Borrelia burgdorferi is the causative agent of Lyme disease, a multisystemic, potentially chronic illness. It is maintained in nature through an infectious cycle between wild mammals and ticks. Like many bacterial pathogens, B. burgdorferi must cope with an array of changing environmental conditions to successfully persist, proliferate and be transmitted between hosts. The bacterial outer surface represents the primary site for interactions with the host. The array of B. burgdorferi outer surface proteins (Osps) has been shown to vary with the infectious cycle. It is likely that these different Osps endow the spirochete with distinct properties that pertain to the disparate environments in which it must survive. Our broad objective is to use a genetic approach to elucidate the molecular mechanisms of adaptation and variation in B. burgdorferi and their roles in the infectious cycle. The switch in B. burgdorferi outer surface protein gene expression that accompanies transmission from the tick vector to the mammalian host in vivo can be at least partly modeled in vitro by a shift in culture temperature. We have demonstrated that the synthesis of a family of at least 10 related outer surface proteins, designated Erp, is increased with this shift in temperature. The erp genes reside in seven separate loci and each of these loci maps to a separate circular plasmid of approximately 32 kb. Although the Erp proteins are highly divergent, the promoter regions of all seven erp loci differ by only a few nucleotides. Transcription of all the erp genes is temperature-regulated in culture. Each erp gene has been individually cloned and expressed as a recombinant fusion protein; all Erp proteins are recognized by infected mouse sera, suggesting they are synthesized by B. burgdorferi in the mammalian environment. Polyclonal rabbit sera have been generated against individual Erp proteins and will be used to analyze Erp synthesis by spirochetes in infected ticks. The recently completed genomic sequence of B. burgdorferi has identified three sigma factors, s70, s54 and RpoS. We are interested in the potential roles of s54 and RpoS, two alternative sigma factors, in the temperature induction and transcriptional regulation of osp genes. We have cloned the borrelial s54 and rpoS genes and are testing whether they can complement mutations in the corresponding genes of other bacteria. To determine if s54 and RpoS are involved in adaptive responses in B. burgdorferi, we will inactivate these genes and test whether such mutants can undergo temperature-induced expression of osp genes in vitro, and completion of the infectious cycle in vivo. We have also begun to characterize phenotypic changes that spirochetes undergo as they enter stationary phase, such as increased resistance to osmotic shock, and will analyze these adaptive responses in RpoS and s54 mutants. Bacterial oligopeptide permeases provide a mechanism for the uptake of small peptides, which not only provide nutrients but often serve as environmental signals that lead to a variety of adaptive responses. We speculate that in B. burgdorferi oligopeptide permease may also be involved in sensing and signaling a response to the changing conditions that accompany transmission between ticks and mammals. We have identified and sequenced the B. burgdorferi locus that encodes all five components of oligopeptide permease. B. burgdorferi has multiple copies of the gene that encodes the peptide binding component, OppA; three reside at the chromsomal locus and two are on plasmids. Northern analyses indicate that each oppA gene is independently transcribed. Induction of a plasmid-encoded oppA gene was observed folowing an increase intemperature. To address the cellular location of the OppA proteins, we have separately expressed each gene and raised polyclonal antisera against individual recombinant proteins. Initial studies indicate OppA is present in both inner and outer membranes. We have begun a genetic analysis of oligopeptide permease in B. burgdorferi to determine its function and role in adaptive responses.
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