Structure of the low-affinity penicillin-binding protein 5 PBP5fm in wild-type and highly penicillin-resistant strains of Enterococcus faecium

Abstract

Among its penicillin-binding proteins (PBPs), Enterococcus faecium possesses a low-affinity PBP5, PBP5fm, which is the main target involved in ␤-lactam resistance. A 7.7-kb EcoRI chromosomal fragment of E. faecium D63r containing the pbp5fm gene was cloned and sequenced. Two open reading frames (ORFs) were found. A 2,037-bp ORF encoded the deduced 73.8-kDa PBP5fm, the amino acid sequences of which were, respectively, 99.8, 78.5, and 62% homologous to those of the low-affinity plasmid-encoded PBP3r of Enterococcus hirae S185r and the chromosome-encoded PBP5 of E. hirae R40 and Enterococcus faecalis 56R. A second 597-bp ORF, designated psrfm, was found 2.3 kb upstream of pbp5fm. It appeared to be 285 bp shorter than and 74% homologous with the regulatory gene psr of E. hirae ATCC 9790. Different clinical isolates of E. faecium, for which a wide range of benzylpenicillin MICs were observed, showed that the increases in MICs were related to two mechanisms. For some strains of intermediate resistance (MICs of 16 to 64 g/ml), the increased level of resistance could be explained by the presence of larger quantities of PBP5fm which had an affinity for benzylpenicillin (second-order rate constant of protein acylation [k ؉2 /K] values of 17 to 25 M ؊1 s ؊1) that remained unchanged. For the two most highly resistant strains, EFM-1 (MIC, 90 g/ml) and H80721 (MIC, 512 g/ml), the resistance was related to different amino acid substitutions yielding very-low-affinity PBP5fm) which were synthesized in small quantities. More specifically, it appeared, with a three-dimensional model of the C-terminal domain of PBP5fm, that the substitutions of Met-485, located in the third position after the conserved SDN triad, by Thr in EFM-1 and by Ala in H80721 were the most likely cause of the decreasing affinity of PBP5fm observed in these strains.The genus Enterococcus, closely related to the genus Streptococcus, is involved in different clinical infections (37). Apart from their physiological properties, enterococci also differ from streptococci in that they generally are naturally 10-to 1,000-fold less susceptible to penicillins than streptococci (37). It was demonstrated that the natural low susceptibility of enterococci to penicillin is linked to the presence of at least one high-molecular-mass penicillin-binding protein (PBP) which has a low affinity for ␤-lactams (1, 17, 50). Enterococcus faecium appears to be the enterococcal species most resistant to ␤-lactam antibiotics, for which there are a wide range of benzylpenicillin MICs (0.5 to Ն 64 g/ml) for clinical isolates (22,30,50). Recently it became obvious that a new population of clinical E. faecium isolates for which the MICs of benzylpenicillin were very high (256 to 512 g/ml) had emerged in different countries (15,22,23,30).Among laboratory mutants and clinical isolates of E. faecium, two mechanisms have been shown to be involved in the high-level resistance to benzylpenicillin. These strains produced either an increased quantity of the essential low-affinity PBP5fm (15, 17) o...

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

“…The second-order rate constant k 2 /K for the acylation of PBP2a from S. aureus by penicillin V is about 16 M -1 s -1 [44]. The corresponding values for PBP5fm from E. faecium strains vary from 15 to 24 M -1 s -1 and they are at least ten times lower for PBP5fm from highly resistant strains [12]. In comparison, a k 2 /K value of 58,000 M -1 s -1 has been reported for the acylation of PBP2x from a sensitive S. pneumoniae strain by benzylpenicillin [45].…”

“…Construction and expression of recombinant PBP5fm molecules DNA encoding PBP5fm was amplified by PCR using pDML517 [12] as template and primers designed to introduce NcoI and NdeI cloning sites, to replace the hydrophobic anchoring peptide and putative polar region by a Met-Gly peptide in positions 32 and 33 and to introduce a Leu-Glu dipeptide at the C terminal end of the protein.…”

“…The presence of such a guanidinium group in b-lactamases is favorable to acylation by b-lactams [40]. Acylation of PBP5fm is very slow (second-order rate constant k 2 /K = 20 M -1 s -1 ) [12]. It can proceed according to a two-step model where Lys425 abstracts a proton from the active-site serine hydroxyl group to promote the nucleophilic attack of the b-lactam carbonyl by the activated serine Og [39,41].…”

“…The mechanism involved in the resistance is the overproduction of the protein [12]. of some clinical isolates highly resistant to ampicillin, not by means of PBP5fm overproduction but by a reduction of the protein affinity for the antibiotic.…”

“…Enterococcus faecium is the most penicillin resistant enterococcal species, with MICs ranging from 0.5 to more than 64 mg/ml for clinical isolates. MICs as high as 256 and 512 mg/ml have been found in very resistant strains [11,12]. Some highly blactam resistant E. faecium strains are also vancomycin resistant [12 -14], although vancomycin and b-lactam antibiotics can act in a synergistic way against vancomycinresistant enterococci [15].…”