One of the most serious contemporary challenges to the treatment of hospital-acquired infections worldwide is the appearance and global spread of staphylococci resistant to all beta-lactam antibiotics (known as methicillin-resistant S. aureus; MRSA). Staphylococci can become resistant to Beta-lactam antibiotics by acquiring a gene (mecA) that encodes a beta-lactam insensitive target enzyme, penicillin binding protein (PBP)2a. This enzyme affords the bacterium the ability to cross-link cell wall and grow while the cell's usual cross-linking enzymes (PBP's) are bound and inactivated by Beta-lactam antibiotics. Most of S. aureus also produce/beta-lactamase encoded by blaZ, which can hydrolyse Beta-lactam antibiotics rendering them inactive. The transcription of mecA and blaZ is regulated by related divergently transcribed two-gene operons mecRl-mecI and blaRl-blaI, respectively that encode a signal transducer (R1) and a repressor (I). The ongoing studies outlined in this proposal are based on previous observations in laboratory strains that demonstrated the coregulation of mecA by both MecI and BlaI Furthermore, these observations could be extended to clinically relevant isolates which escape MecI repression in order to survive/beta-lactam pressure (36). The goal of this proposal is to provide evidence of the existence of additional loci involved in or required for inducible expression of methicillin resistance. These data will provide valuable structure/function information to complement studies investigating the molecular basis of MecR1/BlaR1 and MecI/BlaI signal transduction.
The specific aims are: To Investigate the molecular basis of signal transduction through MecR1/BlaR1 to MecI/Blalby identifying and characterizing """"""""mecR2"""""""" loci - chromosomal genes that may contribute to the induction of mecA/blaZ expression. To test the hypothesis that repression is evolutionarily conserved in order to prevent overproduction of a toxic, regulated gene product. Specifically, we will evaluate if the inducible overexpression of either MecR1/BlaR1 or their corresponding mutants/deletants in the absence of Beta-lactam induction results in a toxic effect to the cell.
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