The Lyme disease spirochete lives without thiamine

Thiamine, or vitamin B1, is vital for the survival of all living things.  One of the biologically functional forms of thiamine, thiamine pyrophosphate (TPP), is essential for the catalytic activity of several critical metabolic enzymes.  For this reason, we must get thiamine from the food that we eat (or the vitamin pills that we swallow).  Microbes obtain the vitamin from their surroundings, but many can also make their own thiamine if it's not available.

It turns out that the Lyme disease spirochete Borrelia burgdorferi does not need thiamine, as described by Zhang and colleagues in Nature Microbiology.  The B. burgdorferi genome lacks the genes encoding the dedicated transporters that bring thiamine into the cell.  The genes encoding the enzymes that produce thiamine are also absent.  Chemical analysis of B. burgdorferi by HPLC failed to detect thiamine or TPP.  Despite lacking the means to make or acquire thiamine, B. burgdorferi grew just fine in culture medium devoid of thiamine.

The researchers conducted stringent tests to verify that B. burgdorferi could live without thiamine.  To remove all traces of thiamine, they introduced the bcmE gene from Clostridium botulinum into the spirochete.  The bcmE gene encodes an enzyme that rapidly breaks down thiamine.  In culture, the spirochete grew at the same rate whether or not it had bcmE.  The bcmE gene did not affect B. burgdorferi's ability to infect mice or to survive in feeding Ixodes scapularis ticks.  The results of these experiments provided strong evidence that B. burgdorferi doesn't need thiamine to infect the tick vector or mouse.

How does B. burgdorferi manage to live without thiamine?  It can do without most of the enzymes that require the TPP coenzyme, but it's less obvious how B. burgdorferi copes without pyruvate dehydrogenase (PDH), a TPP-dependent enzyme that converts pyruvate to acetyl-CoA (see figure).  Acetyl-CoA is an essential precursor to the bacterial cell wall, something that B. burgdorferi obviously needs.  The researchers proposed that B. burgdorferi makes acetyl-CoA by an alternative pathway that starts with acetate.  B. burgdorferi possesses the enzymes acetate kinase (ACK) and phosphate acetyltransferase (PTA), which convert acetate to acetyl-CoA (see figure).

Figure 4 from Zhang et al., 2016.  Enzymes in red (PDC, PDH, and POX) require the TPP coenzyme.  Metabolic pathways found in other bacteria but missing in B. burgdorferi are shown with dashed lines.

B. burgdorferi may not be alone in living without thiamine.  The researchers also looked at the genomes of other bacterial pathogens that are transmitted by arthropods.  Borrelia hermsii (relapsing fever), Rickettsia prowazekii (epidemic typhus), and R. conorii (Mediterranean spotted fever) were missing the genes for thiamine biosynthesis and the enzymes that use thiamine pyrophosphate as a coenzyme.

The presence of the alternative pathway to acetyl-CoA synthesis assumes that acetate is available in the microenvironment surrounding the arthropod-borne pathogen.  According to measurements presented in a 2010 paper, acetate is present in the midgut of fed I. scapularis ticks and in mouse blood.  The B. burgdorferi protein BBA34 may be a transporter that brings acetate into the cell.

References

Zhang K, Bian J, Deng Y, Smith A, Nunez RE, Li MB, Pal U, Yu AM, Qiu W, Ealick SE, & Li C (2016). Lyme disease spirochaete Borrelia burgdorferi does not require thiamin. Nature Microbiology, 2 PMID: 27869793

Xu H, Caimano MJ, Lin T, He M, Radolf JD, Norris SJ, Gherardini F, Wolfe AJ, & Yang XF (2010). Role of acetyl-phosphate in activation of the Rrp2-RpoN-RpoS pathway in Borrelia burgdorferi. PLoS pathogens, 6 (9) PMID: 20862323

Subba Raju BV, Esteve-Gassent MD, Karna SL, Miller CL, Van Laar TA, & Seshu J (2011). Oligopeptide permease A5 modulates vertebrate host-specific adaptation of Borrelia burgdorferi. Infection and immunity, 79 (8), 3407-20 PMID: 21628523

Resurgence of Borrelia burgdorferi in mice a year after antibiotic treatment

As a follow up to my previous post, I would like to say something about several mouse studies from Stephen Barthold's group.  These papers are often cited by those who believe that retreatment is needed in patients who continue to experience symptoms following treatment of Lyme disease with conventional antibiotic regimens.  The assumption is that post-treatment symptoms stem from spirochetes surviving the initial antibiotic therapy.

In the 2008 and 2010 studies (described in detail here and here), Barthold's group gave doxycycline, ceftriaxone, or tigecycline to mice with disseminated Borrelia burgdorferi infection.  As expected, all tissues were culture negative up to three months following antibiotic therapy.  Tissues from untreated mice were culture positive.  However, B. burgdorferi DNA and mRNA were detected by PCR in up to half the treated mice, and microscopy revealed a few intact spirochetes in collagen-rich tissues from these mice.  Ticks allowed to feed on the treated mice even transmitted the spirochetes to other mice (albeit immune deficient ones), where B. burgdorferi DNA was detected by PCR.  Clearly, the spirochetes that survived antibiotic treatment were alive despite being unculturable.

Although live spirochetes remained following antibiotic therapy, there was no evidence that they were capable of causing disease.  Lyme disease is driven by inflammation, but no inflammatory response in the form of infiltrating immune cells were seen in tissues harboring the spirochetes.  A critic of the work also pointed out that the number of spirochetes declined during the 3 months following treatment, implying that any lingering spirochetes would eventually disappear.  It seemed unlikely that a similar phenomenon was responsible for persisting symptoms following treatment of Lyme disease in human patients, who may suffer with disabling symptoms for years.

In 2014 Barthold's group came out with another paper, which I'm discussing here for the first time.  Again, mice with disseminated B. burgdorferi infections were treated with antibiotics, ceftriaxone in this case.  But this time, the mice were left for up to a year before their tissues were examined for the presence of B. burgdorferi.  Control mice were mock treated with saline and examined along with the treated mice.

There weren't any surprises when tissues were tested by culture.  Most of the control mice were culture positive at all time points (2, 4, 8, and 12 months) with both tissues tested, the urinary bladder and the skin where B. burgdorferi was inoculated to initiate infection.  None of the treated mice were culture positive at either site at any time point.

PCR testing for B. burgdorferi DNA was done with tissue obtained from six sites in the mice.  Ticks allowed to feed on the mice were also tested for the presence B. burgdorferi DNA by PCR in a method called xenodiagnosis.  All saline-treated mice were PCR positive in most tissues tested, and most tested positive by xenodiagnosis.

The results with the mice treated with ceftriaxone are shown in the table below.  Each row represents a single mouse.  Note that each tissue homogenate was tested three times.

Table 2 from Hodzic et al., 2014.  "Interval" = time after completion of treatment; "Inoc" = skin from inoculation site; "HB" = heart base; "VM" = ventricular muscle; "QM" = quadriceps muscle; "Tt" = tibiotarsus; "XenoDx" = xenodiagnostic ticks (# ticks testing positive/# ticks placed on mouse).

They saw something remarkable with the mice left for 12 months.  Although few tissues were positive at earlier time points, most tissues extracted from mice a year after treatment tested positive.  6 of the 8 mice also tested positive by xenodiagnosis.  So, instead of eventually disappearing, the spirochetes proliferated starting at some point after 8 months elapsed following treatment.  This resurgence occurred even though the spirochetes remained unculturable.

Barthold's group also looked for evidence of inflammation.  Despite the resurgence of spirochetes, they did not see much evidence of inflammation by microscopy of the tissues 12 months  following antibiotic treatment.  However, the researchers pointed out that no conclusions can be drawn about the ability of the persisting spirochetes to cause disease since inflammation was minimal even in saline-treated mice, which harbored culturable spirochetes.

The researchers next looked for molecular evidence of inflammation.  They measured transcript levels of 18 cytokines in the base of the heart, heart muscle, quadriceps muscle, and leg joint 12 months after treatment with ceftriaxone or saline.  The levels of cytokine transcripts in the two groups were compared to those in age-matched uninfected mice.  Not surprisingly, saline-treated mice had what the authors deemed a "proinflammatory" cytokine profile, most likely due to their ongoing infection.  Antibiotic-treated mice also had a proinflammatory cytokine profile, although it differed from that of the saline-treated mice.  This observation is the first to suggest that the mice were responding to persisting spirochetes that survived antibiotic treatment.

In conclusion, the evidence is convincing that B. burgdorferi persists in mice for a long time after antibiotic treatment.  They don't eventually disappear and may even proliferate.  Whether these unculturable spirochetes are capable of generating an inflammatory condition necessary for disease is less clear, though mice do appear to generate a unique cytokine profile in response to the persisting spirochetes.

Barthold's group caution readers from applying the findings too broadly:

Because of the controversial nature of these findings, they should not be over-interpreted and certainly not translated directly into clinical management of human Lyme borreliosis.

So is there any relevance of these findings to post-treatment symptoms in humans?  I will touch upon this issue in a future post.

Reference

Hodzic E, Imai D, Feng S, & Barthold SW (2014). Resurgence of persisting non-cultivable Borrelia burgdorferi following antibiotic treatment in mice. PLOS One, 9 (1) PMID: 24466286

Related posts

• Chronic Lyme disease in mice?
• Tigecycline fails to eradicate persisting Borrelia burgdorferi
• Long-term antibiotics for those with chronic symptoms that may or may not be related to Lyme disease

Video microscopy of ticks acquiring the Lyme disease spirochete from mice

The bite of an infected Ixodes hard tick transmits the Lyme disease spirochete, Borrelia burgdorferi, to humans.  Ticks acquire B. burgdorferi by feeding on reservoir hosts colonized with the spirochete.  Reservoir hosts include small mammals such as the white-footed mouse, the main reservoir of B. burgdorferi in the northeastern United States.

A study that just came out in The Yale Journal of Biology and Medicine is accompanied by videos of Borrelia burgdorferi being transmitted between mouse and tick.¹  The authors prepared the mice by infecting them with B. burgdorferi genetically modified to express green fluorescent protein.  After waiting two weeks to allow the spirochetes to disseminate, they placed one hungry Ixodes scapularis tick onto an ear of each infected mouse.

Figure 1 from Bockenstedt et al., 2014¹.  A feeding tick (arrow) attached to the ear of a mouse.  The tick is engorged with blood.

Nymphal ticks feed for an average of 2.5 to 8 days.  The meal starts with the tick inserting its barbed feeding apparatus into the skin.  (For a close-up view of this process, head over to the blog Phenomena: Not Exactly Rocket Science.)  The tick releases saliva through the feeding canal into the skin.  Tick saliva contains substances that damage host tissue surrounding the feeding apparatus and pharmacologic agents that inhibit clotting and engage the immune system. Intervals of salivation alternate with ingestion of blood, tissue fluid, and lymph that pool at the feeding site.²

The authors examined the feeding site by two photon intravital microscopy to observe what was happening to the spirochetes. They saw spirochetes in the dermis moving towards the feeding apparatus and disappearing as they presumably got sucked into the feeding canal.  One such spirochete is digitally colored in red in the video below, which was shot 48 hours into the blood meal.  (One hour of video footage was compressed into 30 seconds.)  The feeding apparatus is the green structure near the top of the viewing field.  Previous studies have suggested that ticks acquire B. burgdorferi from skin, not from blood.³  The videos from this study provide support for this notion, according to the authors.

Are the spirochetes mere passengers that get caught in the flow of fluid being drawn into the feeding canal, or are they active participants?  The authors argue for an active role for the spirochetes:

Spirochete movement is unlikely to be due simply to the mechanical flux of tissue fluid as the tick feeds because close examination of individual spirochetes that move toward the hypostome reveals both the oscillating movements that we observe in the absence of tick feeding as well as directional translocation.

References

1.  Bockenstedt LK, Gonzalez D, Mao J, Li M, Belperron AA, & Haberman A (2014). What ticks do under your skin: two-photon intravital imaging of Ixodes scapularis feeding in the presence of the Lyme disease spirochete. The Yale Journal of Biology and Medicine, 87 (1), 3-13 PMID: 24600332

2.   Anderson JF, & Magnarelli LA (2008). Biology of ticks. Infectious Disease Clinics of North America, 22 (2) PMID: 18452797

3.  Nakayama Y, & Spielman A (1989). Ingestion of Lyme disease spirochetes by ticks feeding on infected hosts. The Journal of infectious diseases, 160 (1), 166-7 PMID: 2732513

Escape of the Lyme disease spirochete from the bloodstream involves multiple adhesins and receptors

Microbial pathogens that cause systemic infections often travel within the circulatory system to spread throughout the host.  Eventually, these pathogens exit from the bloodstream to get to their target organ.  The first step of exit, adherence to the inner surface of the vessel wall, is probably the most challenging one because the rapidly flowing blood shoots the microbes through the capillaries.  A recent review described the process akin to "a spider trying to gain a foothold on the wall of a garden hose with the tap turned on full."  Most in vitro studies of adherence involve placement of microbe-mammalian cell cocultures in a stationary incubator.   These static conditions poorly reflect what microbes experience as they are carried throughout the circulatory system.  A better way to study vascular adhesion is to watch the process occur within a live animal.

A Canadian group has been doing just that.  As I explained in this post, they used intravital microscopy to shoot videos of the Lyme disease spirochete, Borrelia burgdorferi, escaping from skin capillaries of living mice (see the earlier post to watch a few of the videos).  The bacteria were genetically engineered to express green florescent protein so that they could be visualized with a fluorescence microscope.

From analyzing the videos, the investigators concluded that escape occurs in a series of steps.  The spirochete first "tethers" itself to the wall.  Within a second (assuming it doesn't let go), the spirochete starts crawling ("dragging") along the inner wall of the capillary.  The crawling spirochete eventually squeezes between the cells in the vessel wall (endothelial cells) to complete its escape.

In a follow-up study (see this post) they showed that expression of bbk32, encoding a B. burgdorferi surface protein, restored the ability of a highly-passaged, nonadherent B. burgdorferi strain to tether and drag along the vessel wall.  BBK32 clings to fibronectin and glycosaminoglycans (GAGs) in vitro.  The Canadian group found that fibronectin, which circulates in the bloodstream, and GAGs, which line the inner surface of capillaries, were necessary for B. burgdorferi to interact with the microvasculature.  Fibronectin, by its ability to anchor itself to GAGs, may serve as a lifeline that allows bacteria with fibronectin-binding proteins to tether themselves to the vessel wall.

Moriarty and colleagues conducted a third study to gain a better molecular understanding of BBK32's role in vascular adhesion.  The study was published in Molecular Microbiology over a year ago (here's the link to the study), but I still think it's worth going over today because of the significance of their findings, not only for Lyme disease but also for other diseases involving bloodstream dissemination of microbial pathogens.

Since separate surfaces of the BBK32 protein are responsible for fibronectin and GAG binding, the investigators wanted to see if BBK32's binding activities were deployed sequentially to carry out tethering and dragging.  Therefore, BBK32 variants defective in binding one or the other host protein were constructed by deleting small segments in each binding region.  The mutant genes encoding the variants were then introduced on plasmids into the highly-passaged, nonadhesive B. burgdorferi strain that they used in their earlier study.  The transformants expressing the BBK32 variants were injected into the bloodstream of mice, and skin capillaries were examined by intravital microscopy so that the investigators could count the tethering and dragging interactions like they did in their two earlier studies.

B. burgdorferi expressing the BBK32 variant defective in fibronectin binding (Δ158-182 in panel A below) underwent fewer tethering interactions than spirochetes expressing full-length BBK32 (BBK32 FL).  On the other hand, the BBK32 variant that bound GAG poorly (Δ45-68) mediated as many tethering interactions as wild-type BBK32.  These results indicate that the first step of adherence to the microvasculature, tethering, involves BBK32 interaction with fibronectin.  The BBK32 variant defective in GAG binding was impaired in promoting the second step of vascular adherence, dragging (panel B).

Figure 5 from Moriarty et al., 2012.  BBK32 Δ45-68 binds GAG poorly; BBK32 Δ158-182 binds fibornectin poorly.

Therefore, the exit pathway involving BBK32 goes as follows.  B. burgdorferi first uses BBK32 to tether to fibronectin, which is anchored to the vessel wall.  For the second step, BBK32 uses a different surface to bind to GAGs, allowing the spirochete to make more extensive contacts with the wall, leading to dragging interactions along the inner surface of the capillary.  Subsequent steps involving sequential contact of other bacterial factors with other host factors results in penetration of the spirochete through the vessel wall.

BBK32 is clearly capable of mediating vascular adhesion of an otherwise nonadherent B. burgdorferi mutant lacking most of its other adhesins.  However, to determine whether BBK32 really has a role in vascular adhesion of infectious B. burgdorferi requires a loss-of-function analysis, as opposed to the gain-of-function experiment just described.  Therefore, the investigators started with an infectious B. burgdorferi strain and knocked out the bbk32 gene.  They injected the bbk32 mutant and wild-type strains into the bloodstream of mice and again counted the number of tethering and dragging interactions in skin capillaries.  Here's where the results get interesting.  In comparison with the wild-type strain, they found that knocking out bbk32 reduced the number of vascular interactions by only 20%, and this reduction wasn't even statistically significant.  This result indicates that the contribution of BBK32 to vascular adhesion in skin capillaries is minor, at best.  Another B. burgdorferi adhesin (or adhesins) is responsible for the majority of vascular interactions in this tissue.

Figure 2B from Moriarty et al., 2012.  "Interactions" is the sum of tethering and dragging interactions counted in skin capillaries.

So where in the host does BBK32 function as a major adhesin?  Since B. burgdorferi is known to colonize joint tissue, the investigators decided to train their microscope on the capillaries supplying blood to the joints.  They saw that the spirochetes underwent the same tethering and dragging interactions that they observed in the skin. When they compared the bbk32 mutant against the infectious strain, they found that BBK32 accounts for roughly half of the tethering and dragging interactions (infectious vs. bbk32 KO in the graph below).  Whatever adhesin or adhesins are responsible for the other 50% do not interact with GAGs since coninjection of large amounts of a fibronectin peptide that binds GAGs failed to reduce the number of early vascular interactions of the bbk32 mutant (bbk32 KO vs. bbk32 KO + FN-C/H II).

Figure 2C from Moriarty et al., 2012.  "Interactions" is the sum of tethering and dragging interactions counted in joint capillaries.

Here's the bottom line:

• BBK32 is capable of mediating the first two steps of B. burgdorferi adhesion to the vasculature.  These steps involve distinct surfaces of BBK32 undgoing sequential contacts with fibronectin and GAGs.
• Whether BBK32 is actually involved in vascular adhesion depends on where the spirochete is located within the host.  For example, BBK32 has a major role in vascular adhesion in the joint, whereas it has little or no role in the skin.
• Clearly, there are other (currently undiscovered) bacterial adhesins and host receptors that promote vascular adhesion and escape.

References

Moriarty TJ, Shi M, Lin YP, Ebady R, Zhou H, Odisho T, Hardy PO, Salman-Dilgimen A, Wu J, Weening EH, Skare JT, Kubes P, Leong J, & Chaconas G (2012). Vascular binding of a pathogen under shear force through mechanistically distinct sequential interactions with host macromolecules. Molecular Microbiology, 86 (5), 1116-31 PMID: 23095033

Coburn J, Leong J, & Chaconas G (2013). Illuminating the roles of the Borrelia burgdorferi adhesins. Trends in Microbiology, 21 (8), 372-9 PMID: 23876218

Related posts

• Watch videos of the Lyme disease spirochete escaping from the bloodstream of live mice!
• The Lyme disease spirochete hijacks fibronectin to escape from the bloodstream

Triggering OspC production in Borrelia burgdorferi during tick feeding: Is temperature the real signal?

The Ixodes tick, the vector of the Lyme disease spirochete, goes months without a meal.  During this time, the Borrelia burgdorferi spirochetes living in its midgut live quiet lives, sipping on the tick's antifreeze to sustain themselves. When the tick finally takes a blood meal from a warm-blooded victim, B. burgdorferi responds by producing a number of new proteins, some of which are needed for transmission to and infection of the mammalian host.  Among these proteins is the outer surface lipoprotein OspC, whose function involves capture of tick (see this post) and mammalian host proteins.  How does B. burgdorferi know when to start making these critical proteins?  The favored model has been that the the warmth of the blood entering the tick triggers B. burgdorferi to make these proteins.  It's been known for almost two decades that B. burgdorferi growing in culture medium produces miniscule amounts of OspC at low temperatures (23º-24ºC) and larger amounts at higher temperatures (32º-37ºC), as shown in the figure below from the classic 1995 report by Tom Schwan and colleagues.

Figure 4 from Schwan et al., 1995.  B. burgdorferi incubated at 24ºC (lanes 2 and 6), transferred from 24ºC to 37ºC (lanes 3 and 7), incubated at 37ºC (lanes 4 and 8), or transferred from 37ºC to 24ºC (lanes 5 and 9).  Panel A, SDS-PAGE gel stained for total proteins with Coomassie  brilliant blue.  Arrow marks location of OspC.  Panel B, Western blot with flagellin antibody (Fla) and OspC antibody.

As reasonable as this model sounds, findings from a recent paper from Brian Stevenson's group (Jutras et al., 2012) challenge the model.  Although not emphasized in earlier papers, the authors noted that B. burgdorferi multiplies much more quickly at higher temperatures.  In their hands, B. burgdorferi proliferated with a doubling time of 32 hours at 23ºC and 12 hours at 34ºC.  As expected, their Western blots showed that more OspC was produced by the spirochetes growing at the higher temperature.  Members of the Erp family of surface proteins, whose levels also rise during tick feeding, were produced at higher levels at the higher temperature as well, as shown in earlier studies.  The investigators devised an experiment to test whether B. burgdorferi could tie OspC and Erp expression to its growth rate instead of temperature.

The standard culture medium for Borrelia is BSK-II with 6% rabbit serum, a complex nutrient-rich concoction.  They made two new formulations of the culture medium to slow the growth rate:  (1) quarter strength BSK-II with the rabbit serum concentration remaining at 6%; (2) full-strength BSK-II with the rabbit serum concentration reduced to 1.2%.  Medium #1 slowed the doubling time at 34ºC to 40 hours, and medium #2 reduced it to 32 hours.  Western blots of the spirochetes harvested from both cultures revealed low levels of the OspC and Erp proteins.  When these spirochetes were inoculated into the standard culture medium (BSKII/6% rabbit serum) and incubated at 34ºC, high levels of the proteins were again detected.  Therefore, B. burgdorferi is capable of adjusting OspC and Erp expression by monitoring its growth rate, even if the surrounding temperature does not change.

The final experiment from the study demonstrates that not even growth rate is the direct signal.  The authors froze B. burgdorferi at -80ºC for at least a month and then inoculated the bacteria into standard culture medium for incubation at 23ºC.  As a control, bacteria being maintained at 34ºC were also transferred to standard culture for incubation at 23ºC.  Both cultures grew with the same doubling time.  Nevertheless, the spirochetes that were revived from the frozen state produced more OspC and Erp proteins that those that were initially maintained at 34ºC.

So what's the real cue?  Going back to the natural life cycle of B. burgdorferi, the spirochetes living in the unfed tick's midgut do not really grow or divide.  The metabolism of B. burgdorferi is slowed by the nutrient-poor conditions in the tick's midgut.  When the tick finally takes a blood meal, the surge of nutrients entering the tick signals B. burgdorferi to rev up its metabolism, triggering production of OspC.  This model would explain why the frozen spirochetes, whose metabolism was undoubtedly slowed, were able to produce large amounts of OspC and Erp proteins when inoculated into standard culture medium at 23ºC, the temperature usually associated with diminished production of the proteins.  The challenge will be to figure out how B. burgdorferi is sensing its metabolic state at the molecular level.

References

Jutras, B.L., Chenail, A.M., & Stevenson, B. (2012). Changes in bacterial growth rate govern expression of the Borrelia burgdorferi OspC and Erp infection-associated surface proteins. Journal of Bacteriology, 195 (4), 757-764 DOI: 10.1128/JB.01956-12

Schwan, T.G., Piesman, J., Golde, W.T., Dolan, M.C., & Rosa, P.A. (1995). Induction of an outer surface protein on Borrelia burgdorferi during tick feeding. Proceedings of the National Academy of Sciences, 92 (7), 2909-2913 DOI: 10.1073/pnas.92.7.2909

Related posts

• Borrelia burgdorferi needs the alternative sigma factor RpoS to flee from the tick's midgut
• The Lyme disease spirochete feasts on tick antifreeze
• A fresh approach towards a Lyme disease vaccine: targeting the tick

An autoantigen targeted during Lyme arthritis

Infection by the spirochete Borrelia burgdorferi, if left untreated, can lead to a form of Lyme disease called Lyme arthritis.  About 10% of Lyme arthritis patients end up with a chronic form that doesn't go away with antibiotic treatment.  Allen Steere's group has long suspected that the antibiotic-refractory form of Lyme arthritis involves an autoimmune process.  This notion seems reasonable since those with antibiotic-refractory Lyme arthritis tend to have certain forms of the HLA-DR gene that are also common among those afflicted with the autoimmune disease rheumatoid arthritis.

Several groups have been searching for autoantigens (self antigens) that could drive the joint inflammation seen in Lyme arthritis patients.  Several candidate protein autoantigens were identified based on their short sequence similarities (molecular mimicry) to a T-cell or antibody epitopes in the B. burgdorferi OspA protein, which is targeted by the immune system in many Lyme arthritis patients, especially those with the antibiotic-refractory form.  However, further studies demonstrated that none of these autoantigens were likely to stimulate a sufficiently robust T-cell or antibody response that could account for the prolonged joint swelling experienced by patients with antibiotic-refractory Lyme disease (see this excellent review article for the complete story).  Therefore, an additional approach is needed to identify additional autoantigen candidates, an approach that does not assume that molecular mimicry underlies antibiotic-refractory Lyme arthritis.

An unbiased approach for finding autoantigens is to gather all of the different self-peptides being displayed by the HLA-DR molecules in the synovial tissue of the swollen joint and then figure out which of these peptides are capable of stimulating T cells.  At one time this approach wasn't possible since the individual peptides presented by HLA-DR molecules are found in such tiny amounts in human tissues, but the sensitivity of today's liquid chromatography/tandem mass spectrometry systems have improved to the point where many of the peptides can now be sorted and sequenced.

Steere's new study, which appeared in January's print issue of Arthritis and Rheumatism, was conducted in collaboration with Catherine Costello's group in Boston University.  The study was a follow-up to an earlier one published two years ago.  The authors extracted the inflamed synovial tissue from the swollen knee of a 12 year old boy suffering from antibiotic-refractory Lyme arthritis (see picture below).  He had gone through three months of antibiotic therapy a year prior to the procedure.  The tissue was culture and PCR negative.  When the patient's HLA-DR genes were examined, he turned out to have a copy of the DRB1*0101 allele, one of the HLA-DRB gene variants that places individuals at a higher risk for antibiotic-refractory Lyme arthritis.

Figure 1A from Drouin et al., 2013

The boy's synovial tissue was ground up, and HLA-DR-specific antibodies were used to capture the HLA-DR molecules with their bound peptides.  The peptides were then analyzed by liquid chromatography/tandem mass spectrometry.  The authors identified 120 different self-peptides from this analysis.  When each peptide was chemically synthesized and mixed with the boy's blood mononuclear cells, one peptide turned out to stimulate proliferation of his T cells.  This peptide came from a human protein called endothelial cell growth factor, or ECGF.

What's the function of ECGF?  The protein stimulates angiogenesis, the sprouting of new blood vessels from pre-existing ones.  Angiogenesis is a general feature of inflammatory arthritis, including Lyme arthritis and rheumatoid arthritis.

The authors went on to examine the T- and B-cell responses to ECGF in other Lyme arthritis patients.  The T-cell response was determined by measuring the amount of interferon-γ secreted by the patients' blood mononuclear cells upon exposure to ECGF in vitro.  In antibiotic-refractory patients, the T-cell response was observed in 38% (14/37) subjects against 30% (8/27) among Lyme arthritis patients who responded to antibiotics. The difference between the two groups was not statistically significant.  The B-cell (antibody) response was examined by ELISA in a larger group of patients.  17% (19/109) of antibiotic-refractory patients and 8% (6/77) of antibiotic-responsive patients had an IgG antibody response against ECGF that was higher than among healthy controls, yet the difference between the antibiotic-refractory and -responsive groups again was not statistically significant (P = 0.09).  So a link between an autoimmune response to ECGF and antibiotic-refractory arthritis was not clear-cut.  However, in support of a link, the authors mentioned that almost all of the Lyme arthritis patients with a T-cell response to ECGF (20/21, 98%) had one of the HLA-DR alleles known to be a risk factor for antibiotic-refractory arthritis.

The authors also looked at the levels of ECGF in the swollen joints of Lyme arthritis patients.  Those with antibiotic-refractory Lyme arthritis had much higher levels of ECGF in their joint fluid (mean 448 ng/ml, 37 subjects) than those whose arthritis responded to antibiotic treatment (mean 154 ng/ml, 19 subjects, P < 0.0001)

Further evidence for a link between an immune response to ECGF and chronic Lyme arthritis came from a group of untreated Lyme disease patients who were followed in the late 1970s, before the cause of Lyme disease was known.  Sera from sequential bleeds were still available from many of these patients.  If an autoimmune process involving ECGF was responsible for the disease, then the immune response to the autoantigen should have appeared before the disease symptoms.  This turned out to be the case.  Six of the seven Lyme arthritis patients who had antibodies against ECGF developed the antibody response before their joints swelled up.  The duration of the arthritis attack was longer in Lyme arthritis patients with an immune response to ECGF, lasting a median of 67 weeks in the seven patients with an ECGF antibody response and only 17 weeks in the 20 Lyme arthritis lacking the response (P = 0.004).

Steere's paper proposes that the immune response to ECGF leads to a persisting, autoimmune form of arthritis in those who have a high level of ECGF in their joint fluid.  In those patients, T cells that recognize ECGF would be activated more easily because of the high levels of ECGF available for phagocytes to engulf, process, and display to the T cells.  These events would lead to a chronic form of arthritis that would persist even when the spirochetes were cleared from the joints by the immune system or antibiotics.  These patients also have a lot of ECGF in their synovial tissue.  Antibody against ECGF could bind to the tissue and trigger attack by complement, contributing to the tissue damage.

Molecular mimicry doesn't appear to be involved in triggering an immune response to ECGF.  The authors were unable to identify any B. burgdorferi proteins that could cross-react with ECGF.

The immune response to ECGF can't be the whole story since most patients with antibiotic-refractory Lyme arthritis don't generate a T-cell or antibody response to the protein.  An autoimmune process in these other patients may involve other self-antigens waiting to be discovered.  Other host and spirochete factors also influence the course of Lyme arthritis (see this post, which gives the spirochete's point of view).


References

Drouin, E.E., Seward, R.J., Strle, K., McHugh, G., Katchar, K., Londoño, D., Yao, C., Costello, C.E., & Steere, A.C. (2013). A novel human autoantigen, endothelial cell growth factor, is a target of T and B cell responses in patients with Lyme disease. Arthritis & Rheumatism, 65 (1), 186-196 DOI: 10.1002/art.37732

Seward, R.J., Drouin, E.E., Steere, A.C., & Costello, C.E. (2010). Peptides presented by HLA-DR molecules in synovia of patients with rheumatoid arthritis or antibiotic-refractory Lyme arthritis. Molecular & Cellular Proteomics, 10 (3) DOI: 10.1074/mcp.M110.002477

A helpful review

Steere, A.C., Drouin, E.E., & Glickstein, L.J. (2011). Relationship between immunity to Borrelia burgdorferi Outer-surface protein A (OspA) and Lyme arthritis. Clinical Infectious Diseases, 52 (Supplement 3) DOI: 10.1093/cid/ciq117

Related post

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

Spirochete research: 2012 in review

Here are some of my favorite spirochete papers from 2012.  Direct access to all research articles (some behind a paywall) is provided via the DOI links.  Where present, the links above the citations lead to my blog posts about the studies.

PATHOGENESIS

Two distinct regions of the Borrelia burgdorferi BBK32 lipoprotein sequentially mediate binding to the vessel wall in vivo during escape of the spirochete from the bloodstream.

• Moriarty TJ, Shi , Lin Y-P, Ebady R, Zhou H, Odisho T, Hardy P-O, Salman-Dilgimen A, Wu J, Weening EH, Skare JT, Kubes P, Leong J, and Chaconas G (December 2012).  Vascular binding of a pathogen under shear force through mechanistically distinct sequential interactions with host macromolecules.  Molecular Microbiology 86(5):1116-1131.  DOI: 10.1111/mmi.12045

The Leptospira interrogans LigB protein protects the spirochete from complement by capturing complement regulatory proteins.

• Castiblanco-Valencia MM, Fraga TR, da Silva LB, Monaris D, Abreu PAE, Strobel S, Jozsi M, Isaac L, and Barbosa AS (March 15, 2012).  Leptospiral immunoglobulin-like proteins interact with human complement regulators factor H, FHL-1, FHR-1, and C4BP.  The Journal of Infectious Diseases 205(6):995-1004.  DOI: 10.1093/infdis/jir875
• Choy HA (July 2012).  Multiple activities of LigB potentiate virulence of Leptospira interrogans: inhibition of alternative and classical pathways of complement.  PLoS One 7(7):e41566. DOI: 10.1371/journal.pone.0041566

A B. burgdorferi lipase with hemolytic activity in vitro:

• Shaw DK, Hyde JA, and Skare JT (January 2012).  The BB0646 protein demonstrates lipase and haemolytic activity associated with Borrelia burgdorferi, the aetiological agent of Lyme disease.  Molecular Microbiology 83(2):319-334.  DOI: 10.1111/j.1365-2958.2011.07932.x

TRANSMISSION

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

• Dunham-Ems SM, Caimano MJ, Eggers CH, and Radolf JD (February 2012).  Borrelia burgdorferi requires the alternative sigma factor RpoS for Dissemination within the vector during tick-to-mammal transmission.  PLoS Pathogens 8(2):e1002532.  DOI: 10.1371/journal.ppat.1002532

MOTILITY

Video microscopy of B. burgdorferi swimming around in gelatin and mouse tissue:

• Harman MW, Dunham-Ems SM, Caimano MJ, Belperron AA, Bockenstedt LK, Fu HC, Radolf JD, and Wolgemuth CW (February 21, 2012).  The heterogeneous motility of the Lyme disease spirochete in gelatin mimics dissemination through tissue.  Proceedings of the National Academy of Sciences USA 109(8):3059-3064.  DOI: 10.1073/pnas.1114362109

L. interrogans sheath protein homologs that are not needed for flagellar sheath formation:

• Lambert A, Picardeau M, Haake DA, Sermswan RW, Srikram A, Adler B, and Murray GA (June 2012).  FlaA proteins in Leptospira interrogans are essential for motility and virulence but are not required for formation of the flagellum sheath.  Infection and Immunity 80(6):2019-2025.  DOI: 10.1128/IAI.00131-12

ULTRASTRUCTURE

A close look at the ultrastructure of Leptospira without the artifacts generated by conventional electron microscopy:

• Raddi G, Morado DR, Yan J, Haake DA, Yang XF, and Liu J (March 2012).  Three-dimensional structures of pathogenic and saprophytic Leptospira species revealed by cryo-electron tomography.  Journal of Bacteriology 194(6):1299-1306.  DOI: 10.1128/JB.06474-11

METAL TOXICITY

B. burgdorferi BicA, a protein that protects the spirochete from the toxic effects of copper and iron:

• Wang P, Lutton A, Olesik J, Vali H, and Li X (December 2012).  A novel iron- and copper-binding protein in the Lyme disease spirochaete.  Molecular Microbiology 86(6):1441-1451.  DOI: 10.1111/mmi.12068

ANTIBIOTIC THERAPY

Inflammatory spirochete debris left behind following antibiotic treatment for Lyme disease

• Bockenstedt LK, Gonzalez DG, Haberman AM, and Belperron AA (July 2, 2012).  Spirochete antigens persist near cartilage after murine Lyme borreliosis therapy.  The Journal of Clinical Investigation 122(7):2652-2660.  DOI: 10.1172/JCI58813

A critical analysis of a study that demonstrated persistence of B. burgdorferi in infected rhesus monkeys that were treated with antibiotics:

• Wormser GP, Baker PJ, O'Connell S, Pachner AR, Schwartz I, and Shapiro ED (July 2012).  Critical analysis of treatment trials of rhesus macaques infected with Borrelia burgdorferi reveals important flaws in experimental design.  Vector-borne and Zoonotic Diseases 12(7):535-538.  DOI: 10.1089/vbz.2012.1012
• Embers ME, Barthold SW, Borda JT, Bowers L, Doyle L, Hodzic E, Jacobs MB, Hasenkampf NR, Martin DS, Narasimhan S, Phillippi-Falkenstein KM, Purcell JE, Ratterree MS, and Philipp MT (January 2012).  Persistence of Borrelia burgdorferi in rhesus macaques following antibiotic treatment of disseminated infection.  PLoS One 7(1):e29914.  DOI: 10.1371/journal.pone.0029914

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

• Wormser GP, Daniels TJ, Bittker S, Cooper D, Wang G, and Pavia CS (March 15, 2012).  Failure of topical antibiotics to prevent disseminated Borrelia burgdorferi infection following a tick bite in C3H/HeJ mice.  The Journal of Infectious Diseases 205(6):991-994.  DOI: 10.1093/infdis/jir382

DIAGNOSTICS

Not so golden?  Microscopic agglutination test for diagnosis of leptospirosis

• Limmathurotsakul D, Turner EL, Wuthiekanun V, Thaipadungpanit J, Suputtamongkol Y, Chierakul W, Smythe LD, Day NPJ, Cooper B, and Peacock SJ (August 1, 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

ECOLOGY

Do nonspiral spirochetes help clean our environment?

• Caro-Quintero A, Ritalahti KM, Cusick KD, Loffler FE, and Konstandtinidis KT (May/June 2012).  The chimeric genome of Sphaerochaeta: Nonspiral spirochetes that break with the prevalent dogma in spirochete biology.  mBio 3(3):e00025-12.  DOI: 10.1128/mBio.00025-12
• Ritalahti KM, Justicia-Leon SD, Cusick KD, Ramos-Hernandez N, Rubin M, Dornbush J, and Loffler FE (January 2012).  Sphaerochaeta globosa gen. nov., sp. nov. and Sphaerochaeta pleomorpha sp. nov., free-living, spherical spirochetes.  International Journal of Systematic and Evolutionary Microbiology 62(Pt 1):210-216.  DOI: 10.1099/ijs.0.023986-0

 

BIOFILM

Biofilms of the Lyme disease spirochete

• Sapi E, Bastian SL, Mpoy CM, Scott S, Rattelle A, Pabbati N, Poruri A, Burugu D, Theophilus PAS, Pham TV, Data A, Dhaliwal NK, MacDonald A, Rossi MJ, Sinha SK, and Luecke DF (October 2012).  Characterization of biofilm formation by Borrelia burgdorferi in vitro.  PLoS One 7(10):e48277.  DOI: 10.1371/journal.pone.0048277

 

HISTORY

Looking for the syphilis spirochete in ancient bones

• Montiel R, Solorzano E, Diaz N,  Alvarez-Sandoval BA, Gonzalez-Ruiz M, Canadas MP, Simoes N, Isidro A, and Malgosa A (May 2012).  Neonate human remains: A window of opportunity to the molecular analysis of syphilis.  PLoS One 7(5):e36371.  DOI: 10.1371/journal.pone.0036371

Presenting flawed studies directly to the public to bypass the scientific peer-review process:

• Armelagos GJ, Zuckerman MK, and Harper KN (March 2012).  The science behind pre-Columbian evidence of syphilis in Europe: research by documentary.  Evolutionary Anthropology 21(2):50-57.  DOI: 10.1002/evan.20340

REVIEWS

Here are two excellent review articles that appeared during the past year:

• Radolf JD, Caimano MJ, Stevenson B, and Hu LT (February 2012).  Of ticks, mice and men: understanding the dual-host lifestyle of Lyme disease spirochaetes.  Nature Reviews Microbiology 10(2):87-99.  DOI: 10.1038/nrmicro2714
• Charon NW, Cockburn A, Li C, Liu J, Miller KA, Miller MR, Motaleb MA, and Wolgemuth CW (2012).  The unique paradigm of spirochete motility and chemotaxis.  Annual Reviews of Microbiology 66:349-370.  DOI: 10.1146/annurev-micro-092611-150145

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 al.  Atomic 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 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

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.

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


Related posts

• Chronic Lyme disease in mice?
• Tigecycline fails to eradicate persisting Borrelia burgdorferi
• The magic of antibiotic tolerance

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.

Do 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

Related post

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

sciseekclaimtoken-4fff6019af15b