The magic of antibiotic tolerance

Bactericidal antibiotics are effective at killing proliferating bacteria as long as they don't carry mutated or acquired genes that encode resistance to the antibiotics. Unfortunately even antibiotic-sensitive bacteria can tolerate antibiotics under some circumstances. Bacteria that are in a nondividing "dormant" state often survive antibiotic exposure.  When the antibiotic is removed and growth resumes, the bacteria regain susceptibility to antibiotics.

At first glance antibiotic tolerance appears to be a passive process in which nondividing cells survive simply because the target of the antibiotic is inactive.  However, this is not correct.  Antibiotic tolerance requires an active response by the bacteria.  The nondividing bacteria that survive antibiotic treatment are called persisters.  Persisters may account for infections that are difficult to eradicate with antibiotics.

Persisters were first identified in 1944 by Joesph Bigger, who was testing the effectiveness of the new miracle drug penicillin on cultures of Staphylococcus.   However, interest in antibiotic tolerance waned as antibiotic resistance came to be a problem.  In the 1980s Harris Moyed revisited the issue of antibiotic tolerance, and his group harnessed the power of bacterial genetics to isolate mutants that exhibited abnormally high frequencies of persister formation and to map the mutations that caused the novel trait.

As Joseph Bigger discovered, very rare persisters can even be identified in growing cultures, around 1 in 100,000 bacteria.  How can these rare bacteria behave so differently from those surrounding them if antibiotic tolerance doesn't involve alterations to the bacterium's DNA?  The current thinking is that the persister state is triggered randomly in a small fraction of proliferating bacteria due to random fluctuations in the expression of a small number of persister genes.  In the rare bacterium (about 1 in 100,000), the persister genes will be expressed at a high enough level to induce the persister state and slow growth of the bacterium.

Certain environmental cues can also enhance the development of persisters.  For example, the fraction of persisters in a culture increases substantially as nutrients are depleted.  At stationary phase, when the bacteria stop increasing in number, at least 1% of the bacterial cells become antibiotic tolerant.

One of the active responses that stimulates persister cell formation is called the stringent response, which rapidly generates the unusual nucleotides ppGpp and pppGpp when bacteria are starving for nutrients.  Historically these derivatives of GDP and GTP were known as "magic spots" because they appeared as novel radioactive spots on thin-layer chromatograms when E. coli starved for amino acids were labeled with ³²P-phosphate. (p)ppGpp has wide-ranging effects on bacterial physiology.  The best-known activity of (p)ppGpp is its attachment, along with the protein DksA, to RNA polymerase during amino acid starvation, causing transcription of rRNA and tRNA genes to cease and transcription of amino acid biosynthetic operons to increase.  This make sense since there's no point in making more ribosomes until more amino acids are available to support protein synthesis and growth of the bacteria.

Magic spot!  Source

(p)ppGpp production is also necessary to generate the rare persisters in cultures of proliferating bacteria.  When the two genes coding for the enzymes that make (p)ppGpp, relA and spoT, are deleted, persister cells become even more rare in growing E. coli cultures.

A recent study published in the journal Science looked at the role of the stringent response in inducing antibiotic tolerance in biofilms, in which nondividing bacteria are embedded in a matrix secreted by the bacteria.  These studies were conducted with E. coli and Pseudomonas aeruginosa strains with deletion mutations in relA and spoT.  When the biofilms formed by the mutant and wild-type strains were treated with different antibiotics, the mutants turned out to be much more sensitive to the antibiotics even though the bacteria were not dividing (data for P. aeruginosa shown in bar graphs below).  Similar results were obtained when standard cultures of the P. aeruginosa mutant and wild-type strains were grown to stationary phase and then treated with antibiotics.

Figure 1E from Nguyen et al.  P. aeruginosa was allowed to form biofilms on polycarbonate membrane filters resting on agar culture medium.  The biofilms were incubated with different antibiotics or with no antibiotic (control).  To determine the number of bacteria surviving antibiotic treatment, the bacteria in the biofilms were dispersed by vigorous mixing and plated onto agar culture medium to determine cfu counts.  +SR, complementation of the ΔrelA ΔspoT mutant with intact copies of relA and spoT; *P ≤ 0.05, ***P ≤ 0.0005 versus wildtype.

Bactericidal antibiotics, regardless of their target, are now known to enhance production of reactive oxygen species (ROS) within bacteria.  If not detoxified by enzymes such as catalase and superoxide dismutase, ROS can fatally damage the bacteria by reacting with their DNA, protein, and lipids.  ROS is generated within bacteria during the course of their normal metabolic activities, even when antibiotics are not present.  When the authors measured the amounts of hydroxyl radical generated within untreated E. coli and P. aeruginosa biofilms, the ΔrelA ΔspoT mutants were burdened with higher levels of hydroxyl radicals than the wild-type strains. Why do the mutant strains have higher levels of hydroxyl radicals?  Further analysis of the bacteria in the biofilms showed that the ΔrelA ΔspoT mutants also had lower levels of catalase and superoxide dismutase activity.  It's possible that bactericidal antibiotics triggered production of lethal amounts of ROS that the mutants could not handle due to their insufficient production of catalase and superoxide dismutase, but this needs to be confirmed by further experimentation using bacterial strains with the superoxide dismutase and catalase genes knocked out.

The Science study also looked at infected laboratory mice treated with antibiotics.  Wild-type and ΔrelA ΔspoT P. aeruginosa strains were grown to stationary phase, and a lethal dose of the bacteria were injected into the abdominal cavity of mice.  Four hours later the animals were treated with the antibiotic ofloxacin.  The antibiotic was more effective at preventing lethal infection by the ΔrelA ΔspoT mutant than those caused by the wild-type strain.  Similarly, ofloxacin was more effective at reducing the number of bacteria in a biofilm chamber implanted underneath the mouse skin when the biofilm was composed of the ΔrelA ΔspoT mutant.

One must keep in mind that multiple pathways to persister cell formation have been identified (see figure below).  Note that for the animal experiments persisters were allowed to develop in the test tube prior to animal inoculation.  It is possible that the stringent response would not be involved at all if persisters were allowed to develop during infection instead.

Figure 4 from Lewis, 2010.  The redundant pathways to persister formation are shown.  FMN, flavin mononucleotide pool; pmf, proton motive force; TAs, toxin/antitoxin modules

Featured paper

Nguyen, D., Joshi-Datar, A., Lepine, F., Bauerle, E., Olakanmi, O., Beer, K., McKay, G., Siehnel, R., Schafhauser, J., Wang, Y., Britigan, B.E., & Singh, P.K. (2011). Active starvation responses mediate antibiotic tolerance in biofilms and nutrient-limited bacteria Science, 334 (6058), 982-986 DOI: 10.1126/science.1211037

Key references

Bigger, J.W. (1944). Treatment of staphylococcal infections with penicillin by intermittent sterilisation The Lancet, 244 (6320), 497-500 DOI: 10.1016/S0140-6736(00)74210-3

Moyed H.S., & Bertrand K.P. (1983). hipA, a newly recognized gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis. Journal of bacteriology, 155 (2), 768-775 PMID: 6348026

Cashel, M., & Gallant, J. (1969). Two compounds implicated in the function of the RC gene of Escherichia coli Nature, 221 (5183), 838-841 DOI: 10.1038/221838a0

Paul, B., Barker, M.M., Ross, W., Schneider, D.A., Webb, C., Foster, J.W., & Gourse, R.L. (2004). DksA: a critical component of the transcription initiation machinery that potentiates the regulation of rRNA promoters by ppGpp and the initiating NTP. Cell, 118 (3), 311-322 DOI: 10.1016/j.cell.2004.07.009

Korch, S.B., Henderson, T.A., & Hill, T.M. (2003). Characterization of the hipA7 allele of Escherichia coli and evidence that high persistence is governed by (p)ppGpp synthesis Molecular Microbiology, 50 (4), 1199-1213 DOI: 10.1046/j.1365-2958.2003.03779.x

Kohanski, M.A., Dwyer, D.J., Hayete, B., Lawrence, C.A., & Collins, J.J. (2007). A common mechanism of cellular death induced by bactericidal antibiotics Cell, 130 (5), 797-810 DOI: 10.1016/j.cell.2007.06.049

Lewis, K. (2010). Persister cells Annual Review of Microbiology, 64 (1), 357-372 DOI: 10.1146/annurev.micro.112408.134306

Related posts

• Chronic Lyme disease in mice?
• Tigecycline fails to eradicate persisting Borrelia burgdorferi

Still no solid evidence for the Old World origin of syphilis

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

Related posts in Spirochetes Unwound

• The origin of syphilis: a phylogenetic approach

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