The overall goal of the project is to understand the regulatory network by means of which staphylococci and streptococci regulate the synthesis of proteins, including virulence factors, that enable the organisms to maintain the state of colonization or to thrive in the hostile in vivo environment during infection.
Specific aims are: 1)To determine the mechanism of target gene activation by agr-RNA.; 2) To identify the effectors of virulence gene regulation (a) that respond to inhibitory environmental signals, (b) that are responsible for activation of certain exoprotein genes during early exponential phase and (c) that are responsible for activation of certain exoprotein genes in response to subinhibitory beta-lactam antibiotics; 3) To characterize environmentally determined patterns of virulence gene expression; 4) To identify the genetic regulators and effectors responsible for a) colonization. b) biofilm formation and maintenance. c) intrageneric bacterial interference in staphylococci; 5) To identify and analyze signaling pathways that regulate the production of streptococcal superantigens and possibly other virulence factors. Subcloning, mutation and deletion analysis coupled with the determination of RNA structure will be used to localize functional domains of the agr-determined RNA that regulates virulence gene expression. New regulatory genes will be identified by transposon mutagenesis plus genome-based cloning. Patterns of gene expression under various conditions and in mutant strains will be determined by means of oligonucleotide arrays based on the S. aureus genome. Known regulatory genes will be tested for their effects on colonization, biofilm formation and interference, and new regulatory genes affecting these phenomena will be identified by transposon mutagenesis. The possibility that streptococcal superantigen genes are autoinduced by peptides similar to those of S. aureus will be investigated by the analysis of culture supernatants. These studies have major implications for the control and treatment of infections due to staphylococci and streptococci and possibly other pathogenic bacteria. The most direct of these implications will involve the possibility of blocking the expression of virulence factors by interfering with the signaling pathways by which they are regulated.
