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.

troponoma pallidum是Syphills的病因学药 传播疾病仍然是公共卫生问题。 这 在处理T. pallidum时遇到的技术困难已提示 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。 我们研究的重点是 出口蛋白的识别和表征，其中一些是 可能是毒力因素。 我们使用了tnphoa插入诱变 识别表达T. pallidum蛋白的几个大肠杆菌克隆 与出口信号合成。 两个最近孤立的克隆，6d2和 4C7，包含T. pallidum DNA插入物，与同源性编码蛋白质的蛋白 细菌ABC传输系统超家族的成员。 这样的系统 调解稀缺营养或出口各种物质的进口， 包括毒力因子。 克隆6D2包含5.5-kb的treponemal DNA 插入大肠杆菌/s的编码蛋白质同源物。伤寒高 - 亲和半乳糖（MG1）运输系统。 克隆4C7包含2.4-kb 编码链球菌的蛋白质同源物的treponemal DNA插入物 Gordonii ABC运输系统。 我们提出的研究的长期目标 是继续使用重组DNA技术以获得更好的 了解粒细胞链球菌的生理学。 在项目期间，我们 提出以下内容； （i）完整的核苷酸序列 将获得和分析P6D2插入物。 克隆将从 与P6D2插入物相邻的DNA基因组DNA文库。 这 将确定连续DNA的核苷酸序列，并确定 分析的推导氨基酸序列。 感兴趣的基因将被亚克隆 并且编码的蛋白质将以定位和 互补分析。 （ii）P4C7的完整核苷酸序列 将获得和分析插入物。 包含连续DNA的克隆 将被鉴定，并确定核苷酸序列，并 分析。 感兴趣的基因将被亚克隆并编码的蛋白质 将进一步表征。 （iii）tnphoa诱变将用于 从我们的T. pallidum基因组DNA库中识别出其他克隆 合成与treponemal生理相关的出口蛋白。 这 将确定和分析基因的核苷酸序列。 这 将评估编码蛋白的细胞位置和功能。 我们提出的研究的结果将提供有关有关的新信息 T. pallidum的基本生物学。 此信息将提供洞察力 开发有效的工具来预防和控制梅毒。 另外，我们使用的方法直接适用 他研究了其他性传播疾病的细菌剂。 这 在我们之间建立了富有成果的思想和技术的交流 UNC STD合作研究中的实验室和其他实验室 中心。 我们预计这种协作互动将进行 在我们的整个项目期间。