Role of class A penicillin-binding proteins in PBP5-mediated beta-lactam resistance in Enterococcus faecalis.

Peptidoglycan polymerization complexes contain multimodular penicillin-binding proteins (PBP) of classes A and B that associate a conserved C-terminal transpeptidase module to an N-terminal glycosyltransferase or morphogenesis module, respectively. In Enterococcus faecalis, class B PBP5 mediates intrinsic resistance to the cephalosporin class of beta-lactam antibiotics, such as ceftriaxone. To identify the glycosyltransferase partner(s) of PBP5, combinations of deletions were introduced in all three class A PBP genes of E. faecalis JH2-2 (ponA, pbpF, and pbpZ). Among mutants with single or double deletions, only JH2-2 DeltaponA DeltapbpF was susceptible to ceftriaxone. Ceftriaxone resistance was restored by heterologous expression of pbpF from Enterococcus faecium but not by mgt encoding the monofunctional glycosyltransferase of Staphylococcus aureus. Thus, PBP5 partners essential for peptidoglycan polymerization in the presence of beta-lactams formed a subset of the class A PBPs of E. faecalis, and heterospecific complementation was observed with an ortholog from E. faecium. Site-directed mutagenesis of pbpF confirmed that the catalytic serine residue of the transpeptidase module was not required for resistance. None of the three class A PBP genes was essential for viability, although deletion of the three genes led to an increase in the generation time and to a decrease in peptidoglycan cross-linking. As the E. faecalis chromosome does not contain any additional glycosyltransferase-related genes, these observations indicate that glycan chain polymerization in the triple mutant is performed by a novel type of glycosyltransferase. The latter enzyme was not inhibited by moenomycin, since deletion of the three class A PBP genes led to high-level resistance to this glycosyltransferase inhibitor.

Comparative analysis of Pseudomonas aeruginosa penicillin-binding protein 7 in the context of its membership in the family of low-molecular-mass PBPs.

The Pseudomonas aeruginosa pbpG gene encoding penicillin-binding protein 7, a homologue of the Escherichia coli gene encoding a DD-endopeptidase, was cloned and sequenced, pbpG was located immediately downstream of the phenylalanine hydroxylase (phh) operon. DNA sequencing revealed an open reading frame of 936 bp (starting with a GTG codon) which encodes a protein of 34,115 Da. N-terminal amino acid sequencing confirmed the presence of a cleavable N-terminal signal peptide of 23 amino acids. Verification that the protein is a penicillin-binding protein was directly demonstrated by labelling with 125I-labelled penicillin X. Inactivation of P. aeruginosa pbpG by interposon mutagenesis resulted in no obvious phenotypic changes, but when P. aeruginosa PbpG was overexpressed in E. coli using a T7 expression system, cell lysis resulted. P. aeruginosa PbpG resembled E. coli PbpG in being associated with the membrane fraction. Two additional members of the PbpG subfamily were identified in the database. P. aeruginosa PbpG shows 63% identity with E. coli penicillin-binding protein 7 (PbpG) and 60% identity with Vibrio cholerae PbpG, but only 23% identity with Haemophilus influenzae PbpG. The PbpG subfamily and three other subfamilies constituting the low-molecular-mass PBP protein family were analysed by multiple alignment of 26 sequences. PbpG exhibited the consensus motifs of other penicillin-binding proteins. Ten anchor residues were identified that are conserved at the family level within the superfamily of serine-active-site penicillin-interacting proteins.

Degradation of penicillin-binding protein 2' in methicillin-resistant Staphylococcus aureus.

We detected a novel protein with [14C]benzylpenicillin (PCG)-binding capacity and a molecular mass of about 60 kD in methicillin-resistant Staphylococcus aureus using a high concentration of [14C]PCG and extending the reaction time. However, the fluorogram showed that the band density of penicillin-binding protein 2' (PBP2') decreased gradually with incubation time. The appearance of the 60-kD protein and the reduction of the band density of PBP2' were stoichiometrically linked, and the binding profiles of beta-lactams for PBP2' and the 60-kD protein corresponded. These results suggested that the 60-kD protein is a degradation product of PBP2'.

Effects of beta-lactamase-mediated antimicrobial resistance: the role of beta-lactamase inhibitors.

Production of beta-lactamase is the most common mechanism of bacterial resistance to beta-lactam antibiotics. Virtually all bacteria have the capability of synthesizing the enzyme. Microorganisms may already possess the native genetic information necessary for beta-lactamase production (i.e., chromosomal), or may acquire the capacity by transfer of DNA from another organism (i.e., plasmid-mediated). The level of beta-lactamase production may be stable and noninducible (constitutive enzyme production), or may be stimulated on exposure to selected beta-lactam antibiotics (inducible enzyme production). Inhibitors such as clavulanic acid and sulbactam prevent antibiotic degradation by the beta-lactamases of many clinically significant pathogens. Therefore, currently available beta-lactam-beta-lactamase-inhibitor combinations exhibit broad spectra of in vitro activity. Ticarcillin-clavulanate possesses clinically significant activity against many bacteria, including streptococci, Staphylococcus aureus, Bacteroides fragilis, and numerous Enterobacteriaceae. Amoxicillin-clavulanate and ampicillin-sulbactam demonstrate clinically significant activity against streptococci (including enterococci), S. aureus, B. fragilis, and some Enterobacteriaceae. Ticarcillin-clavulanate is indicated for treatment of serious infections, including septicemia. Amoxicillin-clavulanate is useful in the treatment of upper respiratory, urinary tract, and skin and soft tissue infections. Ampicillin-sulbactam may be used for treatment of intraabdominal, gynecologic, urinary tract, and skin and soft tissue infections.

Penicillin-binding proteins of penicillin-susceptible and -resistant pneumococci: immunological relatedness of altered proteins and changes in peptides carrying the beta-lactam binding site.

There are several major differences between the penicillin-binding proteins (PBPs) of highly penicillin-resistant and -susceptible strains of pneumococci. The highest-molecular-size PBP 1a (98 kilodaltons [kDa]) of susceptible pneumococci is not detectable in resistant bacteria. Instead, resistant strains contain a PBP of smaller size: 92 and 94 kDa in South African strains 8249 and A95210, respectively, and 96 kDa in New Guinea strain 2955 (S. Zighelboim and A. Tomasz, Antimicrob. Agents Chemother. 17:434-442, 1980). Using antibodies prepared against PBP 1a of penicillin-susceptible pneumococci, we demonstrated that these anomalous-sized proteins in the resistant strains are immunologically related to PBP 1a of penicillin-susceptible bacteria. A second difference between the PBP patterns of strain 8249 and the susceptible pneumococci is that the 78-kDa PBP 2b is not detectable by the radioactive penicillin binding assay in the resistant strain. Using antibodies prepared against PBP 2b of susceptible cells, we demonstrated the presence of PBP 2b in membrane preparations from strain 8249 cells. Thus, the poor detection of this PBP appears to be related to its greatly decreased affinity for the antibiotic molecule. We also compared the patterns of penicillin-labeled peptides derived from PBPs of resistant and susceptible cells during partial proteolysis by V8 protease. Several changes were observable in small peptides carrying the beta-lactam binding site generated from the high Mr (PBP 1a-related) binding proteins. In contrast, no differences in the pattern of penicillin-labeled peptides were seen when the pattern of PBP 2a of susceptible pneumococci was compared with the peptide pattern of PBP 2a from resistant strains. One of the resistant isolates (strain 2955) also had a PBP 3 with a higher-than-normal molecular weight. This protein gave strong positive reaction with antibodies against PBP 3 of susceptible cells. Examination of the pattern of penicilloyl peptides generated from the susceptible and resistant PBP 3s during partial proteolysis revealed only differences which seem to reside distant from the beta-lactam binding site.