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.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...

Natural C. elegans Microbiota Protects against Infection via Production of a Cyclic Lipopeptide of the Viscosin Group

“…We are currently investigating if the effect of MYb115 is mediated by an indirect mechanism-i.e., through reinforcement of intestinal barriers, increased damage repair, or activation of other defense responses. However, we cannot exclude that MYb115 has a direct effect on the Cry toxins released by the BT spores-e.g., by degradation of the toxins-as was previously shown for a protease secreted by the probiotic B. clausii that inhibits the toxic effects of B. cereus toxins in a cytotoxicity assay using mammalian cell lines [20].…”

Bacillus As Potential Probiotics: Status, Concerns, and Future Perspectives

“…The antagonistic activity of aninocoumacin A was also documented against enteric E. faecium and Shigella flexneri. An interesting communication by Ripert et al (2016) revealed that the probiotic B. clausii O/C strain protected vero and Caco-2 cells from the cytotoxic effect of Clostridium difficili and B. cereus toxins. This activity was mediated by serine protease(s) of B. clausii .…”

“…offers higher acid tolerance and better stability during heat processing and low temperature storage ( Bader et al, 2012 ). Additionally, they have also been shown to possess pathogen exclusion, anti-oxidant, antimicrobial, immuno-modulatory ( Lefevre et al, 2015 ; Shobharani et al, 2015 ; Ripert et al, 2016 ) and food fermentation ( Terlabie et al, 2006 ) abilities.…”

Composite genome sequence of Bacillus clausii, a probiotic commercially available as Enterogermina®, and insights into its probiotic properties

“…The intrinsic antibiotic resistance in probiotics is considered advantageous in cases of antibiotics-probiotics combination prescriptions to restore healthy gut [14,15]. The mode of action of B. clausii as a probiotic is not clear, but the strains have been reported to secrete some proteins that are involved in the immunomodulatory mechanism, adaptation and their colonization in the human gastrointestinal tract (GIT) [12,13,[16][17][18]. Uniquely, B. clausii harbor erm (34) gene that imparts the resistance to erythromycin.…”

“…Various clinical trials and molecular studies [8,13,16,[20][21][22][23] have been performed to identify the major features that demarcate B. clausii probiotic strains from other Bacillus spp., but still, the genomic reasons of its probiotic activity have not been reported before. Therefore, we sequenced the composite genome sequence of Bacillus clausii (composite of all four strains of B. clausii used in the probiotic formulation) from Enterogermina®, an oral probiotic, marketed by Sanofi in India.…”