Troponoma pallidum is the etiologic agent of syphills, a sexually transmitted disease that continues to be a public health problem. The technical difficulties encountered in handling T. pallidum have prompted many investigators to use recombinant DNA technology to study this noncultivable pathogen. A major focus of our research is the identification and characterization of exported proteins, some of which are likely to be virulence factors. We have used TnphoA insertion mutagenesis to identify several E. coli clones expressing T. pallidum proteins that are synthesized with export signals. Two recently isolated clones, 6D2 and 4C7, contain T. pallidum DNA inserts that encode proteins with homology to members of a superfamily of bacterial ABC transport systems. Such systems mediate the import of scarce nutrients or the export of various substances, including virulence factors. Clone 6D2 contains a 5.5-kb treponemal DNA insert encoding protein homologs of the E. coli/S. typhimurim high-- affinity galactose (Mg1) transport system. Clone 4C7 contains a 2.4-kb treponemal DNA insert that encodes proteins homologs of a Streptococcus gordonii ABC transport system. The long-term goal of our proposed studies is to continue using recombinant DNA technology to gain a better understanding of the physiology of T. pallidum. For the project period, we are proposing the following; (i) The complete nucleotide sequence of the p6D2 insert will be obtained and analyzed. Clones will be identified from genomic DNA libraries that contain DNA contiguous to the p6D2 insert. The nucleotide sequence of the contiguous DNA will be determined and the deduced amino acid sequences analyzed. Genes of interest will be subcloned and the encoded proteins will be characterized by localization and complementation assays. (ii) The complete nucleotide sequence of the p4C7 insert will be obtained and analyzed. Clones containing contiguous DNA will be identified and the nucleotide sequence will be determined and analyzed. Genes of interest will be subcloned and the encoded proteins will be further characterized. (iii) TnphoA mutagenesis will be used to identify additional clones from our T. pallidum genomic DNA library that synthesize exported proteins relevant to treponemal physiology. The nucleotide sequence of the genes will be determined and analyzed. The cellular location and function of the encoded proteins will be assessed. The results of our proposed studies will provide new information regarding the basic biology of T. pallidum. This information will offer insight towards the development of effective tools to prevent and control syphilis. In addition, the methodology that we are using is directly applicable tot he study of other bacterial agents of sexually transmitted diseases. This has fostered a fruitful exchange of ideas and technology between our laboratory and other laboratories within the UNC STD Cooperative Research Center. We anticipate that this collaborative interaction will be ongoing throughout our project period.
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