The long term goal of this extension is to understand the mechanism of action of complement independent bactericidal antibodies in Borrelia infections. These antibodies are not catalytic, but induce a conformational change on its antigen.CspB. From these studies it was concluded that the antibody shows turnover, and that its effects include an initial change in the antigen which is then propagated through antigen-antigen interactions. These findings satisfied the first hypothesis of the original previous grant proposal. During the last funding period, a DNA microarray of the Borrelia genome, including chromosomal and plasmid genes, was constructed, refined, and validated. The application of microarrays for the analysis of the Borrelia transcriptome can now also be used for global analysis of gene expression patterns. Specifically, the microarray approach will be used to test the hypothesis that the antibody-antigen complex triggers the spirochetal death signals. The in vitro studies will be centered on the fur-lipAoperon, putative hemolysins, and on adaptive gene responses to the action of the antibody. Quality control will include real time quantitative RT-PCR to measure concordance of selected values from the microarray results, and selective individual hybridizations to detect redundant microarray amplification of paraiogous genes. Distribution of expression patterns of replicons, and the functional classification ofdifferentially expressed genes will be done. During the last funding period, complement independent bactericidal antibodies were discovered in mice using the Borrelia relapsing fever model, and proved the hypothesis that bactericidal antibodies are a major form of host defense in infection with Borrelia. It is assumed that the in vivo killing mechanism is similar or identical to the in vitro action. For the next funding period, the role of the B cell in development of the infection, in the clearance of the spirochetemia, and in meningitis will be examined. The role of other elements of the innate immune system will be probed. Spirochetal quorum sensing as a means to clear bacteremia will also be examined.These studies will provide evidence that direct action of antibodies is an overlooked but fundamental mechanism of host defense in infections with Borrelia. and very likely with infections with other bacteria.
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