Expression of the bacterial type III effector DspA/E in Saccharomyces cerevisiae downregulates the sphingolipid biosynthetic pathway leading to growth-arrest

Erwinia amylovora, the bacterium responsible for fire blight, relies on a type III secretion system and a single injected effector, DspA/E, to induce disease in host plants. DspA/E belongs to the widespread AvrE family of type III effectors which suppress plant defense responses and promote bacterial growth followinginfection. Ectopic expression of DspA/E in plant or in Saccharomyces cerevisiae is toxic indicating that DspA/E likely targets a cellular process conserved between yeast and plant. To unravel the mode of action of DspA/E, we screened the Euroscarf, S. cerevisiae library for mutants resistant toDspA/E-induced growth arrest. The most resistant mutants (Δsur4, Δfen1, Δipt1,Δskn1, Δcsg1, Δcsg2, Δorm1, Δorm2) were impaired in the sphingolipid biosynthetic pathway. Exogenously supplied sphingolipid precursors such as the long chain bases(LCBs) phytosphingosine and dihydrosphingosine also suppressed DspA/E-induced yeast growth defect. Expression of DspA/E in yeast downregulated LCBs biosynthesis and induced a rapid decrease in LCB levels,indicating that SPT, the first and rate limiting enzyme of the sphingolipid biosynthetic pathway was repressed. SPT downregulation was mediated by dephosphorylation and activation of Orm proteins that negatively regulate SPT. A Δcdc55 mutation, affecting Cdc55-PP2A protein phosphatase activity, prevented Orm dephosphorylation and suppressed DspA/E-induced growth arrest

Mutational analysis of a predicted double β-propeller domain of the DspA/E effector of Erwinia amylovora

The bacterium Erwinia amylovora causes fire blight, an invasive disease that threatens apple trees, pear trees and other plants of the Rosaceae family. Erwinia amylovora pathogenicity relies on a type III secretion system and on a single effector DspA/E. This effector belongs to the widespread AvrE family of effectors whose biological function is unknown. In this manuscript, we performed a bioinformatic analysis of DspA/E- and AvrE-related effectors. Motif search identified nuclear localization signals, peroxisome targeting signals, endoplasmic reticulum membrane retention signals and leucine zipper motifs, but none of these motifs were present in all the AvrE-related effectors analysed. Protein threading analysis, however, predicted a conserved double β-propeller domain in the N-terminal part of all the analysed effector sequences. We then performed a random pentapeptide mutagenesis of DspA/E, which led to the characterization of 13 new altered proteins with a five amino acids insertion. Eight harboured the insertion inside the predicted β-propeller domain and six of these eight insertions impaired DspA/E stability or function. Conversely, the two remaining insertions generated proteins that were functional and abundantly secreted in the supernatant suggesting that these two insertions stabilized the protein

Excellent ambient oxidation and mineralization of an emerging water pollutant using Pd-doped TiO2_{{2}} photocatalyst and UV-A irradiation

International audienceTiO2_Pd prepared by the incipient wet impregnation (IWI) method was successfully used as a photocatalyst for the degradation of an emerging water pollutant, clofibric acid (CA). It exhibits an improved photoactivity in comparison with different commercial titania in the degradation of CA (25 ppm). The irradiation intensity, photocatalyst dose, CA concentration and influence of water quality and of some salts in the reaction medium were systematically examined to understand their effects on the process efficiency. A total pollutant decomposition and a high mineralization yield (78%) were achieved in 50 and 190 min, respectively, in the optimal conditions, which is very promising for practical applications

The HrpN Effector of Erwinia amylovora , Which Is Involved in Type III Translocation, Contributes Directly or Indirectly to Callose Elicitation on Apple Leaves

Erwinia amylovora is responsible for fire blight of apple and pear trees. Its pathogenicity depends on a type III secretion system (T3SS) mediating the translocation of effectors into the plant cell. The DspA/E effector suppresses callose deposition on apple leaves. We found that E. amylovora and Pseudomonas syringae DC3000 tts mutants or peptide flg22 do not trigger callose deposition as strongly as the dspA/E mutant on apple leaves. This suggests that, on apple leaves, callose deposition is poorly elicited by pathogen-associated molecular patterns (PAMPs) such as flg22 or other PAMPs harbored by tts mutants and is mainly elicited by injected effectors or by the T3SS itself. Callose elicitation partly depends on HrpW because an hrpW-dspA/E mutant elicits lower callose deposition than a dspA/E mutant. Furthermore, an hrpN-dspA/E mutant does not trigger callose deposition, indicating that HrpN is required to trigger this plant defense reaction. We showed that HrpN plays a general role in the translocation process. Thus, the HrpN requirement for callose deposition may be explained by its role in translocation: HrpN could be involved in the translocation of other effectors inducing callose deposition. Furthermore, HrpN may also directly contribute to the elicitation process because we showed that purified HrpN induces callose deposition

Type IV secretion in Gram-negative and Gram-positive bacteria

Type IV secretion systems (T4SSs) are versatile multiprotein nanomachines spanning the entire cell envelope in Gram‐negative and Gram‐positive bacteria. They play important roles through the contact‐dependent secretion of effector molecules into eukaryotic hosts and conjugative transfer of mobile DNA elements as well as contact‐independent exchange of DNA with the extracellular milieu. In the last few years, many details on the molecular mechanisms of T4SSs have been elucidated. Exciting structures of T4SS complexes from Escherichia coli plasmids R388 and pKM101, Helicobacter pylori and Legionella pneumophila have been solved. The structure of the F‐pilus was also reported and surprisingly revealed a filament composed of pilin subunits in 1:1 stoichiometry with phospholipid molecules. Many new T4SSs have been identified and characterized, underscoring the structural and functional diversity of this secretion superfamily. Complex regulatory circuits also have been shown to control T4SS machine production in response to host cell physiological status or a quorum of bacterial recipient cells in the vicinity. Here, we summarize recent advances in our knowledge of ‘paradigmatic’ and emerging systems, and further explore how new basic insights are aiding in the design of strategies aimed at suppressing T4SS functions in bacterial infections and spread of antimicrobial resistances

Analysis of the toxicity of DspA, a protein essential for the pathogenicity of Erwinia amylovora, in the yeast Saccharomyces cerevisiae

La bactérie phytopathogène E. amylovora, est l'agent responsable du Feu bactérien des Spiraeoideae (pommier, poirier, pyracantha), une maladie caractérisée par l'apparition de symptômes nécrotiques des tissus infectés. Le pouvoir pathogène d’E. amylovora repose entre autre sur un système de sécrétion de type III (SSTT) qui permet la sécrétion et l'injection d'effecteurs dans la cellule hôte végétale. Parmi les protéines injectées par le T3SS d'E. amylovora, DspA est essentielle au pouvoir pathogène de la bactérie puisqu’un mutant dspA est non pathogène sur plante (Gaudriault et al., 1997). Le rôle de DspA est dual, d’une part, l’expression de dspA est suffisante pour provoquer des symptômes nécrotiques sur plante et une toxicité chez la levure, d’autre part, DspA est impliquée dans la suppression des réactions de défense telles que la déposition de callose (Degrave et al., 2008; Boureau et al., 2006; Oh et al., 2007; DebRoy et al., 2004). DspA appartient à la famille des effecteurs AvrE qui sont répandus chez les bactéries phytopathogènes et semblent posséder une fonction similaire. Cependant, peu de connaissance existe sur la structure ainsi que la fonction de DspA. L'objectif de ce travail de thèse était de déterminer les domaines ou motifs importants pour la fonction de DspA. Pour cela nous avons choisi d'effectuer une analyse in silico et fonctionnelle de la protéine DspA. L'analyse in silico révèle la présence d'un domaine bêta-propeller au sein de la protéine DspA ainsi que de tous les homologues analysés. De plus, l'analyse fonctionnelle indique que ce domaine est important pour la structure et la fonction de DspA. Dans un second temps, j'ai étudié le mécanisme d'action de DspA dans la levure Saccharomyces cerevisiae. J'ai pu mettre en évidence que l'expression de dspA chez la levure induit un arrêt de croissance et une forte altération du trafic cellulaire. L'étude de mutants de levure suppresseurs de la toxicité de DspA, effectuée avant mon arrivée au laboratoire, montre que les suppresseurs les plus forts sont affectés dans la voie de biosynthèse des sphingolipides, je me suis donc plus particulièrement intéressée au rôle des sphingolipides dans la toxicité générée par DspA. Nos résultats montrent que DspA inhibe la biosynthèse des sphingolipides indirectement via les régulateurs négatifs de la voie, les protéines Orms.Erwinia amylovora is the causative agent of fire blight of Spiraeoideae (apple, pear, pyracantha), a disease characterized by the apparition of necrotic symptoms on infected tissues. The pathogenicity of E. amylovora relies on a functional type III secretion system (T3SS) that allows secretion and injection of effector proteins into the host plant cell. Among these effector proteins injected by the T3SS of E. amylovora, DspA is essential to the bacteria disease process since a dspA mutant is nonpathogenic on plants (Gaudriault et al., 1997). DspA has a dual role; on the one hand dspA expression is sufficient to induce cell death on plants and toxicity on yeast, on the other hand, DspA is involved on suppression of defense reactions like callose deposition (Degrave et al., 2008; Boureau et al., 2006; Oh et al., 2007; DebRoy et al., 2004). DspA belongs to the AvrE familly of type III effectors which are widespread on phytopathogenic bacteria and likely possess a similar function. However, the structure and function of DspA remain unknown. In the first part of my thesis, I attempted to characterize domains or motifs important for the function of DspA. We performed an in silico and a functional analysis of the DspA protein. In silico analysis predicted a bêta-propeller domain in DspA and all the analysed effectors. In the second part of my thesis, I analysed the mechanism of function of DspA in the yeast Saccharomyces cerevisiae. Results showed that expression of dspA in yeast inhibits cell growth and alters the actin cytoskeleton and endocytosis. Screening of the Euroscarf library for mutants resistant to DspA induced toxicity revealed that mutants impaired in the sphingolipid biosynthetic pathway are the best suppressors. Based on this results, I attempted to determine the role of sphingolipids in the toxicity induced by DspA. Results showed that DspA inhibits indirectly the sphingolipid biosynthetic pathway via the negative regulators, Orm proteins

New insights into the role of Bartonella effector proteins in pathogenesis

The facultative intracellular bacteria Bartonella spp. share a common infection strategy to invade and colonize mammals in a host-specific manner. Following transmission by blood-sucking arthropods, Bartonella are inoculated in the derma and then spread, via two sequential enigmatic niches, to the blood stream where they cause a long-lasting intra-erythrocytic bacteraemia. The VirB/VirD4 type IV secretion system (VirB/D4 T4SS) is essential for the pathogenicity of most Bartonella species by injecting an arsenal of effector proteins into host cells. These bacterial effector proteins share a modular architecture, comprising domains and/or motifs that confer an array of functions. Here, we review recent advances in understanding the function and evolutionary origin of this fascinating repertoire of host-targeted bacterial effectors

Étude de la toxicité de DspA, protéine essentielle au pouvoir pathogène d'Erwinia amylovora, chez la levure Saccharomyces cerevisiae

La bactérie phytopathogène E. amylovora, est l'agent responsable du Feu bactérien des Spiraeoideae (pommier, poirier, pyracantha), une maladie caractérisée par l'apparition de symptômes nécrotiques des tissus infectés. Le pouvoir pathogène d E. amylovora repose entre autre sur un système de sécrétion de type III (SSTT) qui permet la sécrétion et l'injection d'effecteurs dans la cellule hôte végétale. Parmi les protéines injectées par le T3SS d'E. amylovora, DspA est essentielle au pouvoir pathogène de la bactérie puisqu un mutant dspA est non pathogène sur plante (Gaudriault et al., 1997). Le rôle de DspA est dual, d une part, l expression de dspA est suffisante pour provoquer des symptômes nécrotiques sur plante et une toxicité chez la levure, d autre part, DspA est impliquée dans la suppression des réactions de défense telles que la déposition de callose (Degrave et al., 2008; Boureau et al., 2006; Oh et al., 2007; DebRoy et al., 2004). DspA appartient à la famille des effecteurs AvrE qui sont répandus chez les bactéries phytopathogènes et semblent posséder une fonction similaire. Cependant, peu de connaissance existe sur la structure ainsi que la fonction de DspA. L'objectif de ce travail de thèse était de déterminer les domaines ou motifs importants pour la fonction de DspA. Pour cela nous avons choisi d'effectuer une analyse in silico et fonctionnelle de la protéine DspA. L'analyse in silico révèle la présence d'un domaine bêta-propeller au sein de la protéine DspA ainsi que de tous les homologues analysés. De plus, l'analyse fonctionnelle indique que ce domaine est important pour la structure et la fonction de DspA. Dans un second temps, j'ai étudié le mécanisme d'action de DspA dans la levure Saccharomyces cerevisiae. J'ai pu mettre en évidence que l'expression de dspA chez la levure induit un arrêt de croissance et une forte altération du trafic cellulaire. L'étude de mutants de levure suppresseurs de la toxicité de DspA, effectuée avant mon arrivée au laboratoire, montre que les suppresseurs les plus forts sont affectés dans la voie de biosynthèse des sphingolipides, je me suis donc plus particulièrement intéressée au rôle des sphingolipides dans la toxicité générée par DspA. Nos résultats montrent que DspA inhibe la biosynthèse des sphingolipides indirectement via les régulateurs négatifs de la voie, les protéines Orms.Erwinia amylovora is the causative agent of fire blight of Spiraeoideae (apple, pear, pyracantha), a disease characterized by the apparition of necrotic symptoms on infected tissues. The pathogenicity of E. amylovora relies on a functional type III secretion system (T3SS) that allows secretion and injection of effector proteins into the host plant cell. Among these effector proteins injected by the T3SS of E. amylovora, DspA is essential to the bacteria disease process since a dspA mutant is nonpathogenic on plants (Gaudriault et al., 1997). DspA has a dual role; on the one hand dspA expression is sufficient to induce cell death on plants and toxicity on yeast, on the other hand, DspA is involved on suppression of defense reactions like callose deposition (Degrave et al., 2008; Boureau et al., 2006; Oh et al., 2007; DebRoy et al., 2004). DspA belongs to the AvrE familly of type III effectors which are widespread on phytopathogenic bacteria and likely possess a similar function. However, the structure and function of DspA remain unknown. In the first part of my thesis, I attempted to characterize domains or motifs important for the function of DspA. We performed an in silico and a functional analysis of the DspA protein. In silico analysis predicted a bêta-propeller domain in DspA and all the analysed effectors. In the second part of my thesis, I analysed the mechanism of function of DspA in the yeast Saccharomyces cerevisiae. Results showed that expression of dspA in yeast inhibits cell growth and alters the actin cytoskeleton and endocytosis. Screening of the Euroscarf library for mutants resistant to DspA induced toxicity revealed that mutants impaired in the sphingolipid biosynthetic pathway are the best suppressors. Based on this results, I attempted to determine the role of sphingolipids in the toxicity induced by DspA. Results showed that DspA inhibits indirectly the sphingolipid biosynthetic pathway via the negative regulators, Orm proteins.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Expression of the bacterial type III effector DspA/E in Saccharomyces cerevisiae downregulates the sphingolipid biosynthetic pathway leading to growth-arrest

Erwinia amylovora, the bacterium responsible for fire blight, relies on a type III secretion system and a single injected effector, DspA/E, to induce disease in host plants. DspA/E belongs to the widespread AvrE family of type III effectors which suppress plant defense responses and promote bacterial growth followinginfection. Ectopic expression of DspA/E in plant or in Saccharomyces cerevisiae is toxic indicating that DspA/E likely targets a cellular process conserved between yeast and plant. To unravel the mode of action of DspA/E, we screened the Euroscarf, S. cerevisiae library for mutants resistant toDspA/E-induced growth arrest. The most resistant mutants (Δsur4, Δfen1, Δipt1,Δskn1, Δcsg1, Δcsg2, Δorm1, Δorm2) were impaired in the sphingolipid biosynthetic pathway. Exogenously supplied sphingolipid precursors such as the long chain bases(LCBs) phytosphingosine and dihydrosphingosine also suppressed DspA/E-induced yeast growth defect. Expression of DspA/E in yeast downregulated LCBs biosynthesis and induced a rapid decrease in LCB levels,indicating that SPT, the first and rate limiting enzyme of the sphingolipid biosynthetic pathway was repressed. SPT downregulation was mediated by dephosphorylation and activation of Orm proteins that negatively regulate SPT. A Δcdc55 mutation, affecting Cdc55-PP2A protein phosphatase activity, prevented Orm dephosphorylation and suppressed DspA/E-induced growth arrest