Etude des mécanismes d'activation transcriptionnelle des gènes de virulence et des fonctions d'adaptation in planta chez la bactérie Ralstonia solanacearum

HrpG est le régulateur central de la virulence chez la bactérie phytopathogène Ralstonia solanacearum. HrpG intègre des signaux environnementaux et hôte-dépendants et induit la transcription du système de sécrétion de type III via HrpB, ainsi que près de 200 gènes indépendants de HrpB. Un régulateur fortement homologue à HrpG a été identifié et caractérisé. Ce régulateur, PrhG, induit également l'expression de hrpB mais uniquement en réponse au signal environnemental. Nous avons montré que HrpG et PrhG sont deux régulateurs directs de hrpB et dont les séquences cis-régulatrices sont partiellement communes. Les études effectuées visent à comprendre comment HrpG et PrhG se partagent le contrôle de l'expression de hrpB et à mieux définir le régulon HrpG pour identifier les fonctions impliquées dans la colonisation et l'adaptation in planta.HrpG is the central virulence regulator of the phytopathogenic bacterium Ralstonia solanacearum. HrpG integrates environmental and host-dependent signals and induces the expression of type III secretion system genes via HrpB, as well as almost 200 genes independent from HrpB. A regulator highly homologous to HrpG was identified and characterised. This regulator, called PrhG, also induces the expression of hrpB, but only upon the environmental signal. We showed that HrpG and PrhG are two direct regulators of hrpB and that they partly require the same cis-regulatory sequences. The objective of our study is to understand how HrpG and PrhG share the control of hrpB expression and to define more precisely HrpG regulon in order to identify functions implicated in colonisation and adaptation to the host environment

Harnessing a versatile robust lactonase for biotechnological applications

Extremozymes have gained considerable interest as they could meet industrial requirements. Among these, SsoPox is a hyperthermostable enzyme isolated from the archaeon Sulfolobus solfataricus1. This enzyme is a lactonase catalyzing the hydrolysis of acyl-homoserine lactones; these molecules are involved in Gram-negative bacterial communication referred to as quorum sensing2. SsoPox exhibits promiscuous phosphotriesterase activity for the degradation of organophosphorous chemicals including insecticides and chemical warfare agents3. Owing to its bi-functional catalytic abilities as well as its intrinsic stability, SsoPox is appealing for many applications, having potential uses in the agriculture, defense, food and health industries. This enzyme have been rationally engineered and highly improved lactonase and phosphotriesterase variants were isolated4. Their biotechnological properties were investigated and their resistance against diverse process-like and operating conditions such as heat resistance, contact with organic solvents, sterilization, storage and immobilization were underlined5. Lactonase improved variants were shown to drastically reduce virulence and biofilm formation in clinical isolates of Pseudomonas aeruginosa and to decrease mortality in rat pneumonia model6,7. The antibiofilm capacity of the enzyme was also proved to be of outmost interest for antifouling applications. Enhanced phosphotriesterase variants were shown to efficiently decontaminate a broad panel of organophosphorus insecticides and were successfully incorporated into filtration devices for bioremediation purposes8. The degradation products generated through enzyme hydrolysis drastically reduced toxicity and increased regeneration capacity in planarian, an original Plathelmintes model. Regarding their tremendous stability these variants are currently used to develop antibacterial medical devices, antifouling paintings and insecticide bioremediation tools. 1. Elias, M. et al. Structural Basis for Natural Lactonase and Promiscuous Phosphotriesterase Activities. J. Mol. Biol. 379, 1017–1028 (2008). 2. Bzdrenga, J. et al. Biotechnological applications of quorum quenching enzymes. Chem. Biol. Interact. (2016). doi:10.1016/j.cbi.2016.05.028 3. Jacquet, P. et al. Current and emerging strategies for organophosphate decontamination: special focus on hyperstable enzymes. Environ. Sci. Pollut. Res. 1–19 (2016). doi:10.1007/s11356-016-6143-1 4. Hiblot, J., Gotthard, G., Elias, M. & Chabriere, E. Differential Active Site Loop Conformations Mediate Promiscuous Activities in the Lactonase SsoPox. PLoS ONE 8, e75272 (2013). 5. Rémy, B. et al. Harnessing hyperthermostable lactonase from Sulfolobus solfataricus for biotechnological applications. Sci. Rep. 6, (2016). 6. Guendouze, A. et al. Effect of quorum quenching lactonase in clinical isolates of Pseudomonas aeruginosa and comparison with quorum sensing inhibitors. Front. Microbiol. 8, (2017). 7. Hraiech, S. et al. Inhaled Lactonase Reduces Pseudomonas aeruginosa Quorum Sensing and Mortality in Rat Pneumonia. PLoS ONE 9, e107125 (2014). 8. Hiblot, J., Gotthard, G., Chabriere, E. & Elias, M. Characterisation of the organophosphate hydrolase catalytic activity of SsoPox. Sci. Rep. 2, (2012)

Lactonase-mediated inhibition of quorum sensing largely alters phenotypes, proteome, and antimicrobial activities in Burkholderia thailandensis E264

IntroductionBurkholderia thailandensis is a study model for Burkholderia pseudomallei, a highly virulent pathogen, known to be the causative agent of melioidosis and a potential bioterrorism agent. These two bacteria use an (acyl-homoserine lactone) AHL-mediated quorum sensing (QS) system to regulate different behaviors including biofilm formation, secondary metabolite productions, and motility.MethodsUsing an enzyme-based quorum quenching (QQ) strategy, with the lactonase SsoPox having the best activity on B. thailandensis AHLs, we evaluated the importance of QS in B. thailandensis by combining proteomic and phenotypic analyses.ResultsWe demonstrated that QS disruption largely affects overall bacterial behavior including motility, proteolytic activity, and antimicrobial molecule production. We further showed that QQ treatment drastically decreases B. thailandensis bactericidal activity against two bacteria (Chromobacterium violaceum and Staphylococcus aureus), while a spectacular increase in antifungal activity was observed against fungi and yeast (Aspergillus niger, Fusarium graminearum and Saccharomyces cerevisiae).DiscussionThis study provides evidence that QS is of prime interest when it comes to understanding the virulence of Burkholderia species and developing alternative treatments

The Janthinobacterium sp. HH01 genome encodes a homologue of the V. cholerae CqsA and L. pneumophila LqsA autoinducer synthases

Janthinobacteria commonly form biofilms on eukaryotic hosts and are known to synthesize antibacterial and antifungal compounds. Janthinobacterium sp. HH01 was recently isolated from an aquatic environment and its genome sequence was established. The genome consists of a single chromosome and reveals a size of 7.10 Mb, being the largest janthinobacterial genome so far known. Approximately 80% of the 5,980 coding sequences (CDSs) present in the HH01 genome could be assigned putative functions. The genome encodes a wealth of secretory functions and several large clusters for polyketide biosynthesis. HH01 also encodes a remarkable number of proteins involved in resistance to drugs or heavy metals. Interestingly, the genome of HH01 apparently lacks the N-acylhomoserine lactone (AHL)-dependent signaling system and the AI-2-dependent quorum sensing regulatory circuit. Instead it encodes a homologue of the Legionella- and Vibrio-like autoinducer (lqsA/cqsA) synthase gene which we designated jqsA. The jqsA gene is linked to a cognate sensor kinase (jqsS) which is flanked by the response regulator jqsR. Here we show that a jqsA deletion has strong impact on the violacein biosynthesis in Janthinobacterium sp. HH01 and that a jqsA deletion mutant can be functionally complemented with the V. cholerae cqsA and the L. pneumophila lqsA genes

Metabolic Adaptation of Ralstonia solanacearum during Plant Infection: A Methionine Biosynthesis Case Study

MetE and MetH are two distinct enzymes that catalyze a similar biochemical reaction during the last step of methionine biosynthesis, MetH being a cobalamin-dependent enzyme whereas MetE activity is cobalamin-independent. In this work, we show that the last step of methionine synthesis in the plant pathogen Ralstonia solanacearum is under the transcriptional control of the master pathogenicity regulator HrpG. This control is exerted essentially on metE expression through the intermediate regulator MetR. Expression of metE is strongly and specifically induced in the presence of plant cells in a hrpG- and metR-dependent manner. metE and metR mutants are not auxotrophic for methionine and not affected for growth inside the plant but produce significantly reduced disease symptoms on tomato whereas disruption of metH has no impact on pathogenicity. The finding that the pathogen preferentially induces metE expression rather than metH in the presence of plant cells is indicative of a probable metabolic adaptation to physiological host conditions since this induction of metE occurs in an environment in which cobalamin, the required co-factor for MetH, is absent. It also shows that MetE and MetH are not functionally redundant and are deployed during specific stages of the bacteria lifecycle, the expression of metE and metH being controlled by multiple and distinct signals

Enzymatic decontamination of paraoxon-ethyl limits long-term effects in planarians

International audienc

PrhG, a Transcriptional Regulator Responding to Growth Conditions, Is Involved in the Control of the Type III Secretion System Regulon in Ralstonia solanacearum▿ †

The ability of Ralstonia solanacearum to cause disease in plants depends on its type III secretion system (T3SS). The expression of the T3SS and its effector substrates is coordinately controlled by a regulatory cascade, at the bottom of which is HrpB. Transcription of the hrpB gene is activated by a plant-responsive regulator named HrpG, which is a master regulator of a wide array of pathogenicity functions in R. solanacearum. We have identified in the genome of strain GMI1000 a close paralog of hrpG (83% overall similarity at the protein level) that we have named prhG. Despite this high similarity, the expression pattern of prhG is remarkably different from that of hrpG: prhG expression is activated after growth of bacteria in minimal medium but not in the presence of host cells, while hrpG expression is specifically induced in response to plant cell signals. We provide genetic evidence that prhG is a transcriptional regulator that, like hrpG, controls the expression of hrpB and the hrpB-regulated genes under minimal medium conditions. However, the regulatory functions of prhG and hrpG are distinct: prhG has no influence on hrpB expression when the bacteria are in the presence of plant cells, and transcriptomic profiling analysis of a prhG mutant revealed that the PrhG and HrpG regulons have only one pathogenicity target in common, hrpB. Functional complementation experiments indicated that PrhG and HrpG are individually sufficient to activate hrpB expression in minimal medium. Rather surprisingly, a prhG disruption mutant had little impact on pathogenicity, which may indicate that prhG has a minor role in the activation of T3SS genes when R. solanacearum grows parasitically inside the plant. The cross talk between pathogenicity regulatory proteins and environmental signals described here denotes that an intricate network is at the basis of the bacterial disease program

: Comment bloquer la communication des bactéries pour inhiber leur virulence ?

La plupart des bactéries utilisent un système de communication, le quorum sensing, fondé sur la sécrétion et la perception de petites molécules appelées autoinducteurs qui leur permettent d’adapter leur comportement en fonction de la taille de la population. Les bactéries mutualisent ainsi leurs efforts de survie en synchronisant entre elles la régulation de gènes impliqués notamment dans la virulence, la résistance aux antimicrobiens ou la formation du biofilm. Des méthodes ont vu le jour pour inhiber cette communication entre bactéries et limiter leurs effets nocifs. Des inhibiteurs chimiques, des anticorps ou encore des enzymes capables d’interférer avec les autoinducteurs ont été développés et se sont montrés efficaces pour diminuer la virulence des bactéries à la fois in vitro et in vivo. Cette stratégie, appelée quorum quenching, a également montré des effets synergiques avec des traitements antibactériens classiques. Il permettrait notamment d’augmenter la sensibilité des bactéries aux antibiotiques. Ceci constitue une piste thérapeutique prometteuse pour lutter contre les infections bactériennes et limiter les conséquences de l’antibiorésistance

Quorum sensing et quorum quenching : Comment bloquer la communication des bactéries pour inhiber leur virulence ?

International audienceMost bacteria use a communication system known as quorum sensing which relies on the secretion and perception of small molecules called autoinducers enabling bacteria to adapt their behavior according to the population size and synchronize the expression of genes involved in virulence, antimicrobial resistance and biofilm formation. Methods have emerged to inhibit bacterial communication and limit their noxious traits. Chemical inhibitors, sequestering antibodies and degrading enzymes have been developed and proved efficient to decrease bacterial virulence both in vitro and in vivo. This strategy, named quorum quenching, also showed synergistic effects with traditional antibacterial treatments by increasing bacterial susceptibility to antibiotics. Thereby quorum quenching constitutes an interesting therapeutic strategy to fight against bacterial infections and limit the consequences of antibiotic resistance.La plupart des bactéries utilisent un système de communication, le quorum sensing, fondé sur la sécrétion et la perception de petites molécules appelées autoinducteurs qui leur permettent d’adapter leur comportement en fonction de la taille de la population. Les bactéries mutualisent ainsi leurs efforts de survie en synchronisant entre elles la régulation de gènes impliqués notamment dans la virulence, la résistance aux antimicrobiens ou la formation du biofilm. Des méthodes ont vu le jour pour inhiber cette communication entre bactéries et limiter leurs effets nocifs. Des inhibiteurs chimiques, des anticorps ou encore des enzymes capables d’interférer avec les autoinducteurs ont été développés et se sont montrés efficaces pour diminuer la virulence des bactéries à la fois in vitro et in vivo. Cette stratégie, appelée quorum quenching, a également montré des effets synergiques avec des traitements antibactériens classiques. Il permettrait notamment d’augmenter la sensibilité des bactéries aux antibiotiques. Ceci constitue une piste thérapeutique prometteuse pour lutter contre les infections bactériennes et limiter les conséquences de l’antibiorésistance