Insertion of Epicatechin Gallate into the Cytoplasmic Membrane of Methicillin-resistant Staphylococcus aureus Disrupts Penicillin-binding Protein (PBP) 2a-mediated β-Lactam Resistance by Delocalizing PBP2

Abstract

Epicatechin gallate (ECg) sensitizes methicillin-resistant Staphylococcus aureus (MRSA) to oxacillin and other β-lactam agents; it also reduces the secretion of virulence-associated proteins, prevents biofilm formation, and induces gross morphological changes in MRSA cells without compromising the growth rate. MRSA is resistant to oxacillin because of the presence of penicillin-binding protein 2a (PBP2a), which allows peptidoglycan synthesis to continue after oxacillin-mediated acylation of native PBPs. We show that ECg binds predominantly to the cytoplasmic membrane (CM), initially decreasing the fluidity of the bilayer, and induces changes in gene expression indicative of an attempt to preserve and repair a compromised cell wall. On further incubation, the CM is reorganized; the amount of lysylphosphatidylglycerol is markedly reduced, with a concomitant increase in phosphatidylglycerol, and the proportion of branched chain fatty acids increases, resulting in a more fluid structure. We found no evidence that ECg modulates the enzymatic activity of PBP2a through direct binding to the protein but determined that PBP2 is delocalized from the FtsZ-anchored cell wall biosynthetic machinery at the septal division site following intercalation into the CM. We argue that many features of the ECg-induced phenotype can be explained by changes in the fluid dynamics of the CM.

Epigallocatechin Gallate Induces Upregulation of the Two-Component VraSR System by Evoking a Cell Wall Stress Response in Staphylococcus aureus

“…It has been proposed that damage inflicted to cell wall peptidoglycan is the main source of the stimuli to which VraR responds due to the tight control of VraS on the phosphorylation state of VraR (1). In addition, radiolabeling has shown that epicatechin gallate (ECG) binds predominantly to the CM, inducing an increase in its fluidity which results in uncoupling of some cell wall components that then cause delocalization of PBP2; as a consequence, the cells become more sensitive to beta-lactams and oxacillin (3). The structural similarity between ECG and EGCG and their common antimicrobial properties (33) tempt us to suggest that binding of EGCG to CM and relocation of PBP2 might be an additional mechanism conferring tolerance to beta-lactams.…”

The direct anti-MRSA effect of emodin via damaging cell membrane

“…As there is an inverse relationship between the intensity of polarized light emitted and fluidity of membrane, the effect of emodin on membrane fluidity was investigated by measuring fluorescence polarization of DPH inserted into the cell membrane (Bernal et al 2010). Briefly, MRSA252 (1.0× 10 6 CFU/mL) was treated with broth; 0.2 % DMSO (vehicle); 0.5, 1, and 2 μg/mL of emodin for 12 h with shaking of 150 rpm at 37°C.…”

Dual Roles of FmtA in Staphylococcus aureus Cell Wall Biosynthesis and Autolysis

“…It is part of the core cell wall stimulon (26). Its expression level is elevated by the presence of cell wall inhibitors and knockout of genes that are directly involved with peptidoglycan biosynthesis (3,23,26,27,42,46). Furthermore, inactivation of fmtA results in a lack of WTAs on S. aureus cell walls and deficiency in biofilm production (4,44).…”

“…Several studies on the genome-wide S. aureus response to cell wall inhibitors have demonstrated that their biological activity causes upregulation of fmtA (3,26,27,41,46). In addition, inactivation of genes involved with cell wall biosynthesis, such as murF, leads to higher levels of fmtA (41).…”