MurAA is required for intrinsic cephalosporin resistance of Enterococcus faecalis

Abstract

Enterococcus faecalis is a low-GC Gram-positive bacterium that is intrinsically resistant to cephalosporins, antibiotics that target cell wall biosynthesis. To probe the mechanistic basis for intrinsic resistance, a library of transposon mutants was screened to identify E. faecalis strains that are highly susceptible to ceftriaxone, revealing a transposon mutant with a disruption in murAA. murAA is predicted to encode a UDP-N-acetylglucosamine 1-carboxyvinyl transferase that catalyzes the first committed step in peptidoglycan synthesis: phosphoenolpyruvate (PEP)-dependent conversion of UDP-N-acetylglucosamine to UDP-N-acetylglucosamine-enolpyruvate. In-frame deletion of murAA, but not its homolog in the E. faecalis genome (murAB), led to increased susceptibility of E. faecalis to cephalosporins. Furthermore, expression of murAA enhanced cephalosporin resistance in an E. faecalis mutant lacking IreK (formerly PrkC), a key kinase required for cephalosporin resistance. Further genetic analysis revealed that MurAA catalytic activity is necessary but not sufficient for this role. Collectively, our data indicate that MurAA and MurAB have distinct roles in E. faecalis physiology and suggest that MurAA possesses a unique property or activity that enables it to enhance intrinsic resistance of E. faecalis to cephalosporins.

Fig 1

Time-kill analysis reveals that fosfomycin and ceftriaxone act synergistically. An overnight culture of WT (OG1RF) was diluted to an OD₆₀₀ of 0.004 and grown in MHB at 37°C with shaking until OD₆₀₀ reached 0.13 to 0.14. Three aliquots were removed and treated with a corresponding antibiotic. Samples were removed at intervals, and viable bacteria were enumerated on MH agar. Antibiotics and concentrations used: 32 μg/ml fosfomycin (squares); 512 μg/ml ceftriaxone (triangles); combination of 32 μg/ml fosfomycin and 512 μg/ml ceftriaxone (circles). Data represent the geometric means ± standard errors from independent experiments.

Fig 2

Cell wall integrity is compromised in the ΔmurAA2 mutant. Bacteria from exponentially growing cultures of plasmid-bearing WT (OG1RF), ΔmurAA2 (DV75-2), and ΔmurAB2 (DV1-4) E. faecalis were collected and split. Aliquots were pretreated with or without lysozyme, as indicated, prior to addition of Laemmli buffer containing 2% SDS. Samples were subjected to SDS-PAGE, and total protein stain was used to assess the extent of lysis. Vector, pDL278p23; P-murAA, pDV27-4.

Fig 3

MurAA is present in the ΔireK mutant. Cultures of WT (CK153) and ΔireK (CK161) mutant with chromosomally borne Strep-tagged murAA alleles were grown in BHI. Whole-cell lysates were prepared and subjected to immunoblot analysis. Membranes were probed with IreK (α-IreK), Strep tag (α-strep), and sigma-70 (α-σ⁷⁰) antibodies.

Fig 4

Purified E. faecalis MurAA, but not MurAA C120S, is catalytically active. His₆-tagged E. faecalis MurAA and MurAA C120S were purified from E. coli BL21(DE3) cells and assayed for UDP-N-acetylglucosamine 1-carboxyvinyl transferase activity. One microgram of freshly purified MurAA (dashed line) was used in the assay. No activity was observed with either 1 μg (full line) or 5 μg (spotted line) of freshly purified MurAA C120S.

Fig 5

Both E. faecalis MurAA and E. coli MurA are catalytically active in E. faecalis lysates. (A) Assay for UDP-N-acetylglucosamine 1-carboxyvinyl transferase activity. Cultures of plasmid-bearing WT (OG1RF) and ΔireK (CK119) E. faecalis strains were grown in MHB supplemented with spectinomycin (100 μg/ml). Whole-cell lysates were assayed for UDP-N-acetylglucosamine 1-carboxyvinyl transferase activity. Data represent the means ± standard errors. Activity was not detected in lysates expressing murAA C120S. (B) Immunoblot analysis. Aliquots were removed from each enzyme reaction and subjected to immunoblot analysis with anti-Strep tag (α-strep; for epitope-tagged MurAA or EcMurA) and anti-sigma-70 (α-σ⁷⁰; loading control) antibodies. Lanes 1 and 4, strains carrying plasmid that expresses E. faecalis MurAA (pDV27-4); lanes 2 and 5, strains carrying plasmid that expresses MurAA C120S (pDV15-1); lanes 3 and 6, strains carrying plasmid that expresses the E. coli MurA (pJMK4).