Role of Class A Penicillin-Binding Proteins in the Expression of β-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 ␤-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 ⌬pbpF ⌬ponA 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 ⌬pbpF ⌬ponA 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 ␤-lactam antibiotics differed as a function of its partner glycosyltransferase.

Ceftaroline Restores Daptomycin Activity against Daptomycin-Nonsusceptible Vancomycin-Resistant Enterococcus faecium

“…Previous work has shown that daptomycin resistance in Enterococcus faecium and Enterococcus faecalis may be accompanied by decreases in membrane fluidity (6,15). Interestingly, our previous characterization of these 2 strains showed no changes in this property between the strains under standard growth conditions (6).…”

“…These phenotypes are part of other studies in our lab on the effects of beta-lactams on bacterial autolysins. We are currently investigating the differential effect of class A penicillin binding protein (PBP) inactivation on susceptibility to daptomycin and antimicrobial peptides and whether ceftaroline and/or ampicillin exert their effects on VRE through these targets (15). Additional evidence by others suggests that low-molecular-weight PBPs, such as ddcP, which influences D-alanine-D-alanine carboxypeptidase activity (16), warrant study as well, particularly with the possible tangential effects on vancomycin MIC by ceftaroline.…”

“…Daptomycin-nonsusceptible enterococci are different from their susceptible counterparts in sensing cell wall stress (via liaFSR) (15,16,18), phospholipid composition (via cls) (20)(21)(22), and increased proclivity to septation (via ezrA) (6). While we did not perform analysis of phospholipid content with ceftaroline therapy, we noticed changes in septation and cell wall thickness induced by ceftaroline.…”

Current Trends of Emergence and Spread of Vancomycin-Resistant Enterococci

“…Penicillin resistance in E. faecium is mediated via point mutations in the housekeeping pbp5 gene leading to reduced penicillin binding to the expressed protein (Jureen et al, 2004;Rice et al, 2004;Rice et al, 2009). Mutated pbp5' was also found as an integral part of conjugative transposons, like Tn5382, thus encoding transferable ampicillin and VanB-type vancomycin resistance (Carias et al, www.intechopen.com 1998; Valdezate et al, 2009).…”

Monofunctional Transglycosylases Are Not Essential for Staphylococcus aureus Cell Wall Synthesis

“…Surprisingly, in some cases, such as the Gram-positive organisms Bacillus subtilis (21), Enterococcus faecalis (1), and Enterococcus faecium (28), it has been shown that strains lacking all class A PBPs are viable and have only slight differences in peptidoglycan composition. In contrast, in Streptococcus pneumoniae, the deletion of class A PBP1a and PBP2a was lethal (13), and in S. aureus, the only class A PBP, PBP2, is essential for viability in methicillin-susceptible strains and in methicillin-resistant strains exposed to ␤-lactam antibiotics (24).…”