Role of class A penicillin-binding proteins in PBP5-mediated beta-lactam resistance in Enterococcus faecalis

Abstract

Peptidoglycan polymerization complexes contain multimodular penicillin-binding proteins (PBP) of classes A and B that associate a conserved C-terminal transpeptidase module to an N-terminal glycosyltransferase or morphogenesis module, respectively. In Enterococcus faecalis, class B PBP5 mediates intrinsic resistance to the cephalosporin class of beta-lactam antibiotics, such as ceftriaxone. To identify the glycosyltransferase partner(s) of PBP5, combinations of deletions were introduced in all three class A PBP genes of E. faecalis JH2-2 (ponA, pbpF, and pbpZ). Among mutants with single or double deletions, only JH2-2 DeltaponA DeltapbpF was susceptible to ceftriaxone. Ceftriaxone resistance was restored by heterologous expression of pbpF from Enterococcus faecium but not by mgt encoding the monofunctional glycosyltransferase of Staphylococcus aureus. Thus, PBP5 partners essential for peptidoglycan polymerization in the presence of beta-lactams formed a subset of the class A PBPs of E. faecalis, and heterospecific complementation was observed with an ortholog from E. faecium. Site-directed mutagenesis of pbpF confirmed that the catalytic serine residue of the transpeptidase module was not required for resistance. None of the three class A PBP genes was essential for viability, although deletion of the three genes led to an increase in the generation time and to a decrease in peptidoglycan cross-linking. As the E. faecalis chromosome does not contain any additional glycosyltransferase-related genes, these observations indicate that glycan chain polymerization in the triple mutant is performed by a novel type of glycosyltransferase. The latter enzyme was not inhibited by moenomycin, since deletion of the three class A PBP genes led to high-level resistance to this glycosyltransferase inhibitor.

FIG. 1.

Schematic representation of vectors and approach used to generate chromosomal deletions. The maps of suicide vector pHS1 (A) and shuttle vector pNJ2 (B) show unique restriction sites used for cloning. The plasmids confer resistance to gentamicin (gent^R) and spectinomycin (spc^R), respectively. (C) Replacement of the pbp genes by the erm erythromycin resistance cassette was generated by a double crossover, as indicated by broken arrows. (D) The erm cassette was removed from the chromosome in two steps. In the first step, integration of plasmid pHS1ΩH1-H2 by a single crossover involving H1 (as represented) or H2 was selected at 42°C on agar containing gentamicin. Integration generated a partial duplication of the locus, since the sequence of the pHS1 vector was flanked by the H1-H2 and H1-erm-H2 alleles. Serial subcultures at the permissive (28°C) and nonpermissive (42°C) temperatures in the absence of antibiotic were used to stimulate the excision and loss of pHS1ΩH1-erm-H2, leaving the H1-H2 allele in the chromosome.

FIG. 2.

PBP profiles of single, double, and triple PBP mutants. Lanes: 1 and 10, JH2-2; 2, JH2-2 ΔponA; 3, JH2-2 ΔpbpF; 4, JH2-2 ΔpbpZ; 5, JH2-2 ΔpbpF ΔpbpZ; 6, JH2-2 ΔponA ΔpbpF; 7, JH2-2 ΔponA ΔpbpZ; 8, JH2-2 ΔponA ΔpbpF ΔpbpZ; 9, JH2-2 Δpbp5; 11, JH2-2/pNJ2Ωpbp5_fs.

FIG. 3.

Structure of E. faecalis muropeptide monomers and dimers. The most abundant muropeptides (e.g., muropeptides 1 and 3) contained two d-Ala residues at the free C-terminal end and two l-Ala residues both at the free N-terminal end and in the cross bridge (boxed). Less-abundant muropeptides (e.g., 9 and 11) contained a tripeptide stem lacking the two C-terminal d-Ala residues. The orientations of the CO→NH peptide bonds are indicated by arrows.