Contribution à l'étude de l'assemblage du Système de Sécrétion de Type III de Shigella flexneri et à la caractérisation du rôle de la protéine IcsB dans l'autophagie

Shigella flexneri is a gram-negative bacterium of the Enterobacteriaceae family, responsible for shigellosis or bacillary dysentery, an invasive colon disease that causes the death of one million individuals a year. Shigella has the ability to invade the colonic or rectal human mucosa, causing an intense inflammatory reaction by destroying the epithelium. Shigella owes its pathogenicity to the virulence plasmid that contains a mxi-spa region encoding the type III secretion system (SST3). The latter makes it possible to inject the effector proteins into the epithelial cells, which destabilizes the cellular signaling pathways in favor of the bacterium.My thesis project focused on the study of virulence factors encoded by genes located in the region of entry of the virulence plasmid. Two types of genes have been studied: 1 / a group of genes named spa: spa40, spa32, spa24, spa29 and spa9 whose products are important in the assembly and operation of SST3 and 2 / the gene icsB which codes for a effector secreted by the SST3 and allows Shigella to escape autophagy. Our experimental results have been the subject of three publications in international journals ("Molecular Microbiology", "Microbiology" and "Microbe and infection") including two as co-first author and one as first author. We have shown that: (1) the association of the proto-channel elements with Spa40 and its interaction with Spa32 plays an important role in the change of specificity of substrates: secretion of the components of the needle (MxiH and MxiI ) to that of the effectors, (2) the associations of the Spa24, Spa9 and Spa29 proto-channel elements with the Mxi-Spa components are important in the consolidation and operation of the export apparatus, (3) the interaction IcsB with host cell cholesterol allows Shigella to protect itself from autophagy.In conclusion, our work has elucidated some of the mysteries used by bacteria of the genus Shigella, namely the mode of assembly of the components of the base of the SST3 and the protection of Shigella against the autophagic cellular response. This work opens new perspectives for studying the SST3 system in other pathogenic bacteria. Secretion systems are conserved in many pathogenic bacteria for humans, animals and plants. Therefore, our contribution goes beyond the scope of Shigella and could lead to the development of new anti-infective drugs.Shigella flexneri est une bactérie à Gram négatif de la famille des entérobactéries, responsable de la shigellose ou dysenterie bacillaire, maladie invasive du colon qui cause la mort d’un million d’individus par an. Shigella a la capacité d’envahir la muqueuse colique ou rectale humaine, en provoquant une intense réaction inflammatoire par la destruction de l’épithélium. Shigella doit sa pathogénicité au plasmide de virulence qui contient une région mxi-spa codant pour le système de sécrétion de type III (SST3). Ce dernier permet d’injecter les protéines effectrices dans les cellules épithéliales ce qui déstabilise les voies de signalisation cellulaires au profit de la bactérie. Mon projet de thèse a porté sur l’étude des facteurs de virulence codés par des gènes localisés dans la région d’entrée du plasmide de virulence. Deux types de gènes ont été étudiés : 1/ un groupe de gènes nommés spa : spa40, spa32, spa24, spa29 et spa9 dont les produits sont importants dans l’assemblage et le fonctionnement du SST3 et 2/ le gène icsB qui code pour un effecteur sécrété par le SST3 et permet à Shigella d’échapper à l’autophagie. Nos résultats expérimentaux ont fait l’objet de trois publications dans des revues internationales («Molecular Microbiology», «Microbiology» et «Microbe and infection») dont deux en tant que co-premier auteur et une en tant que premier auteur. Nous avons ainsi montré que : (1) l’association des éléments du proto-canal avec Spa40 puis son interaction avec Spa32 joue un rôle important dans le changement de spécificité de substrats : de la sécrétion des composants de l’aiguille (MxiH et MxiI) à celle des effecteurs, (2) les associations des éléments du proto-canal Spa24, Spa9 et Spa29 avec les composants Mxi-Spa sont importantes dans la consolidation et le fonctionnement de l’appareil d’exportation, (3) l’interaction d’IcsB avec le cholestérol des cellules hôtes permet à Shigella de se protéger de l’autophagie.En conclusion, nos travaux ont permis d’élucider une partie des mystères utilisés par les bactéries du genre Shigella, à savoir le mode d’assemblage des composants de la base du SST3 et la protection de Shigella contre la réponse cellulaire autophagique. Ce travail ouvre de nouvelles perspectives d’étude du système SST3 chez d’autres bactéries pathogènes. Les systèmes de sécrétion sont conservés chez plusieurs bactéries pathogènes pour l’homme, l’animal et les plantes. Par conséquent, notre contribution dépasse le cadre de Shigella et pourrait aboutir à l’élaboration de nouveaux médicaments anti-infectieux

Invasion of Epithelial Cells Is Correlated with Secretion of Biosurfactant via the Type 3 Secretion System (T3SS) of Shigella flexneri

Biosurfactants are amphipathic molecules produced by many microorganisms, usually bacteria, fungi, and yeasts. They possess the property of reducing the tension of the membrane interfaces. No studies have been conducted on Shigella species showing the role of biosurfactant-like molecules (BLM) in pathogenicity. The aim of this study is to assess the ability of Shigella environmental and clinical strains to produce BLM and investigate the involvement of biosurfactants in pathogenicity. Our study has shown that BLM are secreted in the extracellular medium with EI24 ranging from 80% to 100%. The secretion is depending on the type III secretion system (T3SS). Moreover, our results have shown that S. flexneri, S. boydii, and S. sonnei are able to interact with hydrophobic areas with 17.64%, 21.42%, and 22.22% hydrophobicity, respectively. BLM secretion is totally prevented due to inhibition of T3SS by 100 mM benzoic and 1.5 mg/ml salicylic acids. P. aeruginosa harboring T3SS is able to produce 100% of BLM in the presence or in the absence of both T3SS inhibitors. The secreted BLM are extractable with an organic solvent such as chloroform, and this could entirely be considered a lipopeptide or polypeptide compound. Secretion of BLM allows some Shigella strains to induce multicellular phenomena like “swarming.

Microbiota Landscape of Gut System of Guppy Fish (Poecilia reticulata) Plays an Outstanding Role in Adaptation Mechanisms

Microbial consortium that is present in fish gut systems works together to achieve unknown specific roles. Here, we collected guppy fish from hydrocarbon- and trace metal-contaminated wastewater to assess the relationships between gut microbiota and host fish adaptation. Targeted genes and 16S rRNA amplicon sequencing have been used to identify gut bacteria of guppies. Mineral-conditioned medium contributes to identify bacteria with the ability to grow and/or to tolerate hydrocarbon and trace metals. Additionally, trace metals’ tolerance minimum inhibitory concentration (MIC) of microbiota was evaluated. We first isolated bacteria from the gut system, and we showed that Bacillus spp., Staphylococcus spp., Shigella spp., Salmonella spp, Pseudomonas spp., Citrobacter spp., Salmonella enterica ssp.arizonae sp., Enterobacter spp, and Acinetobacter spp. are part of guppy gut microbiota. Some representative species are able to degrade and/or tolerate gasoline and/or diesel fuel hydrocarbons. Tolerance to trace metals was observed in Gram-positive and Gram-negative bacteria. We showed that minimal inhibitory concentration (MIC) of some microbiota isolated from gut systems has been found including for mercury (Hg) between 2 and 4‰, cobalt (Co) Co (2 and 5‰), zinc (Zn) (9 and 18‰), and plomb (Pb) (22 and 27‰). Zn and Pb were the trace metals for which the rate of tolerance was significantly higher. Finally, we showed that cytochrome c oxidase is not interfering in presence of trace metals. The working consortium showed that bacteria should work together to achieve their best

Escape of intracellular Shigella from autophagy requires binding to cholesterol through the type III effector, IcsB.

Type III secretion systems are present in many pathogenic bacteria and mediate the translocation of bacterial effectors into host cells. Identification of host targets of these effectors is crucial for understanding bacterial virulence. IcsB, a type III secretion effector, helps Shigella to evade the host autophagy defense system by binding to the autophagy protein, Atg5. Here, we show that IcsB is able to interact specifically with cholesterol. The cholesterol binding domain (CBD) of IcsB is located between residues 288 and 351. Specific mutations of single tyrosine residues Y297 or Y340 of IcsB by phenylalanine (F) slightly reduced cholesterol binding, whereas deletion of the entire CBD or double mutation Y297F-Y340F strongly abolished interactions with cholesterol. To determine whether Shigella expressing IcsB variants could evade autophagy as effectively as the wild-type Shigella, we infected MDAMC cells stably expressing the autophagy marker LC3 fused to GFP and bacterial autophagosome formation was quantified using fluorescence microscopy. Mutation Y297F or Y340F slightly impaired IcsB function, whereas complete removal of CBD or mutation Y297F-Y340F significantly impaired autophagy evasion. Furthermore, we report that BopA, the counterpart of IcsB in Burkholderia pseudomallei with similar autophagy-evading properties, contains the CBD domain and is also able to bind cholesterol.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Functional analysis of the large cytoplasmic domain of Shigella Spa40 B4 21 in the assembly and the switch of substrate specificity of the type III secretion system

Contactforum supported by the Royal Flemish Academy of Belgium for Sciences and Arts Jointly organized by the National Committee for Microbiology and the Belgian Society for Microbiologyinfo:eu-repo/semantics/publishe

Production, Partial Purification and Based SDS-PAGE Profiles of Caseinolytic Enzyme in two Bacillus Strains Isolated from Fermented Cassava leaves "Ntoba mbodi" in Congo Brazzaville

Two Bacillus strains isolated from Ntoba mbodi : Bacillus megaterium (B.me NM 02) Bacillus licheniformis (B.li NM01), has shown a significant caseinolytic enzyme activity. We set optimization of growth and enzyme production conditions. Several parameters have been optimized: temperature, pH, various types of media, carbon and nitrogen sources. In both strains, growth is possible from 25 to 60 ° C with an optimum temperature at 37°C for B.li and at 35°C for B.me. Enzyme production was observed from 25 to 55 °C with an optimum temperature of 30 °C for B.li. Enzyme production was observed from 25 to 50 ° C with an optimum temperature of 35 ° C in B.me. For pH, growth and enzyme production can be at 5, 7 and 9 with an optimum at 7 for both strains. The LB medium is better for growth and enzyme production than TSB for B.li and B.me. Among the carbon sources used, fructose is better for growth after 48 hours of incubation in both strains (B.li: 0,93±0,001, B.me: 0,928±0,002), but for enzyme production fructose remains the best carbon source for B.li (14,33±1,24) , while starch is the best for B.me (14.66±1,24). Concerning nitrogen sources, in both strains the best source of growth is the yeast extracts (B.li: 0,969±0,015, B.me: 0,952±0,01). For enzyme production, the two sources can be used for B.li.(14,333±1,247) but for B .me (14,333±0,471) only the yeast extracts is the best as well as for growth and enzyme production. Furthermore, in both strains the produced enzyme was partially purified by using ammonium sulfate precipitation, and SDS-PAGE has been hold, profiles of specific bands are useful to give more information and differentiate the two strains

Synergic Involvements of Microorganisms in the Biomedical Increase of Polyphenols and Flavonoids during the Fermentation of Ginger Juice

Steered fermentation by microorganisms gives great added value in the nutritional quality of local food. Ginger rhizome naturally contains a myriad of bioactive compounds including polyphenol and flavonoids. The aim of this work was to ferment the ginger juice, to evaluate the biochemical parameters of ginger wine, and to understand the involvement of microorganisms in the bioincrease of polyphenol compounds. Titratable acidity and pH values were determined and showed that pH is around 1.6 at the end of the fermentation when the acidity is around 6.431 g/L. Using colorimetric assay, the total polyphenolic and flavonoid compounds were evaluated throughout the fermentation. The variation of the polyphenol and flavonoid concentrations of the unsweetened sample was around 10.18 to 14.64 mg Eq AG/g and 1.394 to 2.224 mg Eq Cat/g Ms, but those from the sweet sample were around 10.82 to 18.34 mg Eq AG/g Ms and 1.311 to 2.290 mg Eq Cat/g. Using one-step PCR, multiplex techniques with specific primers, with yeast-like phenotype 27.27% (6), have been assigned among 22 isolates to Saccharomyces cerevisiae. By using PCR multiplex techniques, Bacillus licheniformis, Bacillus pumilus, Bacillus safensis, and Saccharomyces cerevisiae have been identified. Together with Saccharomyces cerevisiae, we showed that Bacillus sp. are able to secrete enzymatic landscape with some activities up to 50% including cellulase, amylase, pectinase, and protease