Penicillin-binding proteins and cell wall composition in beta-lactam-sensitive and -resistant strains of Staphylococcus sciuri

Abstract

A close homologue of the acquired Staphylococcus aureus mecA gene is present as a native gene in Staphylococcus sciuri. We determined the patterns of penicillin-binding proteins (PBPs) and the peptidoglycan compositions of several S. sciuri strains to explore the functions of this mecA homologue, named pbpD, in its native S. sciuri environment. The protein product of pbpD was identified as PBP4 with a molecular mass of 84 kDa, one of the six PBPs present in representatives of each of three subspecies of S. sciuri examined. PBP4 had a low affinity for nafcillin, reacted with a monoclonal antibody raised against S. aureus PBP2A, and was greatly overproduced in oxacillin-resistant clinical isolate S. sciuri SS37 and to a lesser extent in resistant laboratory mutant K1M200. An additional PBP inducible by oxacillin and corresponding to S. aureus PBP2A was identified in another oxacillin-resistant clinical isolate, S. sciuri K3, which harbors an S. aureus copy of mecA. Oxacillin resistance depended on the overtranscribed S. sciuri pbpD gene in strains SS37 and K1M200, while the resistance of strain K3 depended on the S. aureus copy of mecA. Our data provide evidence that both S. aureus mecA and S. sciuri pbpD can function as resistance determinants in either an S. aureus or an S. sciuri background and that the protein products of these genes, S. aureus PBP2A and S. sciuri PBP4, can participate in the biosynthesis of peptidoglycan, the muropeptide composition of which depends on the bacterium "hosting" the resistance gene.

FIG. 1.

PBP patterns of oxacillin-susceptible and -resistant S. sciuri strains. Membrane proteins were purified from S. sciuri strains grown in antibiotic-free medium (lanes 1 to 9) and from strain K3 grown in the presence of 3, 6, or 9 μg/ml oxacillin (lanes 10 to 12). Membrane preparations (150 μg of proteins) were incubated with a single saturating concentration of [¹⁴C]benzylpenicillin. After SDS-polyacrylamide gel electrophoresis analysis, the gel was exposed to a storage phosphor screen for 2 weeks. PBPs and their corresponding molecular masses are indicated on the left. Protein standard size markers are indicated on the right.

FIG. 2.

Detection by Western blotting of protein products of S. aureus mecA and S. sciuri pbpD. Membrane preparations (80 μg of proteins) were tested by Western blot analysis for the production of proteins that react with a monoclonal antibody raised against S. aureus PBP2A. Membrane proteins were purified from oxacillin-susceptible S. sciuri strains K1, K56, K11, K30, and K3W, from oxacillin-resistant S. sciuri strains K3, K8, K1M200, SS37, and K3 grown in the presence of 3 μg/ml oxacillin, and from S. aureus strains COL and COL-S. The protein products of S. aureus mecA (PBP2A) and S. sciuri pbpD (PBP4) are indicated on the left.

FIG. 3.

Determination of S. sciuri PBP affinity by competition assay. Membrane preparations (150 μg of proteins) purified from strains K1M200 and SS37 were incubated first with nafcillin (20 μg/ml) and then with a saturating concentration of [¹⁴C]benzylpenicillin (lanes 5 and 6). In parallel, the same preparations were incubated with [¹⁴C]benzylpenicillin without preincubation with nafcillin (lanes 3 and 4). PBP patterns of S. aureus strains COL (lane 1) and COL-S (lane 2) are also provided as a comparison. Protein standard size markers are indicated on the right.

FIG. 4.

Oxacillin susceptibility profiles of S. sciuri strains and S. aureus transductants carrying a plasmid-borne copy of the upregulated pbpD gene from S. sciuri strains K1M200 and SS37. (A) S. sciuri strains K1 (open squares and dashed lines), K1M200 (closed squares and solid lines), SS37 (closed triangles and solid lines), K3 (solid circles and solid lines), and K3W (open circles and dashed lines). (B) Oxacillin-susceptible S. aureus recipient COL-S (closed diamonds and dashed lines) and transductants of COL-S carrying plasmid-borne mecA from S. aureus strain COL (TD_COL; closed circles and solid lines); transductants of COL-S carrying plasmid-borne pbpD from S. sciuri strain K1M200 (TD_K1M200; closed squares and solid lines) and from S. sciuri strain SS37 (TD_SS37; closed triangles and solid lines); transductants TD_K1M200 (open squares) and TD_SS37 (open triangles) cured of the plasmid carrying pbpD.

FIG. 5.

Comparison of the muropeptide compositions of peptidoglycans purified from oxacillin-susceptible and -resistant S. sciuri strains. Peptidoglycans were purified from 1-liter cultures grown at 37°C with aeration to an OD of 0.4, digested with mutanolysin, and separated by RP-HPLC. Oxacillin-susceptible S. sciuri strains K1 (A) and K3W (C); oxacillin-resistant S. sciuri strains K1M200 (B), K3 (D), and SS37 (F); and (E) relative amounts of monomeric, dimeric, and highly cross-linked muropeptides are shown.

FIG. 6.

Comparison of the muropeptide compositions of peptidoglycans purified from S. sciuri and S. aureus strains grown in the presence of sub-MICs of oxacillin. Peptidoglycans were purified from 1-liter cultures grown at 37°C with aeration to an OD of 0.4, digested with mutanolysin, and separated by RP-HPLC. S. aureus strain COL grown in the absence (A) or in the presence (B) of 20 μg/ml oxacillin (oxa); S. sciuri strain K1M200 grown in the absence (C) or in the presence (D) of 20 μg/ml oxacillin; S. sciuri strain K3 grown in the absence (E) or in the presence (F) of 3 μg/ml oxacillin to induce the expression of S. aureus mecA are shown.