Unveiling details of the signal transduction pathway on the bla beta-lactam resistance system in Staphylococcus aureus

LLARRULL, L.I.; MOBASHERY, S.

Galveston, Texas

Conferencia; Gordon Research Conferences: New Antibacterial Discovery & Development; 2010

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, which is not inactivated by the beta-lactams. 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 (bla operon). The antibiotic irreversibly acylates the extracellular sensor domain of BlaR1 (BlaRs). BlaRs responds to this acylation by a protein conformational change. It is our goal to elucidate how the signal transmits from the extracellular sensor domain to the cytoplasmic domain, which is proposed to be a metallo-protease, activated in the presence of the antibiotic. We compared the expression of BlaR1 in S. aureus NRS128 in the presence of beta-lactam antibiotics with the expression of recombinant BlaR1 in E. coli, where BlaR1 was detected in the membrane fraction. We also evaluated BlaI proteolysis in the presence of BlaR1. The analysis of the proteolysis patterns observed for BlaR and BlaI in the presence and absence of antibiotics suggest a new interpretation of the relevance of the proteolysis events for the signal transduction pathway, and open new questions on the signal regulation that culminates in manifestation of antibiotic resistance in this unique system. We are also assaying the effect of different mutations on the activity of BlaR1 expressed in E. coli cells, the expression of different truncated versions of BlaR1, and we are studying the interaction of the repressor BlaI with the operator, in order to determine whether the elimination of the dimerization domain in BlaI upon proteolysis eliminates the ability of the repressor to bind to DNA. Our results argue against this hypothesis and instead suggest that BlaI proteolysis gives rise to an unstable fragment which is further degraded both in S. aureus and E. coli cells. The previously proposed site specific cleavage of BlaI would result in further proteolysis and in a substantial decrease in the concentration of the repressor in the cells, which would give rise to the expression of the beta-lactamase.