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ABSTRACTBorrelia burgdorferi, the Lyme disease-causing spirochete, can persistently infect its vertebrate hosts for years. B. burgdorferiis often found associated with host connective tissue, where it interacts with components of the extracellular matrix, including fibronectin. Some years ago, a borrelial surface protein, named BBK32, was identified as a fibronectin-binding protein. However, B. burgdorferiBBK32 mutants are still able to bind fibronectin, indicating that the spirochete possesses additional mechanisms for adherence to fibronectin. We now demonstrate that RevA, an unrelated B. burgdorferiouter surface protein, binds mammalian fibronectin in a saturable manner. Site-directed mutagenesis studies identified the amino terminus of the RevA protein as being required for adhesion to fibronectin. RevA bound to the amino-terminal region of fibronectin. RevA binding to fibronectin was not inhibited by salt or heparin, suggesting that adhesin-ligand interactions are primarily nonionic and occur through the non-heparin-binding regions of the fibronectin amino-terminal domains. revAgenes are widely distributed among Lyme disease spirochetes, and the present studies determined that all RevA alleles tested bound fibronectin. In addition, RevB, a paralogous protein found in a subset of B. burgdorferistrains, also bound fibronectin. We also confirmed that RevA is produced during mammalian infection but not during colonization of vector ticks and determined that revAtranscription is controlled through a mechanism distinct from that of BBK32.

Serological diagnosis of Lyme disease may be complicated by antigenic differences between infecting organisms and those used as test references. Accordingly, it would be helpful to include antigens whose sequences are well conserved by a broad range of Lyme disease spirochetes. In the present study, line blot analyses were performed using recombinant complement regulator-acquiring surface protein 2 (BbCRASP-2) from Borrelia burgdorferi sensu stricto strain B31 and serum samples from human Lyme disease patients from throughout the United States and Germany. The results indicated that a large proportion of the patients had produced antibodies recognizing recombinant BbCRASP-2. In addition, Lyme disease spirochetes isolated from across North America and Europe were found to contain genes encoding proteins with high degrees of similarity to the B. burgdorferi type strain B31 BbCRASP-2, consistent with the high percentage of serologically positive patients. These data indicate that BbCRASP-2 may be valuable for use in a widely effective serological assay.

ABSTRACTThe Lyme disease spirochete, Borrelia burgdorferi, is largely resistant to being killed by its hosts’ alternative complement activation pathway. One possible resistance mechanism of these bacteria is to coat their surfaces with host complement regulators, such as factor H. Five different B. burgdorferiouter surface proteins having affinities for factor H have been identified: complement regulator-acquiring surface protein 1 (BbCRASP-1), encoded by cspA; BbCRASP-2, encoded by cspZ; and three closely related proteins, BbCRASP-3, -4, and -5, encoded by erpP, erpC, and erpA, respectively. We now present analyses of the recently identified BbCRASP-2 and cspZexpression patterns throughout the B. burgdorferiinfectious cycle, plus novel analyses of BbCRASP-1 and erp-encoded BbCRASPs. Our results, combined with data from earlier studies, indicate that BbCRASP-2 is produced primarily during established mammalian infection, while BbCRASP-1 is produced during tick-to-mammal and mammal-to-tick transmission stages but not during established mammalian infection, and Erp-BbCRASPs are produced from the time of transmission from infected ticks into mammals until they are later acquired by other feeding ticks. Transcription of cspZand synthesis of BbCRASP-2 were severely repressed during cultivation in laboratory medium relative to mRNA levels observed during mammalian infection, and cspZexpression was influenced by culture temperature and pH, observations which will assist identification of the mechanisms employed by B. burgdorferito control expression of this borrelial infection-associated protein.

ABSTRACTThe causative agent of Lyme disease, Borrelia burgdorferi, is naturally resistant to its host's alternative pathway of complement-mediated killing. Several different borrelial outer surface proteins have been identified as being able to bind host factor H, a regulator of the alternative pathway, leading to a hypothesis that such binding is important for borrelial resistance to complement. To test this hypothesis, the development of B. burgdorferiinfection was compared between factor H-deficient and wild-type mice. Factor B- and C3-deficient mice were also studied to determine the relative roles of the alternative and classical/lectin pathways in B. burgdorferisurvival during mammalian infection. While it was predicted that B. burgdorferishould be impaired in its ability to infect factor H-deficient animals, quantitative analyses of bacterial loads indicated that those mice were infected at levels similar to those of wild-type and factor B- and C3-deficient mice. Ticks fed on infected factor H-deficient or wild-type mice all acquired similar numbers of bacteria. Indirect immunofluorescence analysis of B. burgdorferiacquired by feeding ticks from the blood of infected mice indicated that none of the bacteria had detectable levels of factor H on their outer surfaces, even though such bacteria express high levels of surface proteins capable of binding factor H. These findings demonstrate that the acquisition of host factor H is not essential for mammalian infection by B. burgdorferiand indicate that additional mechanisms are employed by the Lyme disease spirochete to evade complement-mediated killing.

ABSTRACTDuring the natural mammal-tick infection cycle, the Lyme disease spirochete Borrelia burgdorfericomes into contact with components of the alternative complement pathway. B. burgdorferi, like many other human pathogens, has evolved the immune evasion strategy of binding two host-derived fluid-phase regulators of complement, factor H and factor H-like protein 1 (FHL-1). The borrelial complement regulator-acquiring surface protein 1 (CRASP-1) is a surface-exposed lipoprotein that binds both factor H and FHL-1. Analysis of CRASP-1 expression during the mammal-tick infectious cycle indicated that B. burgdorferiexpresses this protein during mammalian infection, supporting the hypothesized role for CRASP-1 in immune evasion. However, CRASP-1 synthesis was repressed in bacteria during colonization of vector ticks. Analysis of cultured bacteria indicated that CRASP-1 is differentially expressed in response to changes in pH. Comparisons of CRASP-1 expression patterns with those of other infection-associated B. burgdorferiproteins, including the OspC, OspA, and Erp proteins, indicated that each protein is regulated through a unique mechanism.

The growth properties of an Escherichia colistrain carrying a chromosomal deletion of thessuEADCBgenes (formerly designated ycbPONME) indicated that the products of this gene cluster are required for the utilization of sulfur from aliphatic sulfonates. Sequence similarity searches indicated that the proteins encoded by ssuA,ssuB,and ssuCare likely to constitute an ABC type transport system, whereas ssuDand ssuEencode an FMNH2-dependent monooxygenase and an NAD(P)H-dependent FMN reductase, respectively (Eichhorn, E., van der Ploeg, J. R., and Leisinger, T. (1999) J. Biol. Chem.274, 26639–26646). Synthesis of β-galactosidase from a transcriptional chromosomal ssuE′-lacZfusion was repressed by sulfate or cystine and depended on the presence of a functional cblgene, which encodes a LysR-type transcriptional regulator. Electrophoretic mobility shift assays with the ssupromoter region and measurements of β-galactosidase from plasmid-encoded ssuE′-′lacZfusions showed that full expression of the ssuoperon required the presence of a Cbl-binding site upstream of the −35 region. CysB, the LysR transcriptional regulator for the cysgenes, was not required for expression of a chromosomal ssuE′-lacZfusion although the ssupromoter region contained three CysB-binding sites. Integration host factor could also occupy three binding sites in the ssupromoter region but had no influence on expression of a chromosomal ssuE′-lacZfusion.