Higher Prevalence of PldA, a Pseudomonas aeruginosa Trans-Kingdom H2-Type VI Secretion System Effector, in Clinical Isolates Responsible for Acute Infections and in Multidrug Resistant Strains

Pseudomonas aeruginosa can manipulate eukaryotic host cells using secreted effectors delivered by the type III or the type VI Secretion Systems (T3SS and T6SS). The T3SS allows the injection of bacterial effectors (Exo toxins) into eukaryotic cell. P. aeruginosa, encodes three T6SSs, H1-, H2- and H3-T6SS. The H1-T6SS is mainly involved in delivering toxins to kill bacterial competitors. Recently, two T6SS-secreted phospholipases D, PldA (H2-T6SS) and PldB (H3-T6SS), were identified as trans-kingdom virulence effectors, triggering both killing of bacterial competitors and internalization into non-phagocytic cells. We deciphered the prevalence of T3SS and T6SS effectors encoding genes in 185 clinical isolates responsible for infections (septicaemia, pulmonary infections, urinary tract infections, and chronic infections in CF patients), 47 environmental strains, and on 33 carbapenemase-producers. We included 107 complete genomes of P. aeruginosa available in public databases. The prevalence of pldA is increased in clinical isolates responsible for severe acute infection and particularly in multi-drug resistant strains. In contrast, the pldB prevalence was high (96.8%) in all isolates. Regarding T3SS effectors, exoT and exoY are present in nearly all isolates while exoS and exoU were found to be exclusive with a higher prevalence of exoU+ strains in severe acute infections. The hypervirulent exoU+ isolates are more prone to be pldA+, suggesting a role of PldA in virulence. Finally, we observed that extremely drug resistant isolates producing an IMP-type carbapenemase were all pldA+. Our results suggest that PldA might have a role during pulmonary infections and have been co-selected in multidrug resistant strains particularly IMP-producers

An NAD+ Phosphorylase Toxin Triggers Mycobacterium tuberculosis Cell Death.

Toxin-antitoxin (TA) systems regulate fundamental cellular processes in bacteria and represent potential therapeutic targets. We report a new RES-Xre TA system in multiple human pathogens, including Mycobacterium tuberculosis. The toxin, MbcT, is bactericidal unless neutralized by its antitoxin MbcA. To investigate the mechanism, we solved the 1.8 Å-resolution crystal structure of the MbcTA complex. We found that MbcT resembles secreted NAD+-dependent bacterial exotoxins, such as diphtheria toxin. Indeed, MbcT catalyzes NAD+ degradation in vitro and in vivo. Unexpectedly, the reaction is stimulated by inorganic phosphate, and our data reveal that MbcT is a NAD+ phosphorylase. In the absence of MbcA, MbcT triggers rapid M. tuberculosis cell death, which reduces mycobacterial survival in macrophages and prolongs the survival of infected mice. Our study expands the molecular activities employed by bacterial TA modules and uncovers a new class of enzymes that could be exploited to treat tuberculosis and other infectious diseases

Étude d'une nouvelle classe de métallochaperons impliqués dans la résistance à l'intoxication par les métaux chez Mycobacterium tuberculosis

Metals are essential trace elements for all living organisms yet their accumulation is toxic and causes deleterious effects. Immune cells have many mechanisms for the elimination of pathogens. In particular, they are capable of manipulating intracellular metal concentrations, and can poison them by confining them into specialized cell compartments enriched in metals such as copper or zinc. Resistance to intoxication by transition metals has emerged as an important virulence feature of bacterial pathogens. In the major human pathogen Mycobacterium tuberculosis, resistance to zinc is mediated by the P-type ATPase efflux pump CtpC. The ctpC gene is located in operon with Rv3269, a gene encoding a 93-amino acids protein of unknown function. The main objective of my thesis focused on the involvement of Rv3269 in zinc resistance and its relationship with CtpC. Using molecular genetic and biochemical approaches, my thesis work demonstrated that Rv3269 is a novel membrane-bound zinc-binding protein necessary for zinc resistance in M. tuberculosis. Rv3269 and CtpC co-localize in functional membrane microdomains (FMMs) in M. tuberculosis, and Rv3269 contains motifs typical of prokaryotic flotillin (FloT)-like scaffold proteins that are involved in FMM assembly. Genetic disruption of the transmembrane domain, of the zinc-binding domain or of the FloT-like motifs in Rv3269 renders M. tuberculosis highly sensitive to zinc. Similar Rv3269-ctpC-like tandems are present in the genome of other bacteria. Thus, Rv3269 defines an unprecedented class of metallochaperone scaffold-like proteins dedicated to P-ATPase-mediated metal efflux through FMMs in prokaryotes.Les métaux sont des oligo-éléments essentiels pour tous les organismes vivants, néanmoins leur accumulation est toxique et provoque des effets délétères. Les cellules immunitaires sont dotées de nombreux moyens pour l'élimination d'agents pathogènes. Capable de manipuler les concentrations métalliques intracellulaires, elles peuvent en particulier les intoxiquer en les confinant dans des compartiments cellulaires spécialisés riches en métaux, comme le cuivre ou le zinc. La résistance à l'intoxication par les métaux de transition est récemment apparue comme un des mécanismes importants de la virulence des bactéries pathogènes. Chez Mycobacterium tuberculosis, un pathogène majeur responsable de la tuberculose, la résistance au zinc est médiée par une pompe à efflux, l'ATPase de type P CtpC. Le gène ctpC se trouve en opéron avec Rv3269, un gène codant une protéine de 93 acides aminés de fonction inconnue. L'objectif principal de ma thèse s'est concentré sur l'implication de Rv3269 dans la résistance au zinc et sa relation avec CtpC. Par des approches de génétique moléculaire et biochimie, mes travaux de thèse ont permis de démontrer que Rv3269 est elle aussi nécessaire pour la résistance au zinc. Rv3269 contient un motif de fixation du zinc et est associée à la membrane cytoplasmique. Rv3269 et CtpC co-localisent dans des microdomaines membranaires fonctionnels dynamiques au sein de la membrane de M. tuberculosis et Rv3269 présente des motifs typiquement rencontrés chez les flotillines (FloT), des protéines organisatrices de microdomaines membranaires chez les bactéries. L'analyse génétique de Rv3269 a montré que l'altération du domaine transmembranaire, des motifs de type FloT ou du domaine de fixation du zinc ont toutes pour conséquence de rendre M. tuberculosis sensible au zinc. Deux autres P-ATPases de M. tuberculosis sont aussi associées à de petites protéines similaires à Rv3269. Ces petites protéines co-localisent avec Rv3269. Une analyse phylogénétique indique que ce type d'organisation est retrouvé chez d'autres espèces bactériennes. Nous proposons que Rv3269 est le premier exemple décrit d'une nouvelle classe de métallochaperons capables d'organiser des plateformes fonctionnelles dédiées à l'efflux des métaux de transition chez les bactéries

Study of a new class of metallochaperons involved in resistance to metal intoxication in Mycobacterium tuberculosis

Les métaux sont des oligo-éléments essentiels pour tous les organismes vivants, néanmoins leur accumulation est toxique et provoque des effets délétères. Les cellules immunitaires sont dotées de nombreux moyens pour l'élimination d'agents pathogènes. Capable de manipuler les concentrations métalliques intracellulaires, elles peuvent en particulier les intoxiquer en les confinant dans des compartiments cellulaires spécialisés riches en métaux, comme le cuivre ou le zinc. La résistance à l'intoxication par les métaux de transition est récemment apparue comme un des mécanismes importants de la virulence des bactéries pathogènes. Chez Mycobacterium tuberculosis, un pathogène majeur responsable de la tuberculose, la résistance au zinc est médiée par une pompe à efflux, l'ATPase de type P CtpC. Le gène ctpC se trouve en opéron avec Rv3269, un gène codant une protéine de 93 acides aminés de fonction inconnue. L'objectif principal de ma thèse s'est concentré sur l'implication de Rv3269 dans la résistance au zinc et sa relation avec CtpC. Par des approches de génétique moléculaire et biochimie, mes travaux de thèse ont permis de démontrer que Rv3269 est elle aussi nécessaire pour la résistance au zinc. Rv3269 contient un motif de fixation du zinc et est associée à la membrane cytoplasmique. Rv3269 et CtpC co-localisent dans des microdomaines membranaires fonctionnels dynamiques au sein de la membrane de M. tuberculosis et Rv3269 présente des motifs typiquement rencontrés chez les flotillines (FloT), des protéines organisatrices de microdomaines membranaires chez les bactéries. L'analyse génétique de Rv3269 a montré que l'altération du domaine transmembranaire, des motifs de type FloT ou du domaine de fixation du zinc ont toutes pour conséquence de rendre M. tuberculosis sensible au zinc. Deux autres P-ATPases de M. tuberculosis sont aussi associées à de petites protéines similaires à Rv3269. Ces petites protéines co-localisent avec Rv3269. Une analyse phylogénétique indique que ce type d'organisation est retrouvé chez d'autres espèces bactériennes. Nous proposons que Rv3269 est le premier exemple décrit d'une nouvelle classe de métallochaperons capables d'organiser des plateformes fonctionnelles dédiées à l'efflux des métaux de transition chez les bactéries.Metals are essential trace elements for all living organisms yet their accumulation is toxic and causes deleterious effects. Immune cells have many mechanisms for the elimination of pathogens. In particular, they are capable of manipulating intracellular metal concentrations, and can poison them by confining them into specialized cell compartments enriched in metals such as copper or zinc. Resistance to intoxication by transition metals has emerged as an important virulence feature of bacterial pathogens. In the major human pathogen Mycobacterium tuberculosis, resistance to zinc is mediated by the P-type ATPase efflux pump CtpC. The ctpC gene is located in operon with Rv3269, a gene encoding a 93-amino acids protein of unknown function. The main objective of my thesis focused on the involvement of Rv3269 in zinc resistance and its relationship with CtpC. Using molecular genetic and biochemical approaches, my thesis work demonstrated that Rv3269 is a novel membrane-bound zinc-binding protein necessary for zinc resistance in M. tuberculosis. Rv3269 and CtpC co-localize in functional membrane microdomains (FMMs) in M. tuberculosis, and Rv3269 contains motifs typical of prokaryotic flotillin (FloT)-like scaffold proteins that are involved in FMM assembly. Genetic disruption of the transmembrane domain, of the zinc-binding domain or of the FloT-like motifs in Rv3269 renders M. tuberculosis highly sensitive to zinc. Similar Rv3269-ctpC-like tandems are present in the genome of other bacteria. Thus, Rv3269 defines an unprecedented class of metallochaperone scaffold-like proteins dedicated to P-ATPase-mediated metal efflux through FMMs in prokaryotes

Mycobacterium abscessus

International audienceNo abstract availabl

Mycobacterial resistance to zinc poisoning requires assembly of P-ATPase-containing membrane metal efflux platforms

International audienceThe human pathogen Mycobacterium tuberculosis requires a P-1B-ATPase metal exporter, CtpC (Rv3270), for resistance to zinc poisoning. Here, we show that zinc resistance also depends on a chaperone-like protein, PacL1 (Rv3269). PacL1 contains a transmembrane domain, a cytoplasmic region with glutamine/alanine repeats and a C-terminal metal-binding motif (MBM). PacL1 binds Zn2+, but the MBM is required only at high zinc concentrations. PacL1 co-localizes with CtpC in dynamic foci in the mycobacterial plasma membrane, and the two proteins form high molecular weight complexes. Foci formation does not require flotillin nor the PacL1 MBM. However, deletion of the PacL1 Glu/Ala repeats leads to loss of CtpC and sensitivity to zinc. Genes pacL1 and ctpC appear to be in the same operon, and homologous gene pairs are found in the genomes of other bacteria. Furthermore, PacL1 colocalizes and functions redundantly with other PacL orthologs in M. tuberculosis. Overall, our results indicate that PacL proteins may act as scaffolds that assemble P-ATPase-containing metal efflux platforms mediating bacterial resistance to metal poisoning.The human pathogen Mycobacterium tuberculosis requires a metal exporter, CtpC, for resistance to zinc poisoning. Here, the authors show that zinc resistance also depends on a chaperone-like protein that binds zinc ions, forms high-molecular-weight complexes with CtpC in the cytoplasmic membrane, and is required for CtpC function

A Mycobacterium tuberculosis Effector Targets Mitochondrion, Controls Energy Metabolism, and Limits Cytochrome c Exit

International audienceHost metabolism reprogramming is a key feature of Mycobacterium tuberculosis (Mtb) infection that enables the survival of this pathogen within phagocytic cells and modulates the immune response facilitating the spread of the tuberculosis disease. Here, we demonstrate that a previously uncharacterized secreted protein from Mtb, Rv1813c, manipulates the host metabolism by targeting mitochondria. When expressed in eukaryotic cells, the protein is delivered to the mitochondrial intermembrane space and promotes the enhancement of host ATP production by boosting the oxidative phosphorylation metabolic pathway. Furthermore, the release of cytochrome c from mitochondria, an early apoptotic event in response to short-term oxidative stress, is delayed in Rv1813c-expressing cells. This study reveals a novel class of mitochondria targeting effectors from Mtb that might participate in host cell metabolic reprogramming and apoptosis control during Mtb infections. IMPORTANCE In this article, using a combination of techniques (bioinformatics, structural biology, and cell biology), we identified and characterized a new class of effectors present only in intracellular mycobacteria. These proteins specifically target host cell mitochondria when ectopically expressed in cells. We showed that one member of this family (Rv1813c) affects mitochondria metabolism in a way that might twist the immune response. This effector also inhibits the cytochrome c exit from mitochondria, suggesting that it might alter normal host cell apoptotic capacities, one of the first defenses of immune cells against Mtb infection