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

LLARRULL, L.I.; MOBASHERY, S.

Notre Dame, Indiana

Jornada; Chemistry-Biology-Biochemistry Interface Retreat; 2009

Univeristy of Notre Dame

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 to beta-lactam antibiotics 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, which is not inactivated by the beta-lactams (1). The expression of these antibiotic-resistance determinants is induced by the presence of the antibiotic in the milieu. The BlaR1 receptor has been implicated primarily in induction of beta-lactamase expression (2). The antibiotic irreversibly acylates the extracellular sensor domain of BlaR1 (BlaRs) and responds to this acylation by a protein conformational change (3). I proposed a series of studies that would shed light on the microscopic incremental events that culminate to signal transduction and manifestation of antibiotic resistance in this unique system in a very important pathogen. In order to test whether the conformational changes in the sensor domain propagate to the transmembrane and cytoplasmic domains of BlaR1, I proposed the expression and insertion of the complete BlaR1 protein into liposomes, in the presence and in the absence of beta-lactam antibiotics. The analysis of the infrared and circular dichroism spectra of the protein would reveal the nature of the conformational change that the whole protein experiences. I evaluated the expression of BlaR1 using cell-free expression systems in the presence of liposomes and in E. coli cells, where BlaR1 was detected in the membrane fraction. The protein yield was very low even with cell-free expression systems. BlaR1 expressed using both strategies was proteolized, and the proteolysis pattern observed was similar to the one previously seen in S. aureus in the presence of beta-lactam antibiotics (2). 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 blaR operon, both in the presence and absence of beta-lactam antibiotics, which poses new questions on the mechanism of signal transduction.