Project Summary/Abstract Methicillin-resistant Staphylococcus aureus (MRSA) is a problematic human bacterial pathogen, which is broadly resistant to β-lactam antibiotics. This resistance is inducible and is conferred by a set of genes that encode an antibiotic sensor/signal transducer protein, a gene repressor and two resistance determinants (a class A β-lactamases and a unique penicillin-binding protein (PBP) designated as PBP2a). Covalent binding of the β-lactam antibiotic to the surface domain of the sensor/signal transducer protein initiates the induction of resistance, which entails biochemical events on the cytoplasmic side involving proteolysis of the gene repressor and transcription of the genes for the antibiotic-resistance determinants. We address the sequence of events that take place in the cytoplasm in this grant application. We propose in Specific Aim 1 to study two responses that MRSA experiences after the recognition of the antibiotic on the surface. One is a fragmentation of the BlaR1 (the sensor/transducer protein) in its cytoplasmic protease domain, which is believed to activate it (i.e., bring it out of latency). Another is phosphorylation of a protein that we refer to as BlaR2. We propose to document that proteolysis of the cytoplasmic domain initiates the onset of induction. Phosphorylation of BlaR2 also ensues antibiotic recognition on the surface and is essential for the antibiotic response. We propose to identify and characterize BlaR2 and determine the site of its phosphorylation in elucidating a functional link between activation of BlaR1 and phosphorylation of BlaR2. In Specific Aim 2, we would like to build on our preliminary findings on inhibitors of BlaR2 phosphorylation in identification of molecules that could serve as potentiators of β-lactam antibiotics. This class of compounds prevents BlaR2 phosphorylation, which we have documented to result in sensitization of the organism to β-lactam antibiotics. Such a class of molecules has the potential of bring back β-lactam antibiotics from obsolescence in treatment of MRSA infections. MRSA has been with humanity for over 50 years. These studies will shed definitive light on the complex machinery that MRSA strains have evolved for resistance to β-lactam antibiotics.