TREPONEMA PALLIDUM PHYSIOLOGY

梅毒螺旋体生理学

• 批准号: 6099528
• 负责人: LOLA Virginia STAMM
• 金额: --
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6099528
• 关键词: Escherichia coli; Treponema pallidum; adenosinetriphosphatase; bacterial genetics; bacterial proteins; binding proteins; chimeric proteins; computer assisted sequence analysis; cooperative study; genetic library; immunoprecipitation; laboratory rabbit; membrane proteins; membrane transport proteins; microorganism metabolism; molecular cloning; nonblood lipoprotein; nucleic acid sequence; polymerase chain reaction; protein purification; recombinant proteins; virulence; western blottings

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.

梅毒螺旋体是梅毒的病原体，梅毒是一种性病 传播疾病仍然是一个公共卫生问题。 这 处理梅毒螺旋体时遇到的技术困难促使 许多研究者使用重组DNA技术来研究这个 不可培养的病原体。 我们研究的一个主要重点是 输出蛋白质的鉴定和表征，其中一些是 可能是毒力因子。 我们使用了 TnphoA 插入诱变 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。 两个最近分离的克隆，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。 此类系统 调解稀缺营养素的进口或各种物质的出口， 包括毒力因子。 克隆 6D2 含有 5.5 kb 密螺旋体 DNA 插入编码大肠杆菌/S 的蛋白质同源物。伤寒高发—— 亲和半乳糖 (Mg1) 转运系统。 克隆 4C7 包含 2.4-kb 编码链球菌同源蛋白的密螺旋体 DNA 插入片段 gordonii ABC 传输系统。 我们提出的研究的长期目标 就是继续利用重组DNA技术来获得更好的 了解梅毒螺旋体的生理学。 在项目期间，我们 提出以下建议； (i) 完整的核苷酸序列 将获得并分析 p6D2 插入片段。 克隆将被鉴定为 含有与 p6D2 插入片段相邻的 DNA 的基因组 DNA 文库。 这 连续DNA的核苷酸序列将被确定并且 分析推导的氨基酸序列。 感兴趣的基因将被亚克隆 编码的蛋白质将通过定位和表征来表征 互补分析。 (ii) p4C7 的完整核苷酸序列 将获得并分析插入内容。 含有连续 DNA 的克隆 将被鉴定并且核苷酸序列将被确定并且 分析了。 感兴趣的基因将被亚克隆，编码的蛋白质 将进一步表征。 (iii) TnphoA 诱变将用于 从我们的梅毒螺旋体基因组 DNA 文库中鉴定出其他克隆 合成与密螺旋体生理学相关的输出蛋白质。 这 将确定并分析基因的核苷酸序列。 这 将评估编码蛋白质的细胞位置和功能。 我们拟议的研究结果将提供有关以下方面的新信息： 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，我们使用的方法直接适用于 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 北卡罗来纳大学性病合作研究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。