CLONING AND CHARACTERIZATION OF CYTOSOLIC PROTEIN TYROSINE KINASE

志贺氏菌引起的细菌性痢疾的特征是血性和

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.