title: Secreted Compounds of the Probiotic Bacillus clausii Strain O/C Inhibit the Cytotoxic Effects Induced by Clostridium difficile and Bacillus cereus Toxins.
abstract: Although the use of probiotics based on Bacillus strains to fight off intestinal pathogens and antibiotic-associated diarrhea is widespread, the mechanisms involved in producing their beneficial effects remain unclear. Here, we studied the ability of compounds secreted by the probiotic Bacillus clausii strain O/C to counteract the cytotoxic effects induced by toxins of two pathogens, Clostridium difficile and Bacillus cereus, by evaluating eukaryotic cell viability and expression of selected genes. Coincubation of C. difficile and B. cereus toxic culture supernatants with the B. clausii supernatant completely prevented the damage induced by toxins in Vero and Caco-2 cells. The hemolytic effect of B. cereus was also avoided by the probiotic supernatant. Moreover, in these cells, the expression of rhoB, encoding a Rho GTPase target for C. difficile toxins, was normalized when C. difficile supernatant was pretreated using the B. clausii supernatant. All of the beneficial effects observed with the probiotic were abolished by the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF). Suspecting the involvement of a secreted protease in this protective effect, a protease was purified from the B. clausii supernatant and identified as a serine protease (M-protease; GenBank accession number Q99405). Experiments on Vero cells demonstrated the antitoxic activity of the purified protease against pathogen supernatants. This is the first report showing the capacity of a protease secreted by probiotic bacteria to inhibit the cytotoxic effects of toxinogenic C. difficile and B. cereus strains. This extracellular compound could be responsible, at least in part, for the protective effects observed for this human probiotic in antibiotic-associated diarrhea.
authors.affiliation: Université de Bordeaux, UMR 5248, Bordeaux Sciences Agro, Laboratoire de Microbiologie, Gradignan, France.
authors.forename: Gabrielle
authors.initials: G
authors.lastname: Ripert
authors.affiliation: Université de Bordeaux, UMR 5248, Bordeaux Sciences Agro, Laboratoire de Microbiologie, Gradignan, France.
authors.forename: Silvia M
authors.initials: SM
authors.lastname: Racedo
authors.affiliation: Université de Bordeaux, UMR 5248, Bordeaux Sciences Agro, Laboratoire de Microbiologie, Gradignan, France.
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Elie
authors.affiliation: Université de Bordeaux, UMR 5248, Bordeaux Sciences Agro, Laboratoire de Microbiologie, Gradignan, France.
authors.forename: Claudine
authors.initials: C
authors.lastname: Jacquot
authors.affiliation: Université de Bordeaux, UMR 5248, Bordeaux Sciences Agro, Laboratoire de Microbiologie, Gradignan, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Bressollier
authors.affiliation: Université de Bordeaux, UMR 5248, Bordeaux Sciences Agro, Laboratoire de Microbiologie, Gradignan, France maria.urdaci@agro-bordeaux.fr.
authors.forename: Maria C
authors.identifier: 0000-0002-0868-0050
authors.initials: MC
authors.lastname: Urdaci
mesh_terms: D000818:Animals; D001409:Bacillus cereus / Q000472:pathogenicity*; D000070019:Bacillus clausii / Q000378:metabolism*; D001427:Bacterial Toxins / Q000633:toxicity*; D018938:Caco-2 Cells; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002522:Chlorocebus aethiops; D016360:Clostridioides difficile / Q000472:pathogenicity*; D017077:Culture Media, Conditioned / Q000494:pharmacology; D006801:Humans; D007413:Intestinal Mucosa / Q000382:microbiology / Q000473:pathology; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D019936:Probiotics / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000494:pharmacology; D013381:Subtilisins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014709:Vero Cells; D020743:rhoB GTP-Binding Protein / Q000378:metabolism
chemical_list: D001427:Bacterial Toxins; D017077:Culture Media, Conditioned; D011480:Protease Inhibitors; D010664:Phenylmethylsulfonyl Fluoride; C108281:M-protease; D013381:Subtilisins; D020743:rhoB GTP-Binding Protein