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

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

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

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

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

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?

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

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

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

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

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