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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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.

Dehaloccoides 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 H₂.  Dehalococcoides 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