Dorota Czerucka

Saccharomyces boulardii preserves the barrier function and modulates the signal transduction pathway induced in enteropathogenic Escherichia coli-infected T84 cells.

ABSTRACT Use of the nonpathogenic yeast Saccharomyces boulardiiin the treatment of infectious diarrhea has attracted growing interest. The present study designed to investigate the effect of this yeast on enteropathogenic Escherichia coli (EPEC)-associated disease demonstrates that S. boulardii abrogated the alterations induced by an EPEC strain on transepithelial resistance, [3H]inulin flux, and ZO-1 distribution in T84 cells. Moreover, EPEC-mediated apoptosis of epithelial cells was delayed in the presence of S. boulardii. The yeast did not modify the number of adherent bacteria but lowered by 50% the number of intracellular bacteria. Infection by EPEC induced tyrosine phosphorylation of several proteins in T84 cells, including p46 and p52 SHC isoforms, that was attenuated in the presence of S. boulardii. Similarly, EPEC-induced activation of the ERK1/2 mitogen-activated protein (MAP) kinase pathway was diminished in the presence of the yeast. Interestingly, inhibition of the ERK1/2 pathway with the specific inhibitor PD 98059 decreased EPEC internalization, suggesting that modulation of the ERK1/2 MAP pathway might account for the lowering of the number of intracellular bacteria observed in the presence of S. boulardii. Altogether, this study demonstrated that S. boulardii exerts a protective effect on epithelial cells after EPEC adhesion by modulating the signaling pathway induced by bacterial infection.

Experimental effects of Saccharomyces boulardii on diarrheal pathogens

Saccharomyces boulardii is a selected strain of yeast that may have applications in the prevention and treatment of intestinal infections. The animal models and in vitro studies developed to elucidate the mechanisms of this protection are reviewed and discussed.

Saccharomyces boulardii Interferes with Enterohemorrhagic Escherichia coli-Induced Signaling Pathways in T84 Cells

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) infections are associated with the modification of tight-junction permeability and synthesis of proinflammatory cytokine interleukin-8 (IL-8). In a previous study, it was demonstrated that EHEC-induced IL-8 secretion is due to the involvement of the mitogen-activated protein kinase (MAPK), AP-1, and NF-κB pathways. In this study, we investigated the effect of the yeast Saccharomyces boulardii on EHEC infection in T84 cells. For this purpose, cells were (i) incubated with bacteria and yeast at the same time or (ii) incubated overnight with yeast cells that were maintained during infection or eliminated by several washes before infection. Coincubation is sufficient to maintain the transmonolayer electrical resistance (TER) of EHEC-infected cells, whereas the preincubation of cells with the yeast without elimination of the yeast during infection is necessary to significantly decrease IL-8 secretion. We thus analyzed the mechanisms of S. boulardii action. We showed that S. boulardii has no effect on EHEC growth or on EHEC adhesion. Kinetics studies revealed that EHEC-induced myosin light chain (MLC) phosphorylation precedes the decrease of TER. ML-7, an MLC kinase inhibitor, abolishes the EHEC-induced MLC phosphorylation and decrease of TER. Studies show that S. boulardii also abolishes EHEC-induced MLC phosphorylation. We demonstrated that the preincubation of cells with S. boulardii without washes before EHEC infection inhibits NF-κB DNA binding activity, phosphorylation and degradation of IκB-α, and activation of the three members of a MAPK group (extracellular signal-regulated protein kinases 1 and 2, p38, and c-jun N-terminal kinase). These findings demonstrate that S. boulardii exerts a preventive effect on EHEC infection by (i) interfering with one of the transduction pathways implicated in the control of tight-junction structure and (ii) decreasing IL-8 proinflammatory secretion via inhibition of the NF-κB and MAPK signaling pathways in infected T84 cells.

Enterohemorrhagic Escherichia coli Infection Induces Interleukin-8 Production via Activation of Mitogen-Activated Protein Kinases and the Transcription Factors NF-κB and AP-1 in T84 Cells

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) infections are associated with hemorrhagic colitis and the hemolytic-uremic syndrome (HUS). In vivo, elevated plasma levels of the proinflammatory cytokine interleukin-8 (IL-8) in EHEC-infected children are correlated with a high risk of developing HUS. As IL-8 gene transcription is regulated by the transcription factors NF-κB and AP-1, we analyzed the role of these factors in the regulation of IL-8 production after infection of the epithelial intestinal T84 cell line by EHEC. By 6 h of infection, EHEC had induced significant secretion of IL-8 (35.84 ± 6.76 ng/ml versus 0.44 ± 0.04 ng/ml in control cells). EHEC induced AP-1 and NF-κB activation by 3 h of infection. Moreover, the three mitogen-activated protein kinases (MAPK) (ERK1/2, p38, and JNK) were phosphorylated in EHEC-infected T84 cells concomitant with induction of AP-1 DNA binding activity, and IκB-α was phosphorylated and then degraded concomitant with induction of NF-κB DNA binding activity. Pretreatment of cells with the highly specific MEK1/2 inhibitor U0126, the p38 inhibitor SB203580, and/or the proteasome inhibitor ALLN led to inhibition of the IL-8 secretion induced in EHEC-infected T84 cells. These findings demonstrate that (i) EHEC can induce in vitro a potent proinflammatory response by secretion of IL-8 and (ii) the secretion of IL-8 is due to the involvement of MAPK, AP-1, and NF-κB signaling pathways.

Implication of mitogen-activated protein kinases in T84 cell responses to enteropathogenic Escherichia coli infection.

ABSTRACT Enteropathogenic Escherichia coli (EPEC) infection of T84 cells induces a decrease in transepithelial resistance, the formation of attaching and effacing (A/E) lesions, and cytokine production. The purpose of this study was to investigate the ability of EPEC to activate mitogen-activated protein (MAP) kinases in T84 cells and to correlate these signaling pathways with EPEC-induced cell responses. T84 cells were infected with either the wild-type (WT) EPEC strain E2348/69 or two mutants, intimin deletion strain CVD206 (ΔeaeA) and type III secretion apparatus mutant strain CVD452 (ΔescN::aphA). Infection of T84 cells with WT but not mutant EPEC strains induced tyrosine phosphorylation of several proteins in T84 cells, including the p46 and p52 Shc isoforms. Kinetics studies revealed that ERK1/2, p38, and c-Jun N-terminal kinase (JNK) MAP kinases were activated in cells infected with strain E2348/69 but not with the mutant strains. Inhibition of MAP kinases with PD98059 or SB203580 did not affect the EPEC-induced decrease in transepithelial resistance or actin accumulation beneath the WT bacteria, but these two inhibitors significantly decreased interleukin-8 (IL-8) synthesis. We demonstrate that EPEC induces activation of ERK1/2, p38, and JNK cascades, which all depend on bacterial adhesion and expression of the bacterial type III secretion system. ERK1/2 and p38 MAP kinases were equally implicated in IL-8 expression but did not participate in A/E lesion formation or transepithelial resistance modification, indicating that the signaling pathways involved in these events are distinct.

Interaction of Saccharomyces boulardii with Salmonella enterica Serovar Typhimurium Protects Mice and Modifies T84 Cell Response to the Infection

Background Salmonella pathogenesis engages host cells in two-way biochemical interactions: phagocytosis of bacteria by recruitment of cellular small GTP-binding proteins induced by the bacteria, and by triggering a pro-inflammatory response through activation of MAPKs and nuclear translocation of NF-κB. Worldwide interest in the use of functional foods containing probiotic bacteria for health promotion and disease prevention has increased significantly. Saccharomyces boulardii is a non-pathogenic yeast used as a probiotic in infectious diarrhea. Methodology/Principal Findings In this study, we reported that S. boulardii (Sb) protected mice from Salmonella enterica serovar Typhimurium (ST)-induced death and prevented bacterial translocation to the liver. At a molecular level, using T84 human colorectal cancer cells, we demonstrate that incubation with Sb before infection totally abolished Salmonella invasion. This correlates with a decrease of activation of Rac1. Sb preserved T84 barrier function and decreased ST-induced IL-8 synthesis. This anti-inflammatory effect was correlated with an inhibitory effect of Sb on ST-induced activation of the MAPKs ERK1/2, p38 and JNK as well as on activation of NF-κB. Electron and confocal microscopy experiments showed an adhesion of bacteria to yeast cells, which could represent one of the mechanisms by which Sb exerts its protective effects. Conclusions Sb shows modulating effects on permeability, inflammation, and signal transduction pathway in T84 cells infected by ST and an in vivo protective effect against ST infection. The present results also demonstrate that Sb modifies invasive properties of Salmonella.

Effect of Saccharomyces boulardii on cAMP- and Ca2+ -dependent Cl- secretion in T84 cells.

Several reports have confirmed that thecooperative interaction between cAMP- andCa2+-mediated transduction pathways maycontribute to the stimulatory or inhibitory regulationof Cl- secretion in intestinal epithelium.Saccharomyces boulardii has been shown to inhibitcholera toxin-induced secretion in rat jejunum. We haveidentified a 120-kDa protein in medium conditioned bySaccharomyces boulardii that reduces cholera toxin-inducedcAMP in intestinal cells. The present study evaluatedthe effect of medium conditioned by Saccharomycesboulardii on cAMP- and Ca2+-mediatedCl- secretion in T84 cells. Experiments performedwith cAMP agonists revealed that 1 hr of preincubationof cells with medium conditioned by Saccharomycesboulardii was necessary to elicit a 40-50% reduction in receptor (cholera toxin, prostaglandinE2, and vasoactive intestinal polypeptide)and nonreceptor (forskolin) mediated cAMP synthesis and125I- efflux. Secretion induced by carbachol was inhibited when cells werepretreated for 1 hr with medium conditioned bySaccharomyces boulardii despite the absence ofinhibition of Ins (1,4,5)P3. From this studywe conclude that Saccharomyces boulardii exerts aninhibitory effect in vitro on Cl- secretionmediated through both cAMP- and Ca2+-mediatedsignaling pathways.

Modification of Salmonella Typhimurium Motility by the Probiotic Yeast Strain Saccharomyces boulardii

Background Motility is an important component of Salmonella enterica serovar Typhimurium (ST) pathogenesis allowing the bacteria to move into appropriate niches, across the mucus layer and invade the intestinal epithelium. In vitro, flagellum-associated motility is closely related to the invasive properties of ST. The probiotic yeast Saccharomyces boulardii BIOCODEX (S.b-B) is widely prescribed for the prophylaxis and treatment of diarrheal diseases caused by bacteria or antibiotics. In case of Salmonella infection, S.b-B has been shown to decrease ST invasion of T84 colon cell line. The present study was designed to investigate the impact of S.b-B on ST motility. Methodology/Principal Findings Experiments were performed on human colonic T84 cells infected by the Salmonella strain 1344 alone or in the presence of S.b-B. The motility of Salmonella was recorded by time-lapse video microscopy. Next, a manual tracking was performed to analyze bacteria dynamics (MTrackJ plugin, NIH image J software). This revealed that the speed of bacterial movement was modified in the presence of S.b-B. The median curvilinear velocity (CLV) of Salmonella incubated alone with T84 decreased from 43.3 µm/sec to 31.2 µm/sec in the presence of S.b-B. Measurement of track linearity (TL) showed similar trends: S.b-B decreased by 15% the number of bacteria with linear tract (LT) and increased by 22% the number of bacteria with rotator tract (RT). Correlation between ST motility and invasion was further established by studying a non-motile flagella-deficient ST strain. Indeed this strain that moved with a CLV of 0.5 µm/sec, presented a majority of RT and a significant decrease in invasion properties. Importantly, we show that S.b-B modified the motility of the pathogenic strain SL1344 and significantly decreased invasion of T84 cells by this strain. Conclusions This study reveals that S.b-B modifies Salmonellas motility and trajectory which may account for the modification of Salmonellas invasion.

Calpain 2-dependent IκBα degradation mediates CPT-11 secondary resistance in colorectal cancer xenografts†

CPT‐11 (irinotecan), the first‐line chemotherapy for advanced stage colorectal cancer, remains inactive in about half of patients (primary chemoresistance) and almost all initial responders develop secondary resistance after several courses of treatment (8 months on average). Nude mice bearing HT‐29 colon cancer xenografts were treated with CPT‐11 and/or an NF‐κB inhibitor for two courses. We confirm that NF‐κB inhibition potentiated CPT‐11 anti‐tumoural effect after the first course of treatment. However, tumours grew again at the end of the second course of treatment, generating resistant tumours. We observed an increase in the basal NF‐κB activation in resistant tumours and in two resistant sublines, either obtained from resistant HT‐29 tumours (HT‐29R cells) or generated in vitro (RSN cells). The decrease of NF‐κB activation in HT‐29R and RSN cells by stable transfections with the super‐repressor form of IκBα augmented their sensitivity to CPT‐11. Comparing gene expression profiles of HT‐29 and HT‐29R cells, we identified the S100A10/Annexin A2 complex and calpain 2 as over‐expressed potential NF‐κB inducers. SiRNA silencing of calpain 2 but not of S100A10 and/or annexin A2, resulted in a decrease in NF‐κB activation, an increase in cellular levels of IκBα and a partial restoration of the CPT‐11 sensitivity in both HT‐29R and RSN cells, suggesting that calpain 2‐dependent IκBα degradation mediates CPT‐11 secondary resistance. Thus, targeted therapies directed against calpain 2 may represent a novel strategy to enhance the anti‐cancer efficacy of CPT‐11. Copyright

Saccharomyces boulardii Modifies Salmonella Typhimurium Traffic and Host Immune Responses along the Intestinal Tract

Salmonella enterica serovar Typhimurium (ST) is an enteropathogenic Gram-negative bacterium that causes infection following oral ingestion. ST spreads rapidly along the gastrointestinal tract (GIT) and invades the intestinal epithelium to ultimately reach internal body organs. The probiotic yeast Saccharomyces boulardii BIOCODEX (S.b-B) is prescribed for prophylaxis of diarrheal infectious diseases. We previously showed that S.b-B prevents weight loss in ST-infected mice and significantly decreases bacterial translocation to the spleen and liver. This study was designed to investigate the effect of S.b-B on ST migration along the GIT and the impact of the yeast on the hosts early innate immune responses. Bioluminescent imaging (BLI) was used to evaluate the effect of S.b-B on the progression of luminescent Salmonella Typhimurium (ST-lux) in the GIT of mice pretreated with streptomycin. Photonic emission (PE) was measured in GIT extracts (stomach, small intestine, cecum and colon) at various time periods post-infection (PI). PE analysis revealed that, 45 min PI, ST-lux had migrated slightly faster in the mice treated with S.b-B than in the untreated infected animals. At 90 min PI, ST-lux had reached the cecum in both groups of mice. Adhesion of ST to S.b-B was visualized in the intestines of the mice and probably accounts for (1) the faster elimination of ST-lux in the feces, and (2) reduced translocation of ST to the spleen and liver. In the early phase of infection, S.b-B also modifies the hosts immune responses by (1) increasing IFN-γ gene expression and decreasing IL-10 gene expression in the small intestine, and (2) elevating both IFN-γ, and IL-10 mRNA levels in the cecum. BLI revealed that S.b-B modifies ST migration and the host immune response along the GIT. Study findings shed new light on the protective mechanisms of S.b-B during the early phase of Salmonella pathogenesis.