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Importance: Lyme disease is a major tick-borne infection caused by a bacterial pathogen called Borrelia burgdorferi , which is transmitted by ticks and affects hundreds of thousands of people every year. These bacterial pathogens are distinct from other genera of microbes because of their distinct features and ability to transmit a multi-system infection to a range of vertebrates, including humans. Progress in understanding the infection biology of Lyme disease, and thus advancements towards its prevention, are hindered by an incomplete understanding of the microbiology of B. burgdorferi , partly due to the occurrence of many unique borrelial proteins that are structurally unrelated to proteins of known functions yet are indispensable for pathogen survival. We herein report the use of diverse technologies to examine the structure and function of a unique B. burgdorferi protein, annotated as BB0238-an essential virulence determinant. We show that the protein is structurally organized into two distinct domains, is involved in multiplex protein-protein interactions, and facilitates tick-to-mouse pathogen transmission by aiding microbial evasion of early host cellular immunity. We believe that our findings will further enrich our understanding of the microbiology of B. burgdorferi, potentially impacting the future development of novel prevention strategies against a widespread tick-transmitted infection., Competing Interests: The authors declare no conflict of interest.

The RNA polymerase alternative σ factor RpoS in Borrelia burgdorferi (Bb), the Lyme disease pathogen, is responsible for programmatic-positive and -negative gene regulation essential for the spirochete's dual-host enzootic cycle. RpoS is expressed during tick-to-mammal transmission and throughout mammalian infection. Although the mammalian-phase RpoS regulon is well described, its counterpart during the transmission blood meal is unknown. Here, we used Bb-specific transcript enrichment by tick-borne disease capture sequencing (TBDCapSeq) to compare the transcriptomes of WT and ΔrpoS Bb in engorged nymphs and following mammalian host-adaptation within dialysis membrane chambers. TBDCapSeq revealed dramatic changes in the contours of the RpoS regulon within ticks and mammals and further confirmed that RpoS-mediated repression is specific to the mammalian-phase of Bb's enzootic cycle. We also provide evidence that RpoS-dependent gene regulation, including repression of tick-phase genes, is required for persistence in mice. Comparative transcriptomics of engineered Bb strains revealed that the Borrelia oxidative stress response regulator (BosR), a noncanonical Fur family member, and the cyclic diguanosine monophosphate (c-di-GMP) effector PlzA reciprocally regulate the function of RNA polymerase complexed with RpoS. BosR is required for RpoS-mediated transcription activation and repression in addition to its well-defined role promoting transcription of rpoS by the RNA polymerase alternative σ factor RpoN. During transmission, ligand-bound PlzA antagonizes RpoS-mediated repression, presumably acting through BosR.

In this study, we examined the relationship between c-di-GMP and its only known effector protein, PlzA, in Borrelia burgdorferi during the arthropod and mammalian phases of the enzootic cycle. Using a B. burgdorferi strain expressing a plzA point mutant (plzA-R145D) unable to bind c-di-GMP, we confirmed that the protective function of PlzA in ticks is c-di-GMP-dependent. Unlike ΔplzA spirochetes, which are severely attenuated in mice, the plzA-R145D strain was fully infectious, firmly establishing that PlzA serves a c-di-GMP-independent function in mammals. Contrary to prior reports, loss of PlzA did not affect expression of RpoS or RpoS-dependent genes, which are essential for transmission, mammalian host-adaptation and murine infection. To ascertain the nature of PlzA's c-di-GMP-independent function(s), we employed infection models using (i) host-adapted mutant spirochetes for needle inoculation of immunocompetent mice and (ii) infection of scid mice with in vitro-grown organisms. Both approaches substantially restored ΔplzA infectivity, suggesting that PlzA enables B. burgdorferi to overcome an early bottleneck to infection. Furthermore, using a Borrelia strain expressing a heterologous, constitutively active diguanylate cyclase, we demonstrate that 'ectopic' production of c-di-GMP in mammals abrogates spirochete virulence and interferes with RpoS function at the post-translational level in a PlzA-dependent manner. Structural modeling and SAXS analysis of liganded- and unliganded-PlzA revealed marked conformational changes that underlie its biphasic functionality. This structural plasticity likely enables PlzA to serve as a c-di-GMP biosensor that in its respective liganded and unliganded states promote vector- and host-adaptation by the Lyme disease spirochete., Competing Interests: The authors have declared that no competing interests exist.

Borrelia ( Borreliella ) burgdorferi , along with closely related species, is the etiologic agent of Lyme disease. The spirochete subsists in an enzootic cycle that encompasses acquisition from a vertebrate host to a tick vector and transmission from a tick vector to a vertebrate host. To adapt to its environment and persist in each phase of its enzootic cycle, B. burgdorferi wields three systems to regulate the expression of genes: the RpoN-RpoS alternative sigma factor cascade, the Hk1/Rrp1 two-component system and its product c-di-GMP, and the stringent response mediated by Rel Bbu and DksA. These regulatory systems respond to enzootic phase-specific signals and are controlled or fine- tuned by transcription factors, including BosR and BadR, as well as small RNAs, including DsrABb and Bb6S RNA. In addition, several other DNA-binding and RNA-binding proteins have been identified, although their functions have not all been defined. Global changes in gene expression revealed by high-throughput transcriptomic studies have elucidated various regulons, albeit technical obstacles have mostly limited this experimental approach to cultivated spirochetes. Regardless, we know that the spirochete, which carries a relatively small genome, regulates the expression of a considerable number of genes required for the transitions between the tick vector and the vertebrate host as well as the adaptation to each.

During the natural enzootic life cycle of Borrelia burgdorferi (also known as Borreliella burgdorferi ), the bacteria must sense conditions within the vertebrate and arthropod and appropriately regulate expression of genes necessary to persist within these distinct environments. bb0345 of B. burgdorferi encodes a hypothetical protein of unknown function that is predicted to contain an N-terminal helix-turn-helix (HTH) domain. Because HTH domains can mediate protein-DNA interactions, we hypothesized that BB0345 might represent a previously unidentified borrelial transcriptional regulator with the ability to regulate events critical for the B. burgdorferi enzootic cycle. To study the role of BB0345 within mammals, we generated a bb0345 mutant and assessed its virulence potential in immunocompetent mice. The bb0345 mutant was able to initiate localized infection and disseminate to distal tissues but was cleared from all sites by 14 days postinfection. In vitro growth curve analyses revealed that the bb0345 mutant grew similar to wild-type bacteria in standard Barbour-Stoenner-Kelley II (BSK-II) medium; however, the mutant was not able to grow in dilute BSK-II medium or dialysis membrane chambers (DMCs) implanted in rats. Proteinase K accessibility assays and whole-cell partitioning indicated that BB0345 was intracellular and partially membrane associated. Comparison of protein production profiles between the wild-type parent and the bb0345 mutant revealed no major differences, suggesting BB0345 may not be a global transcriptional regulator. Taken together, these data show that BB0345 is essential for B. burgdorferi survival in the mammalian host, potentially by aiding the spirochete with a physiological function that is required by the bacterium during infection., (Copyright © 2020 American Society for Microbiology.)

The transmission, survival, and virulence of Borrelia burgdorferi depend upon the spirochete's ability to modulate its transcriptome as it cycles between its arthropod vector and reservoir host. This complex adaptive process is collectively referred to as "host-adaptation." The paucibacillary nature of borrelial infections precludes the detailed analysis of host adaptation within infected mammalian tissues. To circumvent this limitation, we (J Clin Invest 101:2240-2250, 1998) developed a model system whereby spirochetes are cultivated within dialysis membrane chambers (DMCs) surgically implanted within the peritoneal cavity of a rat. Spirochetes within DMCs are exposed to many, if not all, of the environmental signals and physiological cues required for mammalian host adaptation but are protected from clearance by the host's immune system.

Borrelia burgdorferi is an extreme amino acid (AA) auxotroph whose genome encodes few free AA transporters and an elaborate oligopeptide transport system ( B. burgdorferi Opp [ Bb Opp]). Bb Opp consists of five oligopeptide-binding proteins (OBPs), two heterodimeric permeases, and a heterodimeric nucleotide-binding domain (NBD). Homology modeling based on the crystal structure of liganded Bb OppA4 revealed that each OBP likely binds a distinct range of peptides. Transcriptional analyses demonstrated that the OBPs are differentially and independently regulated whereas the permeases and NBDs are constitutively expressed. A conditional NBD mutant failed to divide in the absence of inducer and replicated in an IPTG (isopropyl-β-d-thiogalactopyranoside) concentration-dependent manner. NBD mutants grown without IPTG exhibited an elongated morphotype lacking division septa, often with flattening at the cell center due to the absence of flagellar filaments. Following cultivation in dialysis membrane chambers, NBD mutants recovered from rats not receiving IPTG also displayed an elongated morphotype. The NBD mutant was avirulent by needle inoculation, but infectivity was partially restored by oral administration of IPTG to infected mice. We conclude that peptides are a major source of AAs for B. burgdorferi both in vitro and in vivo and that peptide uptake is essential for regulation of morphogenesis, cell division, and virulence. IMPORTANCE Borrelia burgdorferi , the causative agent of Lyme disease, is an extreme amino acid (AA) auxotroph with a limited repertoire of annotated single-AA transporters. A major issue is how the spirochete meets its AA requirements as it transits between its arthropod vector and mammalian reservoir. While previous studies have confirmed that the B. burgdorferi oligopeptide transport ( opp ) system is capable of importing peptides, the importance of the system for viability and pathogenesis has not been established. Here, we evaluated the opp system structurally and transcriptionally to elucidate its ability to import a wide range of peptides during the spirochete's enzootic cycle. Additionally, using a novel mutagenesis strategy to abrogate opp transporter function, we demonstrated that peptide uptake is essential for bacterial viability, morphogenesis, and infectivity. Our studies revealed a novel link between borrelial physiology and virulence and suggest that peptide uptake serves an intracellular signaling function regulating morphogenesis and division., (Copyright © 2017 Groshong et al.)

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 σ 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) σ 70 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. IMPORTANCE Borrelia 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., (Copyright © 2017 Grove et al.)

Borrelia burgdorferi, the causative agent of Lyme disease (along with closely related genospecies), is in the deeply branching spirochete phylum. The bacterium is maintained in nature in an enzootic cycle that involves transmission from a tick vector to a vertebrate host and acquisition from a vertebrate host to a tick vector. During its arthropod sojourn, B. burgdorferi faces a variety of stresses, including nutrient deprivation. Here, we review some of the spirochetal factors that promote persistence, maintenance and dissemination of B. burgdorferi in the tick, and then focus on the utilization of available carbohydrates as well as the exquisite regulatory systems invoked to adapt to the austere environment between blood meals and to signal species transitions as the bacteria traverse their enzootic cycle. The spirochetes shift their source of carbon and energy from glucose in the vertebrate to glycerol in the tick. Regulation of survival under limiting nutrients requires the classic stringent response in which RelBbu controls the levels of the alarmones guanosine tetraphosphate and guanosine pentaphosphate (collectively termed (p)ppGpp), while regulation at the tick-vertebrate interface as well as regulation of protective responses to the blood meal require the two-component system Hk1/Rrp1 to activate production of the second messenger cyclic-dimeric-GMP (c-di-GMP)., (© 2016 John Wiley & Sons Ltd.)

Two-component signal transduction systems are the primary mechanisms by which bacteria perceive and respond to changes in their environment. The Hk1/Rrp1 two-component system (TCS) in Borrelia burgdorferi consists of a hybrid histidine kinase and a response regulator with diguanylate cyclase activity, respectively. Phosphorylated Rrp1 catalyzes the synthesis of c-di-GMP, a second messenger associated with bacterial life-style control networks. Spirochetes lacking either Hk1 or Rrp1 are virulent in mice but destroyed within feeding ticks. Activation of Hk1 by exogenous stimuli represents the seminal event for c-di-GMP signaling. We reasoned that structural characterization of Hk1's sensor would provide insights into the mechanism underlying signal transduction and aid in the identification of activating ligands. The Hk1 sensor is composed of three ligand-binding domains (D1-3), each with homology to periplasmic solute-binding proteins (PBPs) typically associated with ABC transporters. Herein, we determined the structure for D1, the most N-terminal PBP domain. As expected, D1 displays a bilobed Venus Fly Trap-fold. Similar to the prototypical sensor PBPs HK29S from Geobacter sulfurreducens and VFT2 from Bordetella pertussis, apo-D1 adopts a closed conformation. Using complementary approaches, including SAXS, we established that D1 forms a dimer in solution. The D1 structure enabled us to model the D2 and D3 domains. Differences in the ligand-binding pockets suggest that each PBP recognizes a different ligand. The ability of Hk1 to recognize multiple stimuli provides spirochetes with a means of distinguishing between the acquisition and transmission blood meals and generate a graded output response that is reflective of the perceived environmental threats., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Borrelia burgdorferi, the Lyme disease spirochete, couples environmental sensing and gene regulation primarily via the Hk1/Rrp1 two-component system (TCS) and Rrp2/RpoN/RpoS pathways. Beginning with acquisition, we reevaluated the contribution of these pathways to spirochete survival and gene regulation throughout the enzootic cycle. Live imaging of B. burgdorferi caught in the act of being acquired revealed that the absence of RpoS and the consequent derepression of tick-phase genes impart a Stay signal required for midgut colonization. In addition to the behavioral changes brought on by the RpoS-off state, acquisition requires activation of cyclic di-GMP (c-di-GMP) synthesis by the Hk1/Rrp1 TCS; B. burgdorferi lacking either component is destroyed during the blood meal. Prior studies attributed this dramatic phenotype to a metabolic lesion stemming from reduced glycerol uptake and utilization. In a head-to-head comparison, however, the B. burgdorferi Δglp mutant had a markedly greater capacity to survive tick feeding than B. burgdorferi Δhk1 or Δrrp1 mutants, establishing unequivocally that glycerol metabolism is only one component of the protection afforded by c-di-GMP. Data presented herein suggest that the protective response mediated by c-di-GMP is multifactorial, involving chemotactic responses, utilization of alternate substrates for energy generation and intermediary metabolism, and remodeling of the cell envelope as a means of defending spirochetes against threats engendered during the blood meal. Expression profiling of c-di-GMP-regulated genes through the enzootic cycle supports our contention that the Hk1/Rrp1 TCS functions primarily, if not exclusively, in ticks. These data also raise the possibility that c-di-GMP enhances the expression of a subset of RpoS-dependent genes during nymphal transmission., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Borrelia burgdorferi, the agent of Lyme disease, is maintained in nature within an enzootic cycle involving a mammalian reservoir and an Ixodes sp. tick vector. The transmission, survival and pathogenic potential of B. burgdorferi depend on the bacterium's ability to modulate its transcriptome as it transits between vector and reservoir host. Herein, we employed an amplification-microarray approach to define the B. burgdorferi transcriptomes in fed larvae, fed nymphs and in mammalian host-adapted organisms cultivated in dialysis membrane chambers. The results show clearly that spirochetes exhibit unique expression profiles during each tick stage and during cultivation within the mammal; importantly, none of these profiles resembles that exhibited by in vitro grown organisms. Profound shifts in transcript levels were observed for genes encoding known or predicted lipoproteins as well as proteins involved in nutrient uptake, carbon utilization and lipid synthesis. Stage-specific expression patterns of chemotaxis-associated genes also were noted, suggesting that the composition and interactivities of the chemotaxis machinery components vary considerably in the feeding tick and mammal. The results as a whole make clear that environmental sensing by B. burgdorferi directly or indirectly drives an extensive and tightly integrated modulation of cell envelope constituents, chemotaxis/motility machinery, intermediary metabolism and cellular physiology. These findings provide the necessary transcriptional framework for delineating B. burgdorferi regulatory pathways throughout the enzootic cycle as well as defining the contribution(s) of individual genes to spirochete survival in nature and virulence in humans., (© 2014 John Wiley & Sons Ltd.)

The spirochete periplasmic flagellum has many unique attributes. One unusual characteristic is the flagellar hook. This structure serves as a universal joint coupling rotation of the membrane-bound motor to the flagellar filament. The hook is comprised of about 120 FlgE monomers, and in most bacteria these structures readily dissociate to monomers (∼ 50 kDa) when treated with heat and detergent. However, in spirochetes the FlgE monomers form a large mass of over 250 kDa [referred to as a high molecular weight complex (HMWC)] that is stable to these and other denaturing conditions. In this communication, we examined specific aspects with respect to the formation and structure of this complex. We found that the Lyme disease spirochete Borrelia burgdorferi synthesized the HMWC throughout the in vitro growth cycle, and also in vivo when implanted in dialysis membrane chambers in rats. The HMWC was stable to formic acid, which supports the concept that the stability of the HMWC is dependent on covalent cross-linking of individual FlgE subunits. Mass spectrometry analysis of the HMWC from both wild type periplasmic flagella and polyhooks from a newly constructed ΔfliK mutant indicated that other proteins besides FlgE were not covalently joined to the complex, and that FlgE was the sole component of the complex. In addition, mass spectrometry analysis also indicated that the HMWC was composed of a polymer of the FlgE protein with both the N- and C-terminal regions remaining intact. These initial studies set the stage for a detailed characterization of the HMWC. Covalent cross-linking of FlgE with the accompanying formation of the HMWC we propose strengthens the hook structure for optimal spirochete motility.

The Lyme disease spirochete Borrelia burgdorferi must differentially express genes and proteins in order to survive in and transit between its tick vector and vertebrate reservoir. The putative DEAH-box RNA helicase, HrpA, has been recently identified as an addition to the spirochete's global regulatory machinery; using proteomic methods, we demonstrated that HrpA modulates the expression of at least 180 proteins. Although most bacteria encode an HrpA helicase, RNA helicase activity has never been demonstrated for HrpAs and the literature contains little information on the contribution of this protein to bacterial physiology or pathogenicity. In this work, we report that B. burgdorferi HrpA has RNA-stimulated ATPase activity and RNA helicase activity and that this enzyme is essential for both mammalian infectivity by syringe inoculation and tick transmission. Reduced infectivity of strains carrying mutations in the ATPase and RNA binding motif mutants suggests that full virulence expression requires both ATPase and coupled helicase activity. Microarray profiling revealed changes in RNA levels of two-fold, or less in an hrpA mutant versus wild-type, suggesting that the enzyme functions largely or exclusively at the post-transcriptional level. In this regard, northern blot analysis of selected gene products highly regulated by HrpA (bb0603 [p66], bba74, bb0241 [glpK], bb0242 and bb0243 [glpA]) suggests a role for HrpA in the processing and translation of transcripts. In addition to being the first demonstration of RNA helicase activity for a bacterial HrpA, our data indicate that the post-transcriptional regulatory functions of this enzyme are essential for maintenance of the Lyme disease spirochete's enzootic cycle.

P66 is a Borrelia burgdorferi surface protein with β₃ integrin binding and channel forming activities. In this study, the role of P66 in mammalian and tick infection was examined. B. burgdorferiΔp66 strains were not infectious in wild-type, TLR2⁻/⁻- or MyD88⁻/⁻-deficient mice. Strains with p66 restored to the chromosome restored near wild-type infectivity, while complementation with p66 on a shuttle vector did not restore infectivity. Δp66 mutants are cleared quickly from the site of inoculation, but analyses of cytokine expression and cellular infiltrates at the site of inoculation did not reveal a specific mechanism of clearance. The defect in these mutants cannot be attributed to nutrient limitation or an inability to adapt to the host environment in vivo as Δp66 bacteria were able to survive as well as wild type in dialysis membrane chambers in the rat peritoneum. Δp66 bacteria were able to survive in ticks through the larva to nymph moult, but were non-infectious in mice when delivered by tick bite. Independent lines of evidence do not support any increased susceptibility of the Δp66 strains to factors in mammalian blood. This study is the first to define a B. burgdorferi adhesin as essential for mammalian, but not tick infection., (© 2012 Blackwell Publishing Ltd.)

The Lyme disease spirochete Borrelia burgdorferi exists in nature in an enzootic cycle that involves the arthropod vector Ixodes scapularis and mammalian reservoirs. To disseminate within and between these hosts, spirochetes must migrate through complex, polymeric environments such as the basement membrane of the tick midgut and the dermis of the mammal. To date, most research on the motility of B. burgdorferi has been done in media that do not resemble the tissue milieus that B. burgdorferi encounter in vivo. Here we show that the motility of Borrelia in gelatin matrices in vitro resembles the pathogen's movements in the chronically infected mouse dermis imaged by intravital microscopy. More specifically, B. burgdorferi motility in mouse dermis and gelatin is heterogeneous, with the bacteria transitioning between at least three different motility states that depend on transient adhesions to the matrix. We also show that B. burgdorferi is able to penetrate matrices with pore sizes much smaller than the diameter of the bacterium. We find a complex relationship between the swimming behavior of B. burgdorferi and the rheological properties of the gelatin, which cannot be accounted for by recent theoretical predictions for microorganism swimming in gels. Our results also emphasize the importance of considering borrelial adhesion as a dynamic rather than a static process.

While the roles of rpoS(Bb) and RpoS-dependent genes have been studied extensively within the mammal, the contribution of the RpoS regulon to the tick-phase of the Borrelia burgdorferi enzootic cycle has not been examined. Herein, we demonstrate that RpoS-dependent gene expression is prerequisite for the transmission of spirochetes by feeding nymphs. RpoS-deficient organisms are confined to the midgut lumen where they transform into an unusual morphotype (round bodies) during the later stages of the blood meal. We show that round body formation is rapidly reversible, and in vitro appears to be attributable, in part, to reduced levels of Coenzyme A disulfide reductase, which among other functions, provides NAD+ for glycolysis. Our data suggest that spirochetes default to an RpoS-independent program for round body formation upon sensing that the energetics for transmission are unfavorable.

Phagocytosis of Borrelia burgdorferi, the causative agent of Lyme disease, is a poorly understood process, despite its importance during the host immune response to infection. B. burgdorferi has been shown to bind to different receptors on the surface of phagocytic cells, including the β(2) integrin, complement receptor 3 (CR3). However, whether these receptors mediate the phagocytosis of the spirochete remains unknown. We now demonstrate that CR3 mediates the phagocytosis of the spirochete by murine macrophages and human monocytes. Interaction of B. burgdorferi with the integrin is not sufficient, however, to internalize the spirochete; phagocytosis requires the interaction of CR3 with the GPI-anchored protein, CD14, independently of TLR/MyD88-induced or inside-out signals. Interestingly, the absence of CR3 leads to marked increases in the production of TNF in vitro and in vivo, despite reduced spirochetal uptake. Furthermore, the absence of CR3 during infection with B. burgdorferi results in the inefficient control of bacterial burdens in the heart and increased Lyme carditis. Overall, our data identify CR3 as a MyD88-independent phagocytic receptor for B. burgdorferi that also participates in the modulation of the proinflammatory output of macrophages. These data also establish a unique mechanism of CR3-mediated phagocytosis that requires the direct cooperation of GPI-anchored proteins.

In little more than 30 years, Lyme disease, which is caused by the spirochaete Borrelia burgdorferi, has risen from relative obscurity to become a global public health problem and a prototype of an emerging infection. During this period, there has been an extraordinary accumulation of knowledge on the phylogenetic diversity, molecular biology, genetics and host interactions of B. burgdorferi. In this Review, we integrate this large body of information into a cohesive picture of the molecular and cellular events that transpire as Lyme disease spirochaetes transit between their arthropod and vertebrate hosts during the enzootic cycle.

In a microarray analysis of the RpoS regulon in mammalian host-adapted Borrelia burgdorferi, bb0728 (cdr) was found to be dually transcribed by the sigma factors σ(70) and RpoS. The cdr gene encodes a coenzyme A disulphide reductase (CoADR) that reduces CoA-disulphides to CoA in an NADH-dependent manner. Based on the abundance of CoA in B. burgdorferi and the biochemistry of the enzyme, CoADR has been proposed to play a role in the spirochaete's response to reactive oxygen species. To better understand the physiologic function(s) of BbCoADR, we generated a B. burgdorferi mutant in which the cdr gene was disrupted. RT-PCR and 5'-RACE analysis revealed that cdr and bb0729 are co-transcribed from a single transcriptional start site upstream of the bb0729 coding sequence; a shuttle vector containing the bb0729-cdr operon and upstream promoter element was used to complement the cdr mutant. Although the mutant was no more sensitive to hydrogen peroxide than its parent, it did exhibit increased sensitivity to high concentrations of t-butyl-hydroperoxide, an oxidizing compound that damages spirochetal membranes. Characterization of the mutant during standard (15% oxygen, 6% CO(2)) and anaerobic (< 1% O(2) , 9-13% CO(2)) cultivation at 37°C revealed a growth defect under both conditions that was particularly striking during anaerobiosis. The mutant was avirulent by needle inoculation and showed decreased survival in feeding nymphs, but displayed no survival defect in unfed flat nymphs. Based on these results, we propose that BbCoADR is necessary to maintain optimal redox ratios for CoA/CoA-disulphide and NAD(+) /NADH during periods of rapid replication throughout the enzootic cycle, to support thiol-disulphide homeostasis, and to indirectly protect the spirochaete against peroxide-mediated membrane damage; one or more of these functions are essential for infection of the mammalian host by B. burgdorferi., (© 2011 Blackwell Publishing Ltd.)

Two-component systems (TCS) are principal mechanisms by which bacteria adapt to their surroundings. Borrelia burgdorferi encodes only two TCS. One is comprised of a histidine kinase, Hk2, and the response regulator Rrp2. While the contribution of Hk2 remains unclear, Rrp2 is part of a regulatory pathway involving the spirochete's alternate sigma factors, RpoN and RpoS. Genes within the Rrp2/RpoN/RpoS regulon function to promote tick transmission and early infection. The other TCS consists of a hybrid histidine kinase, Hk1, and the response regulator Rrp1. Hk1 is composed of two periplasmic sensor domains (D1 and D2), followed by conserved cytoplasmic histidine kinase core, REC, and Hpt domains. In addition to its REC domain, Rrp1 contains a GGDEF motif characteristic of diguanylate cyclases. To investigate the role of Hk1 during the enzootic cycle, we inactivated this gene in two virulent backgrounds. Extensive characterization of the resulting mutants revealed a dramatic phenotype whereby Hk1-deficient spirochetes are virulent in mice and able to migrate out of the bite site during feeding but are killed within the midgut following acquisition. We hypothesize that the phosphorelay between Hk1 and Rrp1 is initiated by the binding of feeding-specific ligand(s) to Hk1 sensor domain D1 and/or D2. Once activated, Rrp1 directs the synthesis of cyclic dimeric GMP (c-di-GMP), which, in turn, modulates the expression and/or activity of gene products required for survival within feeding ticks. In contrast to the Rrp2/RpoN/RpoS pathway, which is active only within feeding nymphs, the Hk1/Rrp1 TCS is essential for survival during both larval and nymphal blood meals.

Borrelia burgdorferi, the spirochetal agent of Lyme disease, is a vector-borne pathogen that cycles between a mammalian host and tick vector. This complex life cycle requires that the spirochete modulate its gene expression program to facilitate growth and maintenance in these diverse milieus. B. burgdorferi contains an operon that is predicted to encode proteins that would mediate the uptake and conversion of glycerol to dihydroxyacetone phosphate. Previous studies indicated that expression of the operon is elevated at 23°C and is repressed in the presence of the alternative sigma factor RpoS, suggesting that glycerol utilization may play an important role during the tick phase. This possibility was further explored in the current study by expression analysis and mutagenesis of glpD, a gene predicted to encode glycerol 3-phosphate dehydrogenase. Transcript levels for glpD were significantly lower in mouse joints relative to their levels in ticks. Expression of GlpD protein was repressed in an RpoS-dependent manner during growth of spirochetes within dialysis membrane chambers implanted in rat peritoneal cavities. In medium supplemented with glycerol as the principal carbohydrate, wild-type B. burgdorferi grew to a significantly higher cell density than glpD mutant spirochetes during growth in vitro at 25°C. glpD mutant spirochetes were fully infectious in mice by either needle or tick inoculation. In contrast, glpD mutants grew to significantly lower densities than wild-type B. burgdorferi in nymphal ticks and displayed a replication defect in feeding nymphs. The findings suggest that B. burgdorferi undergoes a switch in carbohydrate utilization during the mammal to tick transition. Further, the results demonstrate that the ability to utilize glycerol as a carbohydrate source for glycolysis during the tick phase of the infectious cycle is critical for maximal B. burgdorferi fitness.

The genome of Borrelia burgdorferi, the causative agent of Lyme disease, is comprised of a large linear chromosome and numerous smaller linear and circular plasmids. B. burgdorferi exhibits substantial genomic variation, and previous studies revealed genotype-specific variation at the right chromosomal telomere. A correlation has also been established between genotype and invasiveness. The correlation between chromosome length and genotype and between genotype and invasiveness suggested that a gene(s) at the right chromosome telomere may be required for virulence. Of particular interest was bb0844, an RpoS-regulated gene at the right telomere, the expression of which is induced when the spirochete undergoes adaptation to the mammalian host. The structure of the right chromosomal telomere was examined in 53 B. burgdorferi clinical isolates of various genotypes. Four distinct patterns were observed for bb0844: (i) chromosomal localization, (ii) plasmid localization, (iii) presence on both chromosome and plasmid, and (iv) complete absence. These patterns correlated with the B. burgdorferi genotype. On the basis of available sequence data, we propose a mechanism for the genomic rearrangements that accounts for the variability in bb0844 genomic localization. To further explore the role of BB0844 in the spirochete life cycle, a bb0844 deletion mutant was constructed by allelic exchange, and the viability of wild-type and bb0844 deletion mutants was examined in an experimental mouse-tick infection model. The bb0844 mutant was fully infectious in C3H/HeJ mice by either needle inoculation or tick transmission with B. burgdorferi-infected Ixodes scapularis larvae. Naïve larval ticks acquired both wild-type and mutant spirochetes with equal efficiency from B. burgdorferi-infected mice. The results demonstrate that BB0844 is not required for spirochete viability, pathogenicity, or maintenance in the tick vector or the mammalian host. At present, a defined role for BB0844 in B. burgdorferi cannot be ascertained.

Borrelia burgdorferi, the Lyme disease spirochete, dramatically alters its transcriptome and proteome as it cycles between the arthropod vector and mammalian host. During this enzootic cycle, a novel regulatory network, the Rrp2-RpoN-RpoS pathway (also known as the σ(54)-σ(S) sigma factor cascade), plays a central role in modulating the differential expression of more than 10% of all B. burgdorferi genes, including the major virulence genes ospA and ospC. However, the mechanism(s) by which the upstream activator and response regulator Rrp2 is activated remains unclear. Here, we show that none of the histidine kinases present in the B. burgdorferi genome are required for the activation of Rrp2. Instead, we present biochemical and genetic evidence that supports the hypothesis that activation of the Rrp2-RpoN-RpoS pathway occurs via the small, high-energy, phosphoryl-donor acetyl phosphate (acetyl∼P), the intermediate of the Ack-Pta (acetate kinase-phosphate acetyltransferase) pathway that converts acetate to acetyl-CoA. Supplementation of the growth medium with acetate induced activation of the Rrp2-RpoN-RpoS pathway in a dose-dependent manner. Conversely, the overexpression of Pta virtually abolished acetate-induced activation of this pathway, suggesting that acetate works through acetyl∼P. Overexpression of Pta also greatly inhibited temperature and cell density-induced activation of RpoS and OspC, suggesting that these environmental cues affect the Rrp2-RpoN-RpoS pathway by influencing acetyl∼P. Finally, overexpression of Pta partially reduced infectivity of B. burgdorferi in mice. Taken together, these findings suggest that acetyl∼P is one of the key activating molecule for the activation of the Rrp2-RpoN-RpoS pathway and support the emerging concept that acetyl∼P can serve as a global signal in bacterial pathogenesis.

Lyme disease is caused by transmission of the spirochete Borrelia burgdorferi from ticks to humans. Although much is known about B. burgdorferi replication, the routes and mechanisms by which it disseminates within the tick remain unclear. To better understand this process, we imaged live, infectious B. burgdorferi expressing a stably integrated, constitutively expressed GFP reporter. Using isolated tick midguts and salivary glands, we observed B. burgdorferi progress through the feeding tick via what we believe to be a novel, biphasic mode of dissemination. In the first phase, replicating spirochetes, positioned at varying depths throughout the midgut at the onset of feeding, formed networks of nonmotile organisms that advanced toward the basolateral surface of the epithelium while adhering to differentiating, hypertrophying, and detaching epithelial cells. In the second phase of dissemination, the nonmotile spirochetes transitioned into motile organisms that penetrated the basement membrane and entered the hemocoel, then migrated to and entered the salivary glands. We designated the first phase of dissemination "adherence-mediated migration" and provided evidence that it involves the inhibition of spirochete motility by one or more diffusible factors elaborated by the feeding tick midgut. Our studies, which we believe are the first to relate the transmission dynamics of spirochetes to the complex morphological and developmental changes that the midgut and salivary glands undergo during engorgement, challenge the conventional viewpoint that dissemination of Lyme disease-causing spirochetes within ticks is exclusively motility driven.

It is widely believed that innate immune responses to Borrelia burgdorferi (Bb) are primarily triggered by the spirochete's outer membrane lipoproteins signaling through cell surface TLR1/2. We recently challenged this notion by demonstrating that phagocytosis of live Bb by peripheral blood mononuclear cells (PBMCs) elicited greater production of proinflammatory cytokines than did equivalent bacterial lysates. Using whole genome microarrays, we show herein that, compared to lysates, live spirochetes elicited a more intense and much broader transcriptional response involving genes associated with diverse cellular processes; among these were IFN-beta and a number of interferon-stimulated genes (ISGs), which are not known to result from TLR2 signaling. Using isolated monocytes, we demonstrated that cell activation signals elicited by live Bb result from cell surface interactions and uptake and degradation of organisms within phagosomes. As with PBCMs, live Bb induced markedly greater transcription and secretion of TNF-alpha, IL-6, IL-10 and IL-1beta in monocytes than did lysates. Secreted IL-18, which, like IL-1beta, also requires cleavage by activated caspase-1, was generated only in response to live Bb. Pro-inflammatory cytokine production by TLR2-deficient murine macrophages was only moderately diminished in response to live Bb but was drastically impaired against lysates; TLR2 deficiency had no significant effect on uptake and degradation of spirochetes. As with PBMCs, live Bb was a much more potent inducer of IFN-beta and ISGs in isolated monocytes than were lysates or a synthetic TLR2 agonist. Collectively, our results indicate that the enhanced innate immune responses of monocytes following phagocytosis of live Bb have both TLR2-dependent and -independent components and that the latter induce transcription of type I IFNs and ISGs.

Although BBA74 initially was described as a 28-kDa virulence-associated outer-membrane-spanning protein with porin-like function, subsequent studies revealed that it is periplasmic and downregulated in mammalian host-adapted spirochetes. To further elucidate the role of this protein in the Borrelia burgdorferi tick-mammal cycle, we conducted a thorough examination of its expression profile in comparison with the profiles of three well-characterized, differentially expressed borrelial genes (ospA, ospC, and ospE) and their proteins. In vitro, transcripts for bba74 were expressed at 23 degrees C and further enhanced by a temperature shift (37 degrees C), whereas BBA74 protein diminished at elevated temperatures; in contrast, neither transcript nor protein was expressed by spirochetes grown in dialysis membrane chambers (DMCs). Primer extension of wild-type B. burgdorferi grown in vitro, in conjunction with expression analysis of DMC-cultivated wild-type and rpoS mutant spirochetes, revealed that, like ospA, bba74 is transcribed by sigma(70) and is subject to RpoS-mediated repression within the mammalian host. A series of experiments utilizing wild-type and rpoS mutant spirochetes was conducted to determine the transcriptional and translational profiles of bba74 during the tick-mouse cycle. Results from these studies revealed (i) that bba74 is transcribed by sigma(70) exclusively during the larval and nymphal blood meals and (ii) that transcription of bba74 is bracketed by RpoS-independent and -dependent forms of repression that are induced by arthropod- and mammalian host-specific signals, respectively. Although loss of BBA74 does not impair the ability of B. burgdorferi to complete its infectious life cycle, the temporal compartmentalization of this gene's transcription suggests that BBA74 facilitates fitness of the spirochete within a narrow window of its tick phase. A reexamination of the paradigm for reciprocal regulation of ospA and ospC, performed herein, revealed that the heterogeneous expression of OspA and OspC displayed by spirochete populations during the nymphal blood meal results from the intricate sequence of transcriptional and translational changes that ensue as B. burgdorferi transitions between its arthropod vector and mammalian host.

Borrelia burgdorferi is the etiologic agent of Lyme disease, a multisystem inflammatory disorder that principally targets the skin, joints, heart, and nervous system. The role of T lymphocytes in the development of chronic inflammation resulting from B. burgdorferi infection has been controversial. We previously showed that natural killer T (NKT) cells with an invariant (i) TCR alpha chain (iNKT cells) recognize glycolipids from B. burgdorferi, but did not establish an in vivo role for iNKT cells in Lyme disease pathogenesis. Here, we evaluate the importance of iNKT cells for host defense against these pathogenic spirochetes by using Valpha14i NKT cell-deficient (Jalpha18(-/-)) BALB/c mice. On tick inoculation with B. burgdorferi, Jalpha18(-/-) mice exhibited more severe and prolonged arthritis as well as a reduced ability to clear spirochetes from infected tissues. Valpha14i NKT cell deficiency also resulted in increased production of antibodies directed against both B. burgdorferi protein antigens and borrelial diacylglycerols; the latter finding demonstrates that anti-glycolipid antibody production does not require cognate help from Valpha14i NKT cells. Valpha14i NKT cells in infected wild-type mice expressed surface activation markers and produced IFNgamma in vivo after infection, suggesting a participatory role for this unique population in cellular immunity. Our data are consistent with the hypothesis that the antigen-specific activation of Valpha14i NKT cells is important for the prevention of persistent joint inflammation and spirochete clearance, and they counter the long-standing notion that humoral rather than cellular immunity is sufficient to facilitate Lyme disease resolution.

Borrelia burgdorferi must adapt physiologically to two markedly different host milieus and efficiently transit between its mammalian host and arthropod vector during tick feeding. Differential production of lipoproteins is essential for spirochaetes to survive, multiply and migrate within both hosts. Outer-surface protein C (OspC), which is induced during the blood meal, is critical for transmission of Lyme disease spirochaetes by nymphal ticks. Its biological function is poorly understood, however, despite the fact that its crystal structure has been solved. Evidence has accumulated that OspC blocks clearance of spirochaetes following inoculation in skin, and it is thought to do so by facilitating evasion of innate immunity. The study by Liang and co-workers in this edition of Molecular Microbiology extends this work by showing that OspC prevents early elimination and promotes dissemination. Surprisingly, they also show that unrelated borrelial outer-surface lipoproteins can replace these functions in an ospC mutant. They propose that an abundance of lipoprotein(s) is needed to stabilize the borrelial outer membrane against innate defences. This provocative work clearly runs counter to prevailing orthodoxies of bacterial pathogenesis. It also points the way towards future studies that will clarify the 'partially specific' roles of this enigmatic molecule in Lyme disease pathogenesis.

Borrelia burgdorferi (Bb) adapts to its arthropod and mammalian hosts by altering its transcriptional and antigenic profiles in response to environmental signals associated with each of these milieus. In studies presented here, we provide evidence to suggest that mammalian host signals are important for modulating and maintaining both the positive and negative aspects of mammalian host adaptation mediated by the alternative sigma factor RpoS in Bb. Although considerable overlap was observed between genes induced by RpoS during growth within the mammalian host and following temperature-shift, comparative microarray analyses demonstrated unequivocally that RpoS-mediated repression requires mammalian host-specific signals. A substantial portion of the in vivo RpoS regulon was uniquely upregulated within dialysis membrane chambers, further underscoring the importance of host-derived environmental stimuli for differential gene expression in Bb. Expression profiling of genes within the RpoS regulon by quantitative reverse transcription polymerase chain reaction (qRT-PCR) revealed a level of complexity to RpoS-dependent gene regulation beyond that observed by microarray, including a broad range of expression levels and the presence of genes whose expression is only partially dependent on RpoS. Analysis of Bb-infected ticks by qRT-PCR established that expression of rpoS is induced during the nymphal blood meal but not within unfed nymphs or engorged larvae. Together, these data have led us to postulate that RpoS acts as a gatekeeper for the reciprocal regulation of genes involved in the establishment of infection within the mammalian host and the maintenance of spirochetes within the arthropod vector.

Members of the ospE/ospF/elp lipoprotein gene families of Borrelia burgdorferi, the Lyme disease agent, are transcriptionally upregulated in response to the influx of blood into the midgut of an infected tick. We recently have demonstrated that despite the high degree of similarity between the promoters of the ospF (P(ospF)) and ospE (P(ospE)) genes of B. burgdorferi strain 297, the differential expression of ospF is RpoS-dependent, while ospE is controlled by sigma(70). Herein we used wild-type and RpoS-deficient strains of B. burgdorferi and Escherichia coli to analyse transcriptional reporters consisting of a green fluorescent protein (gfp) gene fused to P(ospF), P(ospE), or two hybrid promoters in which the -10 regions of P(ospF) and P(ospE) were switched [P(ospF ) ((E - 10)) and P(ospE) ((F - 10)) respectively]. We found that the P(ospF)-10 region is both necessary and sufficient for RpoS-dependent recognition in B. burgdorferi, while sigma(70) specificity for P(ospE) is dependent on elements outside of the -10 region. In E. coli, sigma factor selectivity for these promoters was much more permissive, with expression of each being primarily due to sigma(70). Alignment of the sequences upstream of each of the ospE/ospF/elp genes from B. burgdorferi strains 297 and B31 revealed that two B31 ospF paralogues [erpK (BBM38) and erpL (BBO39)] have -10 regions virtually identical to that of P(ospF). Correspondingly, expression of gfp reporters based on the erpK and erpL promoters was RpoS-dependent. Thus, the sequence of the P(ospF)-10 region appears to serve as a motif for RpoS recognition, the first described for any B. burgdorferi promoter. Taken together, our data support the notion that B. burgdorferi utilizes sequence differences at the -10 region as one mechanism for maintaining the transcriptional integrity of RpoS-dependent and -independent genes activated at the onset of tick feeding.

While numerous positively regulated loci have been characterized during the enzootic cycle of Borrelia burgdorferi, very little is known about the mechanism(s) involved in the repression of borrelial loci either during tick feeding or within the mammalian host. Here, we report that the alternative sigma factor RpoS is required for the in vivo-specific repression of at least two RpoD-dependent B. burgdorferi loci, ospA and lp6.6. The downregulation of ospA and Ip6.6 appears to require either a repressor molecule whose expression is RpoS dependent or an accessory factor which enables RpoS to directly interact with the ospA and Ip6.6 promoter elements, thereby blocking transcription by RpoD. The central role for RpoS during the earliest stages of host adaptation suggests that tick feeding imparts signals to spirochetes that trigger the RpoS-dependent repression, as well as expression, of in vivo-specific virulence factors critical for the tick-to-mammalian host transition.

In order to be sustained within its enzootic cycle, B. burgdorferi must adapt to two strikingly different environments, the arthropod vector and the mammalian host. The ability to rapidly adapt to environmental changes is therefore presumed to be central to spirochete survival and pathogenic programs. Indeed, it has now been well established that tick feeding initiates extensive changes in both gene expression and protein composition, collectively referred to as "host adaptation," a process that is thought to continue throughout infection. The paucibacillary nature of borrelial infections, however, has hampered our ability to study this bacterium in vivo. To circumvent this limitation, an animal model was developed for obtaining sufficient numbers of organisms to directly examine differential gene expression and antigenic composition of B. burgdorferi within the context of the mammalian host. The DMC model allows for a direct comparison of host-adapted B. burgdorferi and their in vitro-cultivated counterparts.

Lyme disease is a chronic inflammatory disorder caused by the spirochetal bacterium, Borrelia burgdorferi. In vitro evidence suggests that binding of spirochetal lipoproteins to CD14, a pattern recognition receptor expressed on monocytes/macrophages and polymorphonuclear cells, is a critical requirement for cellular activation and the subsequent release of proinflammatory cytokines that most likely contribute to symptomatology and clinical manifestations. To test the validity of this notion, we assessed the impact of CD14 deficiency on Lyme disease in C3H/HeN mice. Contrary to an anticipated diminution in pathology, CD14(-/-) mice exhibited more severe and persistent inflammation than did CD14(+/+) mice. This disparity reflects altered gene regulation within immune cells that may engender the higher bacterial burden and serum cytokine levels observed in CD14(-/-) mice. Comparing their in vitro stimulatory activity, live spirochetes, but not lysed organisms, were a potent CD14-independent stimulus of cytokine production, triggering an exaggerated response by CD14(-/-) macrophages. Collectively, our in vivo and in vitro findings support the provocative notion that: 1) pattern recognition by CD14 is entirely dispensable for elaboration of an inflammatory response to B. burgdorferi, and 2) CD14-independent signaling pathways are inherently more destructive than CD14-dependent pathways. Continued study of CD14-independent signaling pathways may provide mechanistic insight into the inflammatory processes that underlie development of chronic inflammation.

Borrelia burgdorferi, the Lyme disease spirochete, undergoes dramatic changes in antigenic composition as it cycles between its arthropod and mammalian hosts. A growing body of evidence suggests that these changes reflect, at least in part, the need for spirochetes to adapt to the physiological stresses imposed by abrupt changes in environmental conditions and nutrient availability. In many microorganisms, global responses are mediated by master regulators such as alternative sigma factors, with Escherichia coli RpoS (sigmaS) serving as a prototype. The importance of this transcriptional activator in other bacteria, coupled with the report by Hubner et al. (A. Hubner, X. Yang, D. M. Nolen, T. G. Popova, F. C. Cabello, and M. V. Norgard, Proc. Natl. Acad. Sci. USA 98:12724-12729, 2001) demonstrating that the borrelial RpoS ortholog controls expression of OspC and decorin-binding protein A (DbpA), prompted us to examine more closely the roles of RpoS-dependent and -independent differential gene expression in physiological adaptation by the Lyme disease spirochete. We observed that B. burgdorferi rpoS (rpoSBb) was induced following temperature shift and transcript levels were further enhanced by reduced pH (pH 6.8). Using quantitative real-time reverse transcription-PCR (RT-PCR), we demonstrated that, in contrast to its ortholog (rpoSEc) in Escherichia coli, rpoSBb was expressed at significant levels in B. burgdorferi throughout all phases of growth following temperature shift. By comparing a B. burgdorferi strain 297 rpoSBb mutant to its wild-type counterpart, we determined that RpoSBb was not required for survival following exposure to a wide range of environmental stresses (i.e., temperature shift, serum starvation, increased osmolality, reactive oxygen intermediates, and increased or reduced oxygen tension), although the mutant was more sensitive to extremes of pH. While B. burgdorferi strains lacking RpoS were able to survive within intraperitoneal dialysis membrane chambers at a level equivalent to that of the wild type, they were avirulent in mice. Lastly, RT-PCR analysis of the ospE-ospF-elp paralogous lipoprotein families complements earlier findings that many temperature-inducible borrelial loci are controlled in an RpoSBb-independent manner. Together, these data point to fundamental differences between the role(s) of RpoS in B. burgdorferi and that in E. coli. Rather than functioning as a master regulator, RpoSBb appears to serve as a stress-responsive activator of a subset of virulence determinants that, together with the RpoS-independent, differentially expressed regulon, encompass the spirochete's genetic programs required for mammalian host adaptation.

Borrelia burgdorferi, the causative agent of Lyme disease, encodes an RpoS ortholog (RpoS(Bb)) that controls the temperature-inducible differential expression of at least some of the spirochete's lipoprotein genes, including ospC and dbpBA. To begin to dissect the determinants of RpoS(Bb) recognition of, and selectivity for, its dependent promoters, we linked a green fluorescent protein reporter to the promoter regions of several B. burgdorferi genes with well-characterized expression patterns. Consistent with the expression patterns of the native genes/proteins in B. burgdorferi strain 297, we found that expression of the ospC, dbpBA, and ospF reporters in the spirochete was RpoS(Bb) dependent, while the ospE and flaB reporters were RpoS(Bb) independent. To compare promoter recognition by RpoS(Bb) with that of the prototype RpoS (RpoS(Ec)), we also introduced our panel of constructs into Escherichia coli. In this surrogate, maximal expression from the ospC, dbpBA, and ospF promoters clearly required RpoS, although in the absence of RpoS(Ec) the ospF promoter was weakly recognized by another E. coli sigma factor. Furthermore, RpoS(Bb) under the control of an inducible promoter was able to complement an E. coli rpoS mutant, although RpoS(Ec) and RpoS(Bb) each initiated greater activity from their own dependent promoters than they did from those of the heterologous sigma factor. Genetic analysis of the ospC promoter demonstrated that (i) the T(-14) in the presumptive -10 region plays an important role in sigma factor recognition in both organisms but is not as critical for transcriptional initiation by RpoS(Bb) as it is for RpoS(Ec); (ii) the nucleotide at the -15 position determines RpoS or sigma(70) selectivity in E. coli but does not serve the same function in B. burgdorferi; and (iii) the 110-bp region upstream of the core promoter is not required for RpoS(Ec)- or RpoS(Bb)-dependent activity in E. coli but is required for maximal expression from this promoter in B. burgdorferi. Taken together, the results of our studies suggest that the B. burgdorferi and E. coli RpoS proteins are able to catalyze transcription from RpoS-dependent promoters of either organism, but at least some of the nucleotide elements involved in transcriptional initiation and sigma factor selection in B. burgdorferi play a different role than has been described for E. coli.

Borrelia burgdorferi, which causes Lyme disease in humans, has an unusual genome composed of a linear chromosome and up to 21 extrachromosomal elements. Experimental data suggest that two of these elements, linear plasmids lp25 and lp28-1, play essential roles for infectivity in mice. In this study, we prove the essential natures of these two plasmids by selectively displacing lp25 or lp28-1 in an infectious wild-type clone with incompatible shuttle vectors derived from the native plasmids, rendering the respective transformants noninfectious to mice. Conversely, restoration of plasmid lp25 or lp28-1 in noninfectious clones that naturally lack the corresponding plasmid reestablished infectivity in mice. This approach establishes the ability to manipulate the plasmid content of strains by eliminating or introducing entire plasmids in B. burgdorferi and will be valuable in assessing the roles of plasmids even in unsequenced B. burgdorferi strains.

luxS mutants of Borrelia burgdorferi strain 297 naturally colonized their arthropod (Ixodes scapularis) vector, were maintained in ticks throughout the molting process (larvae to nymphs), were tick transmitted to uninfected mice, and elicited histopathology in mice indistinguishable from that induced by wild-type B. burgdorferi.

The outer surface proteins (Osp) A and B are two important lipoproteins of Borrelia burgdorferi, the Lyme disease spirochete. Extensive in vitro studies indicate that ospB shares a common promoter with ospA and thus these two lipoprotein genes are coordinately transcribed. We show here that B. burgdorferi expresses ospB at much higher levels than ospA during experimental murine infection. The ratio of ospA and ospB mRNA transcripts was 3.5:1 in tick-adapted spirochetes while B. burgdorferi matched every ospA mRNA with up to 70 ospB transcripts during murine infection. This was consistent with the analysis of antibody responses to the two lipoproteins, which showed a more frequent OspB response than OspA during chronic murine infection.

Neurological symptoms are common manifestations of Lyme disease; however, the paucibacillary nature of the spirochete in this environment has precluded a molecular analysis of the spirochete in the CNS. We have now adapted differential expression analysis by using a custom-amplified library (DECAL) in conjunction with Borrelia burgdorferi whole-genome and subgenome arrays to examine in vivo gene expression by B. burgdorferi in a non-human primate (NHP) model of neuroborreliosis. The expression profile of B. burgdorferi was examined in the CNS and heart of steroid-treated and immunocompetent NHPs. Eighty-six chromosomal genes and 80 plasmid-encoded genes were expressed at similar levels in the CNS and heart tissue of both immunocompetent and steroid-treated NHPs. The expression of 66 chromosomal genes and 32 plasmid-encoded genes was increased in the CNS of both immunocompetent and steroid-treated NHPs. It is likely that the expression of these genes is governed by physiological factors specific to the CNS milieu. However, 83 chromosomal and 114 plasmid-encoded genes showed contrasting expression profiles in steroid-treated and immunocompetent NHPs. The effect of dexamethasone on the immune status of the host as well as on the host metabolic pathways could contribute to these differences in the B. burgdorferi transcriptome. Results obtained herein underscore the complex interplay of host factors on B. burgdorferi gene expression in vivo. The results provide a global snapshot of the spirochetal transcriptome in the CNS and should spur the design of experiments aimed at understanding the molecular basis of neuroborreliosis.

Borrelia burgdorferi, a spirochaete that causes Lyme borreliosis, contains 21 linear and circular plasmids thought to be important for survival in mammals or ticks. Our results demonstrate that the gene BBE22 encoding a nicotinamidase is capable of replacing the requirement for the 25 kb linear plasmid lp25 during mammalian infection. Transformation of B. burgdorferi lacking lp25 with a shuttle vector containing the lp25 gene BBE22 (pBBE22) restored infectivity in mice to a level comparable to that of wild-type Borrelia. This complementation also restored the growth and host adaptation of lp25-B. burgdorferi in dialysis membrane chambers (DMCs) implanted in rats. A single Cys to Ala conversion at the putative active site of BBE22 abrogated the ability of pBBE22 to re-establish infectivity or growth in DMCs. Additional Salmonella typhimurium complementation studies and enzymatic analysis demonstrated that the BBE22 gene product has nicotinamidase activity and is most probably required for the biosynthesis of NAD. These results indicate that some plasmid-encoded products fulfil physiological functions required in the enzootic cycle of pathogenic Borrelia.

The stringent response is a global bacterial response to nutritional stress mediated by (p)ppGpp. We previously found that both noninfectious Borrelia burgdorferi strain B31 and infectious B. burgdorferi strain N40 produced large amounts of (p)ppGpp during growth in BSK-H medium and suggested that the stringent response was triggered in B. burgdorferi under these conditions. Here we report that (p)ppGpp levels in B. burgdorferi growing in BSK-II or BSK-H medium are not further increased by nutrient limitation or by serine hydroxamate-induced inhibition of protein synthesis and that the presence of (p)ppGpp during growth of N40 in BSK-H medium is not associated with decreased 16S rRNA synthesis. Decreased 16S rRNA synthesis was associated with the decreased growth rate of N40 seen during coculture with tick cells, which are growth conditions that were previously shown to decrease (p)ppGpp levels. One-half as much of the mRNA of the gene encoding the Rel protein of B. burgdorferi (rel(Bbu)) was produced by B31 as by N40 during in vitro growth (2 +/- 0.5 and 4 +/- 0.8 fg of rel(Bbu) mRNA/ng of total Borrelia RNA, respectively). Although the amounts of N40 rel(Bbu) mRNA were identical during growth in vitro and in rat peritoneal chambers, they were markedly decreased during growth in nymphal ticks. In contrast to the lack of change in rel(Bbu) mRNA levels, larger amounts of a 78-kDa protein that was cross-reactive with antibodies to Bacillus subtilis Rel(Bsu) were detected in immunoblots of N40 lysates after growth in rat peritoneal chambers than after growth in vitro. Differences in the level of production of (p)ppGpp between B31 and N40 could not be explained by differences in rel(Bbu) promoters since identical transcriptional start sites 309 nucleotides upstream from the B31 and N40 rel(Bbu) ATG start codon and identical sigma(70)-like promoters were identified by primer extension and sequencing analysis. rel(Bbu) complemented an Escherichia coli CF1693 relA spoT double mutant for growth on M9 minimal medium, and the transformed cells produced rel(Bbu) mRNA. These results indicate that rel(Bbu) is functional and that its transcription and translation and production of (p)ppGpp are affected by environmental conditions in strains N40 and B31. They also suggest that in B. burgdorferi, an organism with few rRNA operons that grows slowly, the role of (p)ppGpp may differ from the classic role played by this molecule in E. coli and that (p)ppGpp may not be responsible for growth rate control.

Borrelia burgdorferi differentially expresses many of the OspE/F/Elp paralogs during tick feeding. These findings, combined with the recent report that stable B. burgdorferi infection of mammals occurs only after 53 h of tick attachment, prompted us to further analyze the expression of the OspE/F/Elp paralogs during this critical period of transmission. Indirect immunofluorescence analysis revealed that OspE, p21, ElpB1, ElpB2, and OspF/BbK2.11 are expressed in the salivary glands of ticks allowed to feed on mice for 53 to 58 h. Interestingly, many of the spirochetes in the salivary glands that expressed abundant amounts of these antigens were negative for OspA and OspC. Although prior reports have indicated that OspE/F/Elp orthologs are surface exposed, none of the individual lipoproteins or combinations of the lipoproteins protected mice from challenge infections. To examine why these apparently surface-exposed lipoproteins were not protective, we analyzed their genetic stability during infection and their cellular locations after cultivation in vitro and within dialysis membrane chambers, mimicking a mammalian host-adapted state. Combined restriction fragment length polymorphism and nucleotide sequence analyses revealed that the genes encoding these lipoproteins are stable for at least 8 months postinfection. Interestingly, cellular localization experiments revealed that while all of these proteins can be surface localized, there were significant populations of spirochetes that expressed these lipoproteins only in the periplasm. Furthermore, host-specific signals were found to alter the expression patterns and final cellular location of these lipoproteins. The combined data revealed a remarkable heterogeneity in populations of B. burgdorferi during tick transmission and mammalian infection. The diversity is generated not only by temporal changes in antigen expression but also by modulation of the surface lipoproteins during infection. The ability to regulate the temporal and spatial expression patterns of lipoproteins throughout infection likely contributes to persistent infection of mammals by B. burgdorferi.

A major obstacle to studying the functions of particular gene products in the mouse-tick infectious cycle of Borrelia burgdorferi has been an inability to knock out genes in pathogenic strains. Here, we investigated conditions for site-directed mutagenesis in B31 MI, the low-passage-number, infectious B. burgdorferi strain whose genome was sequenced. We inactivated several plasmid and chromosomal genes in B31 MI and determined that clones carrying these mutations were not infectious for mice. However, we found extensive heterogeneity among clones and mutants derived from B31 MI based on colony phenotype, growth rate, plasmid content, protein profile, and transformability. Significantly, several B31 MI clones that were not subjected to mutagenesis but that lacked particular plasmids also exhibited defects at various stages in the infectious cycle. Therefore, the high degree of clonal polymorphism within B31 MI complicates the assessment of the contributions of individual genes to the observed phenotypes of the mutants. Our results indicate that B31 MI is not an appropriate strain background for genetic studies in infectious B. burgdorferi, and a well-defined isogenic clone is a prerequisite for targeted mutagenesis. To this end, we derived several wild-type clones from B31 MI that were infectious for mice, and gene inactivation was successful in one of these clones. Due to the instability of the genome with in vitro propagation, careful monitoring of plasmid content of derived mutants and complementation of inactivated genes will be crucial components of genetic studies with this pathogen.

The 32kb circular plasmid (cp32) family of Borrelia burgdorferi has been the subject of intensive investigation because its members encode numerous differentially expressed lipoproteins. As many as nine different cp32s appear to be capable of stable replication within a single spirochaete. Here, we show that a construct (pCE310) containing a 4 kb fragment from the putative maintenance region of a B. burgdorferi CA-11.2A cp32 was capable of autonomous replication in both high-passage B. burgdorferi B31 and virulent B. burgdorferi 297. Deletion analysis revealed that only the member of paralogous family 57 and the adjacent non-coding segment were essential for replication. The PF32 ParA orthologue encoded by the pCE310 insert was almost identical to the PF32 orthologues encoded on the B31 and 297 cp32-3 plasmids. The finding that cp32-3 was selectively deleted in both B31 and 297 transformants carrying pCE310 demonstrated the importance of the PF32 protein for cp32 compatibility and confirmed the prediction that cp32 plasmids expressing identical PF32 paralogues are incompatible. A shuttle vector containing the CA-11.2A cp32 plasmid maintenance region was used to introduce green, yellow and cyan fluorescent protein reporters into B. burgdorferi. Flow cytometry revealed that the green fluorescent protein was well expressed by almost 90% of both avirulent and infectious transformants. In addition to enhancing our understanding of B. burgdorferi plasmid biology, our results further the development of genetic systems for dissecting pathogenic mechanisms in Lyme disease.