective capacity, and sequence conservation of the Treponema pallidum subspecies pallidum Tp92. Journal of Infectious Diseases 181(4):1401-1413. DOI: 10.1086/315399

Cox, D.L., Akins, D.R., Porcella, S.F., Norgard, M.V., and Radolf, J.D. (1995). Treponema pallidum in gel microdroplets: a novel strategy for investigation of treponemal molecular architecture. Molecular Microbiology 15(6):1151-1164.

Image source

Smith, S.G.J, Mahon, V., Lambert, M.A., and Fagan, R.P. (August 2007). A molecular Swiss army knife: OmpA structure, function and expression. FEMS Microbiology Letters 273(1):1-11. DOI: 10.1111/j.1574-6968.2007.00778.x

Designing a Lyme disease vaccine to attack the tick vector

2011-01-01T21:42:00.000-08:00

Conventional vaccines target the surface components or secreted toxins of pathogens. Erol Fikrig's group at Yale University has been exploring an unconventional approach towards developing a vaccine for Lyme disease, which is caused by a tick-borne pathogen. Their recent work, published in the November issue of PLoS Pathogens, demonstrated partial success in protecting laboratory mice by immunization with a protein found in the saliva of the Ixodes tick vector.

Ixodes ticks spend several days feeding on blood while attached to the victim's skin. B. burgdorferi is carried into the victim's skin in the Ixodes tick's saliva starting 3-4 days (on average) after attachment. Tick saliva contains a blend of biological substances that aid the tick in drinking blood from its victim. These substances include cement proteins to keep the tick's feeding apparatus tightly bound to the skin, anti-coagulants to keep the blood flowing into the tick, and anti-inflammatory factors that ward off the local inflammatory response. The activity of these substances also promote transmission of B. burgdorferi from the feeding tick to the victim. Hence a vaccine that targets a saliva component may protect humans from Lyme disease.

A Lyme vaccine that targets the tick has a few advantages over one that targets the spirochete. First, a tick-based Lyme disease vaccine is unlikely to interfere with laboratory diagnosis, which currently relies on detection of antibodies against the Lyme Borrelia spirochete. Second, an effective vaccine that targets the tick may also prevent transmission of other pathogens carried by the Ixodes tick by interfering with tick feeding or with the tick's countermeasures against the host inflammatory response at the feeding site.

In their recent work, the investigators focused their efforts on a salivary protein called tick histamine release factor (tHRF). Because tHRF levels in the salivary glands of feeding Ixodes ticks were higher when B. burgdorferi was present in the tick, they guessed that tHRF was doing something to help transmit B. burgdorferi from the tick to the victim. The authors turned out to be correct. Transmission of B. burgdorferi was impaired when they knocked down the tick's production of tHRF by RNAi.

The investigators went on to test the vaccine potential of tHRF in their mouse model. They passively immunized mice with antiserum raised against tHRF or actively immunized the rodents with recombinant tHRF. Actively immunized mice were also given booster injections with tHRF (the paper did not say how many). Control mice were not immunized. They then challenged the mice with ticks infected with B. burgdorferi. One or three weeks later, tissues were removed from the mice, and the bacterial load of B. burgdorferi in skin, heart, and joints was measured by quantitative PCR.

Their data showed that immunization with tHRF was somewhat effective. Depending on the experiment, B. burgdorferi DNA could not be detected in any of the three tissues in 20-33% of immunized mice, whereas the spirochete's DNA was detected in at least one tissue in all control mice. Even in immunized mice with detectable B. burgdorferi DNA, the levels were often lower than the average level found in the control mice. It would have been nice to know how much inflammation was present in the tissues of the immunized mice. Unfortunately, the histopathology of the tissues was not presented in the paper.

Figure 4, panels E-G from Dai et al., 2010. Bacterial burden in skin (day 7 after challenge) and joint and heart (day 21) was determined by quantitative PCR with flaB primers. Horizonal lines represent the mean value ± SEM. * p < 0.05 and ** p < 0.01. Results were pooled from 3 independent experiments.

tHRF is not the first Lyme vaccine candidate to target a protein found in tick saliva. An earlier report from Fikrig's group demonstrated that active and passive immunization with Salp15, another tick salivary protein, was also somewhat effective in protecting mice from colonization with B. burgdorferi. tHRF was superior to Salp15 in impairing feeding by ticks. Ticks feeding on Salp15-immunized mice were able to complete their blood meal. In contrast, most of the ticks had a hard time feeding on mice immunized with tHRF and could not complete their blood meal, as assessed by tick weights following detachment from the mice.

Although immunization with tHRF and Salp15 prevented colonization in only some mice, Fikrig's work shows for the first time that it may be possible to design a Lyme disease vaccine that targets the tick vector. Ultimately, the most effective vaccine may be a mixture that targets multiple components in both the tick and the spirochete.

References

Dai, J., Narasimhan, S., Zhang, L., Liu, L., Wang, P., & Fikrig, E. (2010). Tick histamine release factor is critical for Ixodes scapularis engorgement and transmission of the Lyme disease agent PLoS Pathogens, 6 (11) DOI: 10.1371/journal.ppat.1001205

Dai, J., Wang, P., Adusumilli, S., Booth, C.J., Narasimhan, S., Anguita, J., & Fikrig, E. (2009). Antibodies against a tick protein, Salp15, protect mice from the Lyme disease agent. Cell Host & Microbe, 6 (5), 482-492 DOI: 10.1016/j.chom.2009.10.006

Related post

A fresh approach towards a Lyme disease vaccine: targeting the tick

How bacteria sort their lipoproteins (Lol!)

2010-11-28T22:18:00.000-08:00

Bacterial lipoproteins are proteins with covalently-attached lipid molecules that anchor the protein to the cytoplasmic or outer membrane. The lipid molecules are attached to the cysteine located at the amino terminus of the lipoprotein. The lipoprotein's protein component, being hydrophilic (water-loving), sticks out from the membrane. Different bacterial lipoproteins participate in a variety of functions, including transport of molecules, stabilization of the cell wall, signal transduction, motility, and interaction with host molecules.

The Lyme disease spirochete Borrelia burgdorferi is exceptional in that a number of different lipoproteins have been found on its surface. Most other bacteria lack (or have few) surface-exposed lipoproteins. To give one example, the figure below shows the arrangement of lipoproteins in the cell envelope of E. coli, home to roughly 90 lipoproteins, none known to be displayed on the surface. Lipoproteins are depicted as colored ovals with the attached squiggles representing the lipid molecules. To perform their functions properly, some lipoproteins must be anchored to the outer leaflet of the inner membrane (blue ovals) whereas the rest must be anchored to the inner leaflet of the outer membrane (red ovals). In both cases, the protein component of the lipoprotein protrudes into the periplasm. The figure also shows the other major category of membrane proteins, the integral membrane proteins, which are embedded in the membrane. There are also proteins that reside in the periplasm, which are not depicted in the figure.

Figure 1 of Narita and Tokuda (2010)

In this post I will describe how lipoproteins are brought to their correct location in the bacterial envelope. I will first describe how lipoproteins are sorted in E. coli since that's where most of the earlier work was conducted. Since many other diderms (bacteria having two membranes) have homologs of the proteins used by E. coli to export and sort lipoproteins, E. coli is a good model for studying localization of lipoproteins. Monoderm bacteria also have lipoproteins, but since they have only one membrane, they don't need to worry about sorting lipoproteins. (I will save the explanation of how lipoproteins get to the bacterial surface for a future post.)

Most proteins to be exported out of the cytoplasm are marked with an amino-terminal signal peptide ≈20 amino acids in length. The sequences of the signal peptides (plus five additional amino acid residues) from two E. coli lipoproteins are shown below. A cytoplasmic membrane protein complex called the Sec translocon transfers proteins harboring the signal peptide to the periplasm, where the signal peptide is lopped off by one of two signal peptidases. Signal peptidase I cleaves off the signal peptide from nonlipoproteins (such as periplasmic or transmembrane outer membrane proteins), and signal peptidase II slices off the signal peptide from lipoproteins.

All lipoproteins harbor a short sequence called a "lipobox" at the end of the signal peptide (underlined in sequences below). The lipobox consensus sequence is -(leu, ala, val)_-4-leu_-3-(ala, ser)_-2-(gly, ala)_-1↓cys₊₁, with the arrow specifying the cleavage site for signal peptidase II and the subscripts denoting positions relative to the cleavage site.

E. coli Braun's lipoprotein (OM) MKATKLVLGAVILGSTLLAG↓CSSNA...

E. coli lpp-28 (IM)           MKLTTHHLRTGAALLLAGILLAG↓CDQSS...

(IM, inner membrane; OM, outer membrane)

The lipobox is recognized by the inner membrane enzyme phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt). Before the signal peptide is removed, Lgt attaches diacylglycerol to the sulfhydryl (-SH) of the lipobox cysteine. After the signal peptide is cleaved off by signal peptidase II, another inner membrane enzyme, apolipoprotein N-acyltransferase (Lnt), attaches a fatty acid molecule to the newly exposed amino (-NH₃) group of the cysteine. Only exported proteins with lipoboxes become lipidated. The lipoprotein remains associated with the inner membrane throughout these processing steps.

The machinery responsible for sorting lipoproteins to the outer membrane is the LolCDE protein complex, a type of ABC transporter that sits in the inner membrane. Lol stands for lipoprotein outer membrane localization. LolCDE recognizes the lipidated cysteine at the amino terminus of lipoproteins. LolCDE loads lipoproteins onto the periplasmic protein LolA, which ferries lipoproteins to the LolB receptor, a lipoprotein that protrudes from the periplasmic face of the outer membrane. After capturing the lipoprotein from LolA, LolB anchors the lipoprotein into the periplasmic layer of the outer membrane.

from figure 3 of Tokuda and Matsuyama (2004)

How does LolCDE know which lipoproteins are supposed to be delivered to the outer membrane and which need to stranded in the inner membrane? For E. coli and other members of the Enterobacteriaceae family of bacteria, the answer is fairly simple. Lipoproteins with aspartate at the +2 position (which follows the lipidated cysteine) remain in the inner membrane.

How does the +2 aspartate prevent transfer of lipoproteins to the outer membrane? It turns out that LolCDE doesn't directly sense the amino acid at the +2 position. Instead, the abundant membrane phospholipid phosphatidylethanolamine (PE) is thought to interfere with LolCDE recognition of the lipidated cysteine when asparatate is at the +2 position. When the side chain carboxyl group (-COO^-) of the +2 aspartate interacts electrostatically with the positively-charged head group of PE, the fatty acids of PE become perfectly positioned to form hydrogen bonds with the lipid molecules attached to the cysteine (see figure below). LolCDE is unable to recognize the amino-terminal cysteine associated with five fatty acid groups (three covalently bound to the cysteine and two from PE). Thus asparatate, when it follows the cysteine, acts indirectly as a Lol avoidance signal. The amino acid at the +3 position can also influence the Lol avoidance signal. For example, negatively-charged amino acids (aspartate and glutamate) at the +3 position strengthen the +2 aspartate Lol avoidance signal by stabilizing the complex between phosphotidylethanolamine and the +2 aspartate (see figure below).

Modified from figure 6 of Tokuda and Matsuyama (2004)

Additional studies with engineered lipoproteins have shown that phenylalanine, tryptophan, tyrosine, lysine, and proline, although rarely found in lipoproteins at the +2 position, can also serve as inner membrane retention signals when asparagine is at the +3 position. Since none of these are negatively-charged amino acids, the mechanism for avoiding LolCDE must differ from those lipoproteins having aspartate at the +2 position.

The nature of the sorting signal differs for bacteria that are not members of Enterobacteriaceae. For example, the three amino acids at positions +2 through +4 dictate whether lipoproteins will remain in the inner membrane of Pseudomonas aeruginosa. For the spirochete B. burgdorferi, a clear rule has yet to emerge from the few studies that have been done. What can be said is that negatively-charged amino acids (aspartate and glutamate) placed within the first several amino acids following the lipidated cysteine sometimes allows the lipoprotein to remain in the membrane. Whether the negatively-charged amino acid functions as an inner membrane retention signal depends on which amino acids are surrounding it. It is not yet possible to simply look at the amino-terminal sequence of B. burgdorferi lipoproteins and confidently predict in which membrane they will be found.

Although the "+2/+3/+4 rule" is useful for predicting whether a newly discovered lipoprotein will be found in the inner or outer membrane, it may not give the complete picture of all of a lipoprotein's features that govern its localization. The rules for sorting lipoproteins were worked out primarily by examining the localization of engineered fusion proteins consisting of the amino termini of lipoproteins (signal peptide with lipobox plus the first several amino acids following the lipobox cysteine) fused to unrelated reporter proteins such as red fluorescent protein (RFP) from corals. For example, placing asp at the +2 position of such a fusion protein would cause RFP to be retained in the inner membrane. Changing the +2 amino acid to serine would cause RFP to be transported to the outer membrane. However, localization of a full-length lipoprotein may not be altered by simply changing its +2 amino acid from aspartate to another amino acid or vice versa. This indicates that the rest of the lipoprotein, the part that's removed when reporters are used, also influences the localization of lipoproteins.

References

TOKUDA, H. (2009). Biogenesis of outer membranes in Gram-negative bacteria. Bioscience, Biotechnology, and Biochemistry, 73 (3), 465-473 DOI: 10.1271/bbb.80778

TOKUDA, H. (2004). Sorting of lipoproteins to the outer membrane in E. coli. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1693 (1), 5-13 DOI: 10.1016/j.bbamcr.2004.02.005

Schulze, R., & Zückert, W. (2006). Borrelia burgdorferi lipoproteins are secreted to the outer surface by default. Molecular Microbiology, 59 (5), 1473-1484 DOI: 10.1111/j.1365-2958.2006.05039.x

The major outer membrane protein of Leptospira interrogans: Not essential for infection?

2010-08-01T01:22:00.000-07:00

Because leptospirosis is a potentially fatal disease, it would be worthwhile to figure out which of the many genes on the two chromosomes of Leptospira express products that are essential for infection.

The lipoprotein LipL32 is the most abundant outer membrane protein found in the outer membrane of pathogenic species of Leptosipra. It's been assumed that LipL32 plays an important role in infections for the following reasons:

LipL32 is found only in pathogenic species of Leptospira. Nonpathogenic species such as L. biflexa lack the gene encoding LipL32.
LipL32 peaks out on the surface of Leptospira, where it is available to interact directly with host molecules.
LipL32 binds (at least weakly) to several components of the extracellular matrix.
Leptospirosis patients generate a strong antibody response against LipL32.
The protein sequence of LipL32 among different species of Leptospira is almost identical.
A lot of metabolic energy must be expended to make the large amounts of LipL32 found in the spirochete.

Although a lipL32 knockout mutant would help scientists figure out whether LipL32 plays an essential role in pathogenesis, targeted gene disruptions are extremely difficult with pathogenic Leptospira. Fortunately, Ben Adler's group at Monash University obtained an insertion mutation in the lipL32 gene of L. interrogans by transposon mutagenesis. This gave the Australians and their collaborators an opportunity to test the role of LipL32 in causing lethal infections in the hamster model of leptospirosis. They first confirmed that the lipL32 mutant failed to express LipL32 by Western blotting the mutant with LipL32 antiserum. I am showing the Coomassie-blue stained protein gel of the whole-cell lysate below so that you can appreciate the abundance of LipL32. It is the most intensely stained band in the control L. interrogans strain, which has its lipL32 gene intact.

Whole-cell lysates of the L. interrogans lipL32 mutant (M933) and a control strain with the transposon in an intergenic region (M777) were run into SDS-acylamide gels and stained (panel A) or analyzed by Western blotting with LipL32 antiserum (panel B). The M777 strain was demonstrated in an earlier study to be lethal for hamsters. (Figure 1 from Murray et al., 2009.)

The survival curves show that the L. interrogans lipL32 mutant was just as lethal to hamsters as the parent L. interrogans with its lipL32 gene intact, irrespective of the infection route. Hence, lipL32 is not necessary for lethal infections of hamsters, at least under the conditions used in this study.


Panel A: Groups of 8 hamsters were inoculated with 1,000 leptospires into the abdominal cavity. Panel B: Groups of 10 hamsters were inoculated with 10⁶ leptospires dropped into the eye. The slight difference in the survival curves was not statistically significant. (Figure 5 from Murray et al., 2009)

Rats are the natural reservoir hosts of L. interrogans. They can carry the spirochete for years in their kidney tubules without showing any signs of illness. LipL32 could have a role in chronic infections. The investigators therefore tested the ability of the lipL32 mutant to establish a chronic infection in laboratory rats. They found that the lipL32 mutant (M933) was able to colonize the rat kidneys as well as the control M777 strain. Kidneys from all 8 rats inoculated with the lipL32 mutant were culture positive.

At first glance it's surprising that lipL32 was not required for acute or chronic infection. The authors pointed out that the function of LipL32 could be copied by other proteins found on the surface of Leptospira. However, the study could have been strengthened by making two changes. First, since the authors were trying to determine whether lipL32 was necessary for chronic infection, the rats should have been allowed to live for at least a few months before their kidneys were cultured. Instead, the infection was allowed to proceed for only 15 days before the rats were sacrificed. Second, they should have measured the bacterial load in the rat kidneys, either by plating serial dilutions of the kidney homogenates for colonies (although I don't know if this is feasible for Leptospira) or by quantitative PCR. Clearly, more work needs to be done before anyone can conclude that LipL32 is not essential for chronic infection.

Featured paper

Murray G.L., Srikram, A., Hoke, D.E., Wunder Jr., E.A., Henry, R., Lo, M., Zhang, K., Sermswan, R.W., Ko, A.I., and Adler, B. (March 2009). Major surface protein LipL32 is not required for either acute or chronic infection with Leptospira interrogans. Infection and Immunity 77(3):952-958. DOI: 10.1128/IAI.01370-08

Related posts

Antigen presentation in the bloodstream: How invariant NKT cells are activated by Lyme disease spirochetes

2010-07-24T17:30:00.000-07:00

The spirochete Borrelia burgdorferi is the tick-borne agent of Lyme disease, which affects the joints, nervous system, and heart. After being deposited into the skin by an infected tick, the spirochete must enter the bloodstream so that it can circulate in the blood to gain access to its target organs.

The host doesn't sit idly as B. burgdorferi establishes an infection. Invariant natural killer (iNKT) cells are one of the tools deployed by the immune system in its battle against the Lyme spirochetes. Scientists know this because B. burgdorferi-infected mice lacking iNKT cells ended up with more spirochetes in their tissues and greater joint swelling than mice with a complete immune system.²

iNKT cells are an odd type of T cell. Like other T cells, iNKT cells have a T cell receptor (TCR), yet they also express protein markers used to identify natural killer (NK) cells. What makes the iNKT cell invariant is its TCR α chain, which comes only in the version dubbed Vα14 in mice and Vα24 in humans. Even the β chain of the TCR of iNKT cells is restricted to three types in mice and just one in humans. The lack of variation is unusual because the α and β TCR chains of conventional αβ T cells come in many forms in each individual, resulting in millions of varieties of TCRs. This enables conventional αβ T cells to recognize a wide range of microbial peptide antigens when displayed by an MHC molecule on the surface of an antigen-presenting cell (see figure below). In contrast, the TCRs of iNKT cells recognize a limited set of glycolipids displayed by the antigen-presenting cell's CD1d molecule, which structurally resembles MHC. So far these glycolipids have been found only in Sphingomonas and B. burgdorferi.

Antigen recognition by T cells. The "X" represents variable T cell receptor chains.
Figure 1 from ref. 3.

The structures of the B. burgdorferi glycolipids recognized by iNKT cells are shown below. BbGL-IIc is recognized by mouse iNKT cells, and BbGL-IIf reacts with human iNKT cells.⁴

Structures of B. burgdorferi glycolipid antigens recognized by iNKT cells. Figure 3d from ref. 3.

iNKT cells are activated when their TCR binds to BbGL-II complexed with CD1d.⁴ The activated iNKT cells secrete cytokines that elicit the appropriate immune response against the spirochetes. How these cytokines promote killing of B. burgdorferi remains unknown.

To view the process of iNKT cell activation, scientists have recently obtained video footage of the early stages of the immune response to Borrelia burgdorferi circulating in the bloodstream of mice.¹ The study by Lee et al., which appeared in the April issue of Nature Immunology, complements two earlier studies that revealed how the Lyme disease spirochete escapes from the bloodstream of mice to invade the surrounding tissues.^5,6

The investigators employed fluorescence video microscopy to watch the immune cells in action following injection of an engineered B. burgdorferi strain expressing green fluorescent protein (GFP) into the bloodstream. Although the spleen is better known for filtering bloodstream pathogens, the liver was selected for observation because iNKT cells make up 30% of the T cells in the liver. In contrast, iNKT cells represent only 2.5% of T cells in the spleen. Moreover, mice missing their spleen were able to limit B. burgdorferi infection as well as mice having a spleen, suggesting that the spleen is not critical in fighting bloodstream B. burgdorferi.

iNKT cells reside in the liver's sinusoids, which are the specialized capillaries that carry blood through the liver. Similar to what other investigators have observed, the authors saw iNKT cells creeping along the inner surface of the liver sinusoids in healthy mice (see video below).

iNKT cells crawling within the liver sinusoids of a mouse genetically altered to express green fluorescent protein (GFP) in iNKT cells. The iNKT cells glow bright green. The elapsed time is shown at the top right. Video 2 from ref. 1.

The investigators wanted to figure out which of the antigen-presenting cells found in the liver presented borrelial glycolipid to iNKT cells. The answer? After the spirochetes were injected into the bloodstream, they were quickly captured by Kupffer cells, the specialized blood-filtering macrophages that also reside in the liver sinusoids (see figure below). Unlike iNKT cells, Kupffer cell remained stationary.

Capture of fluorescent B. burgdorferi (thin green bodies) by Kupffer cells (arrowhead). Kupffer cells are stained red. B. burgdorferi that avoided capture can be seen bound to the endothelium, trying to escape from the bloodstream into the liver tissue (arrow). Figure 2e from ref. 1.

During the next several hours, the captured spirochetes were engulfed and broken up by the Kupffer cells so that BbGL-II could be loaded onto CD1d and displayed on the cell surface. At 8 hours post injection, iNKT cells started to cluster and form stable contacts with Kupffer cells. The iNKT cells were attracted to Kupffer cells churning out the chemokine CXCL9, a potent iNKT cell attractant. The evidence for this was that injection of antibodies against the CXCL9 receptor, located on the iNKT cell surface, blocked clustering of iNKT cells. Interaction of the Kupffer and iNKT cells was accompanied by increased blood and liver levels of the cytokine IFN-γ (interferon-gamma), a sign that the iNKT cells were being activated.

Left panel: Liver 24 hours after injection of a GFP+ strain of B. burgdorferi into the bloodstream of a mouse. Arrows indicate spirochetes (thin green bodies) that were not captured. Kupffer cells are stained a red. The iNKT cells are the large bright green bodies. The bright iNKT clusters overwhelm the faint red Kupffer cells, which are difficult to see. Right panel: To obtain a more convincing image showing contact between Kupffer cells and iNKT cells, a 3D reconstruction of the optical sections through the liver was performed. Rotation of the image reveals interactions between Kupffer and iNKT cells.

Not all spirochetes were captured. The investigators saw B. burgdorferi escaping from the sinusoids into the surrounding liver tissue even as other spirochetes were trapped by nearby Kupffer cells (see figures above). Spirochetes circulating throughout the host probably escaped into other organs in the same manner. Indeed, large amounts of B. burgdorferi DNA were detected by PCR in several organs, including the liver, three days after the spirochetes were injected. Although one doesn't usually think about the effects of Lyme disease on the liver, the authors pointed out that a mild hepatitis is common in Lyme disease patients. In one prospective study, 40% of Lyme disease patients had at least one liver test abnormality.⁷

By now you may be wondering why the liver would devote such a high percentage of its T cells towards recognizing glycolipids that aren't found on most bacteria. One answer is that microbes lacking the proper glycolipids may activate iNKT cells indirectly.³ For example, Salmonella typhimurium uses its LPS to coax antigen-presenting cells into making an endogenous glycolipid that gets presented to the iNKT cell by CD1d.⁸ It is also possible that glycolipids that are recognized by iNKT cells are present in other bacteria but are yet to be discovered.

Featured paper

1. Lee, W.Y., Moriarty, T.J., Wong, C.H.Y., Zhou, H., Strieter, R.M., van Rooijen, N., Chaconas, G., & Kubes, P. (2010). An intravascular immune response to Borrelia burgdorferi involves Kupffer cells and iNKT cells Nature Immunology, 11 (4), 295-302 DOI: 10.1038/ni.1855

Other references

2. Tupin, E., Benhnia, M.R., Kinjo, Y., Patsey, R., Lena, C.J., Haller, M.C., Caimano, M.J., Imamura, M., Wong, C., Crotty, S., Radolf, J.D., Sellati, T.J., and Kronenberg, M. (2008). NKT cells prevent chronic joint inflammation after infection with Borrelia burgdorferi. Proc. Natl. Acad. Sci. USA 105(50):19863-19868. DOI: 10.1073/pnas.0810519105

3. Tupin, E., Kinjo, Y., and Kronenberg, M. (2007). The unique role of natural killer T cells in the response to microorganisms. Nature Reviews Microbiology 5(6):405-417. DOI: 10.1038/nrmicro1657

4. Kinjo, J., Tupin, E., Wu, D., Fujio, M., Garcia-Navarro, R., Benhnia, M. R., Zajonc, D.M., Ben-Menachem, G., Ainge, G.D., Painter, G.F., Khurana, A., Hoebe, K., Behar, S.M., Beutler, B., Wilson, I.A., Tsuji, M., Sellati, T.J., Wong, C., and Kronenberg, M. (2006). Nature Immunology 7(9):978-986. DOI: 10.1038/ni1380

5. Moriarty, T.J., Norman, M.U., Colarusso, P., Bankhead, T., Kubes, P., and Chaconas, G. (June 20, 2008). Real-time high resolution 3D imaging of the Lyme disease spirochete adhering to and escaping from the vasculature of a living host. PLoS Pathogens 4(6):e1000090. DOI: 10.1371/journal.ppat.1000090

6. Norman, M.U., Moriarty, T.J., Dresser, A.R., Millen, B., Kubes, P., and Chaconas, G. (October 3, 2008). Molecular mechanisms involved in vascular interactions of the Lyme disease pathogen in a living host. PLoS Pathogens 4(10):e1000169. DOI: 10.1371/journal.ppat.1000169

7. Horowitz, H.W., Dworkin, B., Forseter, G., Nadelman, R.B., Connolly, C., Luciano, B.B., Nowakowski, J., O'Brien, T.A., Calmann, M., Wormser, G.P. (June 1996). Liver function in early Lyme disease. Hepatology 23(6):1412-1417. DOI: 10.1002/hep.510230617

8. Mattner J., DeBord, K.L., Ismail, N., Goff, R.D., Cantu III, C., Zhou, D., Saint Mezard, P., Wang, V., Gao, Y., Yin, N, Hoebe, K., Schneewind, O., Walker, D., Beutler, B., Teyton, L, Savage, P.B., and Bendelac, A. (March 24, 2005). Exogenous and endogenous glycolipid antigens activate NKT cells during microbial infections. Nature 434(7032):525-529. DOI: 10.1038/nature03408

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Congenital syphilis, upward trend (again) in the United States

2010-06-22T11:43:00.000-07:00

Syphilis can be deadly if passed from an infected mother to her unborn child. The most recent CDC data show that 6.5% of U.S. infants with congenital syphilis (CS) in 2008 were stillborn or died within 30 days of birth.¹

Newborns with CS who are destined to live begin to show signs of disease within the first few weeks of life. The main features of CS in early infancy include fever, skin lesions, enlarged liver and spleen, and a chronic runny nose ("snuffles"), which may be tinged with blood. Bone lesions may lead to Parrot's pseudoparalysis, a condition so painful that the infant will refuse to move the affected extremities. Ongoing damage to bony tissue may later lead to childhood deformities including saddle nose, sabre shins, and Hutchinson incisors (notched central incisors). Other late signs of CS include inflammation of the cornea and sudden hearing loss.

The lesions and deformities associated with congenital syphilis are sparked by Treponema pallidum, a spirochete that can cross the placenta from the mother's bloodstream. The probability of transmission to the fetus depends on how long the mother has been infected with T. pallidum. The risk of transmission is lower in mothers at later stages of syphilis. After crossing the placenta, the spirochete invades the fetal organs. The continuing immune response to persistent T. pallidum infection causes the damage seen in CS. Early treatment of the mother with penicillin, at least 30 days before delivery, is essential to stop the disease.

The rate of congenital syphilis in the United States has started to creep back up after plummeting over two decades.¹ The incidence of congenital syphilis has gone up from 8.2 cases per 100,000 live births in 2005 to 10.1 in 2008 with most of the increase having occurred in the South. CS rates in infants born to black mothers have gone up from 26.6 in 2005 to 34.6 per 100,000 live births in 2008 and now account for half of all CS cases. Since CS is transmitted from mothers with syphilis, CS rates have historically tracked the combined primary and secondary syphilis rate seen in women, which has also started to climb (see figure below). What factors account for the increased incidence of syphilis? In one Alabama county, increased syphilis rates in black women were linked to crack cocaine use and the exchange of sex for money or drugs.² Although more studies are needed to determine whether the same factors are linked to syphilis throughout the South, it should be pointed out that the same factors were associated with the previous syphilis epidemic that peaked in the early 1990s, when there were several thousand yearly cases of CS as opposed to the several hundred seen today.³

Figure from CDC¹

Now that the upward trend in the CS rate has been recognized, public health authorities in partnership with community-based groups must allocate some of their scarce resources to reverse the trend. With prenatal care and prompt treatment, congenital syphilis can be prevented.

1. Centers for Disease Control and Prevention (CDC) (April 16, 2010). Congenital syphilis - United States, 2003-2008. MMWR Morbidity and Mortality Weekly Report 59(14):413-417. link

2. Centers for Disease Control and Prevention (CDC) (May 8, 2009). Primary and secondary syphilis - Jefferson County, Alabama, 2002-2007. MMWR Morbidity and Mortality Weekly Report 58(17):463-467. link

3. Nakashima, A.K., Rolfs, R.T., Flock, M.L., Kilmarx, P., and Greenspan, J.R. (Jan-Feb 1996). Epidemiology of syphilis in the United States, 1941-1993. Sexually Transmitted Diseases 23(1):16-23. PMID: 8801638

Healthy human carriers of the spirochete Leptospira in the Peruvian Amazon

2010-04-30T23:49:00.000-07:00

The spirochete Leptospira is the agent of leptospirosis, a zoonosis that primarily burdens tropical regions of the world. Moist conditions promote the survival of Leptospira in soil and fresh water. Although Leptospira could survive out in wet environments if they had to, they thrive in the kidneys of rats and other maintenance hosts, where they form dense masses lining the inner surface of the kidney tubules. The spirochetes spill into the urine that forms in the tubules, which drain into the bladder. Animals colonized by their "preferred" serovar (immune type) shed Leptospira throughout their lives without ever showing signs of illness. The tainted urine ends up contaminating soil and water with infectious Leptospira.

Humans aren't regarded as long-term carriers of Leptospira. Rather, they are deemed "accidental" (incidental) hosts who may suffer serious complications of acute disease, including kidney failure and lung hemorrhage. Humans get infected when they come into contact with contaminated water or soil or following direct exposure to infectious animal urine or tissue. Leptospira enters through cuts in the skin or mucous membranes. From there the motile spirochete spreads via the bloodstream and invades internal organs, including the kidneys, where they remain for the duration of the disease. Patients typically stop releasing Leptospira into their urine after they recover from the illness, presumably because the spirochetes have been eliminated from their kidneys. However, there have been a few reports of Leptospira excreted in urine months or even years following recovery from leptospirosis. The truth is that no one has ever done a systematic study to determine how common the chronic carrier state is in humans.

A team of investigators from the United States and Peru set out to find long-term carriers of Leptospira. Their study is described in the February issue of PLoS Neglected Tropical Diseases. The authors examined the inhabitants of a rural Amazon village of Padrecocha near the city of Iquitos, Peru, where leptospirosis is endemic. The tropical climate is ideal for the survival of Leptospira in the moist environment favored by the spirochete. Indeed, in an earlier study the authors detected infectious strains of Leptospira in the streams and wells serving the village. Cattle, pigs, dogs, and rats, all potential carriers, freely roam the area.

Ganoza and colleagues wanted to determine what percentage of the villagers were chronic carriers of Leptospira. They first identified villagers who were not recently infected with Leptospira. Out of the 314 healthy villagers enrolled in the study, 102 (32.5%) had no clinical or serological evidence of recent infection; they did not recall experiencing a fever during the previous year (fever is a typical symptom of leptospirosis), and they tested negative for newly-acquired Leptospira infection by IgM ELISA.

The investigators next identified those whose kidney were colonized by Leptospira among the 102 who were not newly infected. Since Leptospira living in the kidney tubules are shed into urine, they screened urine samples by nested PCR using primers targeting the 16S rRNA gene of Leptospira. To exclude false-positive signals, the investigators screened the PCR-generated DNA (amplicon) by dot blot analysis with a Leptospira 16S rRNA probe. Many false positive signals occurred because their Leptospira PCR primers also hybridized to the 16S rRNA gene from Atopobium vaginae, a bacterium recently found to be associated with vaginosis.

When urine from the 102 "long-term" healthy individuals was screened, Leptospira DNA was found in 6 (5.9%). Sequencing of the 16S rRNA gene revealed that the carriers were colonized with L. interrogans, L. fainei, and L. licerasiae. So it turns out that the asymptomatic carrier state is not as rare as initially believed. More than 1 in 20 individuals who had been healthy for at least a year were colonized with Leptospira in their kidneys.

The investigators found seven additional individuals colonized with Leptospira by screening urine from the other 212 individuals in the study. Overall the percentage of shedders of Leptospira among all healthy individuals, irrespective of when they were infected, was 4.1% (13/314). The concentration of Leptospira in the urine of shedders, as measured by quantitative PCR, was low, in the 10²-10⁴/ml range. In contrast, rats may shed up to 10⁸ spirochetes/ml!

The study unearthed another surprise. All 13 individuals who were shedding Leptospira at the time of the study (including the 6 chronic carriers) were women. The proportion of women with Leptospira DNA in their urine (13/13, 100%) was significantly higher than the proportion of women in the group lacking detectable DNA in their urine (199/301, 66%, p = 0.011). This result raises the possibility that women are more likely to become persistent carriers than men. However, the authors pointed out that men were underrepresented in the study sample. Less than one third of the villagers enrolled in the study were men. Most of the other men were away at work when the authors were recruiting people for the study. Agricultural occupations, which bring workers into contact with environmental sources of Leptospira, are well-known risk factors for infection by the spirochete in endemic areas. Hence, male shedders of Leptospira may have been inadvertently excluded from the study.

Another surprising result was that sera from all six chronic carriers failed to agglutinate Leptospira by MAT (microscopic agglutination test), a standard serological test used to check for Leptospira infection whether it occurred recently or years ago. The authors mentioned that this was entirely consistent with old studies failing to detect agglutinating antibodies in the sera of some maintenance host animals excreting Leptospira. However, another possibility is that the "chronic human carriers" may have actually acquired asymptomatic infections very recently. They could have been enrolled in the study before the anti-Leptospira IgM and agglutinating antibodies had enough time the accumulate to the cut-off values selected for the IgM ELISA and MAT, respectively. Although the authors discounted the possibility of newly acquired asymptomatic infections accounting for the seronegativity of the shedders, they recommended a longitudinal study to clarify the issue.

The authors posed several questions raised by their study:

Does persistent Leptospira infection of human kidneys have any subtle effect on their function? If so, is antibiotic treatment warranted?
Are some strains of Leptospira more likely than others to persistently infect the kidneys of humans? .
Can persistent human shedders be a source of transmission of Leptospira to other humans (and animals)?

Future studies will need to include urine cultures to demonstrate that Leptospira shed by human carriers are alive.

In conclusion, this is an important study that challenges the simplistic notion that humans are incidental hosts of Leptospira. The reality appears to be more complicated.

Featured paper

Ganoza, C.A., Matthias, M.A., Saito, M., Cespedes, M., Gotuzzo, E., & Vinetz, J.M. (2010). Asymptomatic renal colonization of humans in the Peruvian Amazon by Leptospira. PLoS Neglected Tropical Diseases, 4 (2) DOI: 10.1371/journal.pntd.0000612

Related paper

Ganoza, C.A., Matthias, M.A., Collins-Richards, D., Brouwer, K.C., Cunningham, C.B., Segura, E.R., Gilman, R.H., Gotuzzo, E., & Vinetz, J.M. (2006). Determining risk for severe leptospirosis by molecular analysis of environmental surface waters for pathogenic Leptospira. PLoS Medicine, 3 (8) DOI: 10.1371/journal.pmed.0030308

A fresh approach towards a Lyme disease vaccine: targeting the tick

2010-03-31T23:53:00.000-07:00

We tend to focus on the pathogen when thinking about how the immune system responds to tick-borne infections. Ticks also provoke an immune response, even those that don't harbor any infectious agent. Animals that are repeatedly bitten by ticks will eventually develop immunity to the tick. There's even a commercial tick vaccine called TickGARD, which protects cattle against infestation by the tick Boophilus microplus. TickGARD is formulated with a protein located in the midgut of B. microplus.

Animals that are immune to ticks also resist infection by some tick-borne pathogens, including Borrelia burgdorferi (Nazario et al., 1998). Vaccines designed from a single tick component may also protect host animals from infectious agents. A tick vaccine formulated with the tick cement protein 64TRP protected mice from being killed by the tick-borne encephalitis virus introduced by its vector, Ixodes ricinus (Labuda et al., 2006).

These earlier studies prompted Erol Fikrig's group at Yale to devise a vaccine that targets an Ixodes tick protein required by B. burgdorferi for a successful infection. They focused on Salp15, a protein found in tick saliva. Salp15 binds to the B. burgdorferi surface protein OspC as the spirochete passes through the salivary gland on its way into the skin of the victim. Salp15 is one of the many bioactive salivary proteins that dampen the immune system to allow the tick to remain attached for several days so that it could complete its blood meal. B. burgdorferi exploits Salp15 to fend off the immune response in the early stages of infection. Since Salp15 coats B. burgdorferi, a vaccine targeting Salp15 could make B. burgdorferi vulnerable to killing by the immune system. Indeed, Salp15 antiserum was able to enhance phagocytosis of Salp15-coated B. burgdorferi by mouse macrophages in vitro.

Despite the promising in vitro results, Salp15 as a vaccine was only partially protective in animal studies. 55-60% of mice actively immunized with Salp15 or passively immunized with Salp15 antiserum ended up infected with B. burgdorferi following challenge with infected Ixodes scapularis ticks. Moreover, ticks were able to feed normally on mice immunized with Salp15, indicating that the animals did not acquire tick immunity.

Where Salp15 shined was in improving the efficacy of another Lyme vaccine. The OspA vaccine requires several doses to achieve maximum protection against B. burgdorferi. When Salp15 was combined with OspA, a single dose of the mixture spared 70% of mice from infection, whereas only 10-20% of mice immunized with a single dose of OspA or OspA plus Salp25D (an irrelevant tick salivary protein) were protected. Thus future Lyme disease vaccines that target a component of the spirochete could also include Salp15 to enhance their protective capacity.

Scientists are undoubtedly examining other tick proteins as potential vaccines against ticks and the pathogens they transmit.

Featured paper

Dai, J., Wang, P., Adusumilli, S., Booth, C.J., Narasimhan, S., Anguita, J., and Fikrig, E. (November 19, 2009). Antibodies against a tick protein, Salp15, protect mice from the Lyme disease agent. Cell Host & Microbe 6:482-492. DOI: 10.1016/j.chom.2009.10.006

Other references

Labuda, M., Trimnell, A.R., Licková, M., Kazimírová, M.,Davies, G.M., Lissina, O., Hails, R.S., and Nuttall, P.A. (April 2006). An antivector vaccine protects against a lethal vector-borne pathogen. PLoS Pathogens 2(4):e27. DOI: 10.1371/journal.ppat.0020027

Nazario, S., Das, S., De Silva, A.M., Deponte, K., Marcantonio, N., Anderson, J.F., Fish, D., Fikrig, E., and Kantor, F.S. (June 1998). Prevention of Borrelia burgdorferi transmission in guinea pigs by tick immunity. American Journal of Tropical Medicine and Hygiene 58(6):780-785. Link

Tigecycline fails to eradicate persisting Borrelia burgdorferi

2010-03-20T20:03:00.000-07:00

Antibiotics are usually successful in treating Lyme disease, especially if administered early. The problem is that some patients continue to experience symptoms even after completing the recommended treatment regimen. Although the current IDSA guidelines assert that the lingering symptoms are not due to persisting Borrelia burgdorferi, the mouse model of Lyme disease clearly demonstrates the survival of live (albeit disabled) spirochetes following treatment with ceftriaxone, one of the antibiotics used to treat disseminated Lyme disease. As I wrote in an earlier post, the key question that must be answered is whether the lingering spirochetes are responsible for the persisting symptoms. If so, a more potent antibiotic that could eliminate all of the spirochetes (or enough of them to allow the immune system to quickly mop up the rest) would be desired.

The newer antibiotic tigecycline was recently approved for treating skin and intra-abdominal infections caused by complex mixtures of bacteria. Tigecycline is a tetracycline antibiotic, which blocks translation of mRNA into proteins by sticking tightly to the 30S ribosome subunit of bacteria. In turns out that tigecycline exhibits greater antimicrobial activity than ceftriaxone (and doxycycline, another Lyme antibiotic) against B. burgdorferi, at least in the test tube.

Barthold and colleagues tested tigecycline to see if it could eradicate B. burgdorferi from persistently infected mice. Groups of mice infected for 4 months with B. burgdorferi were treated with ceftriaxone (10 mice), a low dose of tigecycline (7 mice), or a high dose of tigecycline (9 mice). A control group was sham treated with saline. Three months after treatment was completed, the mice were examined to see if the spirochetes were still in the tissues. As you might expect, B. burgdorferi DNA was detected by PCR at high levels in multiple tissues in all 10 mice that were administered saline, and the spirochetes were successfully cultured from the tissues. In the ceftriaxone group, as shown in an earlier study by Barthold's lab, low levels of B. burgdorferi DNA were detected in leg joints from all 10 mice. Although B. burgdorferi could not be cultured from the ceftriaxone-treated mice, ticks that fed on the mice were able to transmit the spirochetes to immunodeficient (SCID) mice, where B. burgdorferi was detected by PCR at the end of the experiment. Since transmission requires active penetration of B. burgdorferi through several tissue barriers, the spirochetes that remained following antibiotic treatment must have been alive, although they could not be cultured. Moreover, several B. burgdorferi mRNA transcripts were detected in some of the ceftriaxone-treated mice, another hint that the spirochetes remained viable (mRNA, unlike DNA, is extremely labile and would quickly degrade in dead bacteria).

How well did tigecycline work? Despite its heightened potency against B. burgdorferi in test tube experiments and its much longer half-life in mice, tigecycline didn't work any better than ceftriaxone in eliminating the spirochetes, even at the higher dose.

The studies performed by Barthold's group raises several questions:

Would prolonging antibiotic treatment eventually eliminate the spirochetes? Curiously, tigecycline was administered to the mice for only 10 days.
Do the spirochetes that remain following antibiotic treatment cause disease? So far, the answer appears to be, "No." Unlike the saline-treated mice, the antibiotic-treated mice did not exhibit any signs of disease. Necropsies failed to reveal a inflammatory response against the spirochetes remaining in the tissues.
Do the spirochetes that survive antibiotic treatment give rise to disease later? The earlier study by Barthold's group showed that although viable, the spirochetes were slowly diminishing in number in the tissues of mice that were treated with antibiotics. If the mice were followed for a longer period of time, would the disabled spirochetes eventually disappear or revive to elicit a relapse of disease?
Is the mouse model even relevant to human Lyme disease? Borrelia burgdorferi has evolved to persist in the mouse, its natural host, and may act differently in humans. Obviously the experiments presented here can't be performed on humans, but an animal model that is more relevant to human Lyme disease may be more appropriate for addressing the issues raised by Barthold's work.

Featured paper

Barthold, S.W., Hodzic, E., Imai, D.M., Feng, S., Yang, X., and Luft, B.J. (February 2010). Ineffectiveness of tigecycline against persistent Borrelia burgdorferi. Antimicrobial Agents and Chemotherapy 54(2):643-651. DOI: 10.1128/AAC.00788-09

Related paper

Hodzic, E., Feng, S., Holden, K., Freet, K.J., and Barthold, S.W. (May 2008). Persistence of Borrelia burgdorferi following antibiotic treatment in mice. Antimicrobial Agents and Chemotherapy 52(5):1728-1736. DOI: 10.1128/AAC.01050-07

Did spirochetes kill off the Indians in Massachusetts before the Mayflower landed?

2010-02-25T02:09:00.000-08:00

The coast of present-day Massachusetts was inhabited by several Native American tribes in the early 17th century. Fishermen, traders, and explorers from the Old World encountered the Indians during their occasional travel through the area. However by the time the Mayflower landed in Plymouth in 1620 to establish a colony, a mysterious epidemic had ravaged coastal New England, killing up to 90% of the indigenous population during the years 1616 through 1619. Experts have yet to agree on the cause of the epidemic. Smallpox, plague, and yellow fever, all highly lethal diseases, have been blamed.

Native American tribes of southeastern Massachusetts, approx. 1620 (Figure 1 from Marr and Cathey)

An article in the new issue of Emerging Infectious Diseases offers leptospirosis, caused by Leptospira spirochetes, as another possible agent of the 1616-1619 epidemic. This is based not on any new information but on an examination of the lifestyle of the Native Americans of early 17th century New England.

Rats infected with Leptospira may have stowed away in the ships that sailed from Europe to the New World. Because Leptospira lives in the kidney tubules of chronic carriers, infected rats released into the New World would have contaminated their surroundings every time they urinated. Since Leptospira can survive in moist soil and fresh water, indigenous rodents and other animals could have become chronically infected with Leptospira, further spreading the spirochete throughout the region. The Indian lifestyle provided plenty of opportunities for exposure to Leptospira through skin abrasions and swallowing of contaminated water or food. Their high-risk activities included the following:

walking around barefooted
storing food accessible to rodents
swimming and bathing in streams and ponds
working on moist soil to raise and harvest crops

Leptospira has little effect on the health of carrier animals yet can cause humans to fall ill. Many escape with what may be confused with a mild case of the flu, but some end up suffering with life-threatening symptoms. Eyewitnesses of the 1616-1619 epidemic reported that victims were afflicted with skin lesions, severe headaches, yellowing of the skin (likely jaundice), and bloody nose (possibly from lung hemorrhage), which are all symptoms of the severe form of leptospirosis. Even today leptospirosis can be deadly with reported fatality rates of greater than 50% among those with severe lung hemorrhaging.

While the authors should be commended for even considering a disease of a spirochete that is often ignored (at least by those in the developed world), I don't think Leptospira is what killed off the Indians. One strong argument against leptospirosis being the cause of the 1616-1619 epidemic is that Leptospira is not hardy enough to survive the cold winters that Mother Nature inflicts upon New England. Since the fatalities continued through the winter, leptospirosis is unlikely to be the culprit.

Whatever the cause, the epidemic may have been a pivotal event that facilitated English colonization of coastal Massachusetts since the surviving Indians lacked the capacity to resist the newcomers.

Featured paper

Marr, J.S., & Cathey, J.T. (2010). New Hypothesis for Cause of Epidemic among Native Americans, New England, 1616–1619 Emerging Infectious Diseases, 16 (2), 281-286 DOI: 10.3201/eid1602.090276

The Lyme disease spirochete has flagella but doesn't use them to penetrate the gut of the feeding tick

2010-02-12T11:05:00.000-08:00

The Lyme disease agent Borrelia burgdorferi possesses flagella, which are the thin motility structures owned by many members of the bacteria world. Flagella propel bacteria towards their destination by spinning (read this post to see how flagella function in Borrelia). It has been assumed B. burgdorferi spin their flagella whenever they need to move from one location to another. A recent paper in The Journal of Clinical Investigation has demonstrated otherwise, at least for B. burgdorferi in the midgut of a feeding Ixodes (blacklegged) tick.

Borrelia burgdorferi spends much of its life cycle lying dormant in the midgut of Ixodes ticks. The spirochetes lightly pepper the inner surface of the midgut cell lining, with a few spirochetes also hiding between cells. None live at the base of the cells at the basement membrane surrounding the midgut. The spirochetes wake up and multiply only when the tick attaches to an animal or human and imbibes blood. A few days into the blood meal, some spirochetes eventually breech the basement membrane and enter the hemocoel, the fluid-filled space between the tick organs where they must avoid the phagocytes patrolling the area. From there the spirochetes invade the salivary glands, which can then release B. burgdorferi-tainted saliva into the skin of the victim. After completing its satisfying meal of blood, the tick detaches from the skin of the victim, who may end up suffering from Lyme disease.

Dunham-Ems and colleagues wanted to follow the spirochetes in the midgut as ticks took their meal of blood. They engineered a strain of B. burgdorferi expressing green fluorescent protein so that they could watch the spirochetes in the gut by fluorescence microscopy. They allowed ticks with the green B. burgdorferi strain in their midguts to feed on laboratory mice. 24, 48, and 72 hours after the ticks were placed on the mice, the investigators removed the midguts and examined the organ by fluorescence microscopy to see what the spirochetes were doing. Surprisingly, they never saw motile spirochetes in the midgut even though the spirochetes eventually found their way at 72 hours into the hemocoel, where they were highly motile.

If the spirochetes in the midgut remained nonmotile during tick feeding, how did they reach the basement membrane? The few spirochetes that initially populated the midgut multiplied exponentially and formed growing networks of spirochetes on the cell surfaces as the tick drank blood from the mice. By 72 hours the networks eventually coalesced, encasing many gut cells in spirochetes (see the figures below). Spirochetes at the base of the encased cells were poised to penetrate the basement membrane and invade the hemocoel. All of this happened without B. burgdorferi ever spinning its flagella. Only when they broke through into the hemocoel did the flagella start spinning.

Figure 4F-H from Dunham-Ems 2009. Confocal fluorescence microscopy of a midgut from a nymph that fed on a mouse for 72 hours. Panel F shows a network of spirochetes (green) attached to the inner surface of the midgut. An optical section taken 24-26 µm into the lining of the midgut (panel G) reveals aggregates of spirochetes surrounding the cells. Panel H shows that some spirochetes have made it to the basement membrane, which is found 50 µm below the surface. The midgut cell membrane is stained in red. Scale bars = 25 µm. Some of the gut cells are extremely large because they are differentiating as part of the digestion process.

Figure 5 A and B from Dunham-Ems 2009. Silver stain of sections from ticks that fed for 48 hours (panel A) and 72 hours (panel B). The edges of the epithelial cells are easier to see than in the previous figure. Arrows point to aggregates of spirochetes (hairy bodies). At 72 hours at least one cell is encased in spirochetes. Scale bars = 25 µm. Some of the cells are extremely large because they are differentiating as part of the normal digestion process of the tick (dc, differentiated cells; uc, undifferentiated cells).

The investigators also found that something in the tick midgut inhibited the motility of B. burgdorferi. They placed a bit of minced midgut from a tick that had been feeding on a mouse for 72 hours at the edge of a gelatin matrix containing motile fluorescent B. burgdorferi. (Because of their helical shape, spirochetes love to move about in viscous substances such as gelatin.) Most of the spirochetes near the tissue ceased moving and remained motionless throughout the 15 minute viewing period. In contrast, the spirochetes continued moving when mouse blood was placed at the edge of the gelatin matrix.

Why does B. burgdorferi employ a nonmotile mode of penetration of the cell lining of the tick midgut? Is there some advantage for the spirochete to avoid using their flagella? As blood is known to be a powerful chemoattractant for B. burgdorferi, the authors offered the following explanation:

These results, although counterintuitive at first blush, make sense; if blood in the midgut acted as a chemoattractant, spirochetes would never disseminate during feeding.

Hence the "inhibitor" of motility released by the tick gut serves as a signal to the spirochete to not spin their flagella.

To me, this explanation isn't satisfying. It would seem simple for B. burgdorferi to have evolved a regulatory scheme that would allow the spirochete to temporarily uncouple blood chemotaxis from flagellar motility so that they could bore through the gut lining in minutes rather than days. There must be a reason why B. burgdorferi chooses to take its time to penetrate the gut lining.

Perhaps B. burgdorferi delays its journey to the salivary glands to allow the feeding tick to properly prepare the skin, which is an inhospitible environment for both tick and spirochete. As the tick feeds, it releases a brew of anti-immune factors into the skin to protect itself from attack by the immune system. Early arrival of B. burgdorferi to the salivary gland would release the spirochetes into the skin before the anti-immune factors have taken full effect, potentially allowing the host immune system to eliminate the spirochetes before they could establish an infection.

Reference

Dunham-Ems, S.M., Caimano, M.J., Pal, U., Wolgemuth, C.W., Eggers, C.H., Balic, A., & Radolf, J.D. (2009). Live imaging reveals a biphasic mode of dissemination of Borrelia burgdorferi within ticks. Journal of Clinical Investigation. 119(12):3652-3665. DOI: 10.1172/JCI39401

E. coli-like genes in the spirochete Brachyspira hyodysenteriae, the agent of swine dysentery

2010-01-03T17:10:00.000-08:00

The genus Brachyspira comprises at least seven species of anaerobic spirochetes that live in the large intestines of various birds and animals (including humans). One species, Brachyspira hyodysenteriae, causes swine dysentery, a disease that causes economic loss among pig farmers worldwide. Afflicted pigs produce loose stools covered with mucus and blood. In severe cases, necrotic chunks of colon lining are expelled with the stool.

An Australian group sequenced the genome of B. hyodysenteriae strain WA1 to figure out how the spirochete thrives in the complex nutritional environment of the large intestine and induces swine dysentery. They found 2,122 protein-coding genes distributed between a 3,000,694 bp chromosome and a 35,940 bp plasmid. A number of genes encoded degradative enzymes such as proteases, phospholipases, and hemolysins that may or may not account for the damage to the colon observed in swine dysentery cases. Otherwise, no obvious pathogenic mechanism for the disease process could be gleaned from the genome sequence.

Remarkably, half of the proteins encoded in the B. hyodysenteriae genome were most similar in sequence to proteins of Escherichia and Clostridium, genera that are not even on the same branch on the bacterial evolutionary tree as spirochetes. A mere 6.4% of B. hyodysenteriae proteins matched best to proteins of other spirochetes.

From Table 3 of Bellgard 2009.

Among the Escherichia- and Clostridium-like genes, those encoding proteins involved with amino acid and sugar metabolism and transport were over-represented. Since E. coli, Clostridium species, and B. hyodysenteriae all live in the large intestine, the similarity in the proteins may simply reflect convergent evolution that enable the bacteria to metabolize the nutrients available in the colon. The more attractive possibility is that B. hyodysenteriae acquired the genes from the other enteric bacteria by horizontal gene transfer thereby allowing the spirochete to adapt to the complex nutritional environment of the large intestine. At least for the E. coli-like genes, examining their GC content may help distinguish between the two possibilities since the GC content of B. hyodysenteriae is only 27% versus 50% for E. coli.

How can B. hyodysenteriae acquire genes from other enteric bacteria? A commentary in the journal Gut Pathogens raised the possibility that bacteriophage-like elements found in the B. hyodysenteriae genome could be involved, although bacteriophages generally do not transfer DNA between different species of bacteria. Another possibility is that genes could be acquired from other bacteria by conjugation, a form of microbial mating. Although the capacity of B. hyodysenteriae for acquiring DNA from other bacteria by conjugation is unknown, scientists have demonstrated that another spirochete could acquire DNA from E. coli by conjugation in the laboratory setting.

Image source

Sow with piglet, from Wikipedia

References

Bellgard, M.I., Wanchanthuek, P., La, T., Ryan, K., Moolhuijzen, P., Albertyn, Z., Shaban, B., Motro, Y., Dunn, D.S., Schibeci, D., Hunter, A., Barrero, R., Phillips, N.D., and Hampson, D.J. (2009). Genome sequence of the pathogenic intestinal spirochete Brachyspira hyodysenteriae reveals adaptations to its lifestyle in the porcine large intestine. PLoS ONE 4(3):e4641. DOI: 10.1371/journal.pone.0004641

Hampson, D.J. and Ahmed, N. (2009). Spirochaetes as intestinal pathogens: Lessons from a Brachyspira genome. Gut Pathogens 1(1):10. DOI: 10.1186/1757-4749-1-10

Leptospira and E. coli caught in the act

2009-12-28T09:43:00.000-08:00

I found this web photo of E. coli mating with the spirochete Leptospira biflexa in a process called conjugation (image source, Mathieu Picardeau, Pasteur Institute). The donor E. coli cell is transferring a copy of a plasmid bearing antibiotic resistance genes to the recipient spirochete. The DNA is most likely pushed through a pore that forms between the mating pair where the outer membranes come together.

There are many types of plasmids, but only self-transmissible plasmids are capable of transferring copies of themselves to other bacteria by conjugation. These plasmids carry a set of at least 20 genes collectively called tra (transfer), which encode all of the proteins necessary to carry out conjugation. The conjugational proteins assemble into several structures, including the sex pili, which bring the mating pair together, the relaxosome, which processes the DNA for transfer, and the poorly characterized pore through which the DNA traverses. The plasmids can also harbor additional genes that have no role in conjugation, including genes encoding resistance to antibiotics. RP4 is one example of a self-transmissible plasmid that can transfer itself to a wide range of bacteria species. Self-transmissible plasmids have been found in many different bacteria, yet none have been discovered in spirochetes.

Transformation is the microbiologist's favorite genetic tool for delivering DNA of their choosing into bacteria. Unfortunately for those interested in leptospirosis, transformation of disease-causing species of Leptospira such as L. interrogans is difficult. Conjugation employing a laboratory strain of E. coli as a donor provides scientists another route for delivering DNA into Leptospira. For example, the plasmid illustrated below (Figure 1 from Picardeau, 2008) has been used to ferry the Himar1 transposon into Leptospira for random insertional mutagenesis. Many readers may be most familiar with the F conjugational plasmid of E. coli, but the conjugational machinery found on the RP4 self-transmissible plasmid is used here since it is able to deliver DNA to a wide range of bacteria species.

Figure 1 from Picardeau, 2008. The Himar1 transposon consists of the arrowheads and everything in between, including the kanamycin-resistance gene (Km^R). The RP4 oriT element and the genes encoding the C9 tranposase and spectinomycin resistance (Spc^R) lie outside of the transposon.

The critical element of the plasmid is the RP4 oriT sequence where relaxase, a component of the relaxosome, nicks the DNA to initiate the transfer process. The tra genes were removed to permit easy manipulation of the plasmid. To perform conjugation, the plasmid was transformed into a special E. coli strain that encodes the RP4 tra genes on its chromosome. The E. coli cells were then mixed with Leptospira and concentrated onto a filter to facilitate mating. After allowing them to mate for 20 hours, the mating mixture was plated onto Leptospira medium agar plates containing the antibiotic kanamycin to recover Leptospira mutants with the transposon on one of its two chromosomes. The plasmid itself is unable to replicate in Leptospira, so the transposon must hop onto a chromosome following plasmid transfer to enable growth of kanamycin-resistant Leptospira into colonies. The donor E. coli bacteria had been genetically modified to require the nutrient diaminopimelate (DAP) to counterselect the donor on the agar plates, which were lacking DAP.

It is not feasible to screen L. interrogans insertion mutants for a desired phenotype (trait) following a single mating experiment since only a few hundred kanamycin-resistant colonies can be recovered. Tens of thousands of mutants would be necessary to ensure coverage of (almost) all L. interrogans genes.

One application of this genetic tool is to perform multiple mating experiments to generate a library of mutants with insertions of Himar1 in different L. interrogans genes. The sequence of the insertion site of the transposon in the chromosome can be obtained easily with today's sequencing technology. Further experiments can be performed to examine any mutants with insertions in genes that hold the investigator's interest. Several labs have teamed up to embark on a similar approach by delivering the Himar1 transposon into L. interrogans by transformation (see the Murray 2009 paper), which does not yield as many colonies as conjugation.

References

Picardeau, M. (2008). Conjugative transfer between Escherichia coli and Leptospira spp. as a new genetic tool. Applied and Environmental Microbiology 74(1):319-322. DOI: 10.1128/AEM.02172-07

Murray G.L., Morel, V., Cerqueira G.M., Croda, J., Srikram, A., Henry, R., Ko, A.I., Dellagostin, O.A., Bulach, D.M., Sermswan, R.W., Adler, B., and Picardeau, M. (2009). Genome-wide transposon mutagenesis in pathogenic Leptospira species. Infection and Immunity 77(2):810-816. DOI: 10.1128/IAI.01293-08

The genetics of both host and pathogen matter in antibiotic-refractory Lyme arthritis

2009-12-08T16:40:00.000-08:00

The arthritic form of Lyme disease was first reported in the 1970s by Allen Steere, who described the condition in a group of children (and a few adults) residing in and around the town of Lyme, Connecticut. Lyme arthritis can strike when Borrelia burgdorferi introduced into the skin by an Ixodes tick burrows into deeper tissues and ends up in the joints, usually the knee. Swelling results from an inflammatory response to B. burgdorferi residing in the joint. Lyme arthritis is treated with antibiotics, which destroy the bacteria driving inflammation. Unfortunately, arthritic symptoms endure in ~10% of treated patients despite the complete or almost complete eradication of the infection, as determined by negative PCR tests for B. burgdorferi DNA in joint fluid. Such cases are called antibiotic-refractory Lyme arthritis, which can persist for months or sometimes years. In severe cases cartilage and bone erode. Although the pathogenesis of antibiotic-refractory Lyme arthritis could involve persistence of small numbers of B. burgdorferi (or their antigens) in the joints, investigators have been seeking an autoimmune mechanism to explain the prolonged attack on joint tissue by the immune system after the spirochetes have been cleared.

Many autoimmune diseases are linked to variants of HLA (immunity) genes such as those encoding the MHC class II complex. Antibiotic-refractory Lyme arthritis is associated with MHC class II variants that are able to bind to fragments of the B. burgdorferi protein OspA (outer surface protein A) encompassing amino acid residues 165 through 173. Antigen-presenting cells whose MHC class II molecules display OspA_165-173 peptides on their surface stimulate T cells that recognize the OspA peptide. How OspA_165-173-reactive T cells cause autoimmunity has been an area of intensive research, yet a clear answer has not emerged.

One potential pathway to autoimmunity is molecular mimicry, in which a cross-reactive host protein in the joint continues to stimulate OspA_165-173-specific T cells even after the eradication of B. burgdorferi by antibiotics. Although the simplicity of the molecular mimicry model is appealing, exhaustive efforts to find a cross-reactive autoantigen that stimulates OspA_165-173-specific T cells have failed. Moreover, levels of OspA_165-173-reactive T cells decline soon after initiation of antibiotic therapy despite continuing arthritis following treatment. Thus, chronic arthritis does not seem to involve molecular mimicry driven by a cross reaction between the OspA_165-173 epitope and a self-antigen in the joint. It is possible that molecular mimicry involves another B. burgdorferi antigen that is able to bind the MHC class II variants found in genetically susceptible individuals.

Other potential routes to autoimmunity in antibiotic-refractory Lyme arthritis patients emphasize the role of the high levels of key proinflammatory cytokines and chemokines found in their joint fluid, levels even higher than those found in treatment-responsive patients prior to initiation of antibiotic therapy:

In a model known as bystander activation, the immune response to OspA_165-173 (or another B. burgdorferi antigen) causes an excessive inflammatory response that activates other T cells that react to autoantigens in the joint.
The immune system is unable to turn off the intense inflammatory response associated with OspA_165-173 after the spirochetes are cleared from the joint.

Although much attention has been focused on the role of host genetics, a recent study indicates that the genetics of the pathogen could also influence the course of Lyme arthritis. In the July 2009 issue of Arthritis and Rheumatism, Allen Steere and his collaborators showed that antibiotic-refractory Lyme arthritis is associated with different strains of B. burgdorferi. The strains were typed from joint fluid samples collected before or during antibiotic treatment. Among the methods available to group B. burgdorferi isolates, they used the 16S-23S ribosomal RNA intergenic spacer type (RST), of which there are three. Antibiotic-refractory arthritis was defined as joint swelling lasting for at least 3 months after the start of antibiotic treatment. Antibiotic treatment consisted of 8 weeks of oral antibiotics or up to 4 weeks of antibiotics administered intravenously. Joint fluid from all 17 patients in the study tested positive by PCR for B. burgdorferi DNA prior to or during antibiotic treatment.

The authors found that all 7 Lyme arthritis patients infected with RST1 strains had the antibiotic-refractory form. Joint fluid was obtained after antibiotic treatment from 5 of the 7 patients; all 5 samples tested negative for B. burgdorferi DNA by PCR. In contrast, 2 of 6 and 3 of 4 infected with RST2 and RST3 strains, respectively, were successfully treated with antibiotics (see the table below from the Jones et al. 2009 article). A larger number of samples is needed to demonstrate that the difference observed between RST1 and RST2 strains is statistically significant, but there is a clear trend towards RST1 infections having the greatest association with antibiotic treatment failure and RST3 having the least, with RST2 having an intermediate effect. The duration of arthritis also depended on the infecting RST strain.

How do RST1 strains cause arthritis to persist even after the apparent eradication of the spirochetes by the recommended course of antibiotics? The investigators proposed that RST1 strains provoke a stronger inflammatory response in the joint than RST2 or RST3 strains. Coupled with an immune response to OspA_165-173 in genetically susceptible patients, this could cause inflammation to continue at high levels even after elimination of the spirochetes from the joints. RST1 strains may be more likely than the other genotypes to spark intense joint inflammation even in patients who are not genetically prone to antibiotic-refractory arthritis.

In future studies, it would be interesting to see if proinflammatory cytokine levels are related to the RST type that infects the joint. Ultimately, researchers need to identify the B. burgdorferi gene or genes whose variation among the RSTs causes the different treatment outcomes of Lyme arthritis.

Featured paper

Jones, K.L., McHugh, G.A., Glickstein, L.J., & Steere, A.C. (2009). Analysis of Borrelia burgdorferi genotypes in patients with Lyme arthritis: High frequency of ribosomal RNA intergenic spacer type 1 strains in antibiotic-refractory arthritis
Arthritis & Rheumatism, 60 (7), 2174-2182 DOI: 10.1002/art.24812

Other references

Drouin E.E., Glickstein, L., Kwok, W.W., Nepom, G.T., and Steere, A.C. (2008). Human homologues of a Borrelia T cell epitope associated with antibiotic-refractory Lyme arthritis. Molecular Immunology 45(1):180-189. DOI: 10.1016/j.molimm.2007.04.017

Kannian, P., Drouin, E.E., Glickstein, L., Kwok, W.W., Nepom, G.T., and Steere A.C. (2007). Decline in the frequencies of Borrelia burgdorferi OspA_161-175-specific T cells after antibiotic therapy in HLA-DRB1*0401-positive patients with antibiotic-responsive or antibiotic-refractory Lyme arthritis. The Journal of Immunology 179(9):6336-6342.

Shin J.J., Glickstein, L.J., and Steere, A.C. (2007). High levels of inflammatory chemokines and cytokines in joint fluid and synovial tissue throughout the course of antibiotic-refractory Lyme arthritis. Arthritis & Rheumatism 56(4):1325-1335. DOI: 10.1002/art.2241

Steere, A.C., Klitz, W., Drouin, E.E., Falk, B.A., Kwok, W.W., Nepom, G.T., and Baxter-Lowe, L.A. (2006). Antibiotic-refractory Lyme arthritis is associated with HLA-DR molecules that bind a Borrelia burgdorferi peptide. The Journal of Experimental Medicine 203(4):961-971. DOI: 10.1084/jem.20052471

Telomeres without telomerase in Borrelia spirochetes

2009-11-04T15:55:00.000-08:00

You've all heard by now that the 2009 Nobel Prize in Physiology or Medicine will be awarded to Elizabeth Blackburn, Carol Greider, and Jack Szostak. They're the ones who figured out that an enzyme called telomerase combats the shortening that occurs at the ends of the linear chromosomes of eukaryotes (including ours) during each round of DNA replication. Telomerase sticks copies of a short string of nucleotides to the 3' ends of the chromosomal DNA. On the other hand, bacteria do not need telomerase because their chromosomes are circular; they do not have ends that can be shortened.

Spirochetes of the genus Borrelia, which include the agents of Lyme disease and relapsing fever, are an oddity in the bacterial world in that their chromosomes are linear. They also have a large set of linear plasmids. For example, the genome of the Lyme disease agent Borrelia burgdorferi consists of one linear chromosome and 12 linear plasmids, along with 11 circular plasmids. I will refer to the chromosomes and plasmids collectively as replicons. Despite having linear replicons, telomerase is nowhere to be found in Borrelia.

If they lack telomerase, how do Borrelia avoid having the ends of their linear replicons getting pruned during DNA replication? The key lies in the covalently closed hairpin ends of their linear replicons, something that's not found in eukaryotic telomeres. As illustrated in the figure below (figure 1 from Tourand 2003), the hairpin loops allow the replication machinery to copy the nucleotides at the very ends of the telomeres.

The replicated structure shown at the bottom of the figure illustrates that this method of replication creates a new problem. Following DNA replication, the two copies of the replicon end up fused at their ends (L'L and RR'). The fused sequences (telomere junctions) must somehow separate so that one copy of the replicon ends up in each daughter cell as the spirochete lengthens and splits in two.

Fortunately, Borrelia possesses an enzyme designated ResT, a telomere resolvase that cleaves the DNA where the two copies of the replicon are fused and reforms the hairpins at the ends of the new telomeres (see figure below). ResT was discovered by George Chaconas' laboratory in Canada. All of the work with ResT that I describe here was carried out by his group.

Chaconas' group was able to demonstrate the telomere resolvase reaction in vitro by simply mixing purified ResT with DNA containing a telomere junction. The products of the reaction were then analyzed following agarose gel electrophoresis. An example of the assay is shown below. A 4.6 kb piece of DNA containing the telomere junction formed by the left end of the B. burgdorferi linear plasmid lp17 ("L'L") is converted by ResT into the expected 2.6 and 2.0 kb products over a 2 minute time period.

Although the genome sequence of B. burgdorferi was published years ago, the sequence of the telomeres could not be determined back then because of the difficulty in cloning DNA with closed hairpin ends. The sequence of most of the telomeres finally appeared in the literature this year. The telomere sequences are aligned in the table below (figure 6 of Tourand 2009). The end of each telomere is the first nucleotide in each sequence.

The alignment shows that all of the telomeres have a "box 3" sequence. Box 3 is the recognition site for ResT binding. The evidence for this is that ResT binds to box 3 in vitro and changing the nucleotides in box 3 inhibits resolution of the telomere junction by ResT.

The telomeres were grouped based on the box 1 sequence. Type 1 telomeres carry the box 1 sequence TATAAT, and Type 2 telomeres harbor the modified box 1 sequence TATTAT. Type 3 telomeres lack the box 1 motif. When the rates of ResT resolving the different telomere junctions were measured in vitro (last column in alignment above), telomeres that lacked box 1 (Type 3) exhibited the slowest rates, with three telomeres failing to react with ResT. Since these three telomeres are obviously resolved in vivo, their resolution may require additional factors yet to be identified.

References

Tourand, Y., Deneke, J., Moriarty, T.J., & Chaconas, G. (2009). Characterization and in vitro reaction properties of 19 unique hairpin telomeres from the linear plasmids of the Lyme disease spirochete Journal of Biological Chemistry, 284 (11), 7264-7272 DOI: 10.1074/jbc.M808918200

Kobryn, K., & Chaconas, G. (2002). ResT, a telomere resolvase encoded by the Lyme disease spirochete Molecular Cell, 9 (1), 195-201 DOI: 10.1016/S1097-2765(01)00433-6

Tourand, Y., Kobryn, K., & Chaconas, G. (2003). Sequence-specific recognition but position-dependent cleavage of two distinct telomeres by the Borrelia burgdorferi telomere resolvase, ResT Molecular Microbiology, 48 (4), 901-911 DOI: 10.1046/j.1365-2958.2003.03485.x

Tourand, Y., Lee, L., & Chaconas, G. (2007). Telomere resolution by Borrelia burgdorferi ResT through the collaborative efforts of tethered DNA binding domains Molecular Microbiology, 64 (3), 580-590 DOI: 10.1111/j.1365-2958.2007.05691.x

Baby steps towards unraveling transcriptional regulation in the unculturable syphilis spirochete

2009-10-08T23:02:00.000-07:00

I would never select Treponema pallidum as my experimental model if I had to study gene regulation in a spirochete. The main problem is that no one has figured out how to grow T. pallidum in any type of culture medium. T. pallidum can be propagated only by growing the spirochete in the testes of rabbits. Consequently, investigators have not even begun to develop the genetic tools (e.g., gene knock outs, shuttle plasmids) necessary to unravel the regulatory mechanisms that control T. pallidum gene expression.

Despite the limitations imposed by T. pallidum upon those who wish to study gene regulation, a group of syphilis researchers at the University of Washington in Seattle have started to dissect the regulation of several members of the 12-gene tpr (Treponema pallidum repeat) family. No one has figured out what the Tpr proteins do, but syphilis researchers are interested in them in part because they show how the immune response battles T. pallidum infections. For example, antibodies generated against TprK during infection bind to TprK exposed on the surface of T. pallidum and mark them for destruction by macrophages. More recent studies suggest that TprK undergoes antigenic variation (a topic of a future post), which may allow T. pallidum to persist in the host.

The Seattle group's studies on gene regulation have focused on the Subfamily II tpr genes tprE, tprG, and tprJ, as reported in the journal Molecular Microbiology. The sequences upstream of their transcription start sites contain a sequence that closely matches the consensus binding sequence for the E. coli global transcriptional regulator CRP (cAMP regulatory protein), also known as CAP (catabolite activator protein). The T. pallidum genome encodes a CRP homolog designated TP0262. In E. coli and a few other Gram negatives, CRP is an integral component of the complex network of transporter, regulatory, and enzymatic proteins that allow bacteria to selectively metabolize the preferred sugar, usually glucose, from those available in the environment. When glucose is absent, the enzyme adenylate cyclase is activated and synthesizes the second messenger cAMP (cyclic AMP), which turns on CRP by allosteric activation. (Here's a nice description of the allosteric activation of CRP.) The cAMP-CRP complex then binds upstream of various promoters and activates transcription by recruiting RNA polymerase to the promoter. Additional layers of regulation ensure that the genes are transcribed only when the sugar that is to be broken down by the gene products is present.

Because it's not possible to examine gene regulation in T. pallidum, the Seattle group transferred the tpr genes to E. coli, a genetically pliable bacterium. They fused each tpr gene, including the upstream sequences containing the proposed CRP binding site and the promoter, to a gene whose product is easily measurable, green fluorescent protein (gfp). They then introduced the plasmid carrying the gene fusion into an E. coli strain missing its crp gene so that they could measure tpr-driven GFP levels in the presence and absence of a second plasmid expressing TP0262. They found that TP0262 increased tprE'-gfp and tprJ'-gfp fusion expression while decreasing trpG'-gfp expression. The ability of TP0262 to control tpr'-gfp expression was lost when the CRP binding site was removed from the fusion constructions. They also showed that control of the tprJ'-gfp fusion by TP0262 was lost when the adenylate cyclase gene in E. coli was removed, indicating that cAMP was needed to activate TP0262 (data for tprE and tprG were not presented). Their in vitro experiments demonstrated binding of purified recombinant TP0262 to the proposed CRP binding site upstream of the three tpr genes by DNase I protection and gel shift assays.

What was missing from the study, as acknowledged by the authors, were experiments to demonstrate that TP0262 does the same thing in T. pallidum. For future studies, they plan to show that TP0262 is bound upstream of the Subfamily II tpr genes in T. pallidum by chromatin immunoprecipitation, which entails determining the sequence of the segment of DNA that is bound when TP0262 is immunoprecipitated from a T. pallidum extract. Such experiments would not require genetic manipulation or the ability to cultivate T. pallidum. It would only require harvesting a large number of T. pallidum spirochetes from infected rabbits.

What signal does TP0262 respond to? Does it respond to the glucose found in the host? The insightful Commentary by Radolf and Desrosiers sheds some light on the question. They note that T. pallidum is missing the special transporter genes that in E. coli encode the components necessary to link sugar availability to cAMP and CRP. They surmise that TP0262 has thus been freed to regulate genes not related to sugar metabolism, such as the tpr genes. Since CRP is a global transcriptional regulator in other bacteria, it is likely to regulate expression of not only the Subfamily II tpr genes but also additional genes in T. pallidum.

Near the end of their commentary, Radolf and Desrosiers made one comment that stood out:

One of the most important outcomes of the present study is that it will help put to rest the pregenomic view of the syphilis spirochaete as a transcriptionally invariant organism.

Maybe I'm too young to appreciate their point, but I can't believe that there ever was a time when syphilis researchers believed that T. pallidum genes were not regulated!

Featured articles

Giacani, L., Godornes, C., Puray-Chavez, M., Guerra-Giraldez, C., Tompa, M., Lukehart, S.A., & Centurion-Lara, A. (2009). TP0262 is a modulator of promoter activity of tpr Subfamily II genes of Treponema pallidum ssp. pallidum
Molecular Microbiology, 72 (5), 1087-1099 DOI: 10.1111/j.1365-2958.2009.06712.x

Radolf, J.D., & Desrosiers, D.C. (2009). Treponema pallidum, the stealth pathogen, changes, but how?
Molecular Microbiology, 72 (5), 1081-1086 DOI: 10.1111/j.1365-2958.2009.06711.x

Protein census of Leptospira interrogans

2009-08-31T22:39:00.000-07:00

A census of proteins in a bacterial cell was conducted for the first time ever. By "census," I don't mean merely identifying all cellular proteins (which can be accomplished by shotgun tandem mass spectrometry). What I mean is counting the number of copies of every protein. The bacterium targeted for the census was the spirochete Leptospira interrogans. Like the census conducted here in the U.S. every ten years, some proteins were missed. The strategy developed by Malmström and colleagues, as described in the August 6 issue of Nature, allowed them to determine the abundance of 1,864 (or 83%) of the 2,221 proteins that were detectable by tandem mass spectrometry (MS) in Leptospira interrogans that had been grown in standard Leptospira culture medium.

The results of the protein census are compiled in the bar graph below. Proteins with related biological functions were grouped together and are color coded. The "Proteome" bar tabulates the number of different proteins in each group.
The next bar, "Copies per cell (control)," gives you an idea of how much of the protein expression machinery in L. interrogans is directed towards the synthesis of proteins in each functional category. The percentage reflects the amounts and size of the proteins in each category. For example, proteins of unknown function (hypothetical proteins) represent only 12.7% (blue) of the total protein synthesis capacity even though they constitute 30% of the identified proteins and over 40% of all genes in L. interrogans. I would surmise that these hypothetical proteins would account for a more sizable fraction of total protein synthesis under some other condition that L. interrogans would encounter during its life cycle (e.g., during infection).

The other observation noted by the authors is that L. interrogans gears 15% of its protein synthesis effort to make a small number of proteins deemed to be components of the "external encapsulating structure" (green), which is a fancy Gene Ontology term encompassing abundant Leptospira proteins that have been demonstrated to be in the inner or outer membrane. Most of the 15% is accounted for by five outer membrane proteins: LipL32, Loa22, LipL41, LipL21, and LipL36, the functions of which are not entirely clear. The five proteins are among the 10 most abundant proteins in L. interrogans.

The last bar shows the effect of the antibiotic ciprofloxacin ("cipro") on global protein levels in L. interrogans. The most striking change is the massive increase in 15 proteins of unknown function (light blue) leading them to constitute ~20% of the total protein content. As ciprofloxacin is an inhibitor of DNA gyrase, transcription of the genes encoding the 15 proteins may be extremely sensitive to DNA topology.

Did the enormous increase in the copy number of the 15 proteins following ciprofloxacin treatment increase the total number of protein molecules in L. interrogans? The authors found little change in the total cellular protein content:

Interestingly, this large redistribution of the proteome did not significantly change the total cellular protein concentration. Therefore, the large increase in the abundance of [the 15 proteins of unknown function] after ciprofloxacin exposure was compensated by a slight reduction of other high abundant protein classes.... This indicates that in L. interrogans, the cells strive to maintain a certain total number of protein components, that is, a constant cellular proteome concentration.

Featured paper

Malmström, J., Beck M., Schmidt, A., Lange, V., Deutsch, E.W., and Aebersold, R. (2009). Proteome-wide cellular protein concentrations of the human pathogen Leptospira interrogans. Nature 460(7256):762-765. DOI: 10.1038/nature08184

Zebrafish model of leptospirosis: Where's the relevance?

2009-08-20T23:18:00.000-07:00

Scrutinized for the past several decades as a model of embryonic development, the zebrafish has recently been promoted as a vertebrate model for investigating the pathogenesis of infectious diseases. Zebrafish embryos are transparent, allowing microbiologists to readily view the course of infections in real time. Another advantage of the zebrafish model is that it's amenable, at least in theory, to large-scale genetic screens for mutated host or microbial genes that affect the infection process.

Davis and colleagues observed the early stages of zebrafish embryo infection by the spirochete Leptospira interrogans, as described in a recent issue of PLoS Neglected Tropical Diseases. They injected 10-100 spirochetes into the hindbrain of zebrafish embryos at 30 hours post-fertilization, when the innate immune response is fully functional. Macrophages rushed to the hindbrain and engulfed the invaders within the first 4 hours following inoculation of the spirochetes. L. interrogans was also rapidly phagocytosed when injected into the caudal vein.

Figure 1a from Lesley and Ramakrishnan, 2008. A zebrafish embryo 30 hours following fertilization. The hindbrain and caudal vein are indicated with the bracket and arrow, respectively.

Macrophages typically kill and destroy their prey following phagocytosis. However, spirochetes were still observed in the macrophages 24 hours following inoculation, suggesting that L. interrogans can survive inside macrophages.

Their most striking observation was the accumulation of infected macrophages near the dorsal aorta in a region known as the aorta-gonad-mesonephros (AGM), where hematopoietic stem cells are born. This was not a general phenomenon of bacterial infections as macrophages harboring Pseudomonas aeruginosa failed to accumlate at the AGM following its injection into zebrafish embryos.

Figures 2C and 2D from Davis et al., 2009. The embryo was infected with fluorescently stained L. interrogans. 24 hours following infection, most of the spirochetes were found near the dorsal aorta (brackets), with a few scattered around the ventral tail. Scale bar, 300 µm in panel C, 100 µm in panel D.

Here's what the authors concluded in the final sentence of the paper:

The strikingly specific delivery of leptospires to [the AGM] by phagocytes provides insights into pathogenesis by suggesting a novel mechanism for targeting of organs during leptospiral dissemination.

In other words, L. interrogans may be capable of steering macrophages towards specific organs. Once the macrophages arrive at their destination, the spirochetes may escape from the macrophage and colonize the organ.

The challenge for the authors in future studies will be to demonstrate the relevance of the zebrafish model to leptospirosis. Hamsters and guinea pigs are appropriate models for leptospirosis because the pathology and lethality of Leptospira infection in these rodents is similar to what's observed in human leptospirosis patients. The fate of Leptospira that macrophages capture in these rodents differs from what is seen in zebrafish embryos. Leptospira that are found inside macrophages in tissue sections from infected rodents often appear to be disintegrating. Nevertheless, it's possible that a few Leptospira survive phagocytosis and subsequently guide the macrophage towards the target organs.

Featured paper

Davis, J.M., Haake, D.A., & Ramakrishnan, L. (2009). Leptospira interrogans stably infects zebrafish embryos, altering phagocyte behavior and homing to specific tissues PLoS Neglected Tropical Diseases, 3 (6) DOI: 10.1371/journal.pntd.0000463

Other references

Lesley, R. and Ramakrishnan, L. (2008). Insights into early mycobacterial pathogenesis from the zebrafish. Current Opinions in Microbiology 11(3):277-283. DOI: 10.1016/j.mib.2008.05.013

The 14th species of the Lyme disease group of Borrelia

2009-07-28T09:57:00.000-07:00

The cluster of genetically related Borrelia species that includes the Lyme disease spirochete B. burgdorferi is known in the scientific community as Borrelia burgdorferi sensu lato ("in the wider sense"). Three members of B. burgdorferi sensu lato account for most cases of Lyme disease worldwide. They are B. burgdorferi sensu stricto ("in the stricter sense"), B. garinii, and B. afzelii. Several other species of the cluster are suspected of causing a small number of Lyme disease cases in Europe and Asia.

The United States is home to at least four species of B. burgdorferi sensu lato. They are B. burgdorferi sensu stricto (the only species known to cause Lyme disease in the U.S.), B. bissettii, B. andersonii, and B. californiensis. The discovery of a fifth named U.S. species, christened Borrelia carolinensis, was published in the Journal of Clinical Microbiology earlier this year. The new species hails from South Carolina. Most of the isolates were cultured from cotton mice and eastern wood rats, but one isolate was obtained from an Ixodes minor tick feeding on an eastern wood rat. Whether B. carolinensis is capable of inducing Lyme disease is unknown.

The number of named species of B. burgdorferi sensu lato found worldwide now stands at 14:

B. burgdorferi sensu stricto
B. garinii
B. afzelii
B. andersonii
B. bissettii
B. californiensis
B. carolinensis
B. japonica
B. lusitaniae
B. sinica
B. spielmanii
B. tanukii
B. turdi
B. valaisiana

If you read my last post, you will notice that Borrelia lonestari, detected in one case of the Lyme-like illness STARI, is missing from the list. Although STARI clinically resembles a mild form of Lyme disease, genetically B. lonestari is more closely related to the set of Borrelia that causes relapsing fever.

Featured article

Rudenko, N., Golovchenko, M., Grubhoffer, L., and Oliver, J.H. (2009). Borrelia carolinensis sp. nov., a new (14th) member of the Borrelia burgdorferi sensu lato complex from the southeastern region of the United States. Journal of Clinical Microbiology 47(1):134-141. DOI: 10.1128/JCM.01183-08

STARI or Masters disease: More like Lyme than Lyme?

2009-07-19T01:54:00.000-07:00

A tick-borne illness has been masquerading as Lyme disease in the southern United States over the past two decades. Victims first notice the expanding "bulls-eye" skin rash that is similar in appearance to the erythema migrans (EM) of Lyme disease. However, the tick that feeds on the victim is not the Ixodes tick that causes Lyme disease but the Lone Star tick Amblyomma americanum. Moreover, Borrelia burgdorferi, the Lyme disease spirochete, is not the infectious agent. B. burgdorferi has never been successfully cultured from a southern case of the EM-like rash, and sera from most of these patients test negative for Lyme disease by CDC criteria. Lyme disease itself is uncommon in the south as the resident Ixodes ticks rarely feed on humans; most ticks found attached to humans residing in the south are the Lone Star tick, which is unlikely to harbor or transmit B. burgdorferi.

The name bestowed upon the condition, "southern tick-associated rash illness" or STARI, is misleading because the territory of the Lone Star tick has been creeping into the northeastern and northern U.S., where Lyme disease is hyperendemic. The illness has also been dubbed "Masters disease" to honor Dr. Edwin Masters, who passed away last month. Dr. Masters' observations of skin rash patients in his private practice in Cape Girardeau, Missouri sparked the contentious CDC investigation that led to the first detailed description of STARI 14 years ago. You can read about his battles with the CDC in a series of blog posts by Pamela Weintraub, author of the book Cure Unknown, Inside the Lyme Epidemic (a book I hope to read some day).

Fig. 1 from Masters et al., 2008. Lone Star tick territory in green.

Contrary to popular belief, the erythema migrans of most Lyme disease patients does not present as a bull's-eye. In fact, in one study the EM-like rashes in Masters' STARI patients were much more likely to appear as a bull's-eye than the EM of Lyme disease patients from New York. In addition, the STARI patients were less likely than those with Lyme disease to suffer from accompanying symptoms such as joint and muscle aches, fatigue, headache, and stiff neck. A question that remains unresolved is whether arthritic, neurologic, or cardiac symptoms can crop up later, as they do in those afflicted with Lyme disease.

Figure 2 from Masters et al., 2008. EM-like skin lesions in Missouri STARI patients

The agent of STARI has eluded scientists. Borrelia lonestari was suspected at one time when it was detected by PCR in one patient and the Lone Star tick attached to his skin. (The spirochete could not be cultured since it does not grow in Borrelia culture medium.) However, B. lonestari could not be detected in a later study of a series of Masters' STARI patients. Thus, B. lonestari is unlikely to bring about most cases of STARI. The failure to identify the infectious agent of STARI has led some to question whether STARI has an infectious cause.

Masters was convinced that a spirochete, perhaps one closely related to Borrelia burgdorferi, was the agent of STARI. He has offered the following observations as evidence:

Spirochetes have been observed in Lone Star ticks.
Forms resembling spirochetes have been observed by silver staining of the EM-like skin lesions from Masters' STARI patients (see figure below).
Extracts of B. burgdorferi reacted with sera from some STARI patients in ELISA tests, although the sera were Western blot negative according to CDC criteria. This observation indicates that antibodies were elicited against proteins closely related to those found in B. burgdorferi.
Figure 8 from Masters et al., 1998. Silver stain of skin biopsy of an EM-like rash from a Missouri patient showing an apparent spirochete.

Of course these observations are far from definitive proof. Nevertheless, it is possible that a Borrelia spirochete is the culprit of EM-like lesions in the southern U.S. Like B. lonestari, some of these strains may not grow in the standard Borrelia medium used to cultivate B. burgdorferi from the skin rashes of Lyme disease patients. This would account for the inability of investigators to culture the spirochete from the skin rash of STARI patients.

Finally, how is STARI treated? Although the cause of STARI remains unknown, Edwin Masters declared that Lyme-like illness deserves Lyme-like treatment. That is, he recommended that antibiotics be administered to STARI patients according to Lyme treatment guidelines. Establishing whether antibiotics truly help will require a randomized placebo-controlled study.

Featured article

MASTERS, E.J., GRIGERY, C.N., & MASTERS, R.W. (2008). STARI, or Masters Disease: Lone Star Tick–Vectored Lyme-like Illness Infectious Disease Clinics of North America, 22 (2), 361-376 DOI: 10.1016/j.idc.2007.12.010

Other references

Masters, E., Granter, S., Duray, P., and Cordes P. (1998). Physician-diagnosed erythema migrans and erythema migrans-like rashes following Lone Star tick bites. Archives of Dermatology 134(8):955-960.

Wormser G.P., Masters, E., Liveris, D., Nowakowski, J., Nadelman, R. B., Holmgren, D., Bittker, S., Cooper, D., Wang, G., and Schwartz, I. (2005). Microbiologic evaluation of patients from Missouri with erythema migrans. Clinical Infectious Diseases 40(3):423-428. DOI: 10.1086/427289

Wormser G.P., Masters, E., Nowakowski, J., McKenna, D., Holmgren D., Ma, K., Ihde, L., Cavaliere, L.F., and Nadelman, R.B. (2005). Prospective clinical evaluation of patients from Missouri and New York with erythema migrans-like skin lesions. Clinical Infectious Diseases 41(7):958-965. DOI: 10.1086/432935

Role of the second messenger cyclic diguanylate (c-di-GMP) in the Lyme disease spirochete

2009-07-04T16:27:00.000-07:00

Second messengers are the intracellular intermediaries that transmit the signals received from the environment (first messenger) to the cellular machinery that generates the appropriate response. Well known examples of second messengers in mammalian cells include cyclic AMP, cyclic GMP, calcium ion, and inositol triphosphate. A second messenger unique to bacteria is cyclic diguanylate, abbreviated c-di-GMP. First described in the 1980s, c-di-GMP is only now attracting wide interest among those who study signal transduction in bacteria.

Cyclic di-GMP is created from two GTP molecules by diguanylate cyclase and destroyed by phosphodiesterases. Genes encoding the opposing enzymatic activities can be identified by the conserved GGDEF motif in diguanylate cyclases and an EAL or HD-GYP motif in phosphodiesterases. Bacteria modulate the intracellular concentration of c-di-GMP by controlling the amounts and activities of the diguanylate cyclases and phosphodiesterases in response to changes in environmental conditions.

Cyclic di-GMP is best known for promoting the formation of biofilms. Biofilm assembly requires the synthesis and secretion of the special polysaccharides that make up the biofilm matrix and the down-regulation of motility. Both polysaccharide synthesis and the inhibition of motility are modulated by c-di-GMP. The molecule can also affect virulence functions. In many cases, the mechanistic details of how c-di-GMP exerts its effects remain unknown. The molecular target of c-di-GMP includes proteins with the "PilZ" domain (see figure). However, not all proteins bound by c-di-GMP possess the PilZ domain. In some bacteria, c-di-GMP can also bind specific sequences found within the 5' untranslated region of several mRNAs to modulate gene expression.

Figure 1 from Tamayo et al., 2007. DGC, diguanylate cyclase; PDEA, phosphodiesterase A; PDE, phosphodiesterase

The Borrelia burgdorferi gene rrp1 encodes the only protein in the Lyme disease spirochete containing the GGDEF motif. The Rrp1 protein consists of a receiver domain and the GGDEF domain, whose diguanylate cyclase activity requires phosphorylation of the receiver domain. A paper in the March issue of Molecular Microbiology revealed the genes whose expression is affected by Rrp1. The authors compared the transcript profiles (transcriptome) of a B. burgdorferi wild-type and an rrp1 deletion mutant by microarray analysis. It turned out that most of the genes affected by the mutation encode what the authors call the "core" cellular functions of Borrelia burgdorferi. The core functions allow the spirochete to seek out and capture nutrients from the environment, synthesize the building blocks necessary for assembling cellular parts, and extract energy from nutrients to fuel its activities. All bacteria, whether or not they cause disease, possess these core functions. Most transcripts from core genes were increased in the wild-type B. burgdorferi strain relative to the rrp1 mutant. The impaired growth of the rrp1 mutant compared to wild type is consistent with the importance of Rrp1 on the expression of the core functions of B. burgdorferi.

The investigators also found that the rrp1 transcript levels increased 6 fold when ticks harboring B. burgdorferi took a blood meal from mice. High levels of rrp1 mRNA were also maintained in B. burgdorferi growing in culture medium. These observations suggest that more rrp1 transcript is made when the spirochete is awash in nutrients, whether in blood or culture medium. Under these conditions, c-di-GMP signals B. burgdorferi to turn on genes necessary to acquire and metabolize the nutrients.

The protein that directly or indirectly senses changes in nutrient availability is likely to be the histidine kinase encoded by hpk1, the gene that lies immediately upstream of rrp1. The Hpk1 and Rrp1 proteins form a phosphorelay in which phosphate groups swiped from ATP molecules are transferred to the target Rrp1 protein in response to some signal in the environment.

In summary, Rrp1 diguanylate cyclase activity is enhanced at two levels when nutrients become abundant. First, rrp1 transcript levels are increased, leading to more Rrp1 protein being made. Second, the Rrp1 diguanylate cyclase activity is activated by phosphorylation. Increased c-di-GMP levels is the result. The c-di-GMP stimulates increased levels of transcripts emanating primarily from genes encoding the core cellular functions of B. burgdorferi. The question that remains unexplored is how c-di-GMP causes transcript levels to increase. One protein in B. burgdorferi harbors the PilZ domain, but as I mentioned earlier, PilZ is not the only protein domain capable of binding c-di-GMP.

Finally, what does this study reveal about the role of Rrp1 and c-di-GMP in Lyme disease? One possibility is that Rrp1 is involved in tick-to-human transmission and the early stages of the infection:

As I already mentioned, Rrp1 upregulation in B. burgdorferi residing in a tick taking a blood meal may prepare the spirochete to metabolize the nutrients found in the blood as they are transmitted into the skin of the human victim.
Transcripts expressed from several genes encoding factor H-binding proteins (some of the few non-core genes affected by the rrp1 knock out) were at higher levels when Rrp1 was present. Factor H is an inhibitor of the complement system found in our bloodstream. As such, binding of factor H by the spirochete may protect it from being killed by the host complement system. Indeed, the authors demonstrated that the rrp1 mutant was more sensitive to human serum than the wild-type B. burgdorferi strain. However, the significance of this observation is unclear as Borrelia garinii, another agent of Lyme disease, was just as sensitive as the B. burgdorferi rrp1 mutant to human serum. Additionally, earlier studies have shown that factor H is not necessary for successful B. burgdorferi infections (at least in the mouse model of Lyme disease).
The ospC gene, which encodes another protein that may impair immune function during the early stages of infection, was also upregulated by Rrp1.
Several transcripts expressing motility and chemotaxis functions are expressed at higher levels when Rrp1 is present. Motility and chemotaxis are considered to be core functions, but they may also be necessary for B. burgdorferi to establish infection in humans. Note that the proposed effect of c-di-GMP on B. burgdorferi motility is opposite of that found in other bacteria (see figure above).

The obvious experiment to perform is to test whether the rrp1 mutant can cause infection in the mouse model of Lyme disease. Unfortunately, the effect of rrp1 on virulence could not be tested as the authors were unable to knock out the rrp1 gene in an infectious strain of B. burgdorferi.

Featured paper

Rogers, E.A., Terekhova, D., Zhang, H.-M., Hovis, K.M., Schwartz, I., & Marconi, R.T. (2009). Rrp1, a cyclic-di-GMP-producing response regulator, is an important regulator of Borrelia burgdorferi core cellular functions Molecular Microbiology, 71 (6), 1551-1573 DOI: 10.1111/j.1365-2958.2009.06621.x

Image source

Tamayo R., Pratt J.T., and Camilli, A. (2007). Roles of cyclic diguanylate in the regulation of bacterial pathogenesis. Annual Review of Microbiology 61:131-148. DOI: 10.1146/annurev.micro.61.080706.093426

Cholera and spirochetes: Introducing Brachyspira!

2009-06-08T01:44:00.000-07:00

Cholera results in a severe form of diarrhea that can lead to dehydration, shock, and ultimately death without prompt treatment. The disease afflicts the poor in developing countries lacking clean water sources and sanitation infrastructure. Vibrio cholerae is the causative agent and can be viewed by microscopic examination of the so-called "rice-water" stool samples from cholera patients.

rice-water stool from a cholera patient (Figure 1 from Sack et al., 2004)

As reported in a recent issue of Emerging Infectious Diseases, Nelson and colleagues, while examining a cholera outbreak in Bangladesh back in 2006, found that stool samples in over a third of cholera patients contained spirochetes mingling with V. cholerae. Samples were fluorescently stained to aid identification of bacteria. One example is shown below. V. cholerae were visualized with a FITC-conjugated monoclonal antibody to its lipopolysaccharide (in red), and bacterial DNA was stained wtih DAP I (green). Only the merged image is shown below. V. cholerae are the rods with a slight bend and appear yellow (combination of red and green) with a red edge; the spirochetes are the W-shaped forms stained green.

bar = 10 µm

This wasn't the first time spirochetes were observed in rice-water stool. Over a century ago, Theodor Escherich (the discoverer of E. coli) was the first to witness spiral-shaped microbes in fecal samples from cholera victims.

What was the identity of these spirochetes? They were not any of the "Big 3" of Borrelia, Treponema, and Leptospira, which garner the most attention from spirochete researchers (and from the writer of this blog). Nelson and colleagues guessed that they were members of the genus Brachyspira as they are the only spirochetes known to live in the human intestine. They turned out to be correct. They successfully amplified the gene encoding the 16s rRNA with Brachyspira-specific PCR primers. The sequence of the PCR product revealed the spirochetes to be Brachyspira pilosicoli and Brachyspira aalborgi.

Brachyspira account for most cases of human intestinal spirochetosis, defined as the presence of spirochetes in the colon. Although colonization of the large intestine by spirochetes is uncommon in the Western world, up to half of those in developing nations may harbor intestinal spirochetes. A typical example is shown below (click on image for larger version).

Figure 3 from Esteve et al., 2006

The sectioned tissue, which was stained with H&E, was obtained by colonic biopsy. The left panel reveals a fuzzy layer covering the colonic epithelium. These are Brachyspira attached at one end to the lining of the colon. The density of spirochetes can reach up to 1,700 per square millimeter. The right panel shows a colonic biopsy from the same patient after successful treatment with the antimicrobial agent metronidazole. Note that the fuzzy layer has disappeared.

Whether intestinal spirochetes cause disease in humans is unclear. Many people with intestinal spirochetes do not suffer any ill effects, but others endure chronic diarrhea. The mode of transmission of Brachyspira is unknown, but scientists have surmised that ingestion of contaminated water is involved.

The role of Brachyspira in cholera, if any, is even more of a mystery. In the conclusion to their article, Nelson et al. present the hypothesis that intestinal spirochetes exacerbate the already devastating clinical course of cholera.

Featured paper

Nelson, E.J., Tanudra, A., Chowdhury, A., Kane, A.V., Qadri, F., Calderwood, S.B., Coburn, J., Camilli, A. (2009). High Prevalence of Spirochetosis in Cholera Patients, Bangladesh Emerging Infectious Diseases, 15 (4), 571-573 DOI: 10.3201/eid1504.081214

Other references

Esteve, M., Salas, A., Fernandez-Banares, F., Lloreta, J., Marine, M., Gonzalez, C.I., Forne, M., Casalots, J., Santaolalla, R., Espinos, J.C., Munshi, M.A., Hampson, D.J., and Viver, J.M. (2006). Intestinal spirochetosis and chronic watery diarrhea: Clinical and histological response to treatment and long-term follow up. Journal of Gastroenterology and Hepatology 21(8):1326-1333. DOI: 10.1111/j.1440-1746.2006.04150.x

Sack, D.A., Sack, R.B., Nair, G.B., and Siddique, A.K. (2004). Cholera. Lancet 363(9404):223-233. DOI: 10.1016/S0140-6736(03)15328-7

Leptospira heme oxygenase frees iron from heme

2009-05-28T22:27:00.000-07:00

I mentioned in a recent post that iron is an essential trace metal that bacteria must acquire from its surroundings. (From that same post you will also recall that the Lyme disease spirochete B. burgdorferi is a rare exception that doesn't need iron.) Much of the iron in our body is trapped within the center of the heme molecule. Heme itself is not readily accessible as it is bound to host proteins such as hemoglobin. Pathogenic bacteria have evolved sophisticated systems to kidnap heme from host proteins and transport them into the cytoplasm. These complex systems, which include secreted degradative enzymes, heme capturing proteins, and transporter proteins that sit in the membrane, have been examined in numerous bacteria. However, the fate of heme after it is acquired by the bacteria is poorly understood. In some cases, the captured heme may be incorporated into bacterial proteins such as cytochromes, which participate in electron transport. In other cases, bacteria may need to extract the iron trapped in the middle of the heme molecule. Some bacteria possess the enzyme heme oxygenase, which extracts the iron caged within heme by the following reaction (adapted from Scheme 1 in Kikuchi et al., 2005):
Unlike its cousin that causes Lyme disease, the spirochete Leptospira requires iron for growth. Ben Adler's group at the Monash University in Australia isolated a Leptospira interrogans mutant with the transposon TnSC189 inserted into hemO, the gene encoding heme oxygenase. The properties of the hemO mutant is described in two papers in the journal Microbes and Infection. The graph below (figure 1B in Murray et al., 2008) shows that growth of the hemO mutant is impaired, although not completely, when hemoglobin is the sole source of iron in the culture medium. The residual growth of the mutant indicates that L. interrogans may possess another activity that extracts iron from heme.
In their follow-up study, Adler's group demonstrated that the hemO gene was necessary for L. interrogans to fully express its virulence in the hamster model of leptospirosis. For this study they used the hemO mutant and a control L. interrogans strain that had the TnSC189 element inserted in a noncoding region, presumably where gene expression would not be affected. The two strains were injected into the abdominal cavities of separate groups of hamsters, which were then monitored for 14 days. Only 8 of 24 hamsters (33%) survived the challenge with the control strain, whereas 20 of 24 (83%) injected with the hemO mutant survived. The difference in survival rates between the two groups was statistically significant (P = 0.001).

Although the hemO mutant was ineffective at killing hamsters, it was still able to colonize the kidneys of most of the animals. Colonization was assessed by culturing kidney or urine in Leptospira growth medium. The mutant was recovered by culturing of kidney or urine from 17 of 20 hamsters that survived the challenge with the hemO mutant and all 3 that died. These results were similar to what was obtained with hamsters inoculated with the control strain, which was recovered from all 8 animals that survived and all 12 that died. (Not all hamsters were examined for colonization.)

Why was the hemO mutant able to colonize the kidney when it was unable to extract iron from heme? Heme is not the only source of iron in the body. The mutant may have captured one of the other forms of iron present in the host. The genome of L. interrogans encodes several homologs of transporters that the spirochete may use to acquire non-heme sources of iron (Louvel et al., 2006). Since these other iron sources are less abundant than heme, the tissue burden (density of bacteria) of the mutant in the kidneys may have been lower than that of the control strain thereby allowing most of the hamsters challenged with the hemO mutant to survive.

One obvious limitation of the study is that the investigators did not attempt to complement the hemO mutation with a wild-type copy of the gene. However, I should point out that currently no plasmid is available that replicates in L. interrogans, rendering complementation of L. interrogans mutations difficult. The researchers did verify that the gene immediately downstream of hemO was still transcribed in the mutant.

This work is significant for the following reasons. First, although there have been two other studies that have examined the role of Leptospira genes in virulence, this study was the most satisfying to read because it was the first to show that a gene encoding a product of known function has a role in the virulence of Leptospira. Second and perhaps more importantly, it is the first to demonstrate the importance of a bacterial heme oxygenase in virulence.

Featured papers

Murray, G., Ellis, K., Lo, M., & Adler, B. (2008). Leptospira interrogans requires a functional heme oxygenase to scavenge iron from hemoglobin Microbes and Infection, 10 (7), 791-797 DOI: 10.1016/j.micinf.2008.04.010

Murray, G., Srikram, A., Henry, R., Puapairoj, A., Sermswan, R., & Adler, B. (2009). Leptospira interrogans requires heme oxygenase for disease pathogenesis Microbes and Infection, 11 (2), 311-314 DOI: 10.1016/j.micinf.2008.11.014

Other references

Kikuchi, G., Yoshida, T., and Noguchi, M. (2005). Heme oxygenase and heme degradation. Biochemical and Biophysical Research Communications 338(1):558-567. DOI: 10.1016/j.bbrc.2005.08.020

Louvel, H., Bommezzadri S., Zidane, N., Boursaux-Eude, C., Creno, S., Magnier, A., Rouy, Z., Médigue, C., Saint Girons, I., Bouchier, C., and Picardeau, M. (2006). Comparative and functional genomic analyses of iron transport and regulation in Leptospira spp. Journal of Bacteriology 188(22):7893-7904. DOI: 10.1128/JB00711-06

The twentieth species of Leptospira

2009-05-15T00:18:00.000-07:00

A new species of Leptospira was isolated from soil in Johor, Malaysia by researchers at the Universiti Putra Malaysia. The spirochete was dubbed Leptospira kmetyi to honor Emil Kmety, a Slovak bacteriologist who had made numerous contributions to the understanding of the genus Leptospira. L. kmetyi is the twentieth species of Leptospira to be validly published. The sequence of its 16S rRNA gene places L. kmetyi among the "pathogenic" species of Leptospira, as shown in the phylogenetic tree below (Figure 1 from Slack et al., 2009). (Click on the image for a larger version.) Further studies are needed to prove that L. kmetyi is truly pathogenic.

The tree shows that 19 of the species cluster into four major groupings or "clades" within the genus Leptospira as follows:

Pathogenic

L. borgpetersenii
L. weilii
L. alexanderi
L. santarosai
L. noguchii
L. interrogans
L. kirschneri
Leptospira genomospecies 1
L. kmetyi

Novel

L. wolffii

Intermediate

L. fainei
L. broomii
L. inadai

Saprophytic

Leptospira genomospecies 3
L. biflexa
L. wolbachii
Leptospira genomospecies 4
Leptospira genomospecies 5
L. meyeri

The "novel" clade, which currently has L. wolffii as its only member, was first proposed last year in a paper by Slack and colleagues.

The species that was excluded from the phylogenetic analysis is L. licerasiae, which is a recently described member of the intermediate clade (Matthias et al., 2008).

By the way, I have never liked the designation "intermediate" because readers may assume an intermediate pathogenic potential between the pathogenic and saprophytic clades. At least one intermediate member, L. fainei, can cause severe disease, including Weil's syndrome and pulmonary hemorrhage (bleeding of the lungs).

Featured paper

Slack, A.T., Khairani-Bejo, S., Symonds, M.L., Dohnt, M.F., Galloway, R.L., Steigerwalt, A.G., Bahaman, A.R., Craig, S., Harrower, B.J., and Smythe, L.D. (2009). Leptospira kmetyi sp. nov. isolated from an environmental source in Malaysia. International Journal of Systematic and Evolutionary Microbiology 59(4):705-708. DOI: 10.1099/ijs.0.002766-0

Other references

Matthias, M.A., Ricaldi, J.N., Cespedes, M., Diaz, M.M., Galloway, R.L., Saito, M., Steigerwalt, A.G., Patra, K.P., Vidal Ore, C., Gotuzzo, E., Gilman, R.H., Levett, P.N., and Vinetz, J.M. (2008). Human leptospirosis caused by a new, antigenically unique Leptospira associated with a Rattus species reservoir in the Peruvian Amazon. PLOS Neglected Tropical Diseases 2(4):e213. DOI: 10.1371/journal.pntd.0000213

Slack, A.T., Kalambaheti, T., Symonds, M.L., Dohnt, M.F., Galloway, R.L., Steigerwalt, A.G., Chaicumpa, W., Bunyaraksyotin, G., Craig, S., Harrower, B.J., and Smythe, L.D. (2008). Leptospira wolffii sp nov., isolated from a human with suspected leptospirosis in Thailand. International Journal of Systematic and Evolutionary Microbiology 58(10):2305-2308. DOI: 10.1099/ijs.0.64947-0

The manganese transporter of the iron-free Lyme disease spirochete is essential for infection

2009-04-30T23:42:00.000-07:00

Iron is a trace element essential for life. As such, living beings, including humans, must obtain iron from their diet. The same holds true for the bacteria that make us sick. Unfortunately for bacteria, iron is not readily available as it is tied up by various proteins in our body. Consequently, pathogenic bacteria have devised clever strategies to wrest iron away from these proteins.

The Lyme disease spirochete Borrelia burgdorferi lacks the machinery necessary for acquiring iron. In fact, B. burgdorferi can grow in the absence of iron. The reason that B. burgdorferi does not need iron is that the spirochete is missing most of the common metalloproteins that require iron to function. For the few metalloproteins that are present, manganese (Mn) may substitute for iron. This raises the question of how B. burgdorferi acquires Mn.

In the March 3 issue of PNAS, Ouyang and colleagues describe a potential Mn transporter BmtA (Borrelia metal transport protein A) encoded in the B. burgdorferi genome. What is their evidence that BmtA is a Mn transporter?

Analysis of its amino acid sequence indicates that BmtA is a member of the ZIP family of metal transporters. The transporters sit in the membrane and transport metals such as iron, zinc, and manganese across the membrane. Most ZIP family members are predicted to have 8 transmembrane domains with potential metal-binding histidine residues within a "variable region" (see figure below taken from Guerinot 2000) and a signature sequence in the fourth transmembrane domain containing another metal binding histidine. BmtA lacks the his-X-his-X-his (X = any amino acid) metal binding site in the variable region, but it does have the signature sequence.
When the bmtA gene was knocked out, the mutant B. burgdorferi was still able to grow in culture, but it was unable to accumulate Mn in its cytoplasm. The ability to accumulate Mn was restored when a plasmid expressing bmtA was introduced into the mutant.

To show conclusively that BmtA is a Mn transporter, the authors will need to incorporate purified BmtA into an artificial lipid bilayer (liposome) and demonstrate transport of Mn across the membrane.

The investigators next determined whether BmtA was required for infection of the mouse model of Lyme disease. They found that the bmtA mutant was unable to infect mice when injected into the skin. Infection was assessed by culturing heart, joint, and skin tissue removed 4 weeks after inoculation. The effect of the knockout was striking. None of the 66 organs sampled from the 22 mice inoculated with the bmtA mutant were culture positive. On the other hand, all 21 organs obtained from the 7 mice injected with the wild-type B. burgdorferi strain were culture positive. Infectivity was restored when the bmtA gene was introduced on a plasmid back into the bmtA mutant.

Because the bmtA knockout mutant grew in vitro yet was unable to grow in mice, BmtA must be essential for infectivity. Ouyang et al. presented two possible roles of BmtA in virulence in the Discussion of their paper. First, BmtA may be required for the activity of superoxide dismutase, which in B. burgdorferi is predicted to require Mn rather than iron. Superoxide dismutase detoxifies the reactive oxygen species (ROS) generated by phagocytic cells (neutrophils and macrophages) trying to ward off invading bacteria. Indeed, Ouyang et al. demonstrated that knocking out the bmtA gene rendered B. burgdorferi more sensitive to the oxidizing agent t-butyl hydroperoxide.

Second, Mn may contribute to the regulation of Borrelia genes encoding virulence determinants. The authors present as an example the transcriptional regulator BosR, which they state is a "Mn-dependent Fur homolog." This is incorrect as two different research groups have shown Mn to have no effect or even inhibit the activity of BosR. Nevertheless, there may be other Mn-dependent regulators of borrelial gene expression yet to be discovered.

The authors tout BmtA as a discovery that "may lead to new strategies for thwarting Lyme disease." That's probably true, but a word of caution should be expressed here. Any inhibitor of BmtA that's identified in future studies must have high specificity for the borrelial protein since ZIP family proteins are also found in humans.

Featured paper

Ouyang, Z., He, M., Oman, T., Yang, X., & Norgard, M. (2009). A manganese transporter, BB0219 (BmtA), is required for virulence by the Lyme disease spirochete, Borrelia burgdorferi Proceedings of the National Academy of Sciences, 106 (9), 3449-3454 DOI: 10.1073/pnas.0812999106

Other references

Boylan, J.A., Posey, J.E., and Gherardini, F.C. (2003). Borrelia oxidative stress response regulator, BosR: A distinctive Zn-dependent transcriptional activator. Proceedings of the National Academy of Sciences USA 100(20):11684-11689.

Guerinot M.L. (2000). The ZIP family of metal transporters. Biochimica et Biophysica Acta 1465(1-2):190-198.

Posey, J.E. and Gherardini, F.C. (2000). Lack of a role for iron in the Lyme disease pathogen. Science 288(5471):1651-1653.

Does male circumcision protect against syphilis?

2009-04-21T08:31:00.000-07:00

Circumcision has been shown to reduce the risk of men contracting several sexually transmitted infections (STIs). Three randomized controlled trials (RCTs) published over the last few years have demonstrated that removing the foreskin of adult men diminished the risk of HIV infection by at least 50%. An article by Tobian and colleagues in last week's issue of the New England Journal of Medicine revealed a weak protective effect of circumcision against two other infections, herpes simplex virus 2 (HSV-2) and human papilloma virus (HPV), which cause genital herpes and penile warts, respectively. The same study showed no effect of circumcision on acquisition of Treponema pallidum, the agent of syphilis. You can find a nice critical analysis of the study here. Because I am interested in diseases caused by spirochetes, I will focus on the syphilis data.

Tobian et al. conducted two RCTs with similar designs. When the data were combined, they found that 50 of 2083 (2.4%) male adolescents and adults in Uganda who underwent circumcision became infected with T. pallidum over the following 24 month period. Similarly, 45 of 2143 (2.1%) control subjects became infected within the same time period, suggesting that circumcision had no effect on contracting T. pallidum. An editorial in the same journal issue points out that the study may have been underpowered to detect a protective effect (i.e., not enough subjects in the study).

Since the Tobian et al. study failed to give a simple answer to the question, I thought it would be illuminating to look at the older observational studies that examined the effects of male circumcision on syphilis transmission. Fortunately, I found a meta-analysis that compiled data from 14 research papers, most of which described cross-sectional studies.

The meta-analysis presented by Weiss and colleagues revealed a slight protective effect of male circumcision. The relative risks (RRs) along with the 95% confidence intervals (CI) are plotted in the graph. The RR in 11 of the 14 studies were adjusted for potential confounding factors such as age. The summary statistics listed at the bottom of the graph indicate a small protective effect (summary RR, 0.67; 95% CI, 0.54-0.83).

Ideally, all studies included in a meta-analysis would have similar RRs. However, if you look carefully at the graph, you will notice a wide variation in the RRs with some of the 95% confidence intervals failing to overlap. Using standard statistical calculations, the investigators determined that it was unlikely that the variation of the RR among the studies was due to chance (P = 0.01). In other words, differences in how the studies were designed and conducted led to significant variation in the outcomes. Consequently, the authors declared that there was significant heterogeneity among the studies and warned that the summary RR "should be interpreted cautiously."

The authors described one potential source of the heterogeneity. Looking at the plots again, you will note that the Cook and Parker studies demonstrated the largest statistically significant protective effect of male circumcision. Those two studies were conducted in the United States and Australia, respectively, where males are circumcised as infants. In contrast, the two largest studies, authored by Gray and Urassa, showed no effect of circumcision on the risk of becoming infected with T. pallidum. Those studies were conducted in Uganda and Tanzania, respectively, where males are not circumcised until they are adolescents or young adults. Many of the circumcised males examined in the two African studies, which were cross-sectional and case-control studies, could have contracted syphilis before being circumcised. Weiss et al. excluded subjects who were circumcised after their first sexual intercourse or after age eleven, but this information was not available for all studies. This would lead to an underestimate of the protective effects of circumcision.

You may look at the large protective effects of infant circumcision observed in the U.S. and Australian studies (RR = 0.25 and 0.19, respectively) and conclude that mass infant circumcision would be beneficial (at least for protection against syphilis). However, both studies involved men visiting STD clinics, and the results may not apply to the general population in those countries.

References

Tobian, A.A.R., Serwadda, D., Quinn, T.C., Kigozi, G., Gravitt, P.E., Laeyendecker, O., Charvat, B., Ssempijja, V., Riedesel, M., Oliver, A.E., Nowak, R.G., Moulton, L.H., Chen M.Z., Reynolds, S.J., Wawer, M.J., Gray, R.H. (2009). Male circumcision for the prevention of HSV-2 and HPV infections and syphilis. The New England Journal of Medicine 360(13):1298-1309.

Golden M.R. and Wasserheit, J.N. (2009) Prevention of viral sexually transmitted infections--foreskin at the forefront (Editorial). The New England Journal of Medicine 360(13):1349-1350.

Weiss, H.A., Thomas, S.L., Munabi, S.K., and Hayes, R.J. (2006). Male circumcision and risk of syphilis, chancroid, and genital herpes: a systematic review and meta-analysis. Sexually Transmitted Infections 82(2):101-109.

Leptospirosis outbreak among strawberry harvesters in Germany

2009-03-29T00:13:00.000-07:00

Humans can become infected with the spirochete Leptospira if they have wounds that contact contaminated soil or water. Leptospira live in the kidneys of reservoir animals and are released in urine into the environment. The majority of leptospirosis cases occurs in tropical locales, where the warm, moist conditions promote the survival of Leptospira in the environment. Flooding following heavy rainfall can cause large epidemics of leptospirosis in these regions of the world. A research article published in the March 15th issue of Clinical Infectious Diseases by Desai and colleagues describes an unexpected outbreak of leptospirosis among strawberry harvesters in Germany, a country not known for tropical weather. The July 2007 outbreak, while small by the standards of those that occur in tropical countries, was Germany's largest since the 1960s. The authors searched for factors that may have caused the epidemic.

The outbreak occurred among laborers who worked on a strawberry farm near Düren. The strawberry harvesters were seasonal workers from Romania, Slovakia, and Poland. Among the 153 who were employed on the farm during the outbreak, 24 were stricken with leptospirosis, and 13 were hospitalized. Fortunately, no one died. Most who became sick had antibodies against the Leptospira serogroup Grippotyphosa in their blood.

In September 2007, the authors conducted a retrospective cohort study to identify risk factors for acquiring leptospirosis. The strawberry harvesters were questioned about possible sources of exposure to Leptospira before the outbreak, including the presence of wounds, contact with rodents, and consumption of unwashed strawberries. Logistic regression of the data revealed two statistically significant risk factors. First, the odds of the harvesters acquiring leptospirosis increased with the number of days worked with unprotected hand lesions (OR, 1.1; 95% CI, 1.04-1.1). Leptospira probably entered the lesions as the workers reached into the water-logged soil. "Accidental contact with rodents" was listed as the other significant risk factor (OR, 4.8; 95% CI, 1.5-15.9), although the nature of the contact was not described by the authors. Of course recall bias may have skewed the results of the retrospective study. However, there is nothing unusual about these risk factors; contact of skin wounds with moist soil laden with Leptospira and exposure to infected rodents are typical means of acquiring leptospirosis.

The common vole was the main suspect in contaminating the strawberry field with Leptospira. More than 10 mouse holes per square meter were found in the field and surrounding areas when the site was examined in September 2007. Kidneys of voles captured in and near the field were infested with the spirochete. Leptospira was successfully cultured from the kidneys and were identified as members of the serogroup Grippotyphosa, the same serogroup that the antibodies in the patients' blood were targeting.

The strawberry field outbreak is reminiscent of the large epidemics of leptospirosis that sickened thousands of German agriculture workers from the 1920s through the 1960s, with the Leptospira serogroup Grippotyphosa carried by voles and hamsters responsible for many cases. In contrast, most German cases of leptospirosis of late are related to activities outside of work. Recreational activities involving water and exposure at home to infected pets and contaminated soil while gardening are common risk factors. Icterohaemorrhagiae, with rats as the reservoir host, is now the most prevalent serogroup in Germany. So why did a 1960s-style leptospirosis outbreak occur in 2007? The authors propose that the unusual weather in the months preceding the outbreak was a critical factor.

Europe experienced its warmest autumn in 2006 since recordings began, and the warm weather continued through the winter into 2007. In Germany, heavy rainfall accompanied the high temperatures in the months preceding the outbreak. May and June 2007 were Germany's wettest months since 1901. The tropical conditions may have augmented the amount of food available to the voles. In addition, the vole population, which naturally fluctuates every 3-5 years, may have already been at the peak of its cycle in 2007. A population explosion of infected voles may have increased the density of Leptospira residing in the strawberry field. Because the wet, warm conditions were favorable to survival of Leptospira, it is easy to imagine how an outbreak would have occurred among workers who spent days reaching into the contaminated soil with unprotected hands.

In conclusion, the authors present leptospirosis as a potential model for an infectious disease affected by global warming. Only time will tell whether the leptospirosis outbreak caused by the confluence of warm weather and heavy rainfall in a normally temperate region was a statistical anomaly or a sign of things to come.

Featured paper

Desai, S., van Treeck, U., Lierz, M., Espelage, W., Zota, L., Sarbu, A., Czerwinski, M., Sadkowska‐Todys, M., Avdicová, M., Reetz, J., Luge, E., Guerra, B., Nöckler, K., & Jansen, A. (2009). Resurgence of Field Fever in a Temperate Country: An Epidemic of Leptospirosis among Seasonal Strawberry Harvesters in Germany in 2007 Clinical Infectious Diseases, 48 (6), 691-697 DOI: 10.1086/597036

Other references

Jansen, A., Schöneberg, I., Frank, C., Alpers, K., Schneider, T., and Stark, K. (2005). Leptospirosis in Germany, 1962-2003. Emerging Infectious Diseases 11(7):1048-1054.

Pappas, G., Papadimitriou, P., Siozopoulou, V., Christou, L., Akritidis, N. (2008). The globalization of leptospirosis: worldwide incidence trends. International Journal of Infectious Diseases 12(4):351-357.

Rhesus monkeys with Lyme spirochetes in the brain

2009-03-10T23:57:00.000-07:00

Lyme disease has been mentioned in the news a lot lately. First it was a pet chimp mauling his neighbor last month in a Connecticut town. Many news accounts of the assault reported that Travis the chimp suffered from Lyme disease and had been given tea laced with Xanax to calm his erratic behavior prior to the attack. Investigators may never figure out what provoked Travis to attack Charla Nash. Was Lyme disease a factor? Maybe (although if I had to guess, it had more to do with a wild animal being kept as a pet, living where it didn't belong).

This past weekend a human reportedly suffering from long-term Lyme disease gunned down a pastor and injured several parishioners in an Illinois church. As with any topic having to do with chronic Lyme disease, experts disagree on whether Lyme disease can trigger aggression (but I'm pretty sure what the shooter's defense attorney believes). One prominent researcher even questions whether the alleged shooter truly had Lyme disease.

A blogger on Forbes.com doubts whether chimps could be stricken with Lyme disease. In reality, seeing a chimp with Lyme disease should not surprise us at all since another nonhuman primate, the rhesus monkey, has been used for years to study neuroborreliosis, the form of Lyme disease caused by infection of the nervous system. Neuroborreliosis is observed in up to 15% of untreated Lyme disease patients. I will present two studies in which scientists examined infection of the central nervous system of rhesus monkeys by the Lyme disease spirochete, Borrelia burgdorferi.

In a 2000 study by Cadavid and colleagues, the investigators found small numbers of spirochetes in the meninges and spinal cord nerve roots of rhesus monkeys four months following laboratory infection with B. burgdorferi by needle inoculation into the skin (see figure below). In humans, the inflammatory response to the spirochetes in the meninges and nerve roots can cause meningitis (headache, stiff neck, and sensitivity to light) and radiculoneuritis (shooting pains and abnormal skin sensations), respectively.

Figure 2b from Cadavid et al., 2000. A single B. burgdorferi cell in the anterior nerve root stained by immunohistochemistry with anti-B. burgdorferi antibodies.

The investigators found no convincing evidence for the presence of B. burgdorferi in the brain parenchyma of the infected rhesus monkeys. B. burgdorferi was not found in the brain by microscopy. On the other hand, B. burgdorferi DNA was detected in brain tissue by polymerase chain reaction (PCR). This result may suggest that the spirochete is present in the brain at low levels, although the authors believed that spirochetes in the meninges surrounding the brain was the real source of the DNA. It's also possible that the N40 B. burgdorferi strain used by the authors was incapable of invading the brain of rhesus monkeys and that another strain if injected into the skin would have found its way to the brain.

Two conditions associated with neuroborreliosis disrupt brain function in humans. The first is Lyme encephalopathy, which can cause fatigue and problems with concentration and memory. Brain activity is clearly affected in patients with Lyme encephalopathy, yet the underlying pathogenic mechanism remains an enigma. Many patients with this potentially disabling condition do not exhibit the classic signs of central nervous system inflammation such as the presence of white blood cells in their cerebral spinal fluid (CSF), production of antibodies against the infective agent in the CSF, or abnormal brain MRIs. Some neuroborreliosis experts have proposed that these cases of Lyme encephalopathy are a result of "toxic-metabolic" effects of infection elsewhere in the body (such as Lyme arthritis in the joints) spilling into the brain. Nevertheless, a low-level infection of the brain by B. burgdorferi cannot be ruled out. More obvious brain involvement is observed in neuroborreliosis patients with encephalitis, in whom brain lesions are detected by MRI, accompanied by white blood cells and production of anti-B. burgdorferi antibodies in the CSF. Symptoms include slight weakness or paralysis affecting one side of the body, spastic muscles, inability to feel sensations, and bladder dysfunction. In rare cases, patients may experience strokes or seizures. Patients with encephalitis are likely to have spirochetes in their brain, usually near blood vessels.

Lyme disease may also be associated with psychiatric illness, particularly depression. Rare cases of panic attacks, bipolar disorder, mania, obsessive-compulsive disorder, dementia, and violent outbursts have also been reported.

A recent study by Mario Philipp's group in Tulane, published recently in The American Journal of Pathology, described the potential effects of B. burgdorferi on brain cell activity of rhesus monkeys. Spirochetes injected into the skin with a needle or by tick bite are unable to make it into the brain of rhesus monkeys, as I explained above. Therefore, the investigators injected live B. burgdorferi spirochetes directly into the right side of the brains of rhesus monkeys. After two weeks, they found that an average of ~10% of oligodendrocytes at the injection sites had undergone death by apoptosis, as detected by fluorescent TUNEL staining (see figure below). In contrast, fewer than 2% of oligodendrocytes underwent apoptosis when sites in the left side of the brain of the same animals were injected with saline. Oligodendrocytes produce the myelin sheaths that surround the axons of neurons and promote transmission of electric impulses; one would expect the loss of oligodendrocytes to affect neuron function.

The in vivo results corroborated ex vivo experiments in which brain slices from rhesus monkeys were incubated with B. burgdorferi for up to eight hours. In the ex vivo experiments, apoptosis of both oligodendrocytes and neurons were detected by fluorescent TUNEL staining and with antibody against activated caspase 3, a more specific marker of apoptosis. The authors speculated that neurons undergoing apoptosis in vivo were removed by phagocytes and therefore went undetected in the brain injection experiment. The other major brain cell types, microglial cells and astrocytes, were spared from apoptotic death both ex vivo and in vivo.

Figure 4A from Ramesh et al., 2008. Oligodendrocytes are stained red with antibody to S-100. Nuclei of cells undergoing DNA fragmentation, a consequence of apoptosis, are TUNEL stained green. When the images are merged, the nuclei of oligodendrocytes undergoing apoptosis become yellow. B. burgdorferi, which are stained in blue, do not appear to be in physical contact with the oligodendrocytes.

Figure 4B from Ramesh et al., 2008. Percentage of oligodendrocytes undergoing apoptosis from 3 sites of spirochete (A-C) and saline (D-F) injections (click on image for larger view).

The Tulane group also detected myriad cytokines and chemokines such as IL-6, IL-1ß, and CXCL13 being produced in the B. burgdorferi-treated brain slices by immunofluorescence antibody staining and DNA microarray analysis. The authors concluded that B. burgdorferi invasion of the brain elicits production of a brew of inflammatory mediators that promote apoptotic death of oligodendrocytes and neurons. It may be true that brain invasion by B. burgdorferi in cases of human neuroborreliosis leads to production of these cytokines and chemokines, as IL-6, IL-1ß, and CXCL13 have been detected in the CSF of neuroborreliosis patients in earlier studies. However, the apoptotic cells observed by the Tulane group may be the unnatural consequence of small areas of the brain being exposed to extremely large numbers of spirochetes. Each site was injected with 5,000 spirochetes, a number far greater than the rare spirochetes observed by microscopy in the brains of neuroborreliosis patients. For the ex vivo experiments, a 2-mm section of brain was incubated with 20 million spirochetes, again a large burden of bacteria. More convincing would be finding apoptosis of oligodendrocytes and neurons in brain tissues in patients with Lyme encephalitis. Clearly more studies are needed to understand the events that really occur when B. burgdorferi invades the brain.

References

Ramesh, G., Borda, J., Dufour, J., Kaushal, D., Ramamoorthy, R., Lackner, A., & Philipp, M. (2008). Interaction of the Lyme Disease Spirochete Borrelia burgdorferi with Brain Parenchyma Elicits Inflammatory Mediators from Glial Cells as Well as Glial and Neuronal Apoptosis American Journal Of Pathology, 173 (5), 1415-1427 DOI: 10.2353/ajpath.2008.080483

Diego Cadavid, Tim O'Neill, Henry Schaefer, and Andrew R. Pachner (2000). Localization of Borrelia burgdorferi in the Nervous System and Other Organs in a Nonhuman Primate Model of Lyme Disease Laboratory Investigation, 80 (7), 1043-1054

Viewing the arrangement of Borrelia burgdorferi flagella by electron cryotomography

2009-02-22T23:01:00.000-08:00

The most peculiar feature of spirochetes may be the location of their flagella, the thin motility structures that propel bacteria through liquids. Flagella typically extend out from the surface of bacteria into the surroundings. Spirochetes, being not so typical, keep their flagella hidden in the periplasm between the cytoplasmic and outer membranes (see figure). For example, the Lyme disease spirochete Borrelia burgdorferi has 7-11 flagella attached near each end of the "protoplasmic" or cell cylinder, with each flagellum extending through the periplasm towards the center of the spirochete. The flagella impose a flat-wave shape (not a spiral shape!) on B. burgdorferi by wrapping around its protoplasmic cylinder.

from Rosa et al., 2005

How do flagella that are located in the periplasm drive the spirochete through the medium? B. burgdorferi motility is thought to require the rotation of its flagella against the cell cylinder, causing the cell body to gyrate.

B. burgdorferi flagella often appear as a bundle when observed by standard transmission electron microscopy. Here is one such image from a 2000 study revealing at least 10 flagella in a cross section of B. burgdorferi. With the flagella arranged in this manner, it is difficult to imagine how the flagella that are not in direct contact with the cell cylinder could contribute to its gyration.

A study by Charon and colleagues in the January 2009 issue of Journal of Bacteriology suggests that the flagellar bundle is an artifact of the standard techniques used to prepare the samples for electron microscopy. They employed the emerging technique of electron cryotomography to avoid the fixation and staining procedures that often introduce artifacts into samples. Electron cryotomography consists of the following steps:

To preserve structure, the specimen is plunge frozen at -165°C or less. Fixing or staining is not necessary.
While maintaining the sample at the ultralow temperature, 2D projections of the sample are obtained at different angles by transmission electron microscopy.
Computer software assembles the 3D structure of the specimen from the 2D projections.

The software also permits slices of the specimen to be observed without having to actually perform thin sectioning.

Here's a cross-section of B. burgdorferi as viewed by electron cryotomography. Note that the flagella are arranged in a single layer within the periplasm, not in a bundle.

Figure 1 of Charon et al. Bar, 50 nm.
PFs, periplasmic flagella; PS, periplasmic space; PM, plasma (or cytoplasmic) membrane; OM, outer membrane.

A longitudinal slice through the periplasm of B. burgdorferi reveals nine flagella neatly arranged in a parallel fashion along the surface of the protoplasmic cylinder. The authors refer to this array as a "flat ribbon." Each flagellum in the ribbon is separated by ~3 nm, allowing each to rotate in the same direction without interference from neighboring flagella.

Figure 5 of Charon et al. Bar, 200 nm.

3D reconstruction of a section of the spirochete illustrates the flat ribbon of flagella (in red) wrapping around the cell cylinder (in blue). Only a section of the cell cylinder is shown, and the outer membrane has been removed from the image.

These new images support a model for for B. burgdorferi motility that was first described back in the 1990s. In this model, the rotation of the flagella against the cell cylinder generates gyrating waves that progress backwards along the cell body. As explained in the discussion of the Charon et al. paper, it is conceivable that all 7-11 flagella must lie against the cell cylinder as a flat ribbon to exert the force necessary to generate the waves; a flagella bundle may not exert enough force. The torque generated by the rotating flagella causes a counter rotation of the cell cylinder (panel a below). The backward-propagating, gyrating waves push the spirochete through the medium. Flagella arranged in a bundle would not generate enough torque because of potential interference between rotating flagella (panel b).

Figure 8 of Charon et al. a. Flagella arranged in a flat ribbon. b. Flagella arranged in a bundle.

This model also explains why B. burgdorferi moves so well through viscous gel-like material such as the extracellular matrix; the gel provides traction for the backward-progressing waves to drive the spirochete through the medium.

Here's a movie animating B. burgdorferi motility, first presented at a meeting in 2001 .

SOURCE

You can also see real B. burgdorferi gyrating and generating backward-moving waves in a movie embedded in Dr. Nyles Charon's website.

Reference

N. W. Charon, S. F. Goldstein, M. Marko, C. Hsieh, L. L. Gebhardt, M. A. Motaleb, C. W. Wolgemuth, R. J. Limberger, N. Rowe (2009). The Flat-Ribbon Configuration of the Periplasmic Flagella of Borrelia burgdorferi and Its Relationship to Motility and Morphology Journal of Bacteriology, 191 (2), 600-607 DOI: 10.1128/JB.01288-08

The Lyme disease spirochete hijacks fibronectin to escape from the bloodstream

2009-02-07T13:04:00.000-08:00

The glycoprotein fibronectin is a component of the molecular mesh known as the extracellular matrix, which not only provides physical support for our cells but also directs cellular activities during embryonic development, tissue repair, and other processes. High levels of soluble fibronectin (300 μg/ml) are also found in our bloodstream, where it quietly circulates until it is recruited to stabilize clots and promote wound repair.

Fibronectin has a modular organization consisting of binding sites for various matrix and cell surface molecules. Examples include attachment sites for integrins (labeled "Cell" in the figure below) and glycosaminoglycans (labeled "Heparin"), which are exposed on the surface of the endothelial cells that line our blood vessels.

from Wierzbicka-Patynowski, 2003

B. burgdorferi is injected into the skin by an infected tick and spreads outward within the dermis, causing the familiar "bulls-eye" rash in some Lyme disease patients. The spirochete may eventually enter the bloodstream so that it can spread to other tissues. While in the bloodstream, the spirochete is surrounded by fibronectin. In fact, B. burgdorferi attaches to fibronectin in vitro, suggesting a role for fibronectin in Lyme disease.

It turns out that B. burgdorferi exploits the adhesive properties of plasma fibronectin to bind to the vessel wall before escaping into the surrounding tissue. Like their earlier work, which I described in my last post, this follow-up study by Norman and colleagues was conducted with fluorescent B. burgdorferi injected into the veins of live mice. The interactions of the spirochete with the capillary wall were observed by fluorescent intravital microscopy. The earlier study revealed that most of the interactions were transient, lasting for less than a second. B. burgdorferi was also seen crawling (dragging) along the vessel wall, which was followed by escape into the tissue or by stationary adhesion, a more intimate association with the vessel wall. Stationary adhesion could also be followed by extravasation and escape of the spirochete into the tissue. Both stationary adhesion and escape usually occurred between the endothelial cells lining the vessel wall. Each type of interaction (transient, dragging, and stationary adhesion) was quantitated by counting.

In their follow-up study, the authors demonstrated that coinjection of anti-fibronectin antibody and B. burgdorferi into the bloodstream of the mice diminished all catagories of interactions (transient, dragging, and stationary adhesion) by at least 90%. Control antibody (goat IgG) had no effect. These results indicate that fibronectin has a key role in mediating the attachment of B. burgdorferi to the microvasculature. Since fibronectin could potentially bind to GAGs and integrins on endothelial cells, the investigators also coinjected B. burgdorferi with peptides or antibodies known to block attachment of fibronectin to these targets. They found that the GAG-specific peptide reduced the interaction of B. burgdorferi with the vessel wall, whereas the integrin-specific peptide and antibodies had little effect. Thus, fibronectin may serve as a molecular bridge linking B. burgdorferi to GAGs displayed on the endothelial cells lining the blood vessel.

Which B. burgdorferi factor is involved in adherence to the vessel wall in vivo? Past studies had shown that the borrelial protein BBK32, a known fibronectin binding protein, mediated attachment of B. burgdorferi to fibronectin in vitro. Therefore, BBK32 was a logical candidate. To determine whether BBK32 was involved in vascular interactions in the mouse model, the research team employed a noninfectious B. burgdorferi strain that had lost bbk32 and other genes during long-term culture. The noninfectious strain failed to interact with the vasculature in the mouse. However, expression of BBK32 restored the ability of the noninfectious strain to transiently interact and drag along the vessel wall but only partly restored stationary adhesion. This results suggest that although BBK32 plays a role in vascular adherence, other bacterial factors are also involved.

Many pathogens have been shown to interact with fibronectin and GAGs in vitro. However, this study is highly significant as it is the first to demonstrate a role for these host molecules in bacterial adherence to the microvasculature in a living animal. Other spirochetes such as Treponema pallidum and Leptospira also express fibronectin binding proteins. Hence, the mechanism employed by B. burgdorferi to escape from the bloodstream may be similar for all disease-causing spirochetes. Moreover, other microbial pathogens have been shown to stick to fibronectin and GAGs in vitro. Thus, a large number of invasive pathogens may employ similar mechanisms to spread to different tissues via the bloodstream.

M. Ursula Norman, Tara J. Moriarty, Ashley R. Dresser, Brandie Millen, Paul Kubes, George Chaconas (2008). Molecular Mechanisms Involved in Vascular Interactions of the Lyme Disease Pathogen in a Living Host PLoS Pathogens, 4 (10) DOI: 10.1371/journal.ppat.1000169