Role of Penicillin-Binding Protein 5 in Expression of Ampicillin Resistance and Peptidoglycan Structure in Enterococcus faecium

Abstract

The contribution of penicillin-binding protein 5 (PBP 5) to intrinsic and acquired ␤-lactam resistance was investigated by constructing isogenic strains of Enterococcus faecium producing different PBP 5. The pbp5 genes from three E. faecium clinical isolates (BM4107, D344, and H80721) were cloned into the shuttle vector pAT392 and introduced into E. faecium D344S, a spontaneous derivative of E. faecium D344 highly susceptible to ampicillin due to deletion of pbp5 (MIC, 0.03 g/ml). Immunodetection of PBP5 indicated that cloning of the pbp5 genes into pAT392 resulted in moderate overproduction of PBP 5 in comparison to wild-type strains. This difference may be attributed to a difference in gene copy number. Expression of the pbp5 genes from BM4107 (MIC, 2 g/ml), D344 (MIC, 24 g/ml), and H80721 (MIC, 512 g/ml) in D344S conferred relatively low levels of resistance to ampicillin (MICs, 6, 12, and 20 g/ml, respectively). A methionine-to-alanine substitution was introduced at position 485 of the BM4107 PBP 5 by site-directed mutagenesis. In contrast to previous hypotheses based on comparison of nonisogenic strains, this substitution resulted in only a 2.5-fold increase in the ampicillin MIC. The reversed-phase high-performance liquid chromatography muropeptide profiles of D344 and D344S were similar, indicating that deletion of pbp5 was not associated with a detectable defect in cell wall synthesis. These results indicate that pbp5 is a nonessential gene responsible for intrinsic resistance to moderate levels of ampicillin and by itself cannot confer high-level resistance.Enterococcus faecium is intrinsically resistant to moderate levels of ampicillin by production of the low-affinity penicillinbinding protein 5 (PBP 5). Bacterial growth occurs at ␤-lactam concentrations sufficient to inactivate all the other PBPs, suggesting that PBP5 is the only transpeptidase required for peptidoglycan synthesis under such conditions (4,20). Acquired resistance to higher levels of ampicillin in clinical isolates of E. faecium has been associated with increased production of PBP 5 or decreased affinity for the ␤-lactam antibiotics (7,8,10,13,18,20,21). The latter mechanism was inferred from comparison of the pbp5 genes from clinical isolates in which amino acid substitutions at specific positions near or in the conserved motifs of the PBP module were associated with decreased interaction with ␤-lactams and expression of resistance (8,13,18,21). However, the role of the PBP 5 in the level of resistance was not rigorously established since isogenic strains were not constructed. In the present study, pbp5 genes from E. faecium clinical isolates expressing various levels of ampicillin resistance were cloned into a shuttle vector and introduced into E. faecium D344S, a spontaneous mutant in which the chromosomal pbp5 locus is deleted. Expression of the different pbp5 genes in this host resulted in similar low levels of ampicillin resistance, indicating that alterations of PBP 5 alone do not account for acquired high-level ␤-lactam resist...

Inactivation Kinetics of a New Target of β-Lactam Antibiotics

“…Only two of the seven steps led to substitutions in Ldt fm , indicating that modifications of other unknown factors are involved in acquisition of resistance. For the DD-transpeptidases, emergence of ␤-lactam resistance also involves a combination of multiple modifications affecting non-penicillin-binding protein factors, several residues of a given penicillin-binding protein, and sometimes several penicillin-binding proteins (11)(12)(13)(14)(15)(16). The requirement for multiple mutations accounts for the relative robustness of ␤-lactams with respect to the emergence of resistance by target modification.…”

Balance between Two Transpeptidation Mechanisms Determines the Expression of β-Lactam Resistance in Enterococcus faecium

“…Bacterial Strains and Growth Conditions-Parental strain E. faecium D344S is highly susceptible to ampicillin and derives from E. faecium D344 (10) by a spontaneous deletion of pbp5 encoding low-affinity PBP5 (22). E. faecium M1, M2, M3, M4, and M512 are spontaneous mutants of D344S obtained by five successive selection steps on brain heart infusion (Difco) agar containing increasing concentrations of ampicillin as follows.…”

The 2.4-Å crystal structure of the penicillin-resistant penicillin-binding protein PBP5fm from Enterococcus faecium in complex with benzylpenicillin

“…Point mutations in the PBP5fm further reduce the affinity of the protein for b-lactams, leading to very high levels of resistance [13,17]. PBP5fm is thought to be able to take over the role of the other HMMPBPs in peptidoglycan synthesis when these proteins are inhibited by an antibiotic [10,11,18,19]. In E. faecalis, the peptidoglycan synthesized by PBP5 in the presence of benzylpenicillin is slightly different from that produced by the usual PBPs in the absence of benzylpenicillin.…”