Role of the Serine/Threonine kinases PrkC and CD2148 in the physiology of Clostridium difficile

C. difficile (CD) est un bacille à gram-positif anaérobie strict et sporulant. Ce pathogène est la principale cause d’infections nosocomiales intestinales après une antibiothérapie. La contamination des patients s’effectue via l’ingestion de spores de CD. Suite à une prise d’antibiotiques, une dysbiose du microbiote conduit à des changements dans les pools de métabolites favorisant la germination des spores et la croissance de CD dans le tube digestif. CD va alors produire deux toxines entrainant des dommages cellulaires et une forte inflammation ce qui va conduire à des diarrhées ou des colites pseudomembraneuses. Une partie des cellules de CD va aussi former des spores conduisant à la transmission de l’infection. Lors de son cycle infectieux, CD doit s’adapter rapidement aux changements dans son environnement via la régulation de ses différents processus physiologiques. La phosphorylation est une modification post-traductionnelle réversible. Elle est très souvent utilisée pour moduler l’activité de protéines impliquées dans des voies de signalisation. Les Ser/Thr kinases (STKs) bactériennes phosphorylent leurs substrats sur des résidus Ser ou Thr et régulent de nombreux processus tels que la traduction, le métabolisme du carbone, la synthèse de l’enveloppe, la résistance aux antibiotiques, la division cellulaire ou la virulence. De manière intéressante, CD possède deux STKs, PrkC et CD2148, qui n’ont jamais été étudiées. Ces deux STKs possèdent un domaine kinase cytoplasmique ainsi qu’un segment transmembranaire. Seule PrkC présente une partie extracellulaire composée de 2 domaines PASTA. L’inactivation de PrkC affecte la morphologie ainsi que la division cellulaire de CD. Les cellules du mutant ∆prkC sont hétérogènes en taille. Certaines cellules très allongées ne possèdent pas de septum, d’autres cellules ont un septum mal localisé ou plusieurs septa rapprochés pour environ 15% des cellules du mutant ∆prkC comme observé en microscopie électronique. Nos résultats ont montré que le mutant ∆prkC était capable de sporuler et de germer de manière similaire à la souche sauvage mais présente cependant une motilité réduite ainsi qu’une plus grande capacité à former des biofilms. De plus, le mutant ∆prkC montre une plus grande sensibilité aux céphalosporines qui sont des antibiotiques qui favorisent les infections à CD, à certains peptides antimicrobiens et au lysozyme qui sont l’une des premières lignes de défense de l’hôte et aux sels biliaires secondaires tels que le déoxycholate qui sont toxiques pour les cellules végétatives. En outre, la sensibilité accrue du mutant ∆prkC à ces stress d’enveloppe que CD rencontre dans l’intestin lors du cycle infectieux, pourrait être la cause du délai de colonisation observé pour ce mutant chez le hamster. L’inactivation de PrkC affecte donc l’homéostasie de l’enveloppe de CD. Nous avons montré que la composition du peptidoglycane n’est pas différente entre la souche sauvage et le mutant ∆prkC. En revanche, nous avons mis en évidence un relargage plus important du glycopolymère de surface, le PSII, dans le milieu de culture du mutant ∆prkC. Des résultats préliminaires suggèrent que cet acide téichoïque est également plus présent à la surface du mutant ∆prkC et nous réalisons actuellement des expériences complémentaires pour le vérifier. La structure du PSII étant la même chez les deux souches, ces résultats suggèrent que PrkC est impliquée dans les mécanismes de contrôle de la synthèse du PSII, de son exportation ou de son ancrage au peptidoglycane de CD. Au cours de ce travail, nous avons également commencé l’analyse du mutant CD2148::erm. Les premiers phénotypes observés pour ce mutant sont nettement différents de ceux de ∆prkC puisque l’inactivation de CD2148 affecte le processus de sporulation et la séparation des cellules après division cellulaire.Clostridium difficile (CD) is the leading cause of intestinal nosocomial post-antibiotic infections in adults. Exposure to certain antibiotics including cephalosporins induces dysbiosis promoting CD infection. Resistance of CD to these antibiotics is a major concern while resistance mechanisms remain poorly characterized. CD produces two toxins that cause epithelial cell damage and inflammation while additional factors associated to cell surface participate in the colonization process. During infection, CD also encounters several stresses in the gut such as secondary bile salts that are toxic for vegetative cells, antibiotics, antimicrobial peptides released by the host, reactive oxygen and nitrogen species produced during inflammation. Pathogen survival depends on its capacity to rapidly adapt to the host environment. Protein phosphorylation is a reversible post-translational modification employed for signal transduction and regulation. Bacterial Ser/Thr kinases (STKs) regulate numerous physiological processes. In response to specific stimuli, STKs phosphorylate substrates on Ser or Thr residues to trigger the appropriate cellular response. Nothing is known about the role of the two STKs of CD, PrkC and CD2148, in the physiology of this enteropathogen. To investigate their function, we constructed ∆prkC and ∆CD2148 mutants. Cells of the ∆prkC mutant had an increased size and abnormal septa. The ∆prkC mutant also had a reduced motility and formed more biofilms. PrkC inactivation increased sensitivity to antimicrobial compounds that CD may encounter in the gut during infection such as deoxycholate, cephalosporins, cationic antimicrobial peptides and lysozyme. This increased susceptibility was not associated to differences in the structure of peptidoglycan. By contrast, we showed that the ∆prkC mutant released more polysaccharide II (PSII) in the supernatant suggesting a decreased deposition of this glycopolymer to the cell surface in this mutant. Our results also revealed that the ∆prkC mutant had a delay in gut implantation in a hamster model. Finally, we observed that the mutant ∆CD2148 formed chains of cells and sporulated more rapidly than the wild-type strain. Accordingly, key sporulation genes were up-regulated in this mutant. Work is now in progress to detect proteins phosphorylated in vivo using phosphoproteomic approaches and to identify substrates of PrkC and CD2148

Standardization and Validation of Fluorescence-Based Quantitative Assay to Study Human Platelet Adhesion to Extracellular-Matrix in a 384-Well Plate

Platelets play a crucial role in the immunological response and are involved in the pathological settings of vascular diseases, and their adhesion to the extracellular matrix is important to bring leukocytes close to the endothelial cells and to form and stabilize the thrombus. Currently there are several methods to study platelet adhesion; however, the optimal parameters to perform the assay vary among studies, which hinders their comparison and reproducibility. Here, a standardization and validation of a fluorescence-based quantitative adhesion assay to study platelet-ECM interaction in a high-throughput screening format is proposed. Our study confirms that fluorescence-based quantitative assays can be effectively used to detect platelet adhesion, in which BCECF-AM presents the highest sensitivity in comparison to other dyes

Ser/Thr Kinase-Dependent Phosphorylation of the Peptidoglycan Hydrolase CwlA Controls Its Export and Modulates Cell Division in Clostridioides difficile

International audienceCell growth and division require a balance between synthesis and hydrolysis of the peptidoglycan (PG). Inhibition of PG synthesis or uncontrolled PG hydrolysis can be lethal for the cells, making it imperative to control peptidoglycan hydrolase (PGH) activity. The synthesis or activity of several key enzymes along the PG biosynthetic pathway is controlled by the Hanks-type serine/threonine kinases (STKs). In Gram-positive bacteria, inactivation of genes encoding STKs is associated with a range of phenotypes, including cell division defects and changes in cell wall metabolism, but only a few kinase substrates and associated mechanisms have been identified. We previously demonstrated that STK-PrkC plays an important role in cell division, cell wall metabolism, and resistance to antimicrobial compounds in the human enteropathogen Clostridioides difficile . In this work, we characterized a PG hydrolase, CwlA, which belongs to the NlpC/P60 family of endopeptidases and hydrolyses cross-linked PG between daughter cells to allow cell separation. We identified CwlA as the first PrkC substrate in C. difficile . We demonstrated that PrkC-dependent phosphorylation inhibits CwlA export, thereby controlling hydrolytic activity in the cell wall. High levels of CwlA at the cell surface led to cell elongation, whereas low levels caused cell separation defects. Thus, we provided evidence that the STK signaling pathway regulates PGH homeostasis to precisely control PG hydrolysis during cell division. IMPORTANCE Bacterial cells are encased in a PG exoskeleton that helps to maintain cell shape and confers physical protection. To allow bacterial growth and cell separation, PG needs to be continuously remodeled by hydrolytic enzymes that cleave PG at critical sites. How these enzymes are regulated remains poorly understood. We identify a new PG hydrolase involved in cell division, CwlA, in the enteropathogen C. difficile . Lack or accumulation of CwlA at the bacterial surface is responsible for a division defect, while its accumulation in the absence of PrkC also increases susceptibility to antimicrobial compounds targeting the cell wall. CwlA is a substrate of the kinase PrkC in C. difficile . PrkC-dependent phosphorylation controls the export of CwlA, modulating its levels and, consequently, its activity in the cell wall. This work provides a novel regulatory mechanism by STK in tightly controlling protein export

The Ser/Thr kinase PrkC participates in cell wall homeostasis and antimicrobial resistance in Clostridium difficile

International audienceClostridium difficile is the leading cause of antibiotic-associated diarrhea in adults. During infection, C. difficile must detect the host environment and induce an appropriate survival strategy. Signal transduction networks involving serine/threonine kinases (STKs) play key roles in adaptation, as they regulate numerous physiological processes. PrkC of C. difficile is a STK with two PASTA domains. We showed that PrkC is membrane associated and is found at the septum. We observed that deletion of prkC affects cell morphology with an increase in mean size, cell length heterogeneity, and presence of abnormal septa. When compared with the wild-type strain, a ΔprkC mutant was able to sporulate and germinate but was less motile and formed more biofilm. Moreover, a ΔprkC mutant was more sensitive to antimicrobial compounds that target the cell envelope such as the secondary bile salt deoxycholate, cephalosporins, cationic antimicrobial peptides, and lysozyme. This increased susceptibility was not associated with differences in peptidoglycan or polysaccharide II composition. However, the ΔprkC mutant had less peptidoglycan and released more polysaccharide II into the supernatant. A proteomic analysis showed that the majority of C. difficile proteins associated with the cell wall were less abundant in the ΔprkC mutant compared to the wild-type strain. Finally, in a hamster model of infection the ΔprkC mutant had a colonization delay that did not significantly affect overall virulence