We have aim at the elucidation of the molecular mechanisms of resistance to inhibitors of cell wall synthesis in bacteria responsible for severe nosocomial and community-acquired infections. Our STREP was focused on -lactams, the major class of antibiotics in current clinical use, and on resistance due to modifications of the cell wall synthesizing machinery and to production of ß-lactamases, the most prevalent mechanisms in Gram-positive and Gram-negative bacteria, respectively. These studies form a reference to globally assess the modifications of the structure, function, and dynamics of the peptidoglycan assembly pathways responsible for emergence of resistance including the 3 D structure of relevant components and possible targets. It has identify new ß-lactamases, determine their 3 D structure and elucidate different aspects of the regulation of their gene expression and the mechanisms responsible for their mobility.
Antibiotics are not like other drugs in that they act against bacteria and not the human host. Therefore the evolution of resistance under the selective pressure of antibiotics after exposure of populations (human, animal) raises major therapeutical issues. This program addresses the general problem of resistance to antibiotics and concerns the understanding of the mechanisms of resistance, in particular to inhibitors of cell wall synthesis. Among these are the ß-lactams, one of the most important classes of antibiotics, if not the most broadly used antibiotics worldwide. The rates of ß-lactam resistance for many common species found in infections have reached high levels in the community, as well as in the hospital. While In Gram-positive organisms this resistance is mainly due to altered targets, in Gram-negative organisms, acquired resistance to ß-lactams is essentially due to the presence of plasmid-encoded ß-lactamases or the over-expression of chromosome-encoded ß-lactamases. This latter resistance can be enhanced by associated impermeability or efflux mechanisms. Since many pathogens are multiresistant, there will be an eventual limitation in the choice of antibiotics useful for primary treatment and therefore a promotion of a vicious cycle facilitating the emergence of new resistances.[+] Read More
A) Penicillin-binding proteins (PBPs) and critical associated factors. The peptidoglycan is a complex structure the synthesis of which involves multiple coordinated steps in and outside of the cytoplasm. The complete disaccharide-peptide unit linked to the lipid carrier, once translocated through the cytoplasmic membrane, is polymerized by protein complexes involved in cell elongation (elongase) and division (divisome). These complexes include the PBPs (penicillin binding proteins) of classes A and B that belong to the superfamily of the penicilloyl serine transferases and are the targets of the penicillins. According to the modules they contain, they display D,D-transpeptidase, D,D-carboxypeptidase and glycosyltransferase activities Production of D,D-transpeptidases that are inefficiently inactivated by the drugs, commonly referred to as low-affinity PBPs, is the main mechanism responsible for clinically relevant ß-lactam resistance in streptococci, staphylococci, and enterococci.. Due to the complexity of the peptidoglycan assembly pathway, analyses of the mechanisms of resistance has been mainly limited to easily detectable modifications of the drug targets, such as the level of production of the PBPs and their interaction with ß-lactams. However, recent analyses support the view that genes non essential for viability are required for expression of resistance mediated by low-affinity PBPs and other factors. Among these are (i) biosynthetic enzymes adding the side chain to the pentapeptide stem; (ii) regulatory factors, that control as yet unknown responses of the bacteria to the drugs; and (iii) transglycosylases which appear to co-operate in an undefined manner with the D,D-transpeptidase acitvity of low-affinity class B PBPs for peptidoglycan polymerisation in the presence of -lactams. In rare cases, mutations in these chromosomal genes have been detected in resistant bacteria but the extent of such modifications and the role of the encoded protein are largely unknown. Analysis of the role of other components of the divisome and the elongase complexes have not been developed since the metabolism of the lipid-linked peptidoglycan precursors and their delivery to the polymerisation complexes is poorly understood.
B) ß-lactamases. . Among clinical Gram-negative isolates, the major mechanism of resistance to ß-lactam antibiotics is related to the production of hydrolytic enzymes: the ß-lactamases. To counter this problem, the pharmaceutical industry has marketed novel classes of ß-lactams. However, the use of these new drugs was quickly followed by the emergence of new ß-lactamases including those with expanded-spectrum activity many of which evolved from previous existing ß-lactamases. This process involved the three classes of ß-lactamases(classes A, C, and D) belonging to the superfamily of penicilloyl serine transferases, that also includes the PBPs, and the class B enzymes regrouping the metallo-ß-lactamases (Zn++-dependent). While new enzymes are discovered almost every day and have to be explored, the reason of the spreading of several novel enzymes world-wide remains unknown and the factors modulating their expression as well as the vehicles for mobility and spreading of these genes have to be thoroughly studied.
This project focus on the understanding of molecular mechanisms of resistance to ß-lactams and other cell wall inhibitors in clinical Gram-positive and Gram-negative pathogens. We did study different enzymatic properties and structural features of class B PBPs involved in ß-lactam resistance, as well as different auxiliary proteins, among which the class A PBPs and other enzymes involved in the synthesis of substrate structures used by these class A and B PBPs. We found, in the cytoplasmic and in membrane steps of the peptidoglycan synthesis, other critical factors, including regulators, interfering with the expression of resistance to cell wall inhibitors and possible new resistance-conferring targets. The study of ß-lactamases has included an extensive search for, and characterisation of, novel extended-spectrum ß-lactamases and carbapenemases in Gram-negative organisms. For several of these enzymes, gene expression and the molecular basis of their dissemination have been studied. The catalytic properties, the structure-activity relationships and the 3D structure of some of them will were determined with regard to their activities against the antibiotics. The contribution of additional factors, such as outer membrane permeability and efflux pumps, to high-level resistance to ß-lactams was investigated in detail.
This program addresses the general problem of resistance against a class of antibiotics widely used in the community and in hospitals. Any progress, even the smaller ones, in the understanding of these mechanisms will be of benefit to academia, public health and industry. It will increase our knowledge on insufficiently explored and new mechanisms of resistance toward cell wall inhibitors. It will lead to the deciphering and the discovery of novel mechanisms including the role of several auxillary proteins involved in resistance. The isolation of -lactamases, PBPs and other components essential for the expression of resistance to ß-lactams and other cell wall inhibitors, reinforced by knowledge of their 3D structure, will allow for the future setup of assays to screen for new inhibitors and will improve drug design. The knowledge of the sequence of different targets will be used to create databases containing new ß-lactamases and new PBP alterations linked to resistance. Particular mutations responsible for resistance could then be used to develop sequence-based molecular diagnostic tools. Discovery of novel mechanisms of resistance will result in the identification of new phenotypes helpful for the detection of resistance in clinical laboratories. This will aid in the interpretation of susceptibility and resistance to different classes of cell wall inhibitors, in particular ß-lactams.
Finally, transmission and acquisition of resistance by new strains is one of the major factors in resistance dissemination. A better knowledge of these mechanisms should facilitate the recognition of the antibiotics most powerful in selecting for the mechanisms studied. Understanding of the transmission mechanisms is a crucial step in preventing resistance as well as in guiding optimal antibiotic usage.