Role of Class A Penicillin-Binding Proteins in PBP5-Mediated β-Lactam Resistance inEnterococcus faecalis

Abstract

Peptidoglycan polymerization complexes contain multimodular penicillin-binding proteins (PBP) of classesA 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 ␤-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 ⌬ponA ⌬pbpF 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 ␤-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.

Synthesis of Mosaic Peptidoglycan Cross-bridges by Hybrid Peptidoglycan Assembly Pathways in Gram-positive Bacteria

“…Derivatives of expression vector pNJ2 harboring fem and pbp genes (see below) were introduced into E. faecalis JH2Sm::Tn916 (17) by electroporation and transferred by conjugation to E. faecalis JH2-2 (18), E. faecalis JH2-2⌬bppA2 (14), E. faecalis JH2-2⌬pbp5 (17), E. faecium D344S (19), and E. faecium BM4107 (20), as previously described (17). Spectinomycin (60 g/ml) was used in all experiments to counter select loss of the plasmids.…”

“…Plasmid Construction-The open reading frame and ribosome-binding site of the fem and mecA genes of S. aureus and of the pbp5 fm gene of E. faecium were amplified and cloned under the control of the aph-A-3p promoter of the shuttle expression vector pNJ2 (17). Briefly, the mecA and fem genes of S. aureus Mu50 (21) were amplified with the following primers that contained SacI or XbaI restriction sites (underlined): mecA, 5Ј-TTGAGCTCATATAAGGAGGATATTGATG-3Ј and 5Ј-TTTCTAGACGGATTGCTTCACTGTTTTG-3Ј; fmhB, 5Ј-TTGAGCT-CAGGTATTGTTAAATAGAAGG-3Ј and 5Ј-TTTCTAGAGAGCGTTCA-GATTTCAGTCG-3Ј; femA, 5Ј-TTGAGCTCATTAACGAGAGACAAATA-GG-3Ј and 5Ј-TTTCTAGACCTTCCTAAAAAATTCTGTC-3Ј; and femB, 5Ј-TTGAGCTCACAGAATTTTTTAGGAAGGG-3Ј and 5Ј-TTTCTAGAG-CCCTAACATCATTTACATC-3Ј.…”

“…The nucleotide sequence of the inserts in the resulting recombinant plasmids was determined. The construction of the pNJ2 derivative for expression of the pbp5 fs gene of E. faecalis JH2-2 has been previously described (17).…”

“…mecA, pbp5 fs , and pbp5 fm were cloned into the expression vector pNJ2 and introduced into E. faecalis JH2-2⌬pbp5 and E. faecium D344S. The latter hosts are previously characterized mutants susceptible to ␤-lactams because of deletion of their respective species specific pbp5 genes (17,19). Ceftriaxone, a third generation cephalosporin, was used to monitor expression of ␤-lactam resistance, because deletion of the pbp5 genes produced large decreases (Ͼ1000-fold) in the minimal inhibitory concentration of this antibiotic both in E. faecium and E. faecalis.…”

History, Virulence Genes, Identification and Antimicrobial Resistance of Enterococcus faecalis Isolated from Diabetic Foot Patients

“…faecalis chromosome does not comprise any extra glycosyl transferase-related genes, these comments designate that glycan chain polymerization in the triple mutant is did by a novel type of glycosyl transferase. The last enzyme was not reserved by moenomycin, subsequently deletion of the three classes A PBP genes led to high-level resistance to this glycosyl transferase inhibitor (Arbeloa et al, 2004). Enterococci have an intrinsic low vulnerability or resistance to β-lactams.…”