The virulence genes of Staphylococcus aureus are regulated by a novel system that combines quorum sensing with signal transduction. The autoinducing molecules are peptides that may have a cyclic structure. The PI has developed a hypothetical model for how autoinduction works. A prepeptide is synthesized inside the cell, then excreted. During excretion, the peptide is cleaved and cyclized. When a high enough concentration is reached, the peptides bind to the receptor AgrC, probably forming a covalent bond. This changes the conformation of AgrC, starting a signal transduction cascade that activates ArgA and leads to the induction of virulence genes controlled by this system. The autoinducing peptides of one type of S. aureus can inhibit gene expression in other types of S. aureus, a finding that may explain how some strains of S. aureus can prevent others from colonizing a mucosal site. The PI proposes to test this model in a systematic fashion. 1. The PI will determine the mechanism of processing and secretion of the peptides by following intermediates in the intact cell. An attempt will be made to purify the proteins involved in this reaction so that an in vitro system can be developed. 2. The PI will develop a method for producing the final form of the autoinducer, which he believes may be an octapeptide cyclized with a thioester bond. A combination of NMR and X-ray crystallography will be used to prove the structure of the autoinducer and probe the structure of the inducer-receptor complex. 3. The PI will use labeled autoinducer to determine the binding site for this ligand on the receptor protein, AgrC. The PI hopes that this analysis will be facilitated by the covalent bond that may occur between the ligand and receptor. The location of the region to target for more intensive investigation will be obtained by domain swapping and deletion analysis. 4. The PI has noted that the argBCD sequences appear to be chimeric, with some highly conserved and some highly variable regions. He will determine how sequence diversification arises by creating strains with heterologous genes and use an agr-tetK fusion to select for gain of function mutants or start with homologous gene sets and use a spa-tetK fusion to select for loss of function. Only mutations within the region will be examined. 5. The PI has noted a possible relationship between some biotypes of S. aureus and the grouping of the autoinducers. He will determine whether biotypes such as exoprotein pattern, pulsed field signature, and enzyme electrophoresis. A large number of strains from known sites and disease states will be screened. The significance of this is first that arg may regulate the correlated factors and second that it may provide new insights into the factors behind the apparent tissue tropism of many S. aureus strains. 6. The PI will use three different animal models to test the hypothesis that cross inhibition of S. aureus agr genes by autoinducer from another strain. Pairs of different strains will be competed in these models, which test for different S. aureus attributes such as ability to colonize skin and ability to infect deep tissues. Mutants will also be tested in these models to determine the importance of the autoinduction system, and an attempt will be made to detect expression of agr genes in the animal.
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