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. 2007 Jul;153(Pt 7):2093-2103.
doi: 10.1099/mic.0.2007/007807-0.

The beta-lactam-resistance modifier (-)-epicatechin gallate alters the architecture of the cell wall of Staphylococcus aureus

Paul D Stapleton  1 , Saroj Shah  1 , Kerstin Ehlert  2 , Yukihiko Hara  3 , Peter W Taylor  1
Affiliations

The beta-lactam-resistance modifier (-)-epicatechin gallate alters the architecture of the cell wall of Staphylococcus aureus

Paul D Stapleton et al. Microbiology (Reading). 2007 Jul.

Abstract

(-)-Epicatechin gallate (ECg), a component of green tea, sensitizes meticillin-resistant Staphylococcus aureus (MRSA) to beta-lactam antibiotics, promotes staphylococcal cell aggregation and increases cell-wall thickness. The potentiation of beta-lactam activity against MRSA by ECg was not due to decreased bacterial penicillin-binding protein (PBP) 2a expression or ECg binding to peptidoglycan. A 5-10 % reduction in peptidoglycan cross-linking was observed. Reduced cross-linking was insufficient to compromise the integrity of the cell wall and no evidence of PBP2a activity was detected in the muropeptide composition of ECg-grown cells. ECg increased the quantity of autolysins associated with the cell wall, even though the cells were less susceptible to Triton X-100-induced autolysis than cells grown in the absence of ECg. ECg promoted increased lysostaphin resistance that was not due to alteration of the pentaglycine cross-bridge configuration or inhibition of lysostaphin activity. Rather, decreased lysostaphin susceptibility was associated with structural changes to wall teichoic acid (WTA), an acid-labile component of peptidoglycan. ECg also promoted lipoteichoic acid (LTA) release from the cytoplasmic membrane. It is proposed that ECg reduces beta-lactam resistance in MRSA either by binding to PBPs at sites distinct from the penicillin-binding site or by intercalation into the cytoplasmic membrane, displacing LTA from the phospholipid palisade. Thus, ECg-mediated alterations to the physical nature of the bilayer will elicit structural changes to WTA that result in modulation of the cell-surface properties necessary to maintain the beta-lactam-resistant phenotype.

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Figures

Fig. 1
Fig. 1
Influence of exogenous peptidoglycan on modulation of oxacillin susceptibility by ECg and EGCg. (a) Structures of the compounds used in this study. (b) Effect of exogenous peptidoglycan (32 μg ml-1; black symbols) on the capacity of ECg and EGCg to modulate oxacillin resistance in S. aureus BB568. Cells were grown in the absence (○) or presence of 12.5 μg EC ml-1 (◇), 12.5 μg ECg ml-1 (□, ■) or 12.5 μg EGCg ml-1 (△, ▲). White symbols indicate growth in the absence of exogenous peptidoglycan.
Fig. 2
Fig. 2
Effect of green tea catechins on cell aggregation and adherence to glass. (a-c) Gram-staining of EMRSA-15 grown in the absence (a) and presence of (b) EC (12.5 μg ml-1) or (c) ECg (12.5 μg ml-1). (d) EMRSA-15 was grown in the presence or absence of 12.5 μg ECg ml-1 for 16 h, without shaking, and the adherence to the flask wall examined by gently tilting the flask. The reduced density of the ECg-grown culture was due to flocculation as a result of cell clumping and not to inhibition of cell growth.
Fig. 3
Fig. 3
Effect of ECg on Triton X-100-induced autolysis. Triton X-100 was used to stimulate autolysis in S. aureus cells grown in the absence (black symbols) or presence (white symbols) of 12.5 μg ECg ml-1. The four S. aureus isolates investigated were: MSSA isolate BB551 (○, ●) and MRSA isolates BB568 (□, ■), EMRSA-15 (◇, ◆), and EMRSA-16 (△, ▲). Percentage lysis was determined from OD600 measurements and compared to values at time point 0.
Fig. 4
Fig. 4
Effect of ECg on autolysin production in S. aureus BB568. (a) Autolysins were extracted with SDS from the cell walls of S. aureus BB568 grown in MH medium containing various ECg concentrations and separated on SDS-containing polyacrylamide gels containing heat-killed S. aureus cells (zymogram). M is a molecular size maker; lane 1, no ECg; lane 2, 25 μg ECg ml-1; lane 3, 50 μg ECg ml-1; and lane 4, 100 μg ECg ml-1. (b) Zymogram of extracellular autolysin production by S. aureus BB568 grown in the presence of ECg. Lane order (1 to 4) as in (a). (c) Lysis of heat-killed S. aureus cells by extracellular autolysin preparations from cells grown in the absence (■) and presence of ECg (25 μg ml-1, □; 50 μg ml-1, ○; and 100 μg ml-1, ▲).
Fig. 5
Fig. 5
Effect of WTA on the capacity of mutanolysin to hydrolyse peptidoglycan extracted from BB568. Cells were grown in the absence (□) or presence (▲) of 12.5 μg ECg ml-1. (a) Peptidoglycan hydrolysis in the presence of covalently bound WTA. (b) Peptidoglycan hydrolysis after removal of WTA with acid.
Fig. 6
Fig. 6
Effect of ECg on LTA release. Release of [2-3H]glycerol-labelled LTA from S. aureus BB568 grown in the absence (□) or presence of EC (12.5 μg ml-1; ▲), ECg (12.5 μg ml-1; ■), and Triton X-100 (0.02 %, v/v; △).
Fig. 7
Fig. 7
Effect of ECg on the muropeptide composition and degree of cross-linking of peptidoglycan extracted from S. aureus BB568 and EMRSA-16. Purified peptidoglycan preparations from cells grown in the presence or absence of ECg were digested with muramidase and the digestion products separated by HPLC.
Fig. 8
Fig. 8
Effect of ECg on PBPs from S. aureus BB568. (a) PBPs were extracted from cells grown in the absence or presence of 12.5 μg ECg ml-1 and pre-incubated with 12.5 μg ECg ml-1, 600 μg meticillin ml-1, or assay buffer, before PBP labelling with biocillin. Proteins were separated by SDS-PAGE and the PBPs visualized by fluorography. The PBP profile for meticillin-susceptible isolate BB551 is also shown. (b) Relative intensities of the PBP bands in (a); all values are given relative to the intensity of each PBP band grown in the absence of ECg (100 %). Key: white bars, grown in the absence of ECg; hatched bars, grown in the absence of ECg and pre-incubated with ECg before bocillin labelling; black bars, grown in the presence of ECg; grey bars, grown in the presence of ECg and pre-incubated with ECg before bocillin labelling. (c) Membrane proteins extracted from cells grown in ECg were separated on SDS-polyacrylamide gels and PBP2a detected with anti-PBP2a antibody.
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