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Borrelia host adaptation Regulator (BadR) regulates rpoS to modulate host adaptation and virulence factors in Borrelia burgdorferi.

ABSTRACT: The RpoS transcription factor of Borrelia burgdorferi is a 'gatekeeper' because it activates genes required for spirochaetes to transition from tick to vertebrate hosts. However, it remains unknown how RpoS becomes repressed to allow the spirochaetes to transition back from the vertebrate host to the tick vector. Here we show that a putative carbohydrate-responsive regulatory protein, designated BadR (Borrelia host adaptation Regulator), is a transcriptional repressor of rpoS. BadR levels are elevated in B.?burgdorferi cultures grown under in vitro conditions mimicking unfed-ticks and badR-deficient strains are defective for growth under these same conditions. Microarray and immunoblot analyses of badR-deficient strains showed upregulation of rpoS and other factors important for virulence in vertebrate hosts, as well as downregulation of putative tick-specific determinants (e.g. linear plasmid 28-4 genes). DNA-binding assays revealed BadR binds to upstream regions of rpoS. Site-directed mutations in BadR and the presence of phosphorylated sugars affected BadR's binding to the rpoS promoters. badR-deficient B.?burgdorferi were unable to colonize mice. Several putative tick-specific targets have been identified. Our study identified a novel regulator, BadR, and provides a link between nutritional environmental cues utilized by spirochaetes to adaptation to disparate conditions found in the tick and vertebrate hosts.

SUBMITTER: Miller CL

PROVIDER: S-EPMC4828661 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Borrelia host adaptation Regulator (BadR) regulates rpoS to modulate host adaptation and virulence factors in Borrelia burgdorferi.

Miller Christine L CL Karna S L Rajasekhar SL Seshu J J

Molecular microbiology 20130314 1

The RpoS transcription factor of Borrelia burgdorferi is a 'gatekeeper' because it activates genes required for spirochaetes to transition from tick to vertebrate hosts. However, it remains unknown how RpoS becomes repressed to allow the spirochaetes to transition back from the vertebrate host to the tick vector. Here we show that a putative carbohydrate-responsive regulatory protein, designated BadR (Borrelia host adaptation Regulator), is a transcriptional repressor of rpoS. BadR levels are el ...[more]

PMID: 23387366

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Project description:The alternative sigma factor RpoS plays a key role modulating gene expression in Borrelia burgdorferi, the Lyme disease spirochete, by transcribing mammalian host-phase genes and repressing σ70-dependent genes required within the arthropod vector. To identify cis regulatory elements involved in RpoS-dependent repression, we analyzed green fluorescent protein (GFP) transcriptional reporters containing portions of the upstream regions of the prototypical tick-phase genes ospAB, the glp operon, and bba74 As RpoS-mediated repression occurs only following mammalian host adaptation, strains containing the reporters were grown in dialysis membrane chambers (DMCs) implanted into the peritoneal cavities of rats. Wild-type spirochetes harboring ospAB- and glp-gfp constructs containing only the minimal (-35/-10) σ70 promoter elements had significantly lower expression in DMCs relative to growth in vitro at 37°C; no reduction in expression occurred in a DMC-cultivated RpoS mutant harboring these constructs. In contrast, RpoS-mediated repression of bba74 required a stretch of DNA located between -165 and -82 relative to its transcriptional start site. Electrophoretic mobility shift assays employing extracts of DMC-cultivated B. burgdorferi produced a gel shift, whereas extracts from RpoS mutant spirochetes did not. Collectively, these data demonstrate that RpoS-mediated repression of tick-phase borrelial genes occurs by at least two distinct mechanisms. One (e.g., ospAB and the glp operon) involves primarily sequence elements near the core promoter, while the other (e.g., bba74) involves an RpoS-induced transacting repressor. Our results provide a genetic framework for further dissection of the essential "gatekeeper" role of RpoS throughout the B. burgdorferi enzootic cycle.IMPORTANCEBorrelia burgdorferi, the Lyme disease spirochete, modulates gene expression to adapt to the distinctive environments of its mammalian host and arthropod vector during its enzootic cycle. The alternative sigma factor RpoS has been referred to as a "gatekeeper" due to its central role in regulating the reciprocal expression of mammalian host- and tick-phase genes. While RpoS-dependent transcription has been studied extensively, little is known regarding the mechanism(s) of RpoS-mediated repression. We employed a combination of green fluorescent protein transcriptional reporters along with an in vivo model to define cis regulatory sequences responsible for RpoS-mediated repression of prototypical tick-phase genes. Repression of ospAB and the glp operon requires only sequences near their core promoters, whereas modulation of bba74 expression involves a putative RpoS-dependent repressor that binds upstream of the core promoter. Thus, Lyme disease spirochetes employ at least two different RpoS-dependent mechanisms to repress tick-phase genes within the mammal.

Project description:As an enzootic pathogen, the Lyme disease bacterium Borrelia burgdorferi possesses multiple copies of chemotaxis proteins, including two chemotaxis histidine kinases (CHK), CheA1 and CheA2. Our previous study showed that CheA2 is a genuine CHK that is required for chemotaxis; however, the role of CheA1 remains mysterious. This report first compares the structural features that differentiate CheA1 and CheA2 and then provides evidence to show that CheA1 is an atypical CHK that controls the virulence of B. burgdorferi through modulating the stability of RpoS, a key transcriptional regulator of the spirochete. First, microscopic analyses using green-fluorescence-protein (GFP) tags reveal that CheA1 has a unique and dynamic cellular localization. Second, loss-of-function studies indicate that CheA1 is not required for chemotaxis in vitro despite sharing a high sequence and structural similarity to its counterparts from other bacteria. Third, mouse infection studies using needle inoculations show that a deletion mutant of CheA1 (cheA1mut) is able to establish systemic infection in immune-deficient mice but fails to do so in immune-competent mice albeit the mutant can survive at the inoculation site for up to 28 days. Tick and mouse infection studies further demonstrate that CheA1 is dispensable for tick colonization and acquisition but essential for tick transmission. Lastly, mechanistic studies combining immunoblotting, protein turnover, mutagenesis, and RNA-seq analyses reveal that depletion of CheA1 affects RpoS stability, leading to reduced expression of several RpoS-regulated virulence factors (i.e., OspC, BBK32, and DbpA), likely due to dysregulated clpX and lon protease expression. Bulk RNA-seq analysis of infected mouse skin tissues further show that cheA1mut fails to elicit mouse tnf-α, il-10, il-1β, and ccl2 expression, four important cytokines for Lyme disease development and B. burgdorferi transmigration. Collectively, these results reveal a unique role and regulatory mechanism of CheA1 in modulating virulence factor expression and add new insights into understanding the regulatory network of B. burgdorferi.

Dataset Information

Borrelia host adaptation Regulator (BadR) regulates rpoS to modulate host adaptation and virulence factors in Borrelia burgdorferi.

Publications

Borrelia host adaptation Regulator (BadR) regulates rpoS to modulate host adaptation and virulence factors in Borrelia burgdorferi.

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