Métabolisme adaptatif et toxinogenèse de Bacillus cereus F4430/73: implication du système à deux composants ResDE et du système à un composant Fnr

5 annexes 5 p. Diplôme : Dr. d'Universit

Régulation de la toxinogenèse chez Bacillus cereus en réponse à un stress réducteur

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Métabolisme adaptatif et toxinogénèse de Bacillus cereus F4430/73 : implication du système à deux composants ResDE et du régulateur Fnr

Bacillus cereus est une bactérie pathogène opportuniste, responsable de deux types d'intoxications alimentaires. L'une causée par une toxine émétique (céreulide) est à l'origine du syndrome émétique, et l'autre causée par trois entérotoxines (Hbl, Nhe et CytK) est à l'origine du syndrome diarrhéique. Ce dernier est du à la production d'entérotoxines au niveau de l'intestin grêle de l'homme, caractérisé par une atmosphère anaérobie et un bas potentiel d'oxydore duction (POR). L'objectif de cette thèse est de caractériser l'adaptation de B. cereus et d'évaluer sa toxinogenèse face à un environnement anaérobie et réducteur comme souvent rencontré et impliqué dans la virulence d'autres pathogènes bactériens. Nos résultats ont montrés que la souche diarrhéique F4430/73 de B. cereus dispose d'un métabolisme fennentaire très efficace qui lui permet de se développer dans des conditions de bas POR. D'autre part, nous avons montré que la production des entérotoxines est dépendante du métabolisme énergétique. Elle est favorisée par des conditions de croissance fennentaire et est d'autant plus importante que la fermentation est conduite à bas POR (POR=-148 mV). La régulation de l'expression des entérotoxines, en réponse à l'anaérobiose, s'effectue principalement au niveau transcriptionnel. Nous avons démontré l'existence de deux régulateurs contrôlant simultanément les voies fermentaires et la toxinogenèse. Le système à deux composants ResDE agit comme un senseur redox et la protéine à centre Fe-S Fnr agit comme un senseur de la fermentation. Nos résultats suggèrent que le système ResDE et la protéine Fnr appartiennent à une voie de régulation fonctionnant en partie indépendamment du régulateur pléiotrope de virulence PIcR, pour réguler l'expression des gènes des entérotoxines.AIX-MARSEILLE3-BU Sc.St Jérô (130552102) / SudocSudocFranceF

Anaerobic cells of Bacillus cereus F4430/73 respond to low oxidoreduction potential by metabolic readjustments and activation of enterotoxin expression

International audienceGlucose-grown cells of Bacillus cereus respond to anaerobiosis and low extracellular oxidoreduction potentials (ORP), notably by enhancing enterotoxin production. This response involves the ResDE two-component system. We searched the B. cereus genome for other redox response regulators potentially involved in this adaptive process, and we identified one gene encoding a protein predicted to have an amino acid sequence 58% identical (80% similar) to that of the Bacillus subtilis Fnr redox regulator. The fnr gene of the food-borne pathogen B. cereus F4430/73 has been cloned and partially characterized. We showed that fnr was up-regulated during anaerobic fermentation, especially when fermentation occurred at low ORP (under highly reducing conditions). The expression of fnr was down-regulated in the presence of O(2) and nitrate which, unlike fumarate, stimulated the respiratory pathways. The inactivation of B. cereus fnr abolished fermentative growth but only moderately affected aerobic and anaerobic nitrate respiratory growth. Analyses of glucose by-products and the transcription profiles of key catabolic genes confirmed the strong regulatory impact of Fnr on B. cereus fermentative pathways. More importantly, the fnr mutation strongly decreased the expression of PlcR-dependent hbl and nhe genes, leading to the absence of hemolysin BL (Hbl) and nonhemolytic enterotoxin (Nhe) secretion by the mutant. These data indicate that fnr is essential for both fermentation and toxinogenesis. The results also suggest that both Fnr and the ResDE two-component system belong to a redox regulatory pathway that functions at least partially independently of the pleiotropic virulence gene regulator PlcR to regulate enterotoxin gene expression

ResDE-dependent regulation of enterotoxin gene expression in <em>Bacillus cereus</em>: evidences for multiple modes of binding for ResD and interaction with Fnr

International audienceIn the food-borne pathogen Bacillus cereus F4430/73, the production of major virulence factors hemolysin BL (Hbl) and non-hemolytic enterotoxin (Nhe) is regulated through complex mechanisms. The two-component regulatory system ResDE is involved in the activation of hbl and nhe transcription. Here, the response regulator ResD and the sensor kinase ResE were overexpressed and purified, and autophosphorylation of ResE and transphosphorylation of ResD by ResE were demonstrated in vitro. ResD is mainly monomeric in solution regardless of its phosphorylation state. ResD was shown to interact directly with promoter regions (p) of the enterotoxin regulator genes resDE, fnr and plcR and the enterotoxin structural genes nhe and hbl, but with different affinities. Binding of ResD to pplcR, pnhe and phbl was not dependent on ResD phosphorylation status. In contrast, ResD phosphorylation significantly increased interactions between ResD and presDE and pfnr. Taken together, these results showed that phosphorylation of ResD results in a different target expression pattern. Furthermore, ResD and the redox activator Fnr were found to physically interact and simultaneously bind their target DNAs. We propose that unphosphorylated ResD acts as an anti-activator of Fnr while phosphorylated ResD acts as a co-activator of Fnr. Finally, our findings represent the first molecular evidence for the role of ResDE as a sentinel system capable of sensing redox changes and coordinating a response that modulates B. cereus virulenc

Control of Enterotoxin Gene Expression in Bacillus cereus F4430/73 Involves the Redox-Sensitive ResDE Signal Transduction System

In contrast to Bacillus subtilis, the role of the two-component regulatory system ResDE has not yet been investigated in the facultative anaerobe Bacillus cereus. We examined the role of ResDE in the food-borne pathogen B. cereus F4430/73 by constructing resDE and resE mutants. Growth performances, glucose metabolism, and expression of hemolysin BL (Hbl) and nonhemolytic enterotoxin (Nhe) were analyzed in the three strains under distinct oxygenation and extracellular oxidoreduction potential (ORP) conditions. We show that growth and glucose metabolism were only moderately perturbed in both resDE and resE mutants under aerobiosis, microaerobiosis, and anaerobiosis generated under N(2) atmosphere (initial ORP = +45 mV). The major effects of resDE and resE mutations were observed under low-ORP anaerobic conditions generated under hydrogen atmosphere (iORP = −148 mV). These conditions normally favor enterotoxin production in the wild type. The resE mutation was more deleterious to the cells than the resDE mutation, causing growth limitation and strong deregulation of key catabolic genes. More importantly, the resE mutation abolished the production of enterotoxins under all of the conditions examined. The resDE mutation only decreased enterotoxin expression under anaerobiosis, with a more pronounced effect under low-ORP conditions. Thus, the ResDE system was found to exert major control on both fermentative growth and enterotoxin expression, and it is concluded that the ResDE system of B. cereus should be considered an anaerobic redox regulator. The data presented also provide evidence that the ResDE-dependent regulation of enterotoxins might function at least partially independently of the pleiotropic virulence gene regulator PlcR

Characterization of Reactive and Sensitive Skin Microbiota: Effect of Halymenia durvillei (HD) Extract Treatment

After characterization of the reactive skin microbiota, we investigated whether the active Halymenia durvillei (HD), rich in polysaccharides, could modulate this microbiota after 28 days of treatment, act on neuroinflammation parameters, and calm feelings of discomfort and redness. Skin microbiota was assessed using next-generation sequencing experiments (16S RNA gene fragment sequencing) on samples collected from 30 volunteers suffering from reactive, sensitive skin. To evaluate the effect of the HD extract on neuroinflammation, we used an ex vivo model. Finally, an in vivo study was performed using a clinical assessment (blood microcirculation via videocapillaroscopy) of functional signs employing the Sensitive Scale and the soothing effect was evaluated and compared to a placebo treatment. At the phylum level, the samples were mostly composed of Actinobacteria, Proteobacteria, Firmicutes, and Bacteroidetes, which accounted for more than 97% of the total sequencing read in all samples, with no differences before or after treatment with the HD active ingredient. The Shannon Diversity index indicated lower microbial communities compared to healthy skin. Maintenance of the Shannon Diversity index was reported after 28 days of HD active ingredient treatment, wherein microbial communities continued to decrease in number during treatment with the placebo. The average taxonomic composition of associated skin microbial communities showed that reactive skin is characterized by a low proportion of the Chryseobacterium genus compared to a high proportion of the Corynebacterium genus. At the species level, Actinobacteria are mainly represented by Propionibacterium acnes (72.13%) and Corynebacterium kroppenstedtii (13.23%), representing species typically observed in clinical cases of redness, the main criteria for volunteer inclusion. Corynebacterium kroppenstedtii, with increased levels being associated with skin redness, decreased with HD treatment. This decrease coincided with the clinical improvement observed after 7 weeks of treatment. The ex vivo study revealed that the HD extract induced a significant decrease in the expression of TRPV-1 (&minus;67%; p &lt; 0.001) and NK1-R (&minus;43%; p &lt; 0.01) compared to the control after 6 days of treatment. These data support the use of polysaccharides, found in red alga, in the treatment of reactive and sensitive skin related to the modulation of skin microbiota