): The ability to secrete proteins to their surrounding medium is an essential virulence characteristic of most pathogenic bacteria. The major barrier to extracellular protein secretion in gram-negative bacteria is the outer membrane. Accordingly, these bacteria have evolved elaborate pathways for extracellular targeting of proteins. One such protein export mechanism, referred to as the type II secretion pathway is responsible for secretion of proteins that have been previously localized into the bacterial periplasm. The type II pathway which functions in the human pathogen Pseudomonas aeruginosa, is responsible for the secretion of a number of important virulence factors. The machinery of the type II secretion has been previously shown to consist of 12 proteins, encoded by the xcp genes. Additional genes that encode homologues of the Xcp proteins have been identified primarily through the P. aeruginosa genome-sequencing project. The objectives of this proposal are to study the function of the machinery of extracellular protein secretion by defining the structure of the assembled secretion organelle in the cell envelope and to initiate a program for the identification of synthetic chemical compounds that target this pathway.
Three specific aims are proposed to accomplish these objectives. First, an interactive map of all of the components of the type II secretion machinery will be generated, utilizing a combination of approaches including chemical cross-linking, analysis of protein-protein interactions in yeast and a bacterial two hybrid systems and through the use of phage display technology.
The second aim will specially address the structure and function of the outer membrane components of the secretory apparatus. Finally, aided by the use of expression microarray technology, reporter strains will be engineered which will be used as sensitive indicators of a secretion block. These strains will be then used to screen libraries of chemical compounds which specifically inhibit the type II secretion pathway. If successful, the research proposed in this application should provide important new insights into the function of a secretion mechanism which is present in a large number of bacterial pathogens and could lead to the development of potentially new categories of antimicrobial agents.
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