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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