COBRA - Combating resistance to antibiotics by broadening the knowledge on molecular mechanisms behind resistance to inhibitors of cell wall synthesis

Background:

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.

Problem:

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.

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.

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