CLONING AND CHARACTERIZATION OF CYTOSOLIC PROTEIN TYROSINE KINASE

志贺氏菌属引起细菌性痢疾，其特征是血性和 粘液满满的大便。志贺氏菌感染的特征之一是 细菌生长的肠道粘膜细胞的入侵 在细胞内，并从宿主细胞传播到邻近的宿主细胞。宽阔的 各种毒力因子已被描述并被证明与 在感染过程的不同阶段。的表达和活性 志贺氏菌的许多毒力基因受温度调节 和/或渗透压。然而，监管途径的细节 许多毒力基因还没有被彻底阐明。 蛋白激酶和转录调节蛋白的激活是 与真核和原核生物信号通路相关的事件 细胞。酪氨酸磷酸化蛋白最近在 包括志贺氏菌在内的一些细菌物种。此外，蛋白质 在福氏志贺氏菌中观察到酪氨酸激酶活性。我们有 与蛋白酪氨酸激酶活性相关的证据 福氏志贺氏菌的细胞膜及其在这些能力中的可能作用 细菌入侵上皮细胞。这种酪氨酸蛋白的靶标 激酶和其他细胞内酪氨酸蛋白激酶尚未被激活 已确认身份。这项提案的总体目标是克隆和刻画 编码胞质酪氨酸蛋白激酶的基因和底物 酪氨酸蛋白激酶。我们会试着确定这些激酶(S) 与膜相关酪氨酸蛋白形成信号网络 或与其他分子结合。此外，还将启动研究，以 确定这些基因组在福氏志贺氏菌生理学和 入侵。我们将通过识别做出反应的克隆来实现这些目标 对磷酸酪氨酸产生抗体。这些克隆的基因产物将 定位于细胞内。这些基因将被测序并在 以产生抗体为目的的大肠杆菌。这些抗体将是 用来进一步确定基因在细胞内的位置 产品。零突变体将通过两种不同的方法和 将被转移到入侵的志贺氏菌株中，以测试是否或 并不是这些基因产物是致病所必需的。

Shigella species cause bacillary dysentery characterized by bloody and mucous laden stool. One of the hallmarks of Shigella infection is the invasion of intestinal mucosal cells where the bacteria grow intracellularly and spread from host cell to adjacent host cell. A wide variety of virulence factors have been described and shown to be involved in various stages of the infection process. The expression and activity of many of the virulence genes in Shigella are regulated by temperature and/or osmolarity. However, the details of the regulatory pathways for many of the virulence genes have not been thoroughly elucidated. Activation of protein kinases and transcriptional regulatory proteins are events associated with signaling pathways of eukaryotic and prokaryotic cells. Tyrosine phosphorylated proteins have been recently demonstrated in a number of bacterial species including Shigella. Furthermore, protein tyrosine kinase activity has been observed in Shigella flexneri. We have evidence for a protein tyrosine kinase activity associated with the membrane of S. flexneri and its possible role in the ability of these bacteria to invade epithelial cells. Targets of this tyrosine protein kinase and other intracellular tyrosine protein kinases have yet to be identified. The overall goal of this proposal is to clone and characterize genes coding for cytosolic tyrosine protein kinases and substrates of tyrosine protein kinases. We will try to determine if these kinase(s) form a signaling network with the membrane associated tyrosine protein kinase or with other molecules. Furthermore, studies will be initiated to define the roles of these genomes in S. flexneri physiology and in invasion. We will accomplish these goals by identifying clones that react with antibodies to phosphotyrosine. The gene products of these clones will be localized within the cell. The genes will be sequenced and expressed in E. coli for the purpose of antibody production. These antibodies will be used to characterize further the intracellular location of the gene products. Null mutants will be constructed by two separate methods and will be transferred into invasive strains of Shigella to test whether or not these gen products are required for pathogenicity.