e 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 ⌬glp mutant had a markedly greater capacity to survive tick feeding than B. burgdorferi ⌬hk1 or ⌬rrp1 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. Borrelia burgdorferi, the causative agent of Lyme disease, is maintained in nature within an enzootic cycle that involves an arthropod vector and vertebrate reservoir hosts, typically, small rodents and birds (1). In the northeastern United States, the primary vector for B. burgdorferi is the black-legged deer tick, Ixodes scapularis (2, 3). Because B. burgdorferi cannot be passaged transovarially, naive larvae acquire spirochetes by feeding on infected reservoir hosts. Successful colonization of the vector requires B. burgdorferi to establish an intimate association with rapidly differentiating, highly endocytic midgut epithelial cells (4-6). In order to accomplish this feat, spirochetes must resist deleterious substances within the midgut lumen, such as host-and tick-derived innate immune effector molecules, reactive oxygen species (ROS), and salivary enzymes imbibed from the feeding site (4, 7-11). At the same time, B. burgdorferi also must alter its metabolic machinery to exploit the availability of alternative carbon sources (e.g., glycerol, N-acetylglucosamine [GlcNAc], chitobiose) as the supply of ingested glucose diminishes (12-15). During nymphal transmission, spirochetes are almost certai...

Leptospirosis, an emerging zoonotic disease with worldwide distribution, is caused by spirochetes belonging to the genus Leptospira. More than 500,000 cases of severe leptospirosis are reported annually, with >10% of these being fatal. Leptospires can survive for weeks in suitably moist conditions before encountering a new host. Reservoir hosts, typically rodents, exhibit little to no signs of disease but shed large numbers of organisms in their urine. Transmission occurs when mucosal surfaces or abraded skin come into contact with infected urine or urine-contaminated water or soil. In humans, leptospires can cause a variety of clinical manifestations, ranging from asymptomatic or mild fever to severe icteric (Weil's) disease and pulmonary haemorrhage. Currently, little is known about how Leptospira persist within a reservoir host. Prior in vitro studies have suggested that leptospires alter their transcriptomic and proteomic profiles in response to environmental signals encountered during mammalian infection. However, no study has examined gene expression by leptospires within a mammalian host-adapted state. To obtain a more faithful representation of how leptospires respond to host-derived signals, we used RNA-Seq to compare the transcriptome of L. interrogans cultivated within dialysis membrane chambers (DMCs) implanted into the peritoneal cavities of rats with that of organisms grown in vitro. In addition to determining the relative expression levels of “core” housekeeping genes under both growth conditions, we identified 166 genes that are differentially-expressed by L. interrogans in vivo. Our analyses highlight physiological aspects of host adaptation by leptospires relating to heme uptake and utilization. We also identified 11 novel non-coding transcripts that are candidate small regulatory RNAs. The DMC model provides a facile system for studying the transcriptional and antigenic changes associated with mammalian host-adaption, selection of targets for mutagenesis, and the identification of previously unrecognized virulence determinants.

The Treponema denticola FhbB protein contributes to immune evasion by binding factor H (FH). Cleavage of FH by the T. denticola protease, dentilisin, may contribute to the local immune dysregulation that is characteristic of periodontal disease (PD). Although three FhbB phyletic types have been defined (FhbB1, FhbB2, and FhbB3), the in vivo expression patterns and antigenic heterogeneity of FhbB have not been assessed. Here, we demonstrate that FhbB is a dominant early antigen that elicits FhbB type-specific antibody (Ab) responses. Using the murine skin abscess model, we demonstrate that the presence or absence of FhbB or dentilisin significantly influences Ab responses to infection and skin abscess formation. Competitive binding analyses revealed that ␣-FhbB Ab can compete with FH for binding to T. denticola and block dentilisin-mediated FH cleavage. Lastly, we demonstrate that dentilisin cleavage sites reside within critical functional domains of FH, including the complement regulatory domain formed by CCPs 1 to 4. Analysis of the FH cleavage products revealed that they lack cofactor activity. The data presented here provide insight into the in vivo significance of dentilisin, FhbB and its antigenic diversity, and the potential impact of FH cleavage on the regulation of complement activation. P eriodontal disease (PD) of infectious etiology is a significant human and veterinary health concern. PD is a chronic inflammatory disease that degrades tissue and reabsorbs bone that surrounds and supports teeth, leading to endentulism (1). PD results from a dysbiosis of the polymicrobial community of the subgingival crevice. The transition of the bacterial population from predominantly Gram-positive organisms to destructive anaerobic spirochetes and Gram-negative organisms (2-4) is accompanied by local dysregulation of immunoregulatory mechanisms (5-8). Specific periopathogens, including Treponema denticola and Porphyromonas gingivalis, are thought to play leading roles in this process (8)(9)(10). This study is focused on T. denticola, an anaerobic, proteolytic (tissue degrading) spirochete of humans that is a dominant periopathogen and member of the "red microbial complex" (4, 11). T. denticola and other oral treponemes are also key players in root canal and endodontic infections (12, 13).Periopathogens thrive in the subgingival crevice, an anatomical niche bathed in gingival crevicular fluid (GCF). GCF is derived from serum and local tissue exudate (14) and is rich in immune effectors, including activated complement, C-reactive protein, opsonins (C3b and iC3b), anaphylatoxins, and proinflammatory cytokines (reviewed in references 1 and 15). T. denticola evades complement mediated destruction by binding factor H (FH), a negative regulator of the complement system (9, 16-18). FH, an abundant 150-kDa serum glycoprotein (ϳ300 g ml of serum Ϫ1 ) (19,20), consists of 20 imperfect repeat domains (CCPs) of 50 to 60 amino acids each that comprise different functional domains (21-25).The FH binding protein of T. denticola, Fh...

SUMMARY Treponema denticola is an anaerobic spirochete whose abundance in the subgingival crevice correlates with the development and severity of periodontal disease. T. denticola’s ability to survive and thrive in the hostile environment of the periodontal pocket is due, at least in part, to its ability to bind factor H (FH), a negative regulator of the alternative complement pathway. The FH binding protein of T. denticola has been identified as FhbB and its atomic structure has been determined. The interaction of FH with T. denticola is unique in that FH bound to the cell surface is cleaved by the T. denticola protease, dentilisin. It has been postulated that FH cleavage by T. denticola leads to immune dysregulation in periodontal pockets. In this study, we conduct a comparative assessment of the sequence, properties, structure, and ligand binding kinetics of the FhbB proteins of strains 33521 and 35405. The biological outcome of the interaction of these strains with FH could differ significantly as 33521 lacks dentilisin activity. The data presented here offer insight into our understanding of the interactions of T. denticola with the host and its potential to influence disease progression.

The Borrelia burgdorferi bba64 gene product is a surface-localized lipoprotein synthesized within mammalian and tick hosts and is involved in vector transmission of disease. These properties suggest that BBA64 may be a vaccine candidate against Lyme borreliosis. In this study, protective immunity against B. burgdorferi challenge was assessed in mice immunized with the BBA64 protein. Mice developed a high-titer antibody response following immunization with soluble recombinant BBA64 but were not protected when challenged by needle inoculation of culture-grown spirochetes. Likewise, mice passively immunized with an anti-BBA64 monoclonal antibody were not protected against needle-inoculated organisms. BBA64-immunized mice were subjected to B. burgdorferi challenge by the natural route of a tick bite, but these trials did not demonstrate significant protective immunity in either outbred or inbred strains of mice. Lipidated recombinant BBA64 produced in Escherichia coli was assessed for possible improved elicitation of a protective immune response. Although inoculation with this antigen produced a high-titer antibody response, the lipidated BBA64 also was unsuccessful in protecting mice from B. burgdorferi challenge by tick bites. Anti-BBA64 antibodies raised in rats eradicated the organisms, as evidenced by in vitro borreliacidal assays, thus demonstrating the potential for BBA64 to be effective as a protective immunogen. However, passive immunization with the same monospecific rat anti-BBA64 polyclonal serum failed to provide protection against tick bite-administered challenge. These results reveal the challenges faced in not only identifying B. burgdorferi proteins with potential protective capability but also in producing recombinant antigens conducive to preventive therapies against Lyme borreliosis.