Saccharomyces boulardii prevents TNF-alpha-induced apoptosis in EHEC-infected T84 cells

Induction of apoptosis and necrosis by enterohemorrhagic Escherichia coli (EHEC) has been reported in vivo and in vitro, but features of cell death were not noted in those reports. Since tumor necrosis factor-alpha (TNF-alpha) has been implicated in the apoptosis of invasive bacteria, we investigated the role of this cytokine in EHEC-induced apoptosis. We hypothesize that the probiotic yeast strain Saccharomyces boulardii that interferes with EHEC-induced pro-inflammatory pathways delays EHEC-induced apoptosis. By 6 h of infection, flow cytometry analysis of T84 cells demonstrated that 40% of cells were FITC-annexin-V-positive and 40% of cells incorporated both annexin and propidium iodide (PI). Simultaneously, western blot analysis demonstrated that procaspases-8 and -3 were cleaved. Fragmentation of internucleosomal DNA revealed evidence of apoptotic leader formation after 8 and 9 h of infection. Procaspase-9 activation and 3',3-dihexyloxacarbocyanine iodide (DiOC(6)) incorporation were observed at 3 h of infection. In cells preincubated with S. boulardii and infected with EHEC in the presence of yeast, the quantities of procaspases-8, -9 and -3 did not vary, and no DNA fragmentation was observed. The TNF-alpha transcript level and the level of secreted TNF-alpha increased considerably (P<0.001vs control cells) at 6 h of infection in EHEC-alone-infected cells, but were significantly reduced in cells infected in the presence of S. boulardii (P<0.001vs EHEC-alone-infected cells). The presence of anti-TNF-alpha antibody during infection reduced by 30% the level of FITC-annexin V-positive cells. Altogether, these findings demonstrated that: (i) EHEC infection stimulated TNF-alpha synthesis that is implicated in apoptosis of T84 cells; and (ii) S. boulardii induced a decrease in TNF-alpha and related apoptosis in EHEC-infected T84 cells.