Substitutions in PBP2b from β-lactam resistant Streptococcus pneumoniae have different effects on enzymatic activity and drug reactivity

Pneumococcus resists β-lactams by expressing variants of its target enzymes, the penicillin-binding proteins (PBPs), with many amino acid substitutions. Up to 10% of the sequence can be modified. These altered PBPs have a much reduced reactivity with the drugs but retain their physiological activity of cross-linking the peptidoglycan, the major constituent of the bacterial cell wall. However, as β-lactams are chemical and structural mimics of the natural substrate, resistance mediated by altered PBPs raises the following paradox: how PBPs that react poorly with the drugs maintain a sufficient level of activity with the physiological substrate? This question is addressed for the first time in this study, which compares the peptidoglycan cross-linking activity of PBP2b from susceptible and resistant strains with their inhibition by different β-lactams. Unexpectedly, the enzymatic activity of the variants did not correlate with their antibiotic reactivity. This finding indicates that some of the numerous amino acid substitutions were selected to restore a viable level of enzymatic activity by a compensatory molecular mechanism

Nanoscale dynamics of peptidoglycan assembly during the cell cycle of Streptococcus pneumoniae

Dynamics of cell elongation and septation are key determinants of bacterial morphogenesis. These processes are intimately linked to peptidoglycan synthesis performed by macromolecular complexes called the elongasome and the divisome. In rod-shaped bacteria, cell elongation and septation, which are dissociated in time and space, have been well described. By contrast, in ovoid-shaped bacteria, the dynamics and relationships between these processes remain poorly understood because they are concomitant and confined to a nanometer-scale annular region at midcell. Here, we set up a metabolic peptidoglycan labeling approach using click chemistry to image peptidoglycan synthesis by single-molecule localization microscopy in the ovoid bacterium Streptococcus pneumoniae. Our nanoscale-resolution data reveal spatiotemporal features of peptidoglycan assembly and fate along the cell cycle and provide geometrical parameters that we used to construct a morphogenesis model of the ovoid cell. These analyses show that septal and peripheral peptidoglycan syntheses first occur within a single annular region that later separates in two concentric regions and that elongation persists after septation is completed. In addition, our data reveal that freshly synthesized peptidoglycan is remodeled all along the cell cycle. Altogether, our work provides evidence that septal peptidoglycan is synthesized from the beginning of the cell cycle and is constantly remodeled through cleavage and insertion of material at its periphery. The ovoid-cell morphogenesis would thus rely on the relative dynamics between peptidoglycan synthesis and cleavage rather than on the existence of two distinct successive phases of peripheral and septal synthesis

Identification of FtsW as a transporter of lipid-linked cell wall precursors across the membrane

This study identifies FtsW as the flippase that translocates lipid-linked peptidoglycan precursors across the cell membrane during bacterial cell wall synthesis

A Peptidoglycan Fragment Triggers β-lactam Resistance in Bacillus licheniformis

To resist to β-lactam antibiotics Eubacteria either constitutively synthesize a β-lactamase or a low affinity penicillin-binding protein target, or induce its synthesis in response to the presence of antibiotic outside the cell. In Bacillus licheniformis and Staphylococcus aureus, a membrane-bound penicillin receptor (BlaR/MecR) detects the presence of β-lactam and launches a cytoplasmic signal leading to the inactivation of BlaI/MecI repressor, and the synthesis of a β-lactamase or a low affinity target. We identified a dipeptide, resulting from the peptidoglycan turnover and present in bacterial cytoplasm, which is able to directly bind to the BlaI/MecI repressor and to destabilize the BlaI/MecI-DNA complex. We propose a general model, in which the acylation of BlaR/MecR receptor and the cellular stress induced by the antibiotic, are both necessary to generate a cell wall-derived coactivator responsible for the expression of an inducible β-lactam-resistance factor. The new model proposed confirms and emphasizes the role of peptidoglycan degradation fragments in bacterial cell regulation

Caractérisation structurelle et fonctionnelle in vitro de deux protéines de Escherichia coli, DsbA et DsbC, impliquées dans la formation des ponts disulfures in vivo

Les protéines sécrétées contiennent souvent des ponts disulfures qui sont formés très rapidement "in vivo". DsbA et DsbC sont deux protéines solubles du périplasme de "Escherichia coli" qui sont impliquées dans la formation des ponts disulfures. Toutes deux possèdent un site actif comprenant deux cystéines séparées par seulement deux autres résidus. Les activités DsbA et DsbC ont été étudiées "in vitro" sur différents systèmes, notamment sur la formation des ponts disulfures dans la protéine BPTI. Nous avons caractérisé en détail, cinétiquement et à l'équilibre, la chimie inhabituelle des échanges thioldisulfures de ces deux protéines, ce qui permet d'expliquer en partie leurs activités respectives. Il est apparu que DsbA est très efficace pour introduire des ponts disulfures dans des protéines, mais ne catalyse pas leurs réarrangements. Au contraire, DsbC est une isomérase capable de réarranger des ponts disulfures. Il est proposé que ce sont leurs rôles respectifs "in vivo"

Membrane topology of the Streptococcus pneumoniae FtsW division protein

The topology of FtsW from Streptococcus pneumoniae, an essential membrane protein involved in bacterial cell division, was predicted by computational methods and probed by the alkaline phosphatase fusion and cysteine accessibility techniques. Consistent results were obtained for the seven N-terminal membrane-spanning segments. However, the results from alkaline phosphatase fusions did not confirm the hydropathy analysis of the C-terminal part of FtsW, whereas the accessibility of introduced cysteine residues was in agreement with the theoretical prediction. Based on the combined results, we propose the first topological model of FtsW, featuring 10 membrane-spanning segments, a large extracytoplasmic loop, and both N and C termini located in the cytoplasm

Membrane topology of the Streptococcus pneumoniae FtsW division protein

The topology of FtsW from Streptococcus pneumoniae, an essential membrane protein involved in bacterial cell division, was predicted by computational methods and probed by the alkaline phosphatase fusion and cysteine accessibility techniques. Consistent results were obtained for the seven N-terminal membrane-spanning segments. However, the results from alkaline phosphatase fusions did not confirm the hydropathy analysis of the C-terminal part of FtsW, whereas the accessibility of introduced cysteine residues was in agreement with the theoretical prediction. Based on the combined results, we propose the first topological model of FtsW, featuring 10 membrane-spanning segments, a large extracytoplasmic loop, and both N and C termini located in the cytoplasm

Penicillin-binding proteins and beta-lactam resistance

International audienc

The elongation of ovococci.

International audienceThe morphogenesis of ovococci has been reviewed extensively. Recent results have provided new insights concerning the mechanisms of elongation in ovoid bacteria. We present here the proteins involved in the elongation (firmly established and more or less hypothetical) and discuss the relationship between elongation and division of ovococci

Resistance to β-Lactams in Neisseria ssp Due to Chromosomally Encoded Penicillin-Binding Proteins

International audienceNeisseria meningitidis and Neisseria gonorrhoeae are human pathogens that cause a variety of life-threatening systemic and local infections, such as meningitis or gonorrhoea. The treatment of such infection is becoming more difficult due to antibiotic resistance. The focus of this review is on the mechanism of reduced susceptibility to penicillin and other β-lactams due to the modification of chromosomally encoded penicillin-binding proteins (PBP), in particular PBP2 encoded by the penA gene. The variety of penA alleles and resulting variant PBP2 enzymes is described and the important amino acid substitutions are presented and discussed in a structural context