TREPONEMA PALLIDUM PHYSIOLOGY

梅毒螺旋体是梅毒的病原体，是一种性传播疾病 传播疾病仍然是一个公共卫生问题。这个 在处理梅毒螺旋体时遇到的技术困难促使 许多研究人员使用重组DNA技术来研究这一问题 不可培养的病原体。我们研究的一个主要焦点是 出口蛋白质的鉴定和特性，其中一些是 很可能是毒力因素。我们已经使用了TnPhoA插入突变 鉴定几个表达梅毒螺旋体蛋白的大肠杆菌克隆 与输出信号合成。最近分离的两个克隆6D2和 4C7，含有梅毒螺旋体DNA插入片段，编码与 细菌ABC运输系统超家族的成员。这样的系统 调停稀缺养分的进口或各种物质的出口， 包括毒力因素。克隆6D2含有5.5 kb的密螺旋体DNA 插入大肠杆菌/鼠伤寒沙门氏菌HIGH编码蛋白同源物-- 亲和力半乳糖(MG1)转运系统。克隆4C7包含一个2.4 kb的 编码链球菌蛋白同源物的密螺旋体DNA插入片段 Gordonii ABC运输系统。我们建议的研究的长期目标是 是继续使用重组DNA技术来获得更好的 对梅毒螺旋体生理学的认识。在项目期间，我们 建议如下：(I)该病毒的完整核苷酸序列 将获得P6D2插入物并进行分析。克隆将从以下位置标识 包含与p6D2插入相邻的DNA的基因组DNA文库。这个 将确定连续DNA的核苷酸序列，并 对推导的氨基酸序列进行分析。感兴趣的基因将被亚克隆 编码的蛋白质将通过定位和 互补性试验。(Ii)p4C7的完整核苷酸序列 将获得镶件并进行分析。包含连续DNA的克隆 将被鉴定，核苷酸序列将被确定和 分析过了。感兴趣的基因将被亚克隆，编码的蛋白质 将被进一步定性。(Iii)利用TnPhoA诱变来 从我们的梅毒螺旋体基因组DNA文库中鉴定其他克隆 合成与梅毒螺旋体生理相关的出口蛋白质。这个 基因的核苷酸序列将被确定和分析。这个 将对编码蛋白的细胞位置和功能进行评估。 我们建议的研究结果将提供以下方面的新信息 梅毒螺旋体的基本生物学。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，我们正在使用的方法直接适用于TOT 他研究性传播疾病的其他细菌病原体。这 促进了我们之间富有成效的思想和技术交流 北卡罗来纳州大学性病合作研究中心内的实验室和其他实验室 中心。我们预计，这种协作互动将持续进行 在我们的整个项目期间。

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.