VIRULENCE FACTORS IN THE PATHOGENESIS OF SALMONELLOSIS

沙门氏菌病发病过程中的毒力因素

• 批准号: 2060704
• 负责人: JOHNNY Wayne PETERSON
• 金额: $ 18.17万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-09-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2060704
• 关键词: ADP ribosylation; Escherichia coli; G protein; Salmonella; Salmonella food poisoning; Salmonella typhimurium; antibacterial antibody; bacterial DNA; bacterial antigens; cyclic AMP; enterotoxins; fusion gene; gene expression; genetic strain; glutathione transferase; laboratory rabbit; molecular cloning; molecular pathology; nucleic acid sequence; pathologic process; polymerase chain reaction; prostaglandin E; protein purification; site directed mutagenesis; virulence

The overall objective continues to be the probing of the pathogenic mechanism of experimental salmonellosis to define the role of Salmonella enterotoxin (Stn) as a selected virulence factor in the intestinal phase of Salmonella infection. Our past research has enabled us to clone a fragment of Salmonella chromosomal DNA, encoding the stn gene. When expressed with the T7 RNA promoter/polymerase system, cell-free lysates of E. coil, containing Stn, evoked enterotoxic responses in rabbit intestinal loops. We determined the molecular structure of cloned Stn, based on predictions from the nucleotide sequence, which allowed us to localize the stn gene at approximately 90 min on the Salmonella chromosome opposite the hydHG operon. We used site-directed mutagenesis to identify the stn initiation codon, which was a "TTG" rather than the typical "ATG." Further, the unusually high pI of the protein (11.7) confers a strong negative charge on the toxin's surface, causing it to bind to positively charged molecules at pH 7.0, a property that has complicated Stn purification. Our Specific Aims seek to construct an isogenic stn derivative of a virulent Salmonella strain(s) (e.g., TML- R66), for use in determining the precise role of Stn in the pathogenic mechanism (i.e., secretion and/or invasion). Precautions will be taken to avoid secondary alterations in hydHG function. Further, we are striving to improve gene expression and Stn solubility by subcloning the stn gene into selected fusion protein vectors. Total Stn antigen was, increased 64 fold by preparing two fusion proteins [glutathione S- transferase::Stn (Gst::Stn) and thioredoxin A::Stn (TrxA::Stn], and TrxA::Stn increased Stn solubility by 50 fold. With improvements in gene expression of Stn, we anticipate that purification to homogeneity is achievable. The purified protein will be used in experiments to study the molecular mechanism of the enterotoxin in elevating cAMP and PGE2 levels in intestinal cells, as well as to generate a polyclonal antiserum to Stn. Finally, stn genes from selected Salmonella isolates, that differ in intestinal virulence (e.g., fluid accumulation), will be amplified by PCR so that the nucleotide sequence of each can be examined and compared. The proposed studies should provide new and helpful information about the pathogenesis of salmonellosis.

总体目标继续是致病性的探测 实验性沙门氏菌病的机制来定义沙门氏菌的作用 肠毒素（STN）作为肠相的选定毒力因子 沙门氏菌感染。 我们过去的研究使我们能够克隆 沙门氏菌染色体DNA的片段，编码STN基因。 什么时候 用T7 RNA启动子/聚合酶系统表达，无细胞裂解物 含有STN的E.卷轴的诱发肠毒性反应 肠环。我们确定了克隆STN的分子结构， 基于核苷酸序列的预测，这使我们得以 将STN基因在沙门氏菌上定位大约90分钟 染色体与Hydhg操纵子相对。我们使用了定向的诱变 识别STN启动密码子，该密码子是“ TTG”而不是 典型的“ ATG”。此外，蛋白质异常高的PI（11.7） 赋予毒素表面上强烈的负电荷，使其导致 在pH 7.0处与带正电的分子结合，该特性具有 复杂的STN纯化。我们的具体目标寻求建造 毒性沙门氏菌菌株（S）的等源性STN衍生物（例如TML- R66），用于确定STN在病原上的精确作用 机制（即分泌和/或入侵）。将采取预防措施 为了避免HYDHG功能的次要变化。此外，我们是 努力通过亚克隆来提高基因表达和STN溶解度 STN基因进入选定的融合蛋白载体。总抗原是 通过准备两种融合蛋白[谷胱甘肽S-增加64倍 Transferase :: STN（GST :: STN）和Thioredoxin A :: STN（TRXA :: STN]和 TRXA :: STN提高了STN溶解度50倍。基因的改善 STN的表达，我们预计均匀性的纯化是 可以实现。 纯化的蛋白质将用于研究 肠毒素高架营和PGE2中肠毒素的分子机制 肠细胞中的水平以及产生多克隆抗血清的水平 到STN。最后，来自选定沙门氏菌分离株的STN基因，不同 在肠道毒力（例如液体积累）中，将被放大 PCR，因此可以检查并比较每个核苷酸序列。 拟议的研究应提供有关有关的新信息 沙门氏菌病的发病机理。