Jacques Coyette

Analysis of the gene cluster involved in production and immunity of the peptide antibiotic AS‐48 in Enterococcus faecalis

A region of 7.8 kb of the plasmid pMB2 from Enterococcus faecalis S‐48 carrying the information necessary for production and immunity of the peptide antibiotic AS‐48 has been cloned and sequenced. It contains the as‐48A structural gene plus five open reading frames (as‐48B, as‐48C, as‐48C1, as‐48D and as‐48D1 ). Besides As‐48D, all the predicted gene products are basic hydrophobic proteins with potential membrane‐spanning domains (MSDs). None of them shows any homology with protein sequences stored in databanks, except for As‐48D, which shows similarity to the C‐terminal domain of ABC transporters and contains a highly conserved ATP‐binding site. The gene products of as‐48B, as‐48C, as‐48C1 and as‐48D are thought to be involved in AS‐48 production and secretion. The only gene able to provide resistance to AS‐48 by itself is as‐48D1. Immunity also seems to be enhanced at least by the products of as‐48B, as‐48C1 and as‐48D genes. Transcription analysis using probes derived from the different ORFs revealed two large (3.5 and 2.7 kb) mRNAs, suggesting that the different genes are organized in two constitutive operons.

The cyclic structure of the enterococcal peptide antibiotic AS‐48

The complete primary structure of the peptide antibiotic AS‐48 produced by Enterococcus faecalis has been determined by chemical degradation analysis. The cyclic nature of this 70 residues containing peptide was demonstrated by plasma desorption mass analysis of the generated peptides and electrospray ionisation mass analysis of the native polypeptide. As far as we know, this is the first example of an antibiotic protein cyclised by a tail—head peptide bond formation and not by branching of the polypeptide side chains.

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

Abstract: Penicillin-binding proteins (PBPs) are membrane proteins involved in the final stages of peptidoglycan synthesis and represent the targets of β-lactam antibiotics. Enterococci are naturally resistant to these antibiotics because they produce a PBP, named PBP5fm in Enterococcus faecium, with low-level affinity for β-lactams. We report here the crystal structure of the acyl-enzyme complex of PBP5fm with benzylpenicillin at a resolution of 2.4 Å. A characteristic of the active site, which distinguishes PBP5fm from other PBPs of known structure, is the topology of the loop 451–465 defining the left edge of the cavity. The residue Arg464, involved in a salt bridge with the residue Asp481, confers a greater rigidity to the PBP5fm active site. In addition, the presence of the Val465 residue, which points into the active site, reducing its accessibility, could account for the low affinity of PBP5fm for β-lactam. This loop is common to PBPs of low affinity, such as PBP2a from Staphylococcus aureus and PBP3 from Bacillus subtilis. Moreover, the insertion of a serine after residue 466 in the most resistant strains underlines even more the determining role of this loop in the recognition of the substrates.

Resistant penicillin-binding proteins

Abstract. Low-affinity penicillin-binding proteins (PBPs), which participate in the β-lactam resistance of several pathogenic bacteria, have different origins. Natural transformation and recombination events with DNA acquired from neighbouring intrinsically resistant organisms are responsible for the appearance of mosaic genes encoding two or three low-affinity PBPs in highly resistant strains of transformable microorganisms such as Neisseria and Streptococcus pneumoniae. Methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococcal strains possess the mecA determinant gene, which probably evolved within the Staphylococcus genus from a closely related and physiologically functional gene that was modified by point mutations. The expression of mecA is either inducible or constitutive. A stable high-level resistant phenotype requires the synthesis of a normally constituted peptidoglycan. Enterococci have a natural low susceptibility to β-lactams related to the presence of an intrinsic low-affinity PBP. Highly resistant enterococcal strains overexpress this PBP and/or reduce its affinity.

Serine-Type D-Ala-D-Ala Peptidases and Penicillin-Binding Proteins

Publisher Summary This chapter describes serine-type D-Ala-D-Ala peptidases and penicillin-binding proteins. Penicillin is a suicide substrate. Because of the endocyclic nature of the scissile β-lactam amide bond, the leaving group of the enzyme acylation step remains part of the acyl enzyme. The first part only of the transfer cycle is achieved, leading to a long-lived; serine ester-linked acyl(penicilloyl)-enzyme and the enzyme behaves as a penicillin-binding protein (PBP). All bacteria possess an assortment of low and high molecular weight membrane-bound PBPs. The low molecular weight PBPs are single catalytic entities. The bulk of the protein is on the outer face of the plasma membrane and bears a carboxy-terminal extension, the end of which serves as membrane anchor. The low molecular weight PBPs helps to control the extent of wall peptidoglycan cross-linking throughout the life cycle of the cells. The high molecular weight PBPs involved in wall peptidoglycan assembly and cell morphogenesis are multimodule proteins. The bulk of the protein is on the outer face of the membrane and consists of an N-terminal module, fused to a C-terminal, penicillin-binding module.

The penicillin resistance of Enterococcus faecalis JH2-2r results from an overproduction of the low-affinity penicillin-binding protein PBP4 and does not involve a psr-like gene

A penicillin-resistant mutant, JH2-2r (MIC 75 microg ml(-1)), was isolated from Enterococcus faecalis JH2-2 (MIC 5 microg ml(-1)) by successive passages on plates containing increasing concentrations of benzylpenicillin. A comparison of the penicillin-binding protein (PBP) profiles in the two strains revealed a more intensely labelled PBP4 in JH2-2r. Because the sequences of the JH2-2 and JH2-2r pbp4 genes were strictly identical, even in their promoter regions, this intensive labelling could only be associated with an overproduction of the low-affinity PBP4. No psr gene analogous to that proposed to act as a regulator of PBP5 synthesis in Enterococcus hirae and Enterococcus faecium could be identified in the vicinity of pbp4 in E. faecalis JH2-2 and JH2-2r. However, a psr-like gene distant from pbp4 was identified. The cloning and sequencing of that psr-like gene from both E. faecalis strains indicated that they were identical. It is therefore postulated that the PBP4 overproduction in E. faecalis JH2-2r results from the modification of an as yet unidentified factor.

Interaction of Ceftobiprole with the Low-Affinity PBP 5 of Enterococcus faecium

ABSTRACT Ceftobiprole is a new cephalosporin that exhibits a high level of affinity for methicillin-resistant Staphylococcus aureus PBP 2a. It was reported that ceftobiprole did not interact with a mutated form of the low-affinity protein Enterococcus faecium PBP 5 (PBP 5fm) that, when overexpressed, confers a β-lactam resistance phenotype to the bacterium. Our results show that ceftobiprole binds to unmutated PBP 5fm to form a stable acyl-enzyme and that ceftobiprole is able to efficiently kill a penicillin-resistant Enterococcus faecium strain that produces this protein.

Activity of Ceftaroline against Enterococcus faecium PBP5

ABSTRACT The opportunistic human pathogen Enterococcus faecium overproduces the low-affinity PBP5. In clinical strains, mutations in PBP5 further reduce its acylation rate by β-lactams. Previous studies have reported that ceftaroline had poor inhibitory activity against β-lactam-resistant E. faecium strains. In this study, we show that ceftaroline exhibits killing activity against our laboratory-derived ampicillin-resistant E. faecium mutant that overproduces a wild-type PBP5 and that ceftaroline inactivates PBP5 much faster than benzylpenicillin and faster than ceftobiprole.

All Detectable High-Molecular-Mass Penicillin-Binding Proteins Are Modified in a High-Level β-Lactam-Resistant Clinical Isolate of Streptococcus mitis

ABSTRACT All detectable high-molecular-mass penicillin-binding proteins (HMM PBPs) are altered in a clinical isolate of Streptococcus mitis for which the β-lactam MICs are increased from those previously reported in our region (cefotaxime MIC, 64 μg/ml). These proteins were hardly detected at concentrations that saturate all PBPs in clinical isolates and showed, after densitometric analysis, 50-fold-lower radiotracer binding. Resistance was related to mosaic structure in all HMM PBP-coding genes, where critical region replacement was complemented not only by substitutions already reported for the closely related Streptococcus pneumoniae but also by other specific replacements that are presumably close to the active-site serine. Mosaic structure was also presumed in apbp1a-sensitive strain used for comparison, confirming that these structures do not unambiguously imply, by themselves, detectable critical changes in the kinetic properties of these proteins.

Redefining the role of psr in beta-lactam resistance and cell autolysis of Enterococcus hirae.

The contribution of penicillin-binding protein 5 (PBP5) and the PBP5 synthesis repressor (Psr) to the beta-lactam resistance, growth, and cell autolysis of wild-type strain ATCC 9790 and resistant strain R40 of Enterococcus hirae was investigated by disruption or substitution of the corresponding pbp5 and psr genes by Campbell-type recombination. The resulting modifications were confirmed by hybridization and PCR. The low susceptibility of E. hirae to beta-lactams was confirmed to be largely dependent on the presence of PBP5. However, against all expectations, inactivation of psr in ATCC 9790 or complementation of R40 cells with psr did not modify the susceptibility to benzylpenicillin or the growth and cell autolysis rates. These results indicated that the psr gene does not seem to be involved in the regulation of PBP5 synthesis and consequently in beta-lactam resistance or in the regulation of cell autolysis in E. hirae.