The objective of the proposed research is to identify the components and their interrelationships controlling methicillin resistance in Staphylococcus aureus. Methicillin resistant strains of staphylococci produced a novel 78kd penicillin-binding protein (PBP), PBP 2a, that has low binding affinity for beta-lactam antibiotics. PBP 2a is encoded by the mec determinant, for which no equivalent allele exists in susceptible strains. An additional factor besides PBP 2a is necessary for resistance to be expressed fully because heterogeneous strains, which produce PBP 2a, are susceptible at concentrations that do not bind PBP 2a; homogeneous strains, which also produce PBP 2a, are uniformly and highly resistant. The proposed research will test the hypothesis that antibiotic binding to PBPs directly or indirectly mediates all beta-lactam antibiotic effects. Investigation will focus on 1) binding affinities of PBPs from susceptible, heterogeneous, and homogeneous strains of S. aureus for several beta-lactam antibiotics; 2) autolytic processes responsible for cell wall degradation, turnover and autolysis; 3) induction of PBP 2a production, particularly the role of beta-lactamase plasmid; and 4) development of antibody probes for PBPs. Representative strains ranging from fully susceptible to highly resistant will be studied. Binding affinities of PBPs will be determined by competition assay in which membrane proteins are incubated first with non-radioactive beta-lactam antibiotics then with a saturating concentration of 3H-penicillin. Proteins are separated by SDS- polyacrylamide gel electrophoresis and PBPs are detected by fluorography. Amounts of protein are measured by scanning densitometry. Factors that influence autolytic activity, and which may regulate expression of methicillin resistance, will be examined in assays measuring degradation of radiolabelled cell wall. Induction of PBP 2a by binding of inducer to PBPs will be examined and effects of the penicillinase plasmid on induction and resistance will be investigated with paired strains. Similarities in structure common to PBPs and other proteins related to penicillin effects will be probed with polyclonal and monoclonal antibodies. Factors controlling expression of methicillin resistance and mechanisms responsible for penicillin effects may be better understood as a result of these studies.
