TREPONEMA PALLIDUM PHYSIOLOGY

梅毒螺旋体是梅毒的病原体， 传播的疾病仍然是一个公共卫生问题。 的 在处理T.苍白球促使 许多研究人员使用重组DNA技术来研究这一点 不可培养的病原体 我们研究的一个主要重点是 鉴定和表征输出的蛋白质，其中一些是 可能是毒力因子。 我们已经使用TnphoA插入诱变 鉴定了几个E. coli克隆表达T.苍白球蛋白， 与输出信号合成。 两个最近分离的克隆，6D2和 4C 7，含T.苍白球DNA插入物，其编码与 细菌ABC运输系统超家族的成员。 这样的系统 调节稀缺营养物质的输入或各种物质的输出， 包括毒力因子。 克隆6D2含有5.5 kb密螺旋体DNA 插入片段编码E. coli/S. Typhimurim高-- 亲和半乳糖（Mg 1）转运系统。 克隆4C 7含有一个2.4 kb的 编码链球菌同源蛋白质的密螺旋体DNA插入物 gordonii ABC运输系统 我们研究的长期目标是 是继续使用重组DNA技术， 了解T.苍白球 在项目期间，我们 （一）完整的核苷酸序列; 将获得p6 D2插入物并进行分析。 克隆将从 含有与p6 D2插入片段邻接的DNA的基因组DNA文库。 的 将确定连续DNA的核苷酸序列， 分析推导的氨基酸序列。 感兴趣的基因将被亚克隆 并且所编码的蛋白质将通过定位来表征， 互补测定。 (ii)p4 C7的完整核苷酸序列 将获得插入物并进行分析。 含有连续DNA的克隆 将被鉴定，并将确定核苷酸序列， 分析了 感兴趣的基因将被亚克隆，编码的蛋白质 将得到进一步表征。 (iii)TnphoA诱变将用于 从我们的试验中鉴定出更多的克隆。苍白球基因组DNA文库， 合成与密螺旋体生理学相关的输出蛋白。 的 将确定和分析基因的核苷酸序列。 的 将评估编码蛋白质的细胞位置和功能。 我们建议的研究结果将提供有关以下方面的新信息： T.苍白球 这些信息将提供洞察力 致力于开发预防和控制梅毒的有效工具。 此外，我们使用的方法直接适用于 对其他性传播疾病的病原菌的研究。 这 促进了我们之间富有成效的思想和技术交流， 实验室和其他实验室内的CNOSTD合作研究 中心 我们预计，这种协作互动将持续下去， 整个项目期间。

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.