Impact of epicatechin gallate on the structural integrity of the PBP2/PBP2a complex in methicillin resistant Staphylococcus aureus

Introduction: The selective pressure imposed by the misuse and overuse of antibiotics has led to the emergence and dissemination of methicillin resistant Staphylococcus aureus (MRSA), forcing a re-evaluation of therapeutic approaches to the treatment of MRSA infections. Epicatechin gallate (ECg), a constituent of the tea plant Camellia sinensis, has the capacity to abrogate the resistance of MRSA to beta-lactam antibiotics and may be useful as an adjunct to conventional chemotherapy. Current evidence suggests that ECg sensitises resistant strains to beta-lactam agents by disruption of the penicillin binding protein (PBP) complex PBP2/PBP2a at the septal site of cell division following its intercalation into the cytoplasmic membrane (CM) bilayer.;Results: PBP2/PBP2a complexes were identified in both ECg-exposed and control EMRSA-16 cells when SMALPs were pulled down with anti-PBP2 and anti-PBP2a antibodies. Fewer complexes were recovered from ECg exposed cells. Co-immunoprecipitation of SMALPs with antibody against the division scaffold protein FtsZ led to the identification of FtsZ/PBP2/PBP2a complexes. ECg displaced PBP2a from this complex. The PBP2/PBP4 complex was also identified, however there was no difference observed following ECg exposure.;Conclusion: Intercalation of ECg into the MRSA phospholipid palisade led to partial disruption of PBP2a from PBP2/PBP2a and FtsZ/PBP2/PBP2a complexes. The data suggest that ECg-mediated conversion of MRSA to beta-lactam susceptibility may in part be related to loss of functional integrity of the cellular replication machinery. The therapeutic approach with the use of antibiotic resistance modifying agent, such as ECg, in combination with a previously ineffective beta-lactam antibiotic, presents a novel therapy to combat antibiotic resistance in MRSA.;Methods: Styrene maleic acid lipid co-polymer (SMALP) was used to solubilise and extract PBP2/PBP2a membrane complexes from the CM of EMRSA-16 and ECg-exposed cells. Cell walls were partially digested and membrane proteins excised and solubilised with hydrolysed styrene maleic acid (SMA). SMALP particles were visualised by TEM and size distribution determined by dynamic light scattering. Membrane protein complexes were cross-linked within SMALPs, protein complexes recovered by co-immunoprecipitation and the constituents determined by Western blotting and flow cytometry.