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

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

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Baby steps towards unraveling transcriptional regulation in the unculturable syphilis spirochete

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

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

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

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

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

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

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

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

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

Featured articles

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

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