Peptidoglycan biosynthesis-associated enzymatic kinetic characteristics and ß-lactam antibiotic inhibitory effects of different Streptococcus pneumoniae penicillin-binding proteins.

Penicillin-binding proteins (PBPs) include transpeptidases, carboxypeptidases, and endopeptidases for biosynthesis of peptidoglycans in the cell wall to maintain bacterial morphology and survival in the environment. Streptococcus pneumoniae expresses six PBPs, but their enzymatic kinetic characteristics and inhibitory effects on different ß-lactam antibiotics remain poorly understood. In this study, all the six recombinant PBPs of S. pneumoniae displayed transpeptidase activity with different substrate affinities (Km = 1.56-9.11 mM) in a concentration-dependent manner, and rPBP3 showed a greater catalytic efficiency (Kcat = 2.38 s-1) than the other rPBPs (Kcat = 3.20-7.49 × 10-2 s-1). However, only rPBP3 was identified as a carboxypeptidase (Km = 8.57 mM and Kcat = 2.57 s-1). None of the rPBPs exhibited endopeptidase activity. Penicillin and cefotaxime inhibited the transpeptidase and carboxypeptidase activity of all the rPBPs but imipenem did not inhibited the enzymatic activities of rPBP3. Except for the lack of binding of imipenem to rPBP3, penicillin, cefotaxime, and imipenem bound to all the other rPBPs (KD = 3.71-9.35 × 10-4 M). Sublethal concentrations of penicillin, cefotaxime, and imipenem induced a decrease of pneumococcal pbps-mRNA levels (p < 0.05). These results indicated that all six PBPs of S. pneumoniae are transpeptidases, while only PBP3 is a carboxypeptidase. Imipenem has no inhibitory effect on pneumococcal PBP3. The pneumococcal genes for encoding endopeptidases remain to be determined.

A simple screen for murein transglycosylase inhibitors.

A simple assay for detection of compounds that bind to the active site in the transglycosylation domain of the essential bifunctional transglycosylase and transpeptidase penicillin-binding proteins (PBPs) is reported. The method is based on a competition with the specific transglycosylase inhibitor moenomycin. With moenomycin coupled to Affi-Gel beads, a simple filtration procedure allows the amount of labeled PBPs that bind to moenomycin beads in the presence of test substances to be determined. The PBPs can easily be labeled by the covalent binding of penicillin derivatives. Crude membrane extracts can be used as a source for the PBPs, and different kinds of labels for the penicillin-PBP complexes can be used. The assay can be adapted to high-throughput screens.

Peptidyl transferase antibiotics perturb the relative positioning of the 3'-terminal adenosine of P/P'-site-bound tRNA and 23S rRNA in the ribosome.

A range of antibiotic inhibitors that act within the peptidyl transferase center of the ribosome were examined for their capacity to perturb the relative positioning of the 3' end of P/P'-site-bound tRNA and the Escherichia coli ribosome. The 3'-terminal adenosines of deacylated tRNA and N-Ac-Phe-tRNA were derivatized at the 2 position with an azido group and the tRNAs were cross-linked to the ribosome on irradiation with ultraviolet light at 365 nm. The cross-links were localized on the rRNA within extended versions of three previously characterized 23S rRNA fragments F1', F2', and F4' at nucleotides C2601/A2602, U2584/U2585 (F1'), U2506 (F2'), and A2062/C2063 (F4'). Each of these nucleotides lies within the peptidyl transferase loop region of the 23S rRNA. Cross-links were also formed with ribosomal proteins L27 (strong) and L33 (weak), as shown earlier. The antibiotics sparsomycin, chloramphenicol, the streptogramins pristinamycin IA and IIA, gougerotin, lincomycin, and spiramycin were tested for their capacity to alter the identities or yields of each of the cross-links. Although no new cross-links were detected, each of the drugs produced major changes in cross-linking yields, mainly decreases, at one or more rRNA sites but, with the exception of chloramphenicol, did not affect cross-linking to the ribosomal proteins. Moreover, the effects were closely similar for both deacylated and N-Ac-Phe-tRNAs, indicating that the drugs selectively perturb the 3' terminus of the tRNA. The strongest decreases in the rRNA cross-links were observed with pristinamycin IIA and chloramphenicol, which correlates with their both producing complex chemical footprints on 23S rRNA within E. coli ribosomes. Furthermore, gougerotin and pristinamycin IA strongly increased the yields of fragments F2' (U2506) and F4' (U2062/C2063), respectively. The results obtained with an RNAse H approach correlate well with primer extension data implying that cross-linking occurs primarily to the bases. Both sets of data are also consistent with the results of earlier rRNA footprinting experiments on antibiotic-ribosome complexes. It is concluded that the antibiotics perturb the relative positioning of the 3' end of the P/P'-site-bound tRNA and the peptidyl transferase loop region of 23S rRNA.

The mechanism of staphylococci resistance to methicillin: a critical analysis of dominant opinions.

Resistance to methicillin has been reported to be constantly associated with production of a novel penicillin-binding protein (PBP), indicated as PBP2a. This PBP is always present in strains that are homogeneously resistant to the drug, but is also constantly present in the strains heterogeneously resistant, where by far the vast majority of the cells are indeed sensitive to the antibiotic. This apparent lack of correlation between the presence of PBP2a and methicillin resistance has led to the idea, shared by most scientists, that the presence of PBP2a alone cannot explain resistance of staphylococci to methicillin. This confusion has implications also for clinical microbiology. In fact in in-vitro assays, not rarely, strains that are resistant to methicillin appear sensitive to other beta-lactams. However, clinical experience has demonstrated that when infections caused by methicillin-resistant staphylococci, which, in in vitro assays, become sensitive to other beta-lactams, are treated with the latter antibiotics, the incidence of failures is higher than that observed when the same antibiotics are used to treat infections caused by staphylococci sensitive to methicillin. Also in consideration of the fact that the mechanism of resistance to methicillin is not yet understood, many authors recommend to consider methicillin-resistant staphylococci as being in-vivo resistant also to the beta-lactams to which they may become sensitive in in-vitro assays. In the opinion of the authors, the mechanism of staphylococcal resistance to methicillin is reasonably explainable at least in its fundamental aspects. The present dominant opinions concerning methicillin-resistant staphylococci will be critically analyzed on the basis both of data presented in the literature and of data obtained in the authors' laboratory.

Product analysis of in vitro ribosomal protein synthesis for the assessment of kinetic parameters.

For the characterization of the product distribution of in vitro ribosomal protein synthesis a new method is introduced in which radioactively labeled peptides are separated on a reversed-phase HPLC column and detected on line with a flow radioactivity monitor. Employing this procedure the kinetics of product formation under pre-steady-state conditions were measured under a variety of conditions. These measurements yield the intrinsic monomolecular rate constants for peptidyl transfer (greater than 20 s-1) and translocation (rate limiting for elongation). The usefulness of this technique for accuracy measurements is illustrated.