FUNCTIONAL ANALYSIS OF SALMONELLA INVASION PROTEINS

描述（改编自申请人摘要）：沙门氏菌感染 仍然是一个全球性的健康问题， 发达国家的肠胃炎病例超过1000万， 伤寒病例每年在发展中国家。侵入性 沙门氏菌在摄入受污染的食物后进入宿主体内 或水。微生物进入小肠， 特异性地侵入Peyer集合淋巴结的M细胞， 肠子这些细菌具有进入哺乳动物细胞的能力 是由一种独特的机制造成的内化发生 在细菌结合到细胞表面后， 肌动蛋白聚合和膜皱褶。沙门氏菌的能力 入侵宿主细胞的能力受生长条件的调节。高氧 生长条件抑制侵袭表型和低氧生长 诱导细菌入侵的能力。最近有数 氧调节的lacZ融合菌株，其已经失去进入细胞的能力， 鉴定了哺乳动物细胞。很可能是一些， 如果不是全部的话，这些基因直接参与刺激 非吞噬细胞中未鉴定的宿主信号转导途径 其允许致病性沙门氏菌物种的摄取。这些基因将 从一个S.鼠伤寒基因库的补充入侵 非侵入性突变体的缺陷。在分离基因之后， 将确定和分析每个基因DNA序列。定义 非侵入性S.将构建鼠伤寒沙门氏菌突变体， 确定每个基因在M细胞侵袭和小鼠中的重要性 毒性其他工作将集中于确定 细菌入侵蛋白入侵蛋白将从 基因融合载体和纯化的蛋白质将用于提高 兔抗体抗侵袭蛋白抗体将用于 确定沙门氏菌入侵蛋白定位的研究 在进入过程中，以及确定是否有能力 的细菌入侵可以通过与特定的 抗体的另一种研究S.鼠伤寒 宿主细胞将使用一种新描述的遗传分析， 筛选沙门氏菌入侵之间的蛋白质-蛋白质相互作用 蛋白和宿主细胞蛋白。这项工作将有助于 目前对沙门氏菌入侵机制的理解。也是 很可能是研究S.氏菌和 宿主细胞将提供尽可能多的细胞生物学信息 就像它将揭示细菌的致病机制一样。

DESCRIPTION (Adapted from applicant's abstract): Salmonellae infections continue to be a worldwide health problem with approximately 1 million cases of gastroenteritis in developed countries and more than 10 million cases of typhoid fever in developing countries each year. Invasive Salmonella species enter a host following ingestion of contaminated food or water. The organisms move into the small intestine where they specifically invade M cells of Peyer's patches within the small intestine. These bacteria possess the ability to enter mammalian cells by a mechanism which appears to be unique. Internalization occurs following bacterial binding to the surface of a cell, stimulation of actin polymerization, and membrane ruffling. The ability of Salmonella to invade host cells is regulated by growth conditions. High-oxygen growth conditions repress the invasive phenotype and low-oxygen growth induces the ability of the bacteria to invade. Recently, several oxygen-regulated lacZ fusion strains which had lost the ability to enter mammalian cells were identified. It is likely that the products of some, if not all, of these genes are directly involved in stimulating the unidentified host signal transduction pathway in nonphagocytic cells which allows uptake of pathogenic Salmonella species. These genes will be cloned from an S. typhimurium gene bank by complementing the invasion defect of the noninvasive mutants. Following isolation of the genes, the DNA sequence of each gene will be determined and analyzed. Defined noninvasive S. typhimurium mutants will be constructed and used to determine the importance of each gene in M cell invasion and mouse virulence. Other work will concentrate on defining the function of the bacterial invasion proteins. Invasion proteins will be purified from gene fusion vectors and the purified proteins will be used to raise rabbit antibody. The anti-invasion protein antibodies will be used in studies to determine the location of the Salmonella invasion proteins during the entry process, as well as to determine whether the ability of the bacteria to invade can be blocked by preincubation with specific antibody. Another approach to study interactions between S. typhimurium and host cells will use a newly described genetic assay to functionally screen for protein-protein interactions between Salmonella invasion proteins and host cell proteins. This work will contribute to the present understanding of the Salmonella invasion mechanism. It is also likely that the study of the interactions between S. typhimurium and host cells will provide as much information about the biology of cells as it will about the bacterial pathogenic mechanisms.