FUNCTIONAL ANALYSIS OF SALMONELLA INVASION PROTEINS

描述(摘自申请者摘要)：沙门氏菌感染 仍然是一个世界性的健康问题，大约有100万人 发达国家胃肠炎病例和1000多万例 发展中国家每年都有伤寒病例。侵入性 沙门氏菌在摄入受污染的食物后进入宿主 或者水。微生物进入小肠，在那里它们 特异性地侵袭小脑内Peyer‘s斑块的M细胞 肠子。这些细菌具有进入哺乳动物细胞的能力 通过一种似乎是独一无二的机制。内部化发生 在细菌结合到细胞表面后，刺激 肌动蛋白聚合和膜褶皱。沙门氏菌的能力 侵袭宿主细胞受生长条件的调控。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，有几个 失去进入能力的氧调节LacZ融合菌株 对哺乳动物细胞进行鉴定。很可能是一些， 如果不是全部，这些基因都直接参与刺激 非吞噬细胞中未知的宿主信号转导途径 这使得致病的沙门氏菌能够被吸收。这些基因将 通过补充入侵从鼠伤寒沙门氏菌基因库中克隆 非侵袭性突变体的缺陷。在分离出基因之后， 每个基因的DNA序列将被确定和分析。已定义 非侵袭性鼠伤寒沙门氏菌突变体将被构建并用于 确定每个基因在M细胞侵袭和小鼠中的重要性 致命性。其他工作将集中在定义 细菌入侵蛋白。入侵蛋白将从 基因融合载体和纯化的蛋白将用于表达 兔抗体。抗侵袭蛋白抗体将用于 沙门氏菌侵袭蛋白定位的研究 在进入过程中，以及确定是否有能力 可以通过预先孵育特定的细菌来阻止 抗体。研究鼠伤寒沙门氏菌相互作用的另一种方法 宿主细胞将使用一种新描述的基因测试来实现功能 沙门氏菌入侵过程中蛋白质-蛋白质相互作用的筛选 蛋白质和宿主细胞蛋白质。这项工作将有助于 目前对沙门氏菌入侵机制的认识。它也是 很可能对鼠伤寒沙门氏菌和沙门氏菌相互作用的研究 宿主细胞将提供关于细胞生物学的尽可能多的信息 正如它将关于细菌的致病机制。

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.