No 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.T he gut, one of the biggest surfaces of exchange between the body's internal and external environment, is in permanent contact with antigens. Indeed, the intestinal barrier constitutes an important playground for numerous intestinal pathogen bacteria and toxins.Among them, Clostridium difficile, an anaerobic Gram-positive bacterium, is a major cause of diarrhea, pseudomembranous colitis, and septicemia, and it can lead to death. Hospital stay and use of antibiotics are major risk factors for contracting this disease (1). Over the past decade, the frequency and severity of C. difficile infections have increased markedly due to the emergence of socalled hypervirulent strains that overproduce toxins (2-4). The C. difficile toxins modify target cell proteins to cause disassembly of the actin cytoskeleton and induce severe inflammation (5).Expression of several host genes, mainly those involved in cellular processes, cell-cell interactions, apoptosis, and inflammation, is modified during C. difficile infection (6). These include host genes encoding RhoB (regulating actin cytoskeleton and interactions between cells), ZO-1 (tight-junction protein), and ROCK2 (actin cytoskeleton and signaling protein regulator) (7-9).Equally, many other pathogens, such as Salmonella species, E...