Monday, December 31, 2012

Biofilms of the Lyme disease spirochete

Thanks to a recent study published in PLoS One, we now know that free-swimming Borrelia burgdorferi are able to organize themselves into a sedentary community called a biofilm.  This is not too surprising since most other bacteria are capable of the same feat when provided the opportunity.  In fact, outside of the laboratory many bacteria, including those that live on and within us, spend much of their time within biofilms.

Prior to the 1990s biofilms were thought to be blobs of goo containing bacteria randomly distributed throughout their sticky matrix.  In reality, the bacteria and matrix are carefully organized into a complex three-dimensional structure. B. burgdorferi biofilms are no exception.  The organization of B. burgdorferi is apparent even at the earliest stages of biofilm development.  The images below show B. burgdorferi developing into a biofilm on a solid surface.  Instead of randomly associating with each other, the spirochetes organize themselves into "nets" of the type you see hanging from basketball hoops.  The spirochetes come together lengthwise to form the "strands" of the net.  With time, the biofilm thickens as the bacteria form additional layers.  Most of the spaces in the net close up with the rest probably ending up forming a network of channels.  The remaining holes can be seen as pits along the surface of the mature biofilm.  The pits appear to be entry points for the channels, which are thought to circulate nutrients to the members of the community and remove waste products.

Figure 2 from Sapi et alAtomic force microscopy of a developing B. burgdorferi biofilm.
Stalks can also rise up from the surface of microbial biofilms.  One stalk can be seen in this "flyover" along the surface of a mature B. burgdorferi biofilm.

video
Video S2 from Sapi et al.  Composte image from atomic force microscopy.

The matrix of B. burgdorferi biofilms includes DNA and an alginate-like substance bound to calcium.  The images below capture what appears to be matrix being laid down at an early stage of biofilm formation.

Figure 4 from Sapi et al.  Atomic force microscopy of an aggregate of B. burgdorferi in an early stage of biofilm development.  The matrix is colored blue in panel B.

What is the biological significance of B. burgdorferi biofilms?  To answer this question, the authors will need to determine whether B. burgdorferi assembles into biofilms at some point during its life cycle, which involves stages in the tick and vertebrate host.

Reference

Sapi, E., Bastian, S.L., Mpoy, C.M., Scott, S., Rattelle, A., Pabbati, N., Poruri, A., Burugu, D., Theophilus, P.A.S., Pham, T.V., Datar, A., Dhaliwal, N.K., MacDonald, A., Rossi, M.J., Sinha, S.K., & Luecke, D.F. (2012). Characterization of biofilm formation by Borrelia burgdorferi in vitro. PLoS ONE, 7 (10) DOI: 10.1371/journal.pone.0048277

Monday, November 26, 2012

A post-Thanksgiving story of leptospirosis

I'm about half way through 1491, a book that gives readers a view of the Americas before Columbus showed up.  It also describes the devastating impact that foreign infectious diseases had on the native population as Europeans explored the New World.

One chapter tells the story of Tisquantum (Squanto), who lived in the village of Patuxet, one of the many Indian communities thriving along the coast of New England at the time.  In 1614 Thomas Hunt, a British slave trader, kidnapped Tisquantum and other Indians and shipped them to Spain.  Fortunately, Tisquantum was rescued by Spanish priests before he could be sold.  After convincing the priests to let him return home, he left for London, where he learned English while staying at a shipbuilder's home, and eventually made his way back to North America.  As he sailed down the New England shoreline in 1619 on a British ship, he realized that the world familiar to him had vanished.  A mysterious disease had wiped out 90% of the population of coastal New England.  When he arrived at his home village of Patuxet, he found it deserted.  Tisquantum was soon captured and sent to Massasoit, the leader of the Wampanoag confederacy, which encompassed Patuxet.  Massasoit did not trust Tisquantum because of his recent association with the British, yet he would later use him as a translator in a negotiation that turned out to be a pivotal event in American history.

The epidemic had been blamed at one time or another on smallpox, the plague, yellow fever, typhus, and hepatitis.  As I've mentioned before, a recent analysis has added leptospirosis to the list of suspects.  The symptoms and signs of leptospirosis match those reported from first-hand accounts of the mystery ailment.  Here's a post on the Slate website about the epidemic.  I'm glad to see that the story is getting attention from popular news sites.

Leptospirosis can be deadly, but could it account for the devastating 90% motality rate of the 1616-1619 epidemic?  A hypervirulent strain of Leptospira or genetic susceptibility of the Indians could be an explanation.  However, the authors of the study thought that the most critical factor was the Indian lifestyle, which brought them into repeated contact with Leptospira in the environment.  The Europeans who fished nearby were spared because they did not engage in activities that exposed them to Leptospira.  Therefore, only the Indians contracted the illness, according to the hypothesis.

Figure 3 from Marr and Cathey, 2010.

Whatever its cause, it's hard to overstate the significance of the epidemic.  Prior to 1616, the New England native communities traded with the Europeans and even welcomed them for brief stays.  However, all attempts by the foreigners to establish permanent settlements were fiercely resisted.  Coastal New England was well defended by the large native population.  The Wampanoag confederacy became especially hostile towards the Europeans after having their citizens abducted.

By the time the Mayflower landed in Patuxet (Plymouth) in December of 1620, the thinking of the Wampanoag had changed.  Their depleted population was vulnerable to attack by their longtime enemies to the west, the Narragansett, who remained untouched by the epidemic.  To forestall an attack, Massasoit felt that the best course of action was to form an alliance with the Pilgrims rather than expel them.  In the spring of 1621, with Tisquantum serving as the translator, Massasoit arranged a peace treaty with the Pilgrims.

Reference

Marr J.S. & Cathey J.T. (2010). New hypothesis for cause of epidemic among Native Americans, New England, 1616–1619, Emerging Infectious Diseases, 16 (2) 281-286. DOI:

Related post


Thursday, November 8, 2012

Inflammatory spirochete debris left behind following antibiotic treatment for Lyme disease

According to the CDC, 10-20% of Lyme disease patients who have completed antibiotic therapy continue to suffer from symptoms such as joint, muscle, and neurological pain.  The following hypotheses are often presented as possible reasons for the lingering symptoms:  autoimmunity triggered by the infection, tissue damage inflicted by the spirochetes, and (depending on whom you ask) failure of antibiotics to kill all the spirochetes.  A new paper from Linda Bockenstedt's group at Yale proposes that antibiotic treatment of disseminated Borrelia burgdorferi infection leaves behind inflammatory pieces of dead spirochetes that are responsible for the persisting symptoms.

Bockenstedt's group used the mouse model of Lyme disease for the study.  To ensure that the tissues harbored enough B. burgdorferi spirochetes to be visible by intravital microscopy, the mice were genetically deficient in the intracellular signaling protein MyD88.  MyD88 links the recognition of microbial parts by most Toll-like receptors to activation of certain nuclear genes whose products are involved in the inflammatory process.  Mice lacking MyD88 are unable to control the proliferation of a number of bacterial pathogens, including B. burgdorferi.  The load of B. burgdorferi in tissues is about 100-fold higher in MyD88-deficient mice than in mice with a complete immune system.

The spirochetes were genetically altered to express green fluorescent protein (GFP).  The GFP+ B. burgdorferi was introduced into the MyD88-deficient mice by tick inoculation.  21 days later some of the mice were treated for one month with doxycycline, one of the antibiotics used to treat Lyme disease in humans.

The researchers next peered into the thin layer of skin covering the ear by intravital microscopy.  In the mice that were left untreated, they saw lots of spirochetes scurrying about in the dermis.

video

At the deepest depths of the dermis, they noticed immobile specks and patches of green material deposited near the cartilage.  They also saw the deposits in the doxycycline-treated mice.  The material was detected by immunofluorescence of ear sections with antibody against B. burgdorferi up to 10 weeks after antibiotic treatment was completed, indicating that the immune system was unable to clear the deposits.

There was no evidence that any spirochetes survived antibiotic treatment.  The researchers did not see any motile spirochetes in the skin by intravital microscopy.  In addition, tissues were culture negative, ticks that fed on the treated mice were culture negative (xenodiagnosis), and transplantation of skin from the treated mice failed to transmit the infection to recipient mice.  Based on these results, the authors concluded that the deposits were remnants of dead spirochetes.  As expected, untreated mice tested positive by these assays.

Since chronic infection can lead to Lyme arthritis, the investigators also examined the joints.  In another set of mice, the infection was allowed to proceed for four months.  The mice were then treated with the antibiotic ceftriaxone for 18 days.  When the researchers looked in the joints by intravital microscopy, they again saw the green material (see figure below).



Fig. 5 from Bockenstedt et al. showing the surface of the patella where it meets the tendon (enthesis).   Panel A, from mouse infected for 4 months, untreated.  Panel B, from mouse infected for 4 months and then treated with ceftriaxone for 18 days. Scale bar, 30 µm.

A critical issue to address is whether the amorphous material left behind following antibiotic treatment inflames the joints.   The authors could not answer this question directly because of the limitations of the mouse model. Histopathology is unlikely reveal joint inflammation, even in the untreated animals, because laboratory mice do not reliably exhibit joint inflammation so late (4-5 months) during B. burgdorferi infection.  Instead, the authors conducted a test tube experiment to see whether the deposits had inflammatory potential.  They ground up joint tissue from antibiotic-treated mice in buffer and applied the homogenate to cultured mouse macrophages.  The macrophages responded by producing TNF, a key cytokine that promotes inflammation.  The more tissue that was added, the more TNF that was produced by the macrophages.  In contrast, joint tissue from uninfected mice did not promote TNF production by the macrophages.  Therefore, the deposits had the potential to spark inflammation, even after motile spirochetes were eliminated by antibiotics.  The debris would continue to inflame the tissues even after antibiotics killed all live spirochetes, explaining why symptoms persist in ~10% of Lyme arthritis cases even after antibiotic treament.

The relevance of the deposits to Lyme disease in humans could be questioned because the MyD88-deficient mice did not have a complete immune system.  The authors addressed this concern in the Discussion by mentioning a recent study that described a TLR1 variant linked to severe inflammation and treatment failure in Lyme arthritis patients.  Although the gene encoding MyD88 has never been examined in Lyme disease patients, it is conceivable that the TLR1 variant or different forms of other immune genes lead to deposits of Borrelia antigen in the joint and other host tissues.

The authors also addressed the possibility that the deposits are really biofilms, which generally resist killing by antibiotics.  Biofilms are believed to be populated by persister cells, which are in a nondividing state that allows bacteria to tolerate antibiotics.  According to the authors, if the deposits had harbored persister cells, those cells should have resumed growing when conditions became favorable for growth again.  Because the skin and joints from the treated mice were culture negative and because the skin also tested negative by xenodiagnosis and transplantation assays, the authors quickly dismissed the biofilm hypothesis.

Stricly speaking, the authors are correct.  Persister cells should start multiplying again in fresh culture medium.  However, it's hard to dismiss the biofilm hypothesis completely given the known examples of culture-negative chronic infections associated with biofilms (see this review for one example).  Electron microscopy of the joint tissue could reveal whether these deposits are intact spirochetes or debris.

Regardless of their exact nature, deposits of antigen have never been detected within the joints of Lyme arthritis patients.  Allen Steere's group failed to find such deposits in pieces of synovial membrane removed from 26 patients with antibiotic-refractory Lyme arthritis.   The findings of Bockenstedt and colleagues, who detected the deposits in a location outside of the synovial membrane, suggest that Steere's group was looking in the wrong place.


Featured paper

Bockenstedt, L., Gonzalez, D., Haberman, A., & Belperron, A. (2012). Spirochete antigens persist near cartilage after murine Lyme borreliosis therapy Journal of Clinical Investigation, 122 (7), 2652-2660 DOI: 10.1172/JCI58813
 
Helpful references

Bolz DD, Sundsbak RS, Ma Y, Akira S, Kirschning CJ, Zachary JF, Weis JH, and Weis JJ (August 1, 2004).  MyD88 plays a unique role in host defense but not arthritis development in Lyme disease.  The Journal of Immunology 173(3):2003-2010.  Link

Strle K, Shin JJ, Glickstein LJ, and Steere AC (May 2012).  Association of a Toll-like Receptor 1 polymorphism with heightened Th1 inflammatory responses and antibiotic-refractory Lyme arthritis.  Arthritis and Rheumatism 64(5):1497-1507.  DOI: 10.1002/art.34383

Bakaletz LO (October 2007).  Bacterial biofilms in otitis media, evidence and relevance.  The Pediatric Infectious Disease Journal 26(10):S17-S19.  Link

Carlson D, Hernandez J, Bloom BJ, Coburn J, Aversa JM, Steere AC (December 1999).  Lack of Borrelia burgdorferi DNA in synovial samples from patients with antibiotic treatment-resistant Lyme arthritis.  Arthritis and Rheumatism 42(12):2705-2709.  DOI: 10.1002/1529-0131(199912)42:12<2705::aid-anr29>3.0.CO;2-H


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Sunday, July 22, 2012

100 (micro)meter dash

A fun read in the current issue of Nature Reviews Microbiology is an essay entitled "The Microbial Olympics, " just in time for the Summer Olympics.  You will find stories about microbes competing in boxing, javelin, pathogen relay, diving, and other Olympic events.  Flagellated bacteria compete in the 100 micrometer dash, which you can watch below.  (Note that some of the contestants were genetically modified.)

video

Lane assignments:
  1. E. coli chimera (has sodium-driven flagellar motors instead of its normal proton-driven motors)
  2. E. coli (proton-driven flagellar motors)
  3. Vibrio alginolyticus, puller (clockwise-locked flagellum "pulls" cell body from front)
  4. Vibrio alginolyticus, pusher (counterclockwise-locked flagellum "pushes" cell body from back)
  5. Pseudomonas aeruginosa
  6. Rhodobacter sphaeroides
  7. Rhodospirillum rubrum
  8. Yersinia enterocolitica

Reference

Youle M, Rohwer F, Stacy A, Whiteley M, Steel BC, Delalez NJ, Nord AL, Berry RM, Armitage JP, Kamoun S, Hogenhout S, Diggle SP, Gurney J, Pollitt EJG, Boetius A, and Cary SC (August 2012).  Nature Reviews Microbiology 10(8):583-588.  DOI: 10.1038/nrmicro2837

Not so golden? Microscopic agglutination test for diagnosis of leptospirosis

The microscopic agglutination test (MAT) is designated the "gold standard" for the laboratory diagnosis of leptospirosis, a spirochete disease that can cause severe illness if not promptly treated.  Although imperfect, MAT is used as the benchmark when the performance of another diagnostic test for leptospirosis is being assessed.  It is also used to determine the prevalence of leptospirosis in a population.  How imperfect is MAT?  A recent study by Limmathurotsakul and colleagues, published in the journal Clinical Infectious Diseases, claims that its performance is much worse than scientists previously thought.

MAT involves mixing serial dilutions of patient sera with live suspensions of Leptospira.  If agglutinating antibodies against Leptospira are present, the spirochetes will clump.  The clumps can be seen by darkfield microscopy.  Although the idea behind MAT is simple to understand, the technique itself is cumbersome.  Since agglutinating antibodies react best with the specific Leptospira serovar infecting the patient, cultures of at least one serovar from each of the ~20 major Leptospira serogroups must be maintained.  To perform the assay, each serum dilution is mixed individually with a suspension from each culture and examined by microscopy one at a time.  The assay is time consuming, laborious, and potentially hazardous to laboratory personnel.  For these reasons MAT is not routinely employed for diagnostic testing outside of the research setting.

The performance of a diagnostic test is judged by its sensitivity and specificity.  The problem with leptospirosis is figuring out how many actually have the disease so that the sensitivity can be calculated accurately.  Since the sensitivity of culture is poor, researchers rely on antibody tests such as MAT to identify leptospirosis cases.  This approach assumes that the sensitivity and specificity of MAT are 100%.

In general there are two problems with using antibody tests for diagnostics.  The first is that it takes time for the immune system to generate enough antibody that can be detected.  The second problem is that those with previous exposure to the pathogen will test positive even if they are not currently infected.  To minimize these problems, patients with the signs and symptoms of leptospirosis are deemed to have a positive MAT if they fulfill one of the following criteria.
  • At least a four-fold increase in MAT titer between paired sera. 
  • At least a 1:400 MAT titer when only a single specimen is available.  This cutoff is sometimes adjusted based on the prevalence of leptospirosis in the population being examined.

Since some false negative MAT cases can be identified by culture, one way to calculate the sensitivity of MAT is to add the number of MAT-positive and culture-positive (but MAT-negative) cases together to estimate the number of patients with leptospirosis and then calculate the percentage of MAT-positive cases among these patients.  Limmathurotsakul and colleagues performed these calculations with data from their four earlier studies conducted in Thailand.  A total of 413 patients tested positive by MAT or culture (or both).  They found that the sensitivity of MAT was 86%-96% across the four sets of data.  The remaining 4-14% were false negatives, having tested positive by culture but not by MAT.

The authors next calculated the true sensitivity and specificity of MAT with a statistical tool called latent class analysis, which does not assume any perfect gold standard.  Since there is no perfect test, the true disease status of each patient is the unknown or "latent" variable.  Results from multiple diagnostic tests are related to the latent variable using statistical models.  The calculations go beyond the scope of this blog post, but the bottom line is that the true sensitivity and specificity of each diagnostic test can be estimated with these models.  In addition to MAT and culture, the authors tested some of their patients with an immunofluorescence assay (IFA), lateral flow test (LF), and/or PCR. Latent class analysis is more powerful when the diagnostic tests being evaluated detect different features of the infection.  MAT, IFA, and LF are antibody tests, and culture and PCR detect the pathogen itself.

The sensitivity of MAT calculated by this method turned out to be only 49.8%, much lower than the 86%-96% calculated using the standard method that assumes a perfect gold standard.  The sensitivity of culture alone was 10.5%.  Combining culture with MAT did not help much; the sensitivity of the combined approach was only 55.5%.  The low sensitivity of "MAT plus culture" suggests that the specificities calculated for the alternative tests may be underestimated by the standard method.  This is because some of the many false-negative cases may be correctly identified as having leptospirosis by the alternative tests.  This turned out to be the case for two of the tests.   Specificities for all tests were over 95% by latent class analysis.  However, the specificities for PCR (82.5%) and the lateral flow test (70.5%) were lower when "MAT plus culture" was assumed to be the perfect gold standard.

You can see that the accuracy of alternative leptospirosis tests is underestimated when MAT (or MAT plus culture) is assumed to be the perfect reference test.  Another implication of the study is that the prevalence of leptospirosis has been underestimated, at least in Thailand.  The only other study to evaluate the performance of MAT by latent class analysis was conducted by the CDC here in the U.S almost a decade ago.  In contrast to the Limmathurostsakul study, the CDC study determined that the sensitivity of MAT was high, at 98.2%.  There were many differences between the two studies, including the patient population, the alternative tests evaluated, the time interval between collection of paired sera, and the number of serovars included for MAT.  The poor performance of MAT in the Thailand study may therefore not be a universal finding.

References

Limmathurotsakul D, Turner EL, Wuthiekanun V, Thaipadungpanit J, Suputtamongkol Y, Chierakul W, Smythe LD, Day NPJ, Cooper B, Peacock SJ (August 2012).  Fool's gold: why imperfect reference tests are undermining the evaluation of novel diagnostics: a reevaluation of 5 diagnostic tests for leptospirosis.  Clinical Infectious Diseases 55(3):322-331.  DOI: 10.1093/cid/cis403

Bajani MD, Ashford DA, Bragg SL, Woods CW, Aye T, Spiegel RA, Plikaytis BD, Perkins BA, Phelan M, Levett PN, and Weyant RS (February 2003).  Evaluation of four commercially available rapid serologic tests for diagnosis of leptospirosis.  Journal of Clinical Microbiology 41(2):803-809.  DOI:  10.1128/JCM.41.2.803-809.2003

Thursday, July 12, 2012

Borrelia burgdorferi needs the alternative sigma factor RpoS to flee from the tick's midgut

The alternative sigma factor RpoS is a key player in the life cycle of Borrelia burgdorferi, the Lyme disease spirochete.  RpoS directs RNA polymerase to transcribe genes with promoters recognized by the alternative sigma factor.  B. burgdorferi deploys RpoS to directly or indirectly boost transcription of 103 out of its ~1400 genes while inside a mammalian host.  The most famous RpoS-dependent gene is ospC, which encodes a surface protein that enables B. burgdorferi to survive the early stages of infection.  Not surprisingly, RpoS is essential for B. burgdorferi to establish infections in mammals.  On the other hand, B. burgdorferi does not bother to make RpoS while living in the midgut of Ixodes ticks since RpoS-dependent gene products are not needed in this stage of its life cycle.  The rpoS gene is turned on only after the tick attaches to an animal and begins sipping its blood.  B.burgdorferi is transmitted to the victim as the tick feeds.

A study published by Justin Radolf's group in the February issue of PLoS Pathogens showed that RpoS is needed by B. burgdorferi to be transmitted from the tick to a mammal.  Transmission is a multistep process for B. burgdorferi.  Although the spirochetes proliferate to large numbers in the midgut while the tick feeds, only a few of them escape through the wall of the midgut into the hemocoel, the tick's body cavity.  From there the spirochetes invade the salivary glands, which produces the saliva that carries the spirochetes into the victim's skin.  The authors found that B. burgdorferi mutants missing their rpoS gene failed to even make it out of the midgut.  None of the hemolymph samples extracted from the hemocoel of 39 feeding ticks carrying the rpoS mutant were culture positive, whereas the hemolyph from 21 of 25 feeding ticks harboring the wild-type strain were culture positive.

The researchers also viewed the activity of the rpoS mutant in the midgut by fluorescence microscopy.  From an earlier study (described in this blog post), they already knew how wild-type B. burgdorferi behaved within the midgut of feeding ticks.  In brief, the multiplying spirochetes remained firmly attached to the epithelial cells.  A mesh of spirochetes eventually surrounded the cells.  Since an unknown substance in the midgut was inhibiting motility, only the few spirochetes at the base of the epithelial cells detached and managed to wiggle their way into the surrounding hemocoel.

The rpoS mutant behaved quite differently from the wild-type strain in feeding ticks.  Instead of remaining stuck to the surface of the gut epithelial cells, the mutant spirochetes detached and accumulated in the lumen of the midgut.  Since the spirochetes were immotile, they were too far away from the base of the epithelium lining to escape into the hemocoel.

To get a better look of the spirochetes, the researchers examined silver-stained sections of the midgut contents by microscopy.  Here they saw something fascinating.  With the wild-type B. burgdorferi, they saw tufts of spirochetes attached to the epithelial cells, as expected from their earlier studies (panels D and G below).  With the rpoS mutant, they found midguts packed with round bodies (rpoS mutant, panels E and H).  The round bodies were not dead.  When the investigators removed the midguts and released the contents into Borrelia culture medium, the round bodies reverted back to the spiral shape within minutes.

Extracted from Figure 4 of Dunham-Ems et al., 2012.  Midguts of Ixodes ticks after feeding on mice for 72 hour.  Panels D and G, wild-type B. burgdorferi.  Panels E and H, rpoS mutant.  Bars, D and E, 25 µm; G and H, 10 µm.  Source

Spirochetes in culture change shape into round bodies when their nutritional demands fail to be met.  The authors suspected that the rpoS mutant had a metabolic defect that caused the spirochete to round up while rapidly proliferating in the feeding tick's midgut.  They suspected that limited expression of the enzyme coenzyme A disulfide reductase (CoADR) was the source of the metabolic defect since they knew from earlier work that transcription of cdr was partially dependent on RpoS.  (The "housekeeping" sigma factor σ70 also transcribes cdr.)  CoADR couples the oxidation of NADH to NAD+ with the reduction of the disulfide bond linking two molecules of coenzyme A together.  A major role of this reaction is to replenish the NAD+ that is reduced during glycolysis, the primary means for energy generation in B. burgdorferi.

To test their prediction, the researchers knocked out the cdr gene.  Next, they inoculated the mutant into culture medium lacking nutrients needed by B. burgdorferi to grow.  As predicted, they found that starved cdr mutants formed round bodies at an even higher frequency than wild-type B. burgdorferi.  This result supported the notion that the failure of the rpoS mutant to produce enough CoADR is what triggered round bodiy formation in feeding ticks.

ResearchBlogging.orgDo the round bodies serve any biological role in the life cycle of Borrelia burgdorferi, or are they a laboratory artifact generated by knocking the rpoS gene out?  The investigators even saw a few round bodies among the many spiral-shaped spirochetes in feeding ticks harboring wild-type B. burgdorferi. This observation may suggest that round bodies indeed do have a role.  In the final sentence of their paper, the authors leave us to ponder the following: "We propose that round body formation has evolved to support the tick phase of the cycle and predict that there are circumstances, as yet undefined, when spirochetes within the tick resosrt to this survival program on a large scale in order to maintain a population of transmissible organisms."

Main reference

Dunham-Ems SM, Caimano MJ, Eggers CH, & Radolf JD (2012). Borrelia burgdorferi requires the alternative sigma factor RpoS for dissemination within the vector during tick-to-mammal transmission. PLoS pathogens, 8 (2) PMID: 22359504, DOI: 10.1371/journal.ppat.1002532

Other helpful references

Caimano MJ, Iyer R, Eggers CH, Gonzalez C, Morton EA, Gilbert MA, Schwartz I, and Radolf JD (September 2007).  Analysis of the RpoS regulon in Borrelia burgdorferi in response to mammalian host signals provides insight into RpoS function during the enzootic cycle.  Molecular Microbiology 65(5):1193-1217.   DOI: 10.1111/j.1365-2958.2007.05860.x

Eggers CH, Caimano MJ, Malizia RA, Kariu T, Cusack B, Desrosiers DC, Hazlett KRO, Claiborne A, Pal U, and Radolf JD (November 2011).  The coenzyme A disulphide reductase of Borrelia burgdorferi is important for rapid growth throughout the enzootic cycle and essential for infection of the mammalian host.  Molecular Microbiology 82(3):679-697.  DOI: 10.1111/j.1365-2958.2011.07845.x

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Friday, June 22, 2012

Looking for the syphilis spirochete in ancient bones

PCR is a powerful tool that has been used to detect microbial DNA in human remains unearthed by archaeologists.  This approach has helped reveal when and where infectious diseases such as tuberculosis and the plague have afflicted human populations in the past.  With the controversy raging over the question of whether the syphilis spirochete was present in Europe before Columbus sailed to America, one would think that scientists would have tried PCR to detect Treponema pallidum DNA in skeletal remains.  Well, they have, but in almost every case they failed to detect T. pallidum DNA, even in bones bearing the lesions of syphilis.  The problem is that adults who die in the later stages of syphilis do not have many T. pallidum spirochetes in their bones.

On the other hand, spirochetes are relatively abundant in the bones of infants afflicted with congenital syphilis.  Therefore the skeletal remains of the very young may be a better source for detection of T. pallidum DNA by PCR.  As reported in their recent PLoS One article, Montiel and colleagues looked for T. pallidum DNA in skeletal remains gathered from a 16th-17th century crypt in Spain.  The investigators found four infant bones with lesions that were consistent with congenital syphilis.  Since there were two left humeri (specimens ELS551 and ELS558 in the image below), the bones must have belonged to at least two newborns.

Figure 1 from Montiel et al., 2012.  Source.

The PCR reactions were conducted on specimens from both newborns in each of three different laboratories.  Two segments along the T. pallidum chromosome were targeted.  One lab targeted the arp gene, the second lab targeted the 5' UTR of the 15 kDa lipoprotein gene, and the third targeted both sequences.  All attempts but one led to the generation of PCR products.  To confirm that they derived from T. pallidum sequences, the PCR products were either analyzed by restriction digestion or cloned and sequenced.  The 5' UTR of the lipoprotein gene was critical to this effort because its sequence can be used to distinguish the syphilis spirochete from the other disease-causing treponemes.  The PCR products from both newborns turned out to have the Eco47III restriction site that is unique to the syphilis spirochete among modern treponemes.  The molecular analysis therefore supported the diagnosis of congenital syphilis in the two long-deceased infants.

ResearchBlogging.orgThe investigators took special precautions to minimize the risk of contamination, which is always a concern of paleomicrobiologists running PCR reactions.  For example, the experiments were done in laboratories in which Treponema-containing samples had never been handled.  The investigators even excluded positive controls from their PCR reactions.

Scientists are still not certain whether congenital syphilis can be correctly diagnosed by examining bone pathology alone (see pp. 102-103 of this paper for a nice discussion of this issue).  The PCR method will therefore aid scientists wishing to identify skeletal remains afflicted with congenital syphilis.  It also gives paleomicrobiologists hope that PCR methods will help answer the centuries-old question about the origin of syphilis.

References

Montiel R, Solórzano E, Díaz N, Álvarez-Sandoval BA, González-Ruiz M, Cañadas MP, Simões N, Isidro A, & Malgosa A (2012). Neonate human remains: a window of opportunity to the molecular study of ancient syphilis. PloS one, 7 (5) PMID: 22567153

Bouwman, AS, & Brown, TA (2005). The limits of biomolecular palaeopahology: ancient DNA cannot be used to study venereal syphilis Journal of Archaeological Science, 32, 703-713 DOI: 10.1016/j.jas.2004.11.014

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Monday, May 28, 2012

Do nonspiral spirochetes help clean our environment?

Members of the spirochete phylum Spirochaetes are recognized easily by their long spiral shape, which allows their periplasmic flagella to power them through viscous environments.  But scientists are discovering that not all spirochetes share this peculiar shape.  Two bacterial isolates recovered from freshwater sediments in Michigan were spherical and lacked flagella, yet phylogenetic analysis of their 16S rRNA and other genes placed them firmly within the Spirochaetes.  The genus Sphaerochaeta was created to accommodate the new isolates, which were designated Sphaerochaeta globosa and Sphaerochaeta pleomorpha.

Sphaerochaeta pleomorpha viewed by phase contrast microscopy.  Arrowheads point to protrusions.  Panel B shows the round spirochetes organized as "strings of pearls."  Figure 1a and 1b from Ritalahti et al., 2012.

Sphaerochaeta globosa viewed by phase contrast microscopy.  Figure 2a from Ritalahti et al., 2012.

The disease-causing spirochetes such as Borrelia burgdorferi and Leptospira species are shape changers.  Although they are often observed with the familiar spiral morphology, they sometimes morph into nonmotile round bodies when stressed, only to revert to the spiral form when conditions improve (see images below).  Could the Sphaerochaeta strains sprout flagella and morph into the spiral form under the right conditions?  It doesn't appear likely.  Sphaerochaeta retain their round shape under a variety of growth conditions, and their genomes lack motility and chemotaxis genes, including those encoding the components of the flagellum.

The Lyme disease spirochete B. burgdorferi viewed by electron microscopy.  Panel A:  B. burgdorferi in its standard growth medium BSKII, which contains serum.  Panel B:  Most of the spirochetes appear as round bodies after being starved for serum for 48 hours.  Bar, 2 µm.   Figure 1A and 1B from Alban et al., 2000.


Views of B. burgdorferi by phase contrast microscopy.  Panel A: B. burgdorferi starved for serum for 48 hours.  Panel B:  Less than one minute after the culture is replenished with serum, the round bodies convert back to the spiral form.  Bar, 5 µm.  Figure 2A and 2B from Alban et al., 2000.
Sphaerochaeta spirochetes have another unusual property.  Electron microscopy revealed what could be a peptidoglycan-layered cell wall (see image below), yet they grow fine even when high concentrations of ampicillin are dumped into the growth meduim.  The genome sequence revealed the reason for their resistance to the antibiotic.  Although the two Sphaerochaeta strains had the genes necessary to make peptidoglycan, they were missing the genes encoding the enzymes that strengthen the cell wall by cross-linking the peptidoglycan.  These missing enzymes are the targets of β-lactams, the penicillin class of antibiotics that includes ampicillin.  Without the cross-linking enzymes, one may expect the cell wall to be fragile, but it isn't.  The strains grow fine in hypotonic medium, which would have caused the bacteria to burst if they had a weak cell wall.  What strengthens the Sphaerochaeta cell wall to keep it intact under physical strain remains a mystery.


Cell wall architecture of Sphaerochaeta pleomorpha viewed by electron microscopy.  OM, outer membrane; PS, periplasmic space; CW, cell wall.  Figure 1d from Ritalahti et al., 2012.

Even though Sphaerochaeta reside in oxygen-poor environments, they don't live alone.  They are members of a close-knit microbial community that includes bacteria of the genus Dehalococcoides, which respire by reducing organic chlorides instead of oxygen.  Dehalococcoides have attracted attention because of their potential for cleaning up groundwater and other sensitive environments contaminated with chlorinated organic compounds, pollutants that originated mainly from past industrial and agricultural activities.  Although the production of these toxic compounds has ceased in many countries, the pollutants persist in the environment and must be detoxified.  This is where Dehalococcoides bacteria may be beneficial.  They obtain energy by anaerobic respiration of chlorinated organic molecules, which strips off the chloride atoms, rendering the compounds nontoxic.

ResearchBlogging.orgDehaloccoides bacteria do not grow well on their own unless other members of the microbial community are also present.  This indicates that the other microbes provide something that the Dehalococcoides need for optimal growth.  Sphaerochaeta bacteria extract energy from sugars by fermentation, generating a mixture of waste products that include acetate and H2Dehalococcoides have a strict requirement for acetate as a carbon source, and they must use hydrogen as the electron donor for anaerobic respiration of organic chlorides.  Members of Sphaerochaeta may provide these critical substrates to Dehalococcoides.

S. globosa and S. pleomorpha are the best-characterized nonspiral spirochetes, but they were not the first round spirochetes to be found.  A report from 1992 described a round, cold-loving spirochete recovered from Ace Lake in Antarctica.  This spirochete is a member of the genus Spirochaeta, the closest relative of Sphaerochaeta.  More recently, another round nonmotile spirochete, Spirochaeta coccoides, was isolated from the hindgut of a termite.  Based on its genome sequence, reclassification of Spirochaeta coccoides into the genus Sphaerochaeta was proposed recently.  The residence of nonspiral spirochetes in such diverse environments could mean that they are more widespread than we think.


References

Caro-Quintero, A., Ritalahti, K.M., Cusick, K.D., Loffler, F.E., & Konstantinidis, K.T. (2012). The chimeric genome of Sphaerochaeta: Nonspiral spirochetes that break with the prevalent dogma in spirochete biology mBio, 3 (3) DOI: 10.1128/mBio.00025-12

Ritalahti, K.M., Justicia-Leon, S.D., Cusick, K.D., Ramos-Hernandez, N., Rubin, M., Dornbush, J., & Loffler, F.E. (2011). Sphaerochaeta globosa gen. nov., sp. nov. and Sphaerochaeta pleomorpha sp. nov., free-living, spherical spirochaetes INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 62 (1), 210-216 DOI: 10.1099/ijs.0.023986-0

Alban P.S., Johnson P.W., & Nelson D.R. (2000). Serum-starvation-induced changes in protein synthesis and morphology of Borrelia burgdorferi. Microbiology (Reading, England), 146 ( Pt 1), 119-127 PMID: 10658658

Franzmann P.D., & Dobson S.J. (1992). Cell wall-less, free-living spirochetes in Antarctica. FEMS microbiology letters, 76 (3), 289-292 PMID: 1385265

Dröge S., Fröhlich J., Radek R., & König H. (2006). Spirochaeta coccoides sp. nov., a novel coccoid spirochete from the hindgut of the termite Neotermes castaneus. Applied and environmental microbiology, 72 (1), 392-397 PMID: 16391069

Abt, B., Han, C., Scheuner, C., Lu, M., Lapidus, A., Nolan, M., Lucas, S., Hammon, N., Deshpande, S., Cheng, J., Tapia, R., Goodwin, L., Pitluck, S., Liolios, K., Pagani, I., Ivanova, N., Mavromatis, K., Mikhailova, N., Huntemann, M., Pati, A., Chen, A., Palaniappan, K., Land, M., Hauser, L., Brambilla, E., Rohde, M., Spring, S., Gronow, S., Göker, M., Woyke, T., Bristow, J., Eisen, J.A., Markowitz, V., Hugenholtz, P., Kyrpides, N.C., Klenk, H.-P., & Detter, J.C. (2012). Complete genome sequence of the termite hindgut bacterium Spirochaeta coccoides type strain (SPN1T), reclassification in the genus Sphaerochaeta as Sphaerochaeta coccoides comb. nov. and emendations of the family Spirochaetaceae and the genus Sphaerochaet Standards in Genomic Sciences, 6 (2), 194-209 DOI: 10.4056/sigs.2796069

Taş, N., van Eekert, M.H.A., de Vos, W.M., & Smidt, H. (2009). The little bacteria that can - diversity, genomics and ecophysiology of ‘Dehalococcoides’ spp. in contaminated environments Microbial Biotechnology, 3 (4), 389-402 DOI: 10.1111/j.1751-7915.2009.00147.x

Sunday, May 13, 2012

A tick protein helps Lyme disease spirochetes fight complement

Microbial pathogens attempting to establish an infection face the daunting challenge of overcoming the complement system.  To survive the onslaught of complement proteins, pathogenic microbes express surface structures that resist or manipulate the action of complement.  Not surprisingly, many Lyme disease Borrelia strains express proteins ("CRASPs" and "Erps") that ward off complement.  But they also get help from a protein found in the saliva of the Ixodes tick, according to a study that appeared last August in Cell Host & Microbe.

A Bare-bones Review of the Complement System

The complement system consists of ~30 seemingly innocuous proteins floating in our tissue fluids, with the highest concentrations being found in our bloodstream.  Upon activation, they morph sequentially into several protease complexes (see figure below).  Each protease cleaves specific complement components, generating the subunits for the next protease complex in the cascade.  The cascade ends with assembly of the membrane attack complex, a pore that kills the microbe.  It is important to keep in mind that the membrane attack complex is not the only weapon that the complement system uses to kill invading microbes.  Some of the complement fragments generated by the proteases also help ignite inflammation, in part by attracting phagocytes that engulf the target microbe.

The complement system is triggered when a complex of complement proteins bearing a recognition subunit, either C1q, a ficolin, or mannose-binding lectin (MBL), bind to certain microbial surface molecules.  C1q can also attach to the Fc region of antibodies bound to the microbe.  Binding of the recognition subunit to the microbe activates the protease component of the complex.  The protease (C1r/C1s and MASP in the figure below) cleaves the complement components C4 and C2, leading to the formation of another protease complex on the surface of the microbe, a C3 convertase with the composition C4b-C2a.  Although the classical pathway is triggered by C1q and the lectin pathway by MBL and the ficolins, note that both pathways lead to formation of the C3 convertase.

There is also a third pathway.  The alternative pathway is triggered by covalent binding of exposed hydroxyl groups on membrane surfaces to C3b, which is generated by the slow, spontaneous cleavage of C3.  Of course all cell surfaces, whether pathogen or host, bear hydroxyl groups.  To prevent tissue damage, complement regulators quickly inactivate any C3b molecules that end up binding host cells.  C3b molecules that end up bound to the microbe capture factor B, which is subsequently cleaved by the protease factor D, resulting in the formation of another type of C3 convertase, one that comprises the C3b and Bb subunits.

Regardless of its composition, C3 convertase cleaves C3, generating even more C3b, which end up sticking covalently to the microbial surface.  C3b is a key player in the complement system.  C3b flags the unlucky microbe for destruction by phagocytes, which are attracted to the site of infection by soluble protein fragments generated by the complement proteases.  In addition, C3b can also join with either type of C3 convertase, resulting in yet another protease complex called the C5 convertase.  Cleavage of C5 by C5 convertase releases C5b, which assembles the complement proteins C6 through C9 into the lethal membrane attack complex.

A "simplified" view of complement activation.  Ficolins are not shown.  Source


What They Did

In an earlier study, Schuijt and colleagues identified the Ixodes scapularis tick protein TSLPI as a target for antibodies generated by rabbits that were immunized with tick saliva.  Since TSLPI also possessed anti-complement activity, its properties were examined further by the investigators.

Since TSLPI is actually a glycoprotein, the investigators mass produced TSLPI in an insect cell line so that the recombinant protein, like the native one, would be decorated with sugar molecules.  Following production of TSLPI, the glycoprotein was purified for use in the test-tube experiments described below.

Serum contains all the components necessary to execute the complement cascade, including assembly of the membrane attack complex (MAC).  An easy way to test whether a bacterial strain is susceptible to complement-mediated killing by MAC is to see how well it survives in serum.  When a strain of Borrelia garinii, a European Lyme disease species, was mixed with human serum, the MAC assembled on the surface of the spirochete, and the spirochetes perished.  Addition of TSLPI allowed the spirochetes to survive in serum.

As I mentioned earlier, the complement system also has other methods of killing microbes.  Some of the complement components that end up attached to the microbe are recognized by phagocytes attracted to the infection site by the complement cleavage products C3a and C5a.  This activity, known as opsonophagocytosis, is not reflected in the serum killing assay.   To see whether activation of complement produced products that promoted phagocytosis of B. garinii, the investigators added human neutrophils to the mixtures of B. garinii and serum.  The neutrophils engulfed the spirochetes within minutes.  Fewer spirochetes were engulfed when TSLPI was also present, indicating that TSLPI protected B. garinii from opsonophagocytosis.

A U.S. strain of B. burgdorferi was also tested.  Most B. burgdorferi strains survive in human serum unless antibodies against the spirochete are also present.  Therefore, TSLPI was tested with serum pooled from seropositive Lyme disease patients.  TSLPI promoted survival of B. burgdorferi in immune serum.  TSLPI also limited the number of spirochetes that ended up engulfed when neutrophils were added to the immune serum.

The investigators next tried to figure out which of the three complement pathways was being blocked by TSLPI.  Fortunately simple assays are available to recreate the classical pathway with the CH50 assay and the alternative pathway with the AP50 assay.  Both assays are set up so that activation of the complement pathway leads to the assembly of the membrane attack complex in the plasma membrane of red blood cells, causing them to lyse and release their hemoglobin.  The extent of lysis is determined simply by measuring the absorbance of the freed hemoglobin in a spectrophotometer.

In the CH50 assay, human serum is mixed with sheep red blood cells that are coated with anti-sheep red blood cell antibodies.  The classical pathway is triggered when C1q, the recognition subunit of the C1 protease complex, binds the antibody.  In the figure below, TSLPI or BSA (control) was mixed with different dilutions of human serum prior to addition of the antibody-sensitized red blood cells.  Based on the serum killing assay with B. burgdorferi, one may expect the classical pathway to be hindered by TSLPI.   Surprisingly, the results indicated that TSLPI had no effect whatsoever ("control" vs. rTSLPI in graph below).  The anti-C1q and anti-C3 antibodies, additional controls, blocked the classical pathway, as expected.

Fig. 3C from Schuijt et al., 2011.  Effect of TSLPI on the classical complement pathway determined with a CH50 assay.

The AP50 assay is identical to the CH50 assay except for a couple of modifications.  The red blood cells come from rabbits because sheep red blood cells are not sensitive targets for the human alternative complement pathway.  The other critical difference is that the chelator EGTA is added to prevent activation of the classical pathway, which requires calcium for activity.  The results (below) show that TSLPI failed to block the alternative pathway as well.

Fig. 3D from Schuijt et al., 2011.  Effect of TSLPI on the alternative complement pathway determined with an AP50 assay.

No comparable assay is available to examine the lectin pathway.  Instead the investigators added dilutions of human serum to mannan-coated ELISA plates to look at deposition of the C4b protein, a component of C3 convertase.  Mannan is a polysaccharide made up of the simple sugar mannose and is the molecule recognized by MBL.  C4b will be generated only if the lectin pathway is triggered by binding of MBL to mannan.  C4b binds to the mannan coat.  After the incubation, unbound proteins are washed away, and adherence of C4b is detected by doing a standard ELISA with anti-C4 antibody.  The results indicate that TSLPI blocked the deposition of C4b, and thus the lectin pathway, in a dose-dependent manner (see graph below).

Fig. 3E from Schuijt et al., 2011.  Effect of TSLPI on the mannose arm of the lectin-binding pathway determined by ELISA measurement of C4b deposition onto mannan-coated wells.

Additional experiments showed that TSLPI blocked the very first step in the lectin pathway, binding of MBL to mannan.  TSLPI also interfered with the binding of L-ficolin to its target.

To confirm that the lectin pathway killed Borrelia, the authors looked at the killing activity of serum pooled from individuals deficient in MBL.  Such individuals were not hard to find.  According to the authors, about 25% of the population is deficient in MBL!

A lower percentage of B. garinii ended up being killed in MBL-deficient serum than in normal serum.  TSLPI further reduced the killing activity of MBL-deficient sera, suggesting that recognition of B. garinii by ficolin also triggered the lectin pathway.  The balance of the lethal activity of human serum was accounted for by the classical pathway; C1 esterase inhibitor, which blocks both the classical and lectin pathways, completely eliminated the killing activity of human serum.

ResearchBlogging.orgThe investigators next determined whether TSLPI aided B. burgdorferi infection of laboratory mice.  They knocked down TSLPI expression in Ixodes scapularis by RNA interference and used the altered ticks to transmit B. burgdorferi into mice.  As a control, they also had another group of mice that were inoculated with B. burgdorferi with unmodified ticks, which were producing normal levels of TSLPI.  When the altered ticks were used to inoculate B. burgdorferi, the bacterial loads in the heart, joint, and skin (distant from the site of tick feeding) turned out to be much less than when the unaltered ticks were used.  These results demonstrate that TSLPI protected B. burgdorferi from being killed inside the host.

Even though the complement system encounters pathogens early in infection, all of the in vitro B. burgdorferi experiments were done with serum containing anti-B. burdgorferi antibodies, which are not produced until later during infection.  For this reason, the in vitro experiments fail to shed any light on how complement killed B. burgdorferi in the mouse experiment when TSLPI was not around to provide protection.  The authors surmised that B. burgdorferi is susceptible to killing by complement-mediated opsonophagocytosis, which should not require antibody since complement components such as C3b can serve as opsonins.  Unfortunately, the neutrophil phagocytosis experiment with  B. burgdorferi was conducted only with immune serum.

The commentary written by Marconi and McDowell is also worth a look.  Although they concede that the evidence for the lectin pathway playing a crucial role in killing Borrelia is compelling, they point out that other studies have implicated the classical and alternative pathways in controlling Lyme Borrelia infections.  They also wonder how exactly TSLPI, which most likely acts at the site of the tick bite, influenced the bacterial load in tissues distant from the tick bite.  If you're wondering why the mouse experiment wasn't done with B. garinii, Marconi and McDowell explain that tick-animal models for B. garinii infections are not as developed as they are for B. burgdorferi infections.

The bottom line is that the tick salivary glycoprotein TSLPI helps B. burgdorferi and possibly B. garinii establish an infection. The in vitro experiments suggest that TSLPI facilitates infection by protecting Borrelia from being killed by the lectin complement pathway, although it's not clear how the lectin pathway would kill B. burgdorferi if TSLPI was not around to provide protection.


Featured study

Schuijt, T.J., Coumou, J., Narasimhan, S., Dai, J., DePonte, K., Wouters, D., Brouwer, M., Oei, A., Roelofs, J.J.T.H., van Dam, A.P., van der Poll, T., van't Veer, C., Hovius, J.W., & Fikrig, E. (2011). A tick mannose-binding lectin inhibitor interferes with the vertebrate complement cascade to enhance transmission of the Lyme disease agent Cell Host & Microbe, 10 (2), 136-146 DOI: 10.1016/j.chom.2011.06.010

Marconi, R.T., & McDowell, J.V. (2011). Tick salivary proteins offer the Lyme disease spirochetes an easy ride and another way to hide Cell Host & Microbe, 10 (2), 95-96 DOI: 10.1016/j.chom.2011.08.003 (Commentary)


Other references

Schuijt, T.J., Narasimhan, S., Daffre, S., DePonte, K., Hovius, J.W.R., Veer, C., van der Poll, T., Bakhtiari, K., Meijers, J.C.M., Boder, E.T., van Dam, A.P., & Fikrig, E. (2011). Identification and characterization of Ixodes scapularis antigens that elicit tick immunity using yeast surface display PLoS ONE, 6 (1) DOI: 10.1371/journal.pone.0015926


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