The genome sequences of many microorganisms have now been determined. Several of these organisms are the agents of infectious disease The sequences reveal many genes of unknown function. The genome sequence information should enable new approaches to be developed to determine the function of genes and their possible role in pathogenesis. A functional genomics approach will be used to identify proteins important for the Treponema pallidum host-pathogen interaction. T. pallidum is the causative agent of syphilis. The complete genome sequence of this organism has been completed. Several features of T. pallidum make it an excellent system on which to develop and test functional genomics technologies. First, with a size of 1 million base pairs, the genome is one of the smallest known. Second, there are a total of 1031 open reading frames, which makes it feasible to systematically construct libraries containing each open reading frame in a relatively short period of time. Finally, little is known of the biology or pathogenesis of this organism because a continuous culture system is not available. This severely limits the experimental options for study of the organism. Therefore, new approaches are needed to understand gene function in T. pallidum. During the previous funding period, we have used a topoisomerase-based method to clone PCR products encoding 1008 of the 1031 open reading frames identified in the genome sequence of T. pallidum. In addition, the plasmid vector system used for cloning the open reading frames, the univector system, permits the rapid conversion of the original plasmid clone set to other functional vectors containing various promoters or tag sequences. The conversion to functional vectors is based on a single step Cre-loxP site-specific recombination reaction. Using Cre-loxP recombination, the T. pallidum clone set has been converted to specialized vectors for large scale protein expression, phage display and two-hybrid analysis. These plasmid collections will be used in a functional genomics approach to i) identify proteins involved in adhesion to host cells, ii) systematically identify T. pallidum antigenic proteins, and iii) establish a large-scale protein-protein interaction network among periplasmic and surface localized proteins.
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