SummaryTwo-component systems (TCS) are universal among bacteria and play critical roles in gene regulation. Our understanding of the contributions of TCS in the biology of the Borrelia is just now beginning to develop. Borrelia burgdorferi, a causative agent of Lyme disease, harbours a TCS comprised of open reading frames (ORFs) BB0419 and BB0420. BB0419 encodes a response regulator designated Rrp1, and BB0420 encodes a hybrid histidine kinase-response regulator designated Hpk1. Rrp1, which contains a conserved GGDEF domain, undergoes phosphorylation and produces the secondary messenger, cyclic diguanylate (c-di-GMP), a critical signaling molecule in numerous organisms. However, the regulatory role of the Rrp1-Hpk1 TCS and c-di-GMP signaling in Borrelia biology are unexplored. In this study, the distribution, conservation, expression and potential global regulatory capability of Rrp1 were assessed. rrp1 was found to be universal and highly conserved among isolates, co-transcribed with hpk1, constitutively expressed during in vitro cultivation, and significantly upregulated upon tick feeding. Allelic exchange replacement and microarray analyses revealed that the Rrp1 regulon consists of a large number of genes encoded by the core Borrelia genome (linear chromosome, linear plasmid 54 and circular plasmid 26) that encode for proteins involved in central metabolic processes and virulence mechanisms including immune evasion.

In North America, tick-borne relapsing fever (TBRF) is caused by the spirochete species Borrelia hermsii, Borrelia parkeri, and Borrelia turicatae. We previously demonstrated that some isolates of B. hermsii and B. parkeri are capable of binding factor H and that cell-bound factor H can participate in the factor I-mediated cleavage of C3b. Isolates that bound factor H expressed a factor H-binding protein (FHBP) that we estimated to be approximately 19 to 20 kDa in size and thus, pending further characterization, temporarily designated FHBP19. Until this report, none of the FHBPs of the TBRF spirochetes had been characterized. Here we have recovered the gene encoding the FHBP of B. hermsii YOR from a lambda ZAP II library and determined its sequence. The gene encodes a full-length protein of 22.7 kDa, which after processing is predicted to be 20.5 kDa. This protein, which we redesignate factor H-binding protein A (FhbA), is unique to B. hermsii. Two-dimensional pulsed-field gel electrophoresis and hybridization analyses revealed that the B. hermsii gene encoding FhbA is a single genetic locus that maps to a linear plasmid of approximately 220 kb. The general properties of FhbA were also assessed. The protein was found to be surface exposed and lipidated. Analysis of the antibody response to FhbA in infected mice revealed that it is antigenic during infection, indicating expression during infection. The identification and characterization of FhbA provides further insight into the molecular mechanisms of pathogenesis of the relapsing fever spirochetes.

In North America tick-borne relapsing fever is caused by Borrelia hermsii, Borrelia parkeri, and Borrelia turicatae (5). These pathogens are transmitted to humans through the bites of infected Ornithodoros ticks (5). The hallmark features of tick-borne relapsing fever are cyclic, high-level spirochetemias (10 7 spirochetes ml blood Ϫ1

BBA68 (BbCRASP-1) of the Lyme disease spirochetes binds human factor H (FH) and FH-like protein 1 (FHL-1).Here we assess transcription of the BBA68 gene and production of BBA68 in infected mice and humans using real-time reverse transcriptase PCR and immunoblotting. The species specificity of FH binding to BBA68 was also tested. The data suggest that BBA68 does not play an important role in immune evasion in animals.The Lyme disease spirochete, Borrelia burgdorferi, can bind the human complement regulatory proteins, factor H (FH) and FH-like protein 1 (FHL-1) (1,8,11,16). FH and FHL-1 serve as cofactors in the factor I-mediated cleavage of C3b (18). C3b is an important opsonin and central component of the complement system. The binding of FH and FHL-1 to the bacterial surface is thought to locally increase the efficiency of C3b cleavage, locally downregulate the alternative complement cascade, and inhibit C3b-mediated opsonophagocytosis (30). The FH/FHL-1 binding proteins (FHBPs) identified in B. burgdorferi B31MI include the OspE proteins (BBL39, BBN38, and BBP38) and BBA68 (also referred to as BbCRASP-1) (1,8,10,11,15,16). Analyses of in vitro-cultivated bacteria indicate that BBA68 is the dominant FHBP and is the only paralog of the 14-member protein family 54 (The Institute for Genome Research designation) to exhibit human FH/FHL-1 binding activity (10, 15). Inactivation of BBA68 increases sensitivity to complement in vitro, and the introduction of BBA68 into Borrelia garinii strains that lack the gene decreases their serum sensitivity (3). While it is widely held that BBA68 plays an important role in immune evasion, earlier studies provided evidence that it may not be expressed during infection (2,17,23,25). In fact, Tokarz et al. demonstrated that, when human blood was added to actively growing cultures, BBA68 and BBA69 gene transcription was downregulated 1.8-fold. In addition, Brooks et al. demonstrated a sevenfold reduction in BBA68 transcript levels in spirochetes cultivated in dialysis membrane chambers implanted into the peritoneal cavities of rats (2, 23). The goals of this study were to further investigate the putative contribution of BBA68 in immune evasion in humans and other animals. In summary, the data indicate that the BBA68 gene is not transcribed during infection in mice and does not elicit an antibody response in mice and humans. In addition, BBA68 does not bind to FH produced by animals other than humans, and hence it is not likely to be involved in FH-mediated immune evasion in its natural mammalian reservoirs. Collectively, the data suggest that BBA68 does not carry out an important function directly in infected humans and animals.As one approach to assessing production of BBA68 during infection we screened for an antibody response to BBA68 in infected mice and humans. Since an understanding of the potential heterogeneity of BBA68 is an essential first step in interpreting immunoblot analyses and identifying the appropriate test antigen to be employed in the screening, we first assessed...

SINC, a secreted effector of Chlamydia psittaci, uniquely targets the inner nuclear membrane of infected cells and uninfected neighbors. Candidate partners include ELYS, lamin B1, and four proteins of the inner nuclear membrane, suggesting that SINC interacts with host proteins that control nuclear structure, signaling, and chromatin organization.