Functional analysis of the N-deacetylases of Clostridium difficile

Clostridium difficile est une bactérie anaérobie sporulante responsable de 15 à 25% des diarrhées post-antibiotiques. Les N-déacétylases sont largement distribuées parmi les bactéries à Gram positif et elles sont impliquées dans différentes fonctions de surface. L'analyse du génome de C. difficile montre que 13 gènes codent pour des N-déacétylases potentielles, et nous avons caractérisé l’ensemble de ces N-déacétylases.Le peptidoglycane de la cellule végétative de C. difficile est N-déacétylé sur 93% des glucosamines, et cette modification participe à la résistance de la bactérie au lysozyme, un composant majeur de l’immunité innée. Nous avons identifié les N-déacétylases PgdA, PgdB et PdaV responsables de cette N-déacétylation, et nous avons évalué leur impact au sein de la virulence de C. difficile. Nous avons également défini le rôle de deux N-déacétylases NagA dans le recyclage du peptidoglycane.Le peptidoglycane de la spore, ou cortex, a été analysé lors de ce travail et sa structure chez C. difficile est atypique par rapport au cortex décrit pour d’autres espèces bactériennes. Nous avons défini les N-déacétylases responsables de la N-déacétylation de la glucosamine du cortex. Nous avons également caractérisé les deux N-déacétylases PdaA1 et PdaA2 responsables de la synthèse des δ-lactames, une modification spécifique du cortex, ainsi que leur influence dans la virulence de C. difficile. Dans ce cadre, nous avons montré que les δ-lactames ont un rôle physiologique plus large pour C. difficile que chez Bacillus subtilis. De plus, nous avons identifié deux N-déacétylases potentiellement impliquées dans la synthèse de ce cortex.À travers ces résultats, ce travail apporte de nouvelles connaissances dans le rôle des N-déacétylases bactériennes.Clostridium difficile is an anaerobic and spore-forming bacteria responsible for 15 to 25% of post-antibiotic diarrhea. N-deacetylases are largely distributed among Gram positive bacteria and are involved in many surface processes. C. difficile genome analysis showed that 13 genes potentially encode N-deacetylases. In this work, we have characterized all of these enzymes.The vegetative cell peptidoglycan of C. difficile is deacetylated on 93% its glucosamine, and this modification is involved in the resistance of C. difficile against lysozyme, a major component of the innate immunity. We identified the N-deacetylases PgdA, PgdB and PdaV responsible for this N-deacetylation, and we assessed their impact on C. difficile virulence. The role of two N-deacetylases involved in peptidoglycan recycling has also been assessed.The spore peptidoglycan, known as the cortex, has also been characterized during this work, and its structure is atypical in C. difficile compared to other bacterial species. We showed that N-deacetylation of the glucosamine is present in the cortex peptidoglycan, and we identified the N-deacetylases responsible for this modification. Additionally, we characterized the N-deacetylases PdaA1 and PdaA2 responsible for the synthesis of muramic-δ-lactams, a cortex specific modification, as well as their impact on C. difficile virulence. In his context, we determined that muramic-δ-lactams have a broader role in C. difficile compared to their role in Bacillus subtilis. Moreover, two N-deacetylases involved in cortex synthesis have been identified.This work adds a contribution in the knowledge of the roles of bacterial N-deacetylases

The invasive pathogen Yersinia pestis disrupts host blood vasculature to spread and provoke hemorrhages

International audienceYersinia pestis is a powerful pathogen with a rare invasive capacity. After a flea bite, the plague bacillus can reach the bloodstream in a matter of days giving way to invade the whole organism reaching all organs and provoking disseminated hemorrhages. However, the mechanisms used by this bacterium to cross and disrupt the endothelial vascular barrier remain poorly understood. In this study, an innovative model of in vivo infection was used to focus on the interaction between Y . pestis and its host vascular system. In the draining lymph nodes and in secondary organs, bacteria provoked the porosity and disruption of blood vessels. An in vitro model of endothelial barrier showed a role in this phenotype for the pYV/pCD1 plasmid that carries a Type Three Secretion System. This work supports that the pYV/pCD1 plasmid is responsible for the powerful tissue invasiveness capacity of the plague bacillus and the hemorrhagic features of plague

Altered spore cortex impairs virulence in <i>C. difficile</i>

International audienceSpores are produced by many organisms as the result of a survival mechanism, triggered under several types of adverse environmental conditions. They are multi-layered structures, composed of a compressed dehydrated inner core, surrounded by the inner membrane, a germ cell-wall, a peptidoglycan layer known as the cortex, an outer membrane, a proteinaceous external coat, and for some species the outermost layer called the exosporium. This study focuses on the spore cortex of Clostridium difficile, a Gram-positive spore-forming, toxin-producing anaerobic bacterium that can colonize the intestinal tracts of humans, considered as the leading cause of hospital and community-acquired antibiotic-associated diarrhea. Given the highly original structure described for the vegetative cell peptidoglycan of C. difficile, and notably the particularly high level of N-deacetylation and its impact on host-pathogen interactions, we focused on the cortex N-deacetylases, and especially the N-deacetylase responsible for muramic lactam synthesis in C. difficile. Moreover, given the central role of spores in the physiopathology of C. difficile infection, we also investigated the contribution of cortex structure in C. difficile virulence, presenting the first study connecting cortex structure and virulence. In this context, we provide the fine structure of C. difficile cortex and the characterization of pdaA as the N-deacetylase responsible for muramic lactams synthesis

Muramic-δ-lactams are involved in <i>C. difficile</i> sporulation, germination and virulence

Background and aimsSpores are produced by many organisms as the result of a survival mechanism, triggered under several environmental conditions. They are multi-layered structures, one of which is a peptidoglycan layer known as the cortex. The cortex peptidoglycan has been described for several organisms, including B. subtilis and C. perfringens, but has yet to be published for C. difficile. Compared to the vegetative cell peptidoglycan, the cortex peptidoglycan possesses a unique, modified sugar called muramic-δ-lactam, synthesized by at least two enzymes: an amidase CwlD and an N-deacetylase PdaA. In this work, we analyzed the C. difficile cortex structure, we characterized the N-deacetylase involved in muramic-δ-lactam synthesis and investigated the impact of muramic-δ-lactams on C. difficile physiology and virulence.MethodsThe cortex of C. difficile 630∆erm and pdaA mutant strains were analyzed using UHPLC coupled HRMS. Germination was assessed through optical density monitoring of spore suspensions after addition of taurocholate. Spore resistance properties were investigated by enumeration of spore suspensions after treatment with ethanol, hydrogen peroxide or heat. Sporulation was studied in liquid cultures after 72H of growth. Morphology of both strains was assessed through transmission electron microscopy. ResultsThe cortex analysis revealed several differences between the B. subtilis and C. difficile cortex structures. For instance, only 24% of muropeptides in C. difficile carried muramic-δ-lactams, compared to 50% of muropeptides in B. subtilis. Analysis of the cortex from the pdaA mutant showed minor traces of muramic-δ-lactams (0.4% of all muropeptides). Investigation of the consequences of this decrease in muramic-δ-lactams in the pdaA mutant showed a decreased sporulation rate, an altered germination, and a decreased heat-resistance. In a virulence assay, the pdaA mutant also showed a delayed virulence.ConclusionsSurprisingly, our results suggest a much broader impact for muramic-δ-lactams in C. difficile compared to previously characterized model organisms, such as B. subtilis. Our results highlight a novel factor linking both the germination and sporulation processes, and provide an insight into a new strategy to target C. difficile and its dissemination by targeting enzymes involved in cortex synthesis