Outer Surface Lipoprotein Layer Homeostasis and Gene Regulation in Borrelia burgdorferi

The outer surface lipoprotein (osp) layer forms an interface between the internal and the external environment of the Lyme disease spirochete, Borrelia burgdorferi. The homeostatic maintenance of the osp layer effectuates adaptation of B. burgdorferi as it gets transmitted from the tick vector to a mammalian host and vice-versa. However, the regulation of the outer surface lipoproteins (osps) is still a conundrum for borrelia scientists. Part of this dissertation inquires about the homeostatic maintenance of the osp layer. We found that the deletion of the dominantly expressed tick phase osp, OspA, induces expression of two other osps. OspD, and BBJ41. Also, increased expression of OspC was seen in borrelia mutants lacking OspA, OspD, and BBJ41. These results suggest constant osp layer maintenance, irrespective of the presence or the absence of the dominant Osps, like OspA and OspC. Furthermore, our conclusive electron microscopic study demonstrates that the overall density of the osp layer remains identical in wild type and mutant B. burgdorferi, lacking either several osps or the dominantly expressed OspA. OspA is abundantly expressed on the borrelial surface as it persists in an unfed tick. A blood meal causes rapid downregulation of OspA as B.burgdorferi prepares to infect the mammalian host. The downregulation of OspA is speculated to be regulated by an unknown repressor protein. The remaining part of this dissertation pertains to the investigation of this unknown repressor protein for ospA. The borrelia oxidative stress regulator protein, BosR, has been attributed with an indirect role in OspA downregulation. However, due to its homolgy with a family of transcriptional repressors, BosR is more likely to cause direct repression of OspA. Therefore, we investigated the direct interaction of BosR and the ospA regulatory region. The DNA binding experiments demonstrated that borrelia oxidative stress regulator, BosR, binds directly to the cisI and cisII regulatory regions of ospA promoter. Thus, conclusively, BosR acts as a repressor protein which causes OspA downregulation in B. burgdorferi

BosR (BB0647) Controls the RpoN-RpoS Regulatory Pathway and Virulence Expression in Borrelia burgdorferi by a Novel DNA-Binding Mechanism

In Borrelia burgdorferi (Bb), the Lyme disease spirochete, the alternative σ factor σ54 (RpoN) directly activates transcription of another alternative σ factor, σS (RpoS) which, in turn, controls the expression of virulence-associated membrane lipoproteins. As is customary in σ54-dependent gene control, a putative NtrC-like enhancer-binding protein, Rrp2, is required to activate the RpoN-RpoS pathway. However, recently it was found that rpoS transcription in Bb also requires another regulator, BosR, which was previously designated as a Fur or PerR homolog. Given this unexpected requirement for a second activator to promote σ54-dependent gene transcription, and the fact that regulatory mechanisms among similar species of pathogenic bacteria can be strain-specific, we sought to confirm the regulatory role of BosR in a second virulent strain (strain 297) of Bb. Indeed, BosR displayed the same influence over lipoprotein expression and mammalian infectivity for strain Bb 297 that were previously noted for Bb strain B31. We subsequently found that recombinant BosR (rBosR) bound to the rpoS gene at three distinct sites, and that binding occurred despite the absence of consensus Fur or Per boxes. This led to the identification of a novel direct repeat sequence (TAAATTAAAT) critical for rBosR binding in vitro. Mutations in the repeat sequence markedly inhibited or abolished rBosR binding. Taken together, our studies provide new mechanistic insights into how BosR likely acts directly on rpoS as a positive transcriptional activator. Additional novelty is engendered by the facts that, although BosR is a Fur or PerR homolog and it contains zinc (like Fur and PerR), it has other unique features that clearly set it apart from these other regulators. Our findings also have broader implications regarding a previously unappreciated layer of control that can be involved in σ54–dependent gene regulation in bacteria

The oligopeptide ABC transporter OppA4 negatively regulates the virulence factor OspC production of the Lyme disease pathogen

Borrelia burgdorferi sensu lato, the agent of Lyme disease, exists in nature through a complex enzootic life cycle that involves both ticks and mammals. The B. burgdorferi genome encodes five Oligopeptide ABC transporters (Opp) that are predicted to be involve in transport of various nutrients. Previously, it was reported that OppA5 is important for the optimal production of OspC, a major virulence factor of B. burgdorferi. In this study, possible role of another Oligopeptide ABC transporter, OppA4 in ospC expression was investigated by construction of an oppA4 deletion mutant and the complemented strain. Inactivation of oppA4 resulted an increased production of OspC, suggesting that OppA4 has a negative impact on ospC expression. Expression of ospC is controlled by Rrp2-RpoN-RpoS, the central pathway essential for mammal infection. We showed that increased ospC expression in the oppA4 mutant was due to an increased rpoS expression. We then further investigated how OppA4 negatively regulates this pathway. Two regulators, BosR and BadR, are known to positively and negatively, respectively, regulate the Rrp2-RpoN-RpoS pathway. We found that deletion of oppA4 resulted in an increased level of BosR. Previous reports showed that bosR is mainly regulated at the post-transcriptional level by other factors. However, OppA4 appears to negatively regulate bosR expression at the transcriptional level. The finding of OppA4 involved in regulation of the Rrp2-RpoN-RpoS pathway further reinforces the importance of nutritional virulence to the enzootic cycle of B. burgdorferi

Investigation of ospC Expression Variation among Borrelia burgdorferi Strains

Outer surface protein C (OspC) is the most studied major virulence factor of Borrelia burgdorferi, the causative agent of Lyme disease. The level of OspC varies dramatically among B. burgdorferi strains when cultured in vitro, but little is known about what causes such variation. It has been proposed that the difference in endogenous plasmid contents among strains contribute to variation in OspC phenotype, as B. burgdorferi contains more than 21 endogenous linear (lp) and circular plasmids (cp), and some of which are prone to be lost. In this study, we analyzed several clones isolated from B. burgdorferi strain 297, one of the most commonly used strains for studying ospC expression. By taking advantage of recently published plasmid sequence of strain 297, we developed a multiplex PCR method specifically for rapid plasmid profiling of B. burgdorferi strain 297. We found that some commonly used 297 clones that were thought having a complete plasmid profile, actually lacked some endogenous plasmids. Importantly, the result showed that the difference in plasmid profiles did not contribute to the ospC expression variation among the clones. Furthermore, we found that B. burgdorferi clones expressed different levels of BosR, which in turn led to different levels of RpoS and subsequently, resulted in OspC level variation among B. burgdorferi strains

Microarray-Based Comparative Genomic and Transcriptome Analysis of Borrelia burgdorferi

Borrelia burgdorferi, the spirochetal agent of Lyme disease, is maintained in nature in a cycle involving a tick vector and a mammalian host. Adaptation to the diverse conditions of temperature, pH, oxygen tension and nutrient availability in these two environments requires the precise orchestration of gene expression. Over 25 microarray analyses relating to B. burgdorferi genomics and transcriptomics have been published. The majority of these studies has explored the global transcriptome under a variety of conditions and has contributed substantially to the current understanding of B. burgdorferi transcriptional regulation. In this review, we present a summary of these studies with particular focus on those that helped define the roles of transcriptional regulators in modulating gene expression in the tick and mammalian milieus. By performing comparative analysis of results derived from the published microarray expression profiling studies, we identified composite gene lists comprising differentially expressed genes in these two environments. Further, we explored the overlap between the regulatory circuits that function during the tick and mammalian phases of the enzootic cycle. Taken together, the data indicate that there is interplay among the distinct signaling pathways that function in feeding ticks and during adaptation to growth in the mammal

Two Distinct Mechanisms Govern RpoS-mediated Repression of Tick-phase Genes During Mammalian Host Adaptation by Borrelia burgdorferi, the Lyme Disease Spirochete

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 sigma(70)-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) sigma(70) promoter elements had significantly lower expression in DMCs relative to growth in vitro at 37 degrees 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

Activation of the RpoN-RpoS regulatory pathway during the enzootic life cycle of Borrelia burgdorferi

<p>Abstract</p> <p>Background</p> <p>The maintenance of <it>Borrelia burgdorferi </it>in its complex tick-mammalian enzootic life cycle is dependent on the organism's adaptation to its diverse niches. To this end, the RpoN-RpoS regulatory pathway in <it>B. burgdorferi </it>plays a central role in microbial survival and Lyme disease pathogenesis by up- or down-regulating the expression of a number of virulence-associated outer membrane lipoproteins in response to key environmental stimuli. Whereas a number of studies have reported on the expression of RpoS and its target genes, a more comprehensive understanding of when activation of the RpoN-RpoS pathway occurs, and when induction of the pathway is most relevant to specific stage(s) in the life cycle of <it>B. burgdorferi</it>, has been lacking.</p> <p>Results</p> <p>Herein, we examined the expression of <it>rpoS </it>and key lipoprotein genes regulated by RpoS, including <it>ospC</it>, <it>ospA</it>, and <it>dbpA</it>, throughout the entire tick-mammal infectious cycle of <it>B. burgdorferi</it>. Our data revealed that transcription of <it>rpoS</it>, <it>ospC</it>, and <it>dbpA </it>is highly induced in nymphal ticks when taking a blood meal. The RpoN-RpoS pathway remains active during the mammalian infection phase, as indicated by the sustained transcription of <it>rpoS </it>and <it>dbpA </it>in <it>B. burgdorferi </it>within mouse tissues following borrelial dissemination. However, <it>dbpA </it>transcription levels in fed larvae and intermolt larvae suggested that an additional layer of control likely is involved in the expression of the <it>dbpBA </it>operon. Our results also provide further evidence for the downregulation of <it>ospA </it>expression during mammalian infection, and the repression of <it>ospC </it>at later phases of mammalian infection by <it>B. burgdorferi</it>.</p> <p>Conclusion</p> <p>Our study demonstrates that the RpoN-RpoS regulatory pathway is initially activated during the tick transmission of <it>B. burgdorferi </it>to its mammalian host, and is sustained during mammalian infection.</p

A Molecular Genetic Investigation of the Regulation of Outer Surface Protein C in Borrelia Burgdorferi: Identification and Characterization of the Novel Regulatory Protein BBD18

Lyme disease, caused by Borrelia burgdorferi, is the most prevalent arthropod-borne disease in the U.S. In nature, B. burgdorferi is maintained in an enzootic cycle between Ixodes ticks and mammalian hosts. In order to cause infection in mammals, B. burgdorferi spirochetes within a feeding tick sense environmental changes and subsequently alter their gene expression and protein profiles. As part of this adaptation, spirochetes within the tick midgut downregulate outer surface protein (Osp) A and upregulate OspC. Although OspC is an essential B. burgdorferi virulence factor needed for early mammalian infection, it is also a target for the mammalian acquired immune response, and thus, OspC is repressed soon after B. burgdorferi establishes infection. It has been shown that the central Rrp2/RpoN/RpoS pathway is essential for the upregulation of OspC and other genes important for mammalian infection. However, many key aspects in the strict regulation of OspC remain undefined. In order to investigate the complex regulatory mechanisms controlling OspC expression, we adapted the lacZ reporter system from Escherichia coli for use in B. burgdorferi. Using this lacZ system and other molecular genetic approaches, we identified BBD18 as a novel factor that has the potential to negatively regulate OspC. Expression of BBD18 in B. burgdorferi repressed transcription of ospC and abrogated infection in mice. We determined that BBD18 likely interfaces with the central Rrp2/RpoN/RpoS pathway and exerts its effect on OspC through the repression of RpoS. Structural modeling indicated that BBD18 has a putative DNA-binding motif, and site-directed mutagenesis within this domain abrogated BBD18\u27s ability to repress OspC and prevent infection of mice. We propose that the BBD18 protein acts in concert with other regulatory factors to precisely control gene regulation in B. burgdorferi during the enzootic cycle

Absence of \u3ci\u3esodA\u3c/i\u3e Increases the Levels of Oxidation of Key Metabolic Determinants of \u3ci\u3eBorrelia burgdorferi\u3c/i\u3e

Borrelia burgdorferi, the causative agent of Lyme disease, alters its gene expression in response to environmental signals unique to its tick vector or vertebrate hosts. B. burgdorferi carries one superoxide dismutase gene (sodA) capable of controlling intracellular superoxide levels. Previously, sodA was shown to be essential for infection of B. burgdorferi in the C3H/HeN model of Lyme disease. We employed two-dimensional electrophoresis (2-DE) and immunoblot analysis with antibodies specific to carbonylated proteins to identify targets that were differentially oxidized in the soluble fractions of the sodA mutant compared to its isogenic parental control strain following treatment with an endogenous superoxide generator, methyl viologen (MV, paraquat). HPLC-ESI-MS/MS analysis of oxidized proteins revealed that several proteins of the glycolytic pathway (BB0057, BB0020, BB0348) exhibited increased carbonylation in the sodA mutant treated with MV. Levels of ATP and NAD/NADH were reduced in the sodA mutant compared with the parental strain following treatment with MV and could be attributed to increased levels of oxidation of proteins of the glycolytic pathway. In addition, a chaperone, HtpG (BB0560), and outer surface protein A (OspA, BBA15) were also observed to be oxidized in the sodA mutant. Immunoblot analysis revealed reduced levels of Outer surface protein C (OspC), Decorin binding protein A (DbpA), fibronectin binding protein (BBK32), RpoS and BosR in the sodA mutant compared to the control strains. Viable sodA mutant spirochetes could not be recovered from both gp91/phox−⁄− and iNOS deficient mice while borrelial DNA was detected in multiple tissues samples from infected mice at significantly lower levels compared to the parental strain. Taken together, these observations indicate that the increased oxidation of select borrelial determinants and reduced levels of critical pathogenesis-associated lipoproteins contribute to the in vivo deficit of the sodA mutant in the mouse model of Lyme disease. This study, utilizing the sodA mutant, has provided insights into adaptive capabilities critical for survival of B. burgdorferi in its hosts

Changes in Bacterial Growth Rate Govern Expression of the \u3cem\u3eBorrelia burgdorferi\u3c/em\u3e OspC and Erp Infection-Associated Surface Proteins

The Lyme disease spirochete controls production of its OspC and Erp outer surface proteins, repressing protein synthesis during colonization of vector ticks but increasing expression when those ticks feed on vertebrate hosts. Early studies found that the synthesis of OspC and Erps can be stimulated in culture by shifting the temperature from 23°C to 34°C, leading to a hypothesis that Borrelia burgdorferi senses environmental temperature to determine its location in the tick-mammal infectious cycle. However, borreliae cultured at 34°C divide several times faster than do those cultured at 23°C. We developed methods that disassociate bacterial growth rate and temperature, allowing a separate evaluation of each factor\u27s impacts on B. burgdorferi gene and protein expression. Altogether, the data support a new paradigm that B. burgdorferi actually responds to changes in its own replication rate, not temperature per se, as the impetus to increase the expression of the OspC and Erp infection-associated proteins