Study of the anti-oxidant and anti-inflammatory activities of bacterial secondary metabolites produced directly in water, in acellular and cellular models and by the establishment of an original model of intestinal barrier

L'objectif principal de ce travail de Thèse consiste à étudier le pouvoir antioxydant et anti-inflammatoire de métabolites secondaires rejetés directement dans l'eau par certaines bactéries isolées de torrents ou de sources de moyenne montagne et pouvant être ingérés par les autochtones. Différents modèles sont développés, soit de type acellulaire, permettant de visualiser les effets directs des extraits bactériens testés sur les espèces réactives de l'oxygène, soit de type cellulaire permettant d'étudier l'impact de ces extraits sur les voies de transduction du signal et les systèmes de production des espèces réactives de l’oxygène de cellules macrophagiques placées en simple culture ou interagissant avec d'autres cellules de la muqueuse intestinale. Dans ce dernier cas, il s'agit de mettre au point un modèle de barrière intestinale original pouvant être dédié à l'étude de l'impact des extraits sur les processus inflammatoires intestinaux. Un travail d'identification bactérienne recherchant de nouvelles espèces est réalisé, faisant intervenir la spectrométrie de masse, la spectrométrie de masse en tandem, l'étude de l'acide ribonucléique 16 S et celle du génome. Les résultats obtenus ont donné lieu à publications.The main objective of this thesis work was to study the antioxidant and anti-inflammatory properties of secondary metabolites released directly into water by some bacteria isolated from torrents or spring water from mid-mountain that can be ingested by aboriginal people. Different models were used or developed for these purposes, either of pharmacological cell-free type, making it possible to visualize the scavenging effects of bacterial extracts tested on reactive oxygen species, or of cellular type in order to study the impact of these extracts on the signal transduction pathways and reactive oxygen species production systems of macrophagic cells placed in single culture or interacting with other cells from the intestinal mucosa. In the latter case, the aim was to develop an original intestinal barrier model that can be dedicated to studying the impact of extracts on intestinal inflammatory processes. A bacterial identification work looking for new species was carried out in this thesis project, which involved mass spectrometry, tandem mass spectrometry, the study of 16S ribonucleic acid and that of the genome. The results obtained have given rise to publications

A Review on Enterocin DD14, the Leaderless Two-Peptide Bacteriocin with Multiple Biological Functions and Unusual Transport Pathway

Enterocin DD14 (EntDD14) is a two-peptide leaderless bacteriocin (LLB) produced by Enterococcus faecalis 14, a human strain isolated from meconium. Studies performed on EntDD14 enabled it to show its activity against Gram-positive bacteria such as Listeria monocytogenes, Clostridium perfringens, Enterococcus faecalis, and Staphylococcus aureus. EntDD14 was also shown to potentiate the activity of different antibiotics such as erythromycin, kanamycin, and methicillin when assessed against methicillin-resistant Staphylococcus aureus (MRSA) in vitro and in vivo in the NMRI-F holoxenic mouse model. Additionally, EntDD14 has an antiviral activity and decreased the secretion of pro-inflammatory IL-6 and IL-8 in inflamed human intestinal Caco-2 cells. The genome of E. faecalis 14 was sequenced and annotated. Molecular tools such as Bagel4 software enabled us to locate a 6.7kb-EntDD14 cluster. Transport of EntDD14 outside of the cytoplasm was shown to be performed synergistically by a channel composed of two pleckstrin-homology-domain-containing proteins, namely DdE/DdF and the ABC transporter DdGHIJ. This latter could also protect the bacteriocinogenic strain against extracellular EntDD14. Here, we focus on academic data and potential therapeutic issues of EntDD14, as a model of two-peptide LLB

Cytotoxicity and Identification of Antibacterial Compounds from Baillonella toxisperma Bark Using a LC-MS/MS and Molecular Networking Approach

International audienceBaillonella toxisperma is a medicinal plant used in northern Gabon to treat microbial diseases. It is a plant well-known by local populations, but very few studies have focused on the molecules responsible for the antibacterial activities of B. toxisperma. This study proposes a dereplication strategy based on molecular networking generated from HPLC-ESI-Q/TOF data, allowing investigation of the molecules responsible for the antibacterial activity of B. toxisperma. From this strategy, eighteen compounds were putatively identified. All of these compounds belonged mainly to five families of natural compounds, including phenylpropanolamines, stilbenes, flavonoids, lignans and phenolic glycosides. The chemical study carried out from the bark of B. toxisperma allowed us to identify, for the first time, compounds such as resveratrol and derivatives, epicatechin, epigallocatechin and epigallocatechin gallate. In addition, antibacterial activity (diffusion method and microdilution) and cytotoxicity (Cell Counting Kit-8 (CCK-8 Assay)) in vitro were evaluated. The crude ethanolic extract, as well as the fractions of B. toxisperma, showed significant antibacterial activity. However, the ethanolic fractions F2 and F4 presented high antibacterial activity compared to the crude extract. Cytotoxicity studies on colon-cancer cells (Caco-2) and human keratinocyte cells (HaCaT) showed moderate cytotoxicity in both cell types. This study clearly shows the therapeutic potential of the ethanolic extract of the bark of B. toxisperma and provides information on the phytochemical composition and bioactive compounds of the plant

Evaluation of Antiradical and Antioxidant Activities of Lipopeptides Produced by Bacillus subtilis Strains

International audienceThis study investigated the antiradical and antioxidant potential of the three families of lipopeptides (i.e., surfactin, mycosubtilin, and plipastatin/fengycin) produced by Bacillus subtilis strains. The antiradical/antioxidant activities of highly purified lipopeptides were studied in acellular models using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, superoxide anion ( O 2 . - ), hydrogen peroxide, (H 2 O 2 ) and hydroxyl radical (HO . ). At a lipopeptide concentration of 500 mg.L −1 , the maximum inhibition of DPPH reached 22.88% (obtained for plipastatin). Moreover, the scavenging effects of O 2 . - , H 2 O 2 , and HO . at the highest concentration tested (250 mg.L −1 ) were found to be 6, 21, and 3% for surfactin, 19, 9, and 15% for mycosubtilin, 21, 18, and 59% for plipastatin, 21, 31, and 61% for the mixture of surfactin/plipastatin, and 13, 16, and 15% for the mixture of surfactin/mycosubtilin, respectively. These results showed that plipastatin was the best candidate due to its antioxidant activities

Fluorescent Pseudomonas strains from mid-mountain water able to release antioxidant proteins directly into water

Little is known about fluorescent Pseudomonas and investigations are needed to help us better understand how their species work. The aim was here to mimic what naturally occurs in environmental water containing strains isolated from mid-mountain water samples and identified as Pseudomonas fluorescens by conventional biochemical techniques. Three strains were cultured before being directly inoculated into distilled water. Surprisingly, the three cell-less extracts obtained after spinning the bacterial suspensions showed strong in vitro anti-oxidative effects against superoxide anion and hydroxyl radical but with discrepancies. The extracts obtained were found to contain antioxidant proteins among other stress proteins that were released by viable bacteria. They were identified using tandem/mass spectrometry and showed different profiles in sodium-dodecyl sulfate polyacrylamide gel electrophoresis. Bacterial identification was deepened using 16S ribonucleic acid and genome sequencing analyses to explain the differences observed between strains

Dataset on phenotypic characterization, on protein and genome analysis of three fluorescent Pseudomonas strains from mid-mountain water

International audienceThe identification of non-fermentative Gram negative bacilli from run-off and spring water, including fluorescent Pseudomonas is very complex and investigations are needed to contribute to the systematic of these bacteria. In this dataset, the phenotypical profiles of three strains isolated from Vosges mountains first identified as Pseudomonas fluorescens were determined using APIⓇ 50 CH galleries. Then, the identification of their proteins released directly into water was carried out using tandem/mass spectrometry after separating proteins on native two-dimensional polyacrylamide gels. Finally, genotypic analysis data is presented, that illustrates biodiversity in this fluorescent bacterial group. This data is referred by a research article entitled "Fluorescent Pseudomonas strains from mid-mountain water able to release antioxidant proteins directly into water"

Synthetic Flavonoid BrCl-Flav—An Alternative Solution to Combat ESKAPE Pathogens

ESKAPE pathogens are considered as global threats to human health. The discovery of new molecules for which these pathogens have not yet developed resistance is a high medical priority. Synthetic flavonoids are good candidates for developing new antimicrobials. Therefore, we report here the potent in vitro antibacterial activity of BrCl-flav, a representative of a new class of synthetic tricyclic flavonoids. Minimum inhibitory/bactericidal concentration, time kill and biofilm formation assays were employed to evaluate the antibacterial potential of BrCl-flav. The mechanism of action was investigated using fluorescence and scanning electron microscopy. A checkerboard assay was used to study the effect of the tested compound in combination with antibiotics. Our results showed that BrCl-flav displayed important inhibitory activity against all tested clinical isolates, with MICs ranging between 0.24 and 125 µg/mL. A total kill effect was recorded after only 1 h of exposing Enterococcus faecium cells to BrCl-flav. Additionally, BrCl-flav displayed important biofilm disruption potential against Acinetobacter baumannii. Those effects were induced by membrane integrity damage. BrCl-flav expressed synergistic activity in combination with penicillin against a MRSA strain. Based on the potent antibacterial activity, low cytotoxicity and pro-inflammatory effect, BrCl-flav has good potential for developing new effective drugs against ESKAPE pathogens

Mycosubtilin, a multifaceted lipopeptide from Bacillus subtilis for crop protection : an overview of the last decade results obtained from lab to the field

International audienceBacterial lipopeptides, and particularly those produced by the Bacillus subtilis group, have been the subjectof a great deal of scientific research over the past few years, as they display interesting biological activities(biosurfactant, antitumoral, antimicrobial, …). Among these lipopeptides, mycosubtilin belonging to the iturin family,has been deeply studied as a joint work from our academic and private research teams. This lipopeptide consists ofa heptapeptide linked to a fatty acid chain of variable isomery and length ranging from 15 to 18 carbons. It is asecondary metabolite produced by a nonribosomal peptide synthetase, a completely original biosyntheticmechanism.Through four European projects (INTERREG IV PhytoBio, INTERREG V SmartBioControl – Bioprod andBioprotect, BBI BIOSMART, and SME Instrument), we have been able to address various scientific and economicissues that make this molecule of biological origin a promising candidate to get a place in the agroecologicaltransition for integrated plant protection without chemical pesticides. In this presentation, we will first discuss thedevelopment of the production of this molecule from the laboratory scale to the semi‐industrial scale. Then, themultiple activities of mycosubtilin will be presented, by showing the results of antifungal bioactivity found at thelaboratory scale and which demonstrates its exceptional spectrum of activity. Results obtained in greenhouse on thepathosystems lettuce‐Bremia lactucae, wheat‐Zymoseptoria tritici, and tomato‐Botrytis cinerea will be exposed.Moreover, the efficacy of a mycosubtilin formulation (alone or in combination with another lipopeptide) will bedemonstrated in field conditions on the pathosystems apple‐Venturia inaequalis, lettuce‐Bremia lactucae, andtomato‐Botrytis cinerea. Moreover, the biostimulant effect of a formulation containing mycosubtilin will bepresented, particularly on vegetable crops (lettuce and cucumber). Finally, we will discuss the results of cytotoxicity,toxicity and ecotoxicity in comparison with chemical molecules currently on the market

Mycosubtilin, a multifaceted lipopeptide from Bacillus. subtilis for plant crop protection: an overview of the last decade results obtained from lab to the field.

International audienc