Etudes in vivo des propriétés probiotiques et anti-Candida albicans de Lactobacillus rhamnosus Lcr35®

National audienceLes probiotiques sont définis par la FAO et l’OMS comme des microorganismes vivants conférant des effets bénéfiques à l’hôte comme par exemple Lactobacillus rhamnosus Lcr35®. Ce dernier est notamment commercialisé pour ses propriétés anti-Candida albicans. De plus, ses propriétés sont potentialisées par des composés inclus dans les formulations. Cependant, comme pour la plupart des probiotiques, les mécanismes d’action de Lcr35® ne sont pas encore décrits. Les identifier devient aujourd’hui une priorité. Dans ce but, des études in vivo utilisant un organisme modèle, Caenorhabditis elegans, ont été réalisées : (i) étude de la survie du nématode en présence du probiotique potentialisé ou non, (ii) étude des effets préventifs et curatifs de Lcr35® contre une infection à Candida chez C. elegans et (iii) approches transcriptomiques pour identifier les voies de signalisation impliquées dans les effets anti-Candida de Lcr35®.La pathogénicité de C. albicans est généralement liée à la filamentation induisant la destruction des cellules de l’hôte. De Barros et al. (2018) ont montré que C. elegans, prétraité avec L. paracasei, était moins sensible à une infection à C. albicans. Cependant, une réduction significative de la filamentation n’a pas été observée. De ce fait, des expérimentations sur la réduction de la filamentation de C. albicans par Lcr35® seront réalisées

Environmental Toxicity and Antimicrobial Efficiency of Titanium Dioxide Nanoparticles in Suspension

International audienceThe aim of this work was to evaluate the photokilling efficiency of synthesized titanium dioxide nanoparticles in suspension. Two strains of Escherichia coli, Lactobacillus casei rhamnosus and Staphylococcus aureus were used as probes to test the photokilling activities of the nanoparticles. The toxicity effects of TiO 2 nanoparticles on the environment were determined by a standard test using gram-negative bioluminescent bacteria Vibrio fischeri. The antimicrobial activity of these nanoparticles (NPs) was then investigated versus NPs concentration, UV irradiation time and mi-cro-organism strains. We evaluated the LC50 values of the nanoparticles suspension by counting the Colony-Forming Units. Results highlighted the differences in bacteria sensitivity facing photo-killing treatment induced by the irradiation of anatase TiO 2 nanoparticles suspension. At the concentration of 1 g·L −1 TiO 2 , tested bacteria were killed after 30 minutes of photo-treatment. Using different TiO 2 concentrations, the Staphylococcus aureus gram-positive/catalase-positive bacteria were more resistant than gram-negative/catalase-positive ones or gram-positive/catalase-negative bacteria. An effect of UV irradiation was evaluated by the quantification of hydrogen peroxide generated by the photolysis of water molecules in presence of the nanoparticles with or without the most resistant bacterium (S. aureus). After 30 minutes with UV irradiation in these two conditions , the concentration of hydrogen peroxide was 35 µM in presence of 1.2 g·L −1 TiO 2 suspension. This result suggested that the resistance mechanism of S. aureus was not due to an extracelullar H 2 O 2 enzymatic degradation

Caractérisation in vivo des mécanismes probiotiques de Lactobacillus rhamnosus Lcr35®

National audienceLa levure Candida albicans est un pathogène opportuniste retrouvée dans la flore commensale de 80% de la population. Elle est l’agent d’un grand nombre d’infections digestives et gynécologiques appelées candidoses. La prise d’antifongique, bien qu’efficace, peut entrainer une dysbiose favorable à une récidive ultérieure. Lactobacillus rhamnosus Lcr35® est un probiotique dont les propriétés antimicrobiennes, notamment anti-C. albicans, ont été démontrées à la fois in vitro et lors d’essais cliniques. Cependant, les mécanismes moléculaires expliquant ces propriétés n’ont pas encore été décrits. L’objectif de ma thèse est d’identifier ces mécanismes en utilisant un modèle in vivo physiologiquement proche de l’Homme, le ver Caenorhabditis elegans.Ce nématode modèle sera présenté suivi des différentes stratégies expérimentales mises en œuvre : (i) l’étude du temps de vie en présence du probiotique ou du pathogène, (ii) l’étude des effets préventifs et curatifs du probiotique vis-à-vis d’une infection fongique puis (iii) des études omiques (transcriptomique et métabolomique).Les premiers résultats ont mis en évidence que Lcr35® induit un effet positif sur la durée de vie de C. elegans à l’inverse de C. albicans qui induit un effet négatif. De plus, un effet protecteur du probiotique vis-à-vis d’une infection à C. albicans est observé. En effet, en présence de Lcr35®, le nématode résiste à l’infection et voit son temps de vie augmenter. En perspective, une étude transcriptomique de la réponse globale du ver à l’administration du probiotique et/ou du pathogène est envisagée dans le but d’identifier les mécanismes moléculaires responsables des effets observés

A potential genomic biomarker for the detection of polycyclic aromatic hydrocarbon pollutants: multidrug resistance gene 49 in Drosophila melanogaster.

International audiencePolycyclic aromatic hydrocarbons (PAHs) are a major source of air, water, and soil pollution. The multidrug resistance (mdr)/permeability glycoprotein (P-gp) complex is implicated in the multidrug resistance pattern developed against various drugs and xenobiotics, including polycyclic aromatic hydrocarbons. In order to develop a genomic biomarker, we investigated the response of the mdr49 gene (mdr49) of Drosophila melanogaster to PAHs. Structural analysis of mdr49-PA, which is the putative protein expressed from Drosophila mdr49 gene, demonstrated that this transmembrane protein indeed belongs to the adenosine triphosphate-binding cassette transporter superfamily. Polymerase chain reaction (PCR) and real-time PCR analysis revealed that the mdr49 gene is expressed continuously at all the stages of fly development, including embryos, pupae, larvae, and adults, as well as in embryonic Drosophila S12 cells. In the adult fly, the mdr49 gene was expressed in all the analyzed segments (head, thorax, and abdomen) and organs (olfactory and sexual organs). The quantification of mdr49 transcripts by real-time PCR in adult flies exposed to benzo[a]pyrene over time or in presence of increasing concentrations of this pollutant showed a clear dose-dependent response. Similarly, mdr49 gene expression increased after adult flies were exposed to structurally varied PAHs. The detection of tested PAHs by Drosophila P-gp efflux pump was checked by flow cytometry

Curative Treatment of Candidiasis by the Live Biotherapeutic Microorganism Lactobacillus rhamnosus Lcr35 in the Invertebrate Model Caenorhabditis elegans: First Mechanistic Insights

International audienceThe resistance of Candida albicans to conventional drug treatments, as well as the recurrence phenomena due to dysbiosis caused by antifungal treatments, have highlighted the need to implement new therapeutic methodologies. The antifungal potential of live biotherapeutic products (LBP) has already been demonstrated using preclinical models (cell cultures, laboratory animals). Understanding their mechanisms of action is strategic for the development of new therapeutics for humans. In this study, we investigated the curative anti-C. albicans properties of Lactobacillus rhamnosus Lcr35 ® using the in vitro Caco-2 cell and the in vivo Caenorhabditis elegans models. We showed that Lcr35 ® does inhibit neither the growth (p = 0.603) nor the biofilm formation (p = 0.869) of C. albicans in vitro. Lcr35 ® protects the animal from the fungal infection (+225% of survival, p < 2 × 10-16) even if the yeast is detectable in its intestine. In contrast, the Lcr35 ® cell-free supernatant does not appear to have any antipathogenic effect. At the mechanistic level, the DAF-16/Forkhead Box O transcription factor is activated by Lcr35 ® and genes of the p38 MAP Kinase signaling pathway and genes involved in the antifungal response are upregulated in presence of Lcr35 ® after C. albicans infection. These results suggest that the LBM strain acts by stimulating its host via DAF-16 and the p38 MAPK pathway

Investigation into In Vitro and In Vivo Caenorhabditis elegans Models to Select Cheese Yeasts as Probiotic Candidates for their Preventive Effects against Salmonella Typhimurium

International audienceThe design of multiscale strategies integrating in vitro and in vivo models is necessary for the selection of new probiotics. In this regard, we developed a screening assay based on the investigation of the potential of yeasts from cheese as probiotics against the pathogen Salmonella Typhimurium UPsm1 (ST). Two yeasts isolated from raw-milk cheese (Saccharomyces cerevisiae 16, Sc16; Debaryomyces hansenii 25, Dh25), as well as S. cerevisiae subspecies boulardii (CNCM I-1079, Sb1079), were tested against ST by applying in vitro and in vivo tests. Adherence measurements to Caco-2 and HT29-MTX intestinal cells indicated that the two tested cheese yeasts presented a better adhesion than the probiotic Sb1079 as the control strain. Further, the Dh25 was the cheese yeast most likely to survive in the gastrointestinal tract. What is more, the modulation of the TransEpithelial Electrical Resistance (TEER) of differentiated Caco-2 cell monolayers showed the ability of Dh25 to delay the deleterious effects of ST. The influence of microorganisms on the in vivo model Caenorhabditis elegans was evaluated by measuring the longevity of the worm. This in vivo approach revealed that this yeast increased the worm's lifespan and protected it against ST infection, confirming that this in vivo model can be useful for screening probiotic cheese yeasts

Drosophila melanogaster P-glycoprotein : A membrane detoxification system toward polycyclic aromatic hydrocarbon pollutants

International audiencePolycyclic aromatic hydrocarbons (PAHs) are well-known ubiquitous environmental contaminants. Permeability glycoprotein (P-gp) is a transmembrane detoxification efflux pump transporting various lipophilic xenobiotics, such as PAHs, out of the cells. The existence of a P-gp detoxification system inducible by PAHs was investigated in Drosophila melanogaster. Western blot experiments showed that D. melanogaster expressed a 140-kDa P-gp in S12 cells, embryos, and adult flies. Permeability glycoprotein was expressed in adult flies in the head, abdomen, and thorax and sublocalized in the sexual and olfactory organs. Flow cytometry experiments using Drosophila S12 cells in the presence of PAHs and target P-gp drug compounds revealed that Drosophila P-gp acted as an efflux detoxification pump. In Drosophila exposed to benzo[a]pyrene or to ambient air polluted by higher or lower PAH concentrations, P-gp expression was clearly showed a dose-dependent increase response. The P-gp induction was detected both in adult flies and in different fly parts, such as the head, thorax, and antennae. Drosophila P-gp acts as a membrane barrier against PAH pollutants