Role of class A penicillin-binding proteins in the expression of beta-lactam resistance in Enterococcus faecium

Abstract

Peptidoglycan is polymerized by monofunctional d,d-transpeptidases belonging to class B penicillin-binding proteins (PBPs) and monofunctional glycosyltransferases and by bifunctional enzymes that combine both activities (class A PBPs). Three genes encoding putative class A PBPs (pbpF, pbpZ, and ponA) were deleted from the chromosome of Enterococcus faecium D344R in all possible combinations in order to identify the glycosyltransferases that cooperate with low-affinity class B Pbp5 for synthesis of peptidoglycan in the presence of beta-lactam antibiotics. The viability of the triple mutant indicated that glycan strands can be polymerized independently from class A PBPs by an unknown glycosyltranferase. The susceptibility of the DeltapbpF DeltaponA mutant and triple mutants to extended spectrum cephalosporins (ceftriaxone and cefepime) identified either PbpF or PonA as essential partners of Pbp5 for peptidoglycan polymerization in the presence of the drugs. Mass spectrometry analysis of peptidoglycan structure showed that loss of PonA and PbpF activity led to a minor decrease in the extent of peptidoglycan cross-linking by the remaining PBPs without any detectable compensatory increase in the participation of the L,D-transpeptidase in peptidoglycan synthesis. Optical density measurements and electron microscopy analyses showed that the DeltapbpF DeltaponA mutant underwent increased stationary-phase autolysis compared to the parental strain. Unexpectedly, deletion of the class A pbp genes revealed dissociation between the expression of resistance to cephalosporins and penicillins, although the production of Pbp5 was required for resistance to both classes of drugs. Thus, susceptibility of Pbp5-mediated peptidoglycan cross-linking to different beta-lactam antibiotics differed as a function of its partner glycosyltransferase.

FIG. 1.

Southern hybridization of class A pbp genes in parent and mutant strains. BglII-digested genomic DNA of the E. faecium D344R and of the mutants carrying deletions of class A pbp genes in all possible combinations was analyzed by Southern blot hybridization using a mixture of internal fragments of pbpF, pbpZ, and ponA to generate the probe. Lane 1, D344R; lane 2, D344R (ΔpbpF); lane 3, D344R(ΔponA); lane 4, D344R (ΔpbpZ); lane 5, D344R (ΔpbpF ΔponA); lane 6, D344R (ΔpbpF ΔpbpZ); lane 7, D344R (ΔpbpZ ΔponA); lane 8, D344R (ΔpbpF ΔpbpZ ΔponA); lane 9, empty lane; lane 10, D344R.

FIG. 2.

Population analyses of the E. faecium D344R and of the mutants carrying deletions of class A pbp genes. Dilutions of exponentially growing cultures were plated on agar containing twofold increasing concentrations of ceftriaxone to enumerate CFU.

FIG. 3.

Induction of ceftriaxone resistance by penicillin in E. faecium D344R(ΔponA ΔpbpF ΔpbpZ). The disc diffusion assay shows blunting of the inhibition zone around the disk containing ceftriaxone (bottom) in the vicinity of the disk containing penicillin (top).

FIG. 4.

Analysis of peptidoglycan structure. Lactoyl-peptides from E. faecium D344R (A) and from the ΔpbpF ΔponA double mutant (B) were analyzed by rp-HPLC (mAU, absorbance unit [10³] at 210 nm). (E) The content of individual peaks was identified by mass spectrometry. The calculated and observed monoisotopic masses are indicated. The relative abundances were determined by integration of the absorbance at 210 nm. The structure of the peptidoglycan fragments labeled with asterisks were determined by tandem mass spectrometry. An rp-HPLC analysis was also performed for reduced disaccharide-peptides from D344R (C) and the ΔpbpF ΔponA mutants (D) that were characterized by mass spectrometry (F).

FIG. 5.

Transmission electron micrographs of stationary-phase cultures of E. faecium D344R (A) and of the D344R (ΔpbpF ΔponA) double-deletion mutant (B). The exponential phases of growth cultures of D344R (C) and of the mutant (D) were also analyzed.

FIG. 6.

OD₆₀₀ values for cell cultures of wild type, double mutant, and PbpF-complemented double mutant grown in BHI broth without ceftriaxone (kanamycin was used to maintain the plasmid in the complemented strain).