Elucidation of the Molecular Processes in antibiotic Resistance of Methicillin-resistant Staphylococcus aureus

LLARRULL, L.I.; MOBASHERY, S.

Chicago, Illinois

Conferencia; XXVIII Midwest Enzyme Chemistry Conference; 2008

University of Chicago

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a globally important pathogen that is resistant to all classes of commercially available beta-lactam antibiotics. The basis for resistance is acquisition of a pair of signal sensing/transducing systems that unleash two separate and complementary antibiotic resistance mechanisms: the production of a beta-lactamase, and the expression of a novel penicillin-binding protein. The expression of these antibiotic-resistance determinants is induced by the presence of the antibiotic in the milieu. It was proposed that the BlaR1 receptor, involved primarily in induction of beta-lactamase expression, is a novel kind of sensor protein (a protease) and is an interesting target for inhibition. We have expressed BlaR1 using cell-free expression systems in the presence of liposomes, and in E. coli cells, where BlaR1 was detected in the membrane fraction. BlaR1 expressed using both strategies was proteolyzed, and the proteolysis pattern observed was similar to the one previously observed in S. aureus in the presence of beta-lactam antibiotics. This indicates that BlaR1 is prone to digestion by E. coli proteases. The pattern of proteolysis bands was not modified in the presence of antibiotics. Hence, in E. coli, acylation of the sensor domain of BlaR1 by the antibiotics does not activate BlaR1 autoproteolysis. However, ProtK proteolysis assays suggest that BlaR1 and acylated BlaR1 present different conformations. BlaR1 was capable of proteolyzing BlaI, the repressor of the bla operon both in the presence and absence of beta-lactam antibiotics, which poses new questions on the mechanism of signal transduction.