Modulation de la virulence de Yersinia pestis impliquant le locus de la flagelline et un processus de variation de phase

En tant que source de pandémies mondiales, la peste a tué des millions de personnes au cours de trois grandes vagues pandémiques depuis l'Antiquité jusqu'à nos jours. L'agent causal Yersinia pestis est une bactérie Gram-négative présentant l'une des virulences les plus extrêmes dans la nature. Bien que très similaire sur le plan génétique, son ancêtre récent Yersinia pseudotuberculosis diffère par son mode de vie et par sa virulence, ce qui fait du genre Yersinia un modèle fascinant pour étudier l'évolution de la pathogenèse bactérienne. L'armement pathogène commun aux deux espèces repose principalement sur la présence d'un Système de Sécrétion de Type 3 (T3SS) et d'un Ilot de Haute Pathogénicité (HPI). Alors que le HPI chromosomique confère une capacité élevée à capturer le fer, le T3SS est codé sur le plasmide pYV/pCD1 et englobe à la fois un système d'injection de protéines appelé injectisome et une série d'effecteurs appelés Yersinia Outer Proteins (Yops). Une relation évolutive existe entre l'injectisome et l'appareil flagellaire, dont le fonctionnement repose également sur une sécrétion de type III. Y. pseudotuberculosis est motile grâce à un flagelle alors que Y. pestis ne l'est pas. Un petit nombre de mutations, dont un décalage de cadre dans le régulateur maître du système flagellaire flhD, explique le manque de motilité du bacille de la peste, malgré la présence d'un système flagellaire presque complet. Dans ce travail, nous avons étudié le rôle dans la peste du locus fliC codant pour la signature bactérienne flagelline, une partie du système flagellaire que l'on croyait inactive chez Y. pestis. De manière très intéressante, nous avons mis en évidence un rôle du locus fliC dans la pathogenèse, en partie dû à une régulation de plusieurs facteurs de virulence, dont la pseudocapsule F1 et le T3SS. Nous montrons aussi dans ce travail que les Yersinia pathogènes peuvent moduler leur virulence au moyen d'un processus de commutation phénotypique impliquant un réarrangement génétique dans le gène yscP, un gène dont le produit YscP contrôle la sécrétion de type 3, et l'émergence ultérieure d'un phénotype T3S négatif. Nous émettons l'hypothèse que dans la nature, la sous-population de Y. pestis T3S négatif contribue au maintien d'une infection de faible niveau entre les épizooties. Enfin, des études sur les systèmes flagellaires nous ont également conduit à identifier un phénomène de motilité jusque-là non décrit chez Y. pseudotuberculosis. Ce type de motilité se produit principalement à 37°C (la température de l'hôte), alors qu'il est réprimé à basse température. Il suit donc un modèle de dépendance à la température qui est opposé à celui précédemment observé pour les mécanismes de 'swimming' et de 'swarming'.As a source of worldwide pandemics, plague has killed millions of people during three major pandemic waves from antique times to present. The causative agent Yersinia pestis is a Gram-negative bacterium exhibiting one of the most extreme virulence in nature. Although very similar at the genetic level, its recent ancestor Yersinia pseudotuberculosis differ in lifestyle and virulence, which makes the Yersinia genus a fascinating model for studying the evolution of bacterial pathogenesis. The pathogenic armament common to the two species relies mostly on the presence of a Type 3 Secretion System (T3SS) and a High Pathogenicity Island (HPI). Whereas the chromosomic HPI confers a high capacity to capture iron, the T3SS is encoded on the pYV/pCD1 plasmid and encompasses both a protein injection system called injectisome, and a series of effectors called Yersinia Outer Proteins (Yops). An evolutionary relationship exists between the injectisome and the flagellar apparatus, the functioning of which also relies on type III secretion. Y. pseudotuberculosis is capable of flagellum-dependent motility whereas Y. pestis it is not. A small number of mutations, including a frameshift in the master regulator of the flagellar system flhD, explains the lack of motility in the plague bacillus, albeit the presence of an almost complete flagellar system (homologous to that of Y. pseudotuberculosis). In this work, we have studied the role in plague of the fliC locus, encoding for the bacterial signature flagellin, a part of the flagellar system previously thought to be inactive in Y. pestis. Very interestingly, we have revealed a role for the fliC locus in pathogenesis, partly due to a regulation of several virulence factors, including the F1 pseudocapsule and T3SS. In this work, we also show that pathogenic Yersinia can modulate their virulence by means of a phenotypic switching process involving a genetic rearrangement in the yscP gene, a gene whose product YscP controls Type 3 Secretion, and the subsequent emergence of a T3S-negative phenotype. We hypothesize that in nature, the T3S-negative Y. pestis subpopulation contributes to the maintenance of low-level infection between epizootics. Finally, studies on flagellar systems has also led us to identify a previously undescribed motility phenomenon in Y. seudotuberculosis. This type of motility predominantly occurs at 37°C (the host temperature), whereas it is repressed at low temperatures. It thus follows a temperature-dependency pattern which is opposite to the previously observed for swimming and swarming

Sélection génétique par les pandémies de peste

Revue pédagogique à l'intention des enseignants en sciences du second degré, aux scientifiques du domaine et plus largement aux lecteurs intéressés.DoctoralLiving organisms best fitted to survive and reproduce are the winners of the natural selection process. Infectious diseases are thought to exert a strong selection pressure on the immune system, thus contributing to eliminate deleterious genes and to positively select genetic variants providing a survival advantage. Because of the enormous death toll payed by mankind to historical plague pandemics, the highly pathogenic bacteria Yersinia pestis probably favored the selection of such genetic variants among survivors. Methods to evidence these effects have considerably evolved in the last decades with the onset of mass genome sequencing and exploding computation capacities, favoring the development of population genetics approaches such as genome-wide association studies (GWAS) over a priori candidate-gene approaches. In this review, early and recent studies and hypotheses on natural selection by plague are gathered and the related immune mechanisms described, when available. Several genes which may have been selected by plague are associated with susceptibility to inflammatory or autoimmune diseases, highlighting the impact of mutations affecting immunity-related genes. Finally, how effects of natural selection on the pathogen itself and its animal hosts mirrors those in man is proposed.Les organismes vivants les mieux adaptés à survivre et se reproduire sont les grands gagnants du processus de sélection naturelle. Les maladies infectieuses exercent une forte pression de sélection sur le système immunitaire, contribuant à éliminer des allèles délétères et à sélectionner positivement les variants amenant un avantage en termes de survie. Du fait que l’humanité a payé un tribut énorme à la peste en nombre de morts durant les pandémies historiques, la bactérie Yersinia pestis, hautement pathogène, a probablement favorisé la sélection de tels variants génétiques chez les survivants. Les méthodes pour analyser ces effets ont considérablement évolué durant les dernières décennies avec l’arrivée du séquençage de génome à haut débit, et l’explosion de la puissance de calcul des ordinateurs, amenant les approches de génétique des populations telles que les études d’association pangénomiques à prendre le pas sur les approches ciblées sur des gènes candidats déterminés a priori. Dans cette revue, les études anciennes et récentes et les hypothèses sur la sélection naturelle par la peste sont présentées et les mécanismes immunitaires correspondants sont décrits lorsqu’ils sont connus. Une série d’allèles qui auraient été sélectionnés par la peste sont associés à une sensibilité accrue à d’autres maladies, mettant l’accent sur l’impact des mutations affectant les gènes liés à l’immunité. Finalement, les effets de la sélection naturelle sur le pathogène lui-même et les animaux réservoirs sont présentés

Mitochondrial ascorbic acid prevents mitochondrial O2.- formation, an event critical for U937 cell apoptosis induced by arsenite through both autophagic-dependent and independent mechanisms

A 16 h exposure of U937 cells to 2.5 µM arsenite promotes superoxide ( O2.-) formation and inhibition of the activity of aconitase, a O2.- sensitive enzyme. Both responses were abolished by the complex I inhibitor rotenone, or by the respiration-deficient phenotype. Interestingly, a similar suppressive effect was mediated by a short term pre-exposure to a low concentration of l-ascorbic acid (AA), previously shown to be actively taken up by the cells and by their mitochondria. The mitochondrial origin of O2.- was confirmed by fluorescence microscopy studies, whereas different approaches failed to detect a contribution of NADPH oxidase. Under similar conditions, arsenite induced autophagy as well as a decline in mitochondrial membrane potential resulting in delayed (48 h) apoptosis. Importantly, all these events turned out to be sensitive to treatments associated with prevention of O2.- formation, including AA, and were only partially blunted by inhibitors of autophagy. As a final note, the toxic effects mediated by O2.- were entirely dependent on its conversion to H2 O2 . AA-sensitive mitochondrial O2.- formation is therefore involved in autophagy and apoptosis induced by arsenite in U937 cells, although part of the lethal response appears mediated by an autophagy-independent mechanism. © 2016 BioFactors, 2016

The study of the mechanism of arsenite toxicity in respiration-deficient cells reveals that NADPH oxidase-derived superoxide promotes the same downstream events mediated by mitochondrial superoxide in respiration-proficient cells

We herein report the results from a comparative study of arsenite toxicity in respiration-proficient (RP) and -deficient (RD) U937 cells. An initial characterization of these cells led to the demonstration that the respiration-deficient phenotype is not associated with apparent changes in mitochondrial mass and membrane potential. In addition, similar levels of superoxide (O2(.-)) were generated by RP and RD cells in response to stimuli specifically triggering respiratory chain-independent mitochondrial mechanisms or extramitochondrial, NADPH-oxidase dependent, mechanisms. At the concentration of 2.5μM, arsenite elicited selective formation of O2(.-) in the respiratory chain of RP cells, with hardly any contribution of the above mechanisms. Under these conditions, O2(.-) triggered downstream events leading to endoplasmic reticulum (ER) stress, autophagy and apoptosis. RD cells challenged with similar levels of arsenite failed to generate O2(.-) because of the lack of a functional respiratory chain and were therefore resistant to the toxic effects mediated by the metalloid. Their resistance, however, was lost after exposure to four fold greater concentrations of arsenite, coincidentally with the release of O2(.-) mediated by NADPH oxidase. Interestingly, extramitochondrial O2(.-) triggered the same downstream events and an identical mode of death previously observed in RP cells. Taken together, the results obtained in this study indicate that arsenite toxicity is strictly dependent on O2(.-) availability that, regardless of whether generated in the mitochondrial or extramitochondrial compartments, triggers similar downstream events leading to ER stress, autophagy and apoptosis

Alpha-tubulin acetylation in Trypanosoma cruzi: a dynamic instabilityof microtubules is required for replication and cell cycle progression

Trypanosomatids have a cytoskeleton arrangement that is simpler than what is found in most eukaryotic cells. However, it is precisely organized and constituted by stable microtubules. Such microtubules compose the mitotic spindle during mitosis, the basal body, the flagellar axoneme and the subpellicular microtubules, which are connected to each other and also to the plasma membrane forming a helical arrangement along the central axis of the parasite cell body. Subpellicular, mitotic and axonemal microtubules are extensively acetylated in Trypanosoma cruzi. Acetylation on lysine (K) 40 of a-tubulin is conserved from lower eukaryotes to mammals and is associated with microtubule stability. It is also known that K40 acetylation occurs significantly on flagella, centrioles, cilia, basal body and the mitotic spindle in eukaryotes. Several tubulin posttranslational modifications, including acetylation of K40, have been cataloged in trypanosomatids, but the functional importance of these modifications for microtubule dynamics and parasite biology remains largely undefined. The primary tubulin acetyltransferase was recently identified in several eukaryotes as Mec-17/ATAT, a Gcn5-related N-acetyltransferase. Here, we report that T. cruzi ATAT acetylates a-tubulin in vivo and is capable of autoacetylation. TcATAT is located in the cytoskeleton and flagella of epimastigotes and colocalizes with acetylated a-tubulin in these structures. We have expressed TcATAT with an HA tag using the inducible vector pTcINDEX-GW in T. cruzi. Over-expression of TcATAT causes increased levels of the alpha tubulin acetylated species, induces morphological and ultrastructural defects, especially in the mitochondrion, and causes a halt in the cell cycle progression of epimastigotes, which is related to an impairment of the kinetoplast division. Finally, as a result of TcATAT over-expression we observed thatFil: Alonso, Victoria Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología Molecular y Celular de Rosario. Laboratorio de Biología y Bioquímica de Trypanosoma cruzi (IBR-CONICET); Argentina.Fil: Alonso, Victoria Lucía. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina.Fil: Carloni, Mara Emilia. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina.Fil: Silva Gonçalves, Camila. Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Ultraestrutura Celular Hertha Meyer; Brazil.Fil: Silva Gonçalves, Camila. Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens; Brazil.Fil: Martínez Peralta, Gonzálo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología Molecular y Celular de Rosario. Laboratorio de Biología y Bioquímica de Trypanosoma cruzi (IBR-CONICET); Argentina.Fil: Martínez Peralta, Gonzálo. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina.Fil: Chesta, Maria Eugenia. Universidad Nacional de Rosario. Facultad de Ciencias Médicas; Argentina.Fil: Pezza, Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología Molecular y Celular de Rosario. Laboratorio de Biología y Bioquímica de Trypanosoma cruzi (IBR-CONICET); Argentina.Fil: Tavernelli, Luis Emilio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología Molecular y Celular de Rosario. Laboratorio de Biología y Bioquímica de Trypanosoma cruzi (IBR-CONICET); Argentina.Fil: Motta, Maria Cristina M. Universidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Ultraestrutura Celular Hertha Meyer; Brazil.Fil: Motta, María Cristina M. Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens; Brazil.Fil: Serra, Esteban Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología Molecular y Celular de Rosario. Laboratorio de Biología y Bioquímica de Trypanosoma cruzi (IBR-CONICET); Argentina.Fil: Serra, Esteban Carlos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina