Adherent Bacteria and Parasiticidal Secretion Products of Human Cervicovaginal Microbiota-Associated Lactobacillus gasseri Confer Non-Identical Cell Protection against Trichomonas vaginalis-Induced Cell Detachment.

Trichomonas vaginalis, a protozoan parasite specific to the human genital tract, is one of the most common sexually transmitted pathogens. Its pathogenicity is strongly associated with its expression of a broad array of proteases triggering cytotoxic effects in host epithelial cells. Vaginal microbiota-associated Lactobacillus, including those of L. gasseri in particular, can counteract T. vaginalis pathogenesis, but the mechanisms involved have yet to be clarified. T. vaginalis strain G3 (Tv G3) cytotoxicity was assessed by examining cell morphology, cell detachment, and fluorescent labeling of the F-actin cytoskeleton and immunolabeling of vinculin-position focal adhesions (FAs) by confocal laser scanning electron microscopy on confluent cervicovaginal epithelial HeLa cell monolayers. The inhibitory effects of bacterial cells and secreted products of L. gasseri ATCC 9857 and KS 120.1 on the Tv G3 viability and parasite deleterious effects on HeLa cells were investigated. Pre-adhering L. gasseri cells delayed but did not inhibit Tv G3-induced cell detachment, F-actin cytoskeleton disorganization and the disappearance of vinculin-positive focal FAs. L. gasseri KS 120.1 secretion products had a rapid parasiticide activity by killing time- and concentration-dependent Tv G3 parasites after direct contact. By killing Tv G3 parasites already associated with the epithelial cells, secretion products have abolished parasite-induced cell detachment. Our findings suggest that vagina microbiota-associated L. gasseri creates a physical barrier and exerts pharmacological-type mechanisms to counteract the deleterious cytotoxic effects of T. vaginalis.

Microbiota tryptophan metabolism induces aryl hydrocarbon receptor activation and improves alcohol-induced liver injury.

OBJECTIVE: Chronic alcohol consumption is an important cause of liver-related deaths. Specific intestinal microbiota profiles are associated with susceptibility or resistance to alcoholic liver disease in both mice and humans. We aimed to identify the mechanisms by which targeting intestinal microbiota can improve alcohol-induced liver lesions. DESIGN: We used human associated mice, a mouse model of alcoholic liver disease transplanted with the intestinal microbiota of alcoholic patients and used the prebiotic, pectin, to modulate the intestinal microbiota. Based on metabolomic analyses, we focused on microbiota tryptophan metabolites, which are ligands of the aryl hydrocarbon receptor (AhR). Involvement of the AhR pathway was assessed using both a pharmacological approach and AhR-deficient mice. RESULTS: Pectin treatment modified the microbiome and metabolome in human microbiota-associated alcohol-fed mice, leading to a specific faecal signature. High production of bacterial tryptophan metabolites was associated with an improvement of liver injury. The AhR agonist Ficz (6-formylindolo (3,2-b) carbazole) reduced liver lesions, similarly to prebiotic treatment. Conversely, inactivation of the ahr gene in alcohol-fed AhR knock-out mice abrogated the beneficial effects of the prebiotic. Importantly, patients with severe alcoholic hepatitis have low levels of bacterial tryptophan derivatives that are AhR agonists. CONCLUSIONS: Improvement of alcoholic liver disease by targeting the intestinal microbiota involves the AhR pathway, which should be considered as a new therapeutic target.

Small Trafficking Inhibitor Retro-2 Disrupts the Microtubule-Dependent Trafficking of Autophagic Vacuoles.

Autophagy is a catabolic recycling process by which a cell degrades its own constituents to contribute to cell homeostasis or survival. We report that the small trafficking inhibitor Retro-2 impairs microtubule-dependent vacuolar trafficking in autophagy. Retro-2 induced autophagy and promoted the dramatic cytoplasmic accumulation of large autophagosomes. Moreover, Retro-2 decreased the spreading of autophagosomes within the cytoplasm of nutrient-starved cells. In addition, Retro-2 abolished autolysosomes formation. We show that these effects arise from hitherto unsuspected disassembly activity of the small molecule on the cellular microtubule network, which is known to act as a key regulator of vacuolar trafficking of the autophagy pathway.

The macrophage microtubule network acts as a key cellular controller of the intracellular fate of Leishmania infantum.

The parasitophorous vacuoles (PVs) that insulate Leishmania spp. in host macrophages are vacuolar compartments wherein promastigote forms differentiate into amastigote that are the replicative form of the parasite and are also more resistant to host responses. We revisited the biogenesis of tight-fitting PVs that insulate L. infantum in promastigote-infected macrophage-like RAW 264.7 cells by time-dependent confocal laser multidimensional imaging analysis. Pharmacological disassembly of the cellular microtubule network and silencing of the dynein gene led to an impaired interaction of L. infantum-containing phagosomes with late endosomes and lysosomes, resulting in the tight-fitting parasite-containing phagosomes never transforming into mature PVs. Analysis of the shape of the L. infantum parasite within PVs, showed that factors that impair promastigote-amastigote differentiation can also result in PVs whose maturation is arrested. These findings highlight the importance of the MT-dependent interaction of L. infantum-containing phagosomes with the host macrophage endolysosomal pathway to secure the intracellular fate of the parasite.

Key role of the macrophage microtubule network in the intracellular lifestyle of Leishmania amazonensis.

We conducted a study to decipher the mechanism of the formation of the large communal Leishmania amazonensis-containing parasitophorous vacuole (PV) and found that the macrophage microtubule (MT) network dynamically orchestrates the intracellular lifestyle of this intracellular parasite. Physical disassembly of the MT network of macrophage-like RAW 264.7 cells or silencing of the dynein gene, encoding the MT-associated molecular motor that powers MT-dependent vacuolar movement, by siRNA resulted in most of the infected cells hosting only tight parasite-containing phagosome-like vacuoles randomly distributed throughout the cytoplasm, each insulating a single parasite. Only a minority of the infected cells hosted both isolated parasite-containing phagosome-like vacuoles and a small communal PV, insulating a maximum of two to three parasites. The tight parasite-containing phagosome-like vacuoles never matured, whereas the small PVs only matured to a small degree, shown by the absence or faint acquisition of host-cell endolysosomal characteristics. As a consequence, the parasites were unable to successfully complete promastigote-to-amastigote differentiation and died, regardless of the type of insulation.

Diverse Expression of Antimicrobial Activities Against Bacterial Vaginosis and Urinary Tract Infection Pathogens by Cervicovaginal Microbiota Strains of Lactobacillus gasseri and Lactobacillus crispatus.

We aimed to analyze the strain-by-strain expression of a large panel of antimicrobial activities counteracting the virulence mechanisms of bacterial vaginosis-associated Prevotella bivia CI-1 and Gardnerella vaginalis 594, pyelonephritis-associated Escherichia coli CFT073, and recurrent cystitis- and preterm labor-associated IH11128 E. coli by Lactobacillus gasseri and Lactobacillus crispatus clinical strains, and L. gasseri ATCC 9857 and KS 120.1, and L. crispatus CTV-05 strains isolated from the cervicovaginal microbiota of healthy women. All L. gasseri and L. crispatus strains exerted antimicrobial activity by secreted lactic acid, which killed the microbial pathogens by direct contact. Potent bactericidal activity was exerted by a very limited number of resident L. gasseri and L. crispatus strains showing the specific ability to a strain to produce and release antibiotic-like compounds. These compounds eradicated the microbial pathogens pre-associated with the surface of cervix epithelial cells, providing efficient protection of the cells against the deleterious effects triggered by toxin-producing G. vaginalis and uropathogenic E. coli. Furthermore, these compounds crossed the cell membrane to kill the pre-internalized microbial pathogens. In addition, all L. gasseri and L. crispatus cells exhibited another non-strain specific activity which inhibited the association of microbial pathogens with cervix epithelial cells with varying efficiency, partially protecting the cells against lysis and detachment triggered by toxin-producing G. vaginalis and uropathogenic E. coli. Our results provide evidence of strain-level specificity for certain antimicrobial properties among cervicovaginal L. gasseri and L. crispatus strains, indicating that the presence of a particular species in the vaginal microbiota is not sufficient to determine its benefit to the host. A full repertory of antimicrobial properties should be evaluated in choosing vaginal microbiota-associated Lactobacillus isolates for the development of live biotherapeutic strategies.

Inhibitors of retrograde trafficking active against ricin and Shiga toxins also protect cells from several viruses, Leishmania and Chlamydiales.

Medical countermeasures to treat biothreat agent infections require broad-spectrum therapeutics that do not induce agent resistance. A cell-based high-throughput screen (HTS) against ricin toxin combined with hit optimization allowed selection of a family of compounds that meet these requirements. The hit compound Retro-2 and its derivatives have been demonstrated to be safe in vivo in mice even at high doses. Moreover, Retro-2 is an inhibitor of retrograde transport that affects syntaxin-5-dependent toxins and pathogens. As a consequence, it has a broad-spectrum activity that has been demonstrated both in vitro and in vivo against ricin, Shiga toxin-producing O104:H4 entero-hemorrhagic E. coli and Leishmania sp. and in vitro against Ebola, Marburg and poxviruses and Chlamydiales. An effect is anticipated on other toxins or pathogens that use retrograde trafficking and syntaxin-5. Since Retro-2 targets cell components of the host and not directly the pathogen, no selection of resistant pathogens is expected. These lead compounds need now to be developed as drugs for human use.

A gastrointestinal anti-infectious biotherapeutic agent: the heat-treated Lactobacillus LB.

Experimental in vitro and in vivo studies support the hypothesis that heat-treated, lyophilized Lactobacillus acidophilus LB cells and concentrated, neutralized spent culture medium conserve the variety of pharmacological, antimicrobial activities of the live probiotic strain against several infectious agents involved in well-established acute and persistent watery diarrhoea and gastritis. Heat-treated cells and heat-stable secreted molecules trigger multiple strain-specific activities explaining the therapeutic efficacy of L. acidophilus LB. This review discusses the current body of knowledge on the antimicrobial mechanisms of action exerted by L. acidophilus LB demonstrated in in vitro and in vivo experimental studies, and the evidence for the therapeutic efficacy of this anti-infectious biotherapeutic agent proved in randomized clinical trials for the treatment of acute and persistent watery diarrhoea associated with several intestinal infectious diseases in humans.

Leishmania hijacking of the macrophage intracellular compartments.

Leishmania spp., transmitted to humans by the bite of the sandfly vector, are responsible for the three major forms of leishmaniasis, cutaneous, diffuse mucocutaneous and visceral. Leishmania spp. interact with membrane receptors of neutrophils and macrophages. In macrophages, the parasite is internalized within a parasitophorous vacuole and engages in a particular intracellular lifestyle in which the flagellated, motile Leishmania promastigote metacyclic form differentiates into non-motile, metacyclic amastigote form. This phenomenon is induced by Leishmania-triggered events leading to the fusion of the parasitophorous vacuole with vesicular members of the host cell endocytic pathway including recycling endosomes, late endosomes and the endoplasmic reticulum. Maturation of the parasitophorous vacuole leads to the intracellular proliferation of the Leishmania amastigote forms by acquisition of host cell nutrients while escaping host defense responses.

Cell line-dependent cytotoxicity of poly(isobutylcyanoacrylate) nanoparticles coated with chitosan and thiolated chitosan: Insights from cultured human epithelial HeLa, Caco2/TC7 and HT-29/MTX cells.

Nanoparticles composed of poly(isobutylcyanoacrylate) core coated with a mixture of chitosan and thiolated chitosan have already shown promising results in terms of mucoadhesion and permeation enhancement properties of pharmaceutical active drugs delivered via mucosal routes. In the present work, the cytotoxicity of these nanoparticles was first investigated using direct contact assay on undifferentiated human cervix epithelial HeLa cells. The results showed strong toxicity in HeLa cells for the two investigated concentrations 25 and 50 µg/mL. The cytotoxic effect was mainly attributed to the poly(isobutylcyanoacrylate) core since no significant differences in nanoparticle cytotoxicity were reported when nanoparticle shell composition was modified by adding chitosan or thiolated chitosan. In contrast, lower nanoparticle toxicity was reported using human fully-differentiated enterocyte-like Caco-2/TC7, and fully-differentiated mucus-secreting HT-29/MTX cells forming monolayer in culture mimicking an intestinal epithelial barrier. This study demonstrated that the toxicity of poly(isobutylcyanoacrylate) nanoparticles is highly cell line-dependent.

Thermosensitive and mucoadhesive pluronic-hydroxypropylmethylcellulose hydrogel containing the mini-CD4 M48U1 is a promising efficient barrier against HIV diffusion through macaque cervicovaginal mucus.

To be efficient, vaginal microbicide hydrogels should form a barrier against viral infections and prevent virus spreading through mucus. Multiple particle tracking was used to quantify the mobility of 170-nm fluorescently labeled COOH-modified polystyrene particles (COOH-PS) into thermosensitive hydrogels composed of amphiphilic triblock copolymers with block compositions EOn-POm-EOn (where EO refers to ethylene oxide and PO to propylene oxide) containing mucoadhesive hydroxypropylmethylcellulose (HPMC). COOH-PS were used to mimic the size and the surface charge of HIV-1. Analysis of COOH-PS trajectories showed that particle mobility was decreased by Pluronic hydrogels in comparison with cynomolgus macaque cervicovaginal mucus and hydroxyethylcellulose hydrogel (HEC; 1.5% by weight [wt%]) used as negative controls. Formulation of the peptide mini-CD4 M48U1 used as an anti-HIV-1 molecule into a mixture of Pluronic F127 (20 wt%) and HPMC (1 wt%) did not affect its anti-HIV-1 activity in comparison with HEC hydrogel. The 50% inhibitory concentration (IC50) was 0.53 µg/ml (0.17 µM) for M48U1-HEC and 0.58 µg/ml (0.19 µM) for M48U1-F127-HPMC. The present work suggests that hydrogels composed of F127-HPMC (20/1 wt%, respectively) can be used to create an efficient barrier against particle diffusion in comparison to conventional HEC hydrogels.

Antisecretory factor peptide AF-16 inhibits the secreted autotransporter toxin-stimulated transcellular and paracellular passages of fluid in cultured human enterocyte-like cells.

Both the endogenous antisecretory factor (AF) protein and peptide AF-16, which has a sequence that matches that of the active N-terminal region of AF, inhibit the increase in the epithelial transport of fluid and electrolytes induced by bacterial toxins in animal and ex vivo models. We conducted a study to investigate the inhibitory effect of peptide AF-16 against the increase of transcellular passage and paracellular permeability promoted by the secreted autotransporter toxin (Sat) in a cultured cellular model of the human intestinal epithelial barrier. Peptide AF-16 produced a concentration-dependent inhibition of the Sat-induced increase in the formation of fluid domes, in the mucosal-to-serosal passage of D-[1-(14)C]mannitol, and in the rearrangements in the distribution and protein expression of the tight junction (TJ)-associated proteins ZO-1 and occludin in cultured human enterocyte-like Caco-2/TC7 cell monolayers. In addition, we show that peptide AF-16 also inhibits the cholera toxin-induced increase of transcellular passage and the Clostridium difficile toxin-induced effects on paracellular permeability and TJ protein organization in Caco-2/TC7 cell monolayers. Treatment of cell monolayers by the lipid raft disorganizer methyl-ß-cyclodextrin abolished the inhibitory activity of peptide AF-16 at the transcellular passage level and did not modify the effect of the peptide at the paracellular level.

Anti-infective activities of lactobacillus strains in the human intestinal microbiota: from probiotics to gastrointestinal anti-infectious biotherapeutic agents.

A vast and diverse array of microbial species displaying great phylogenic, genomic, and metabolic diversity have colonized the gastrointestinal tract. Resident microbes play a beneficial role by regulating the intestinal immune system, stimulating the maturation of host tissues, and playing a variety of roles in nutrition and in host resistance to gastric and enteric bacterial pathogens. The mechanisms by which the resident microbial species combat gastrointestinal pathogens are complex and include competitive metabolic interactions and the production of antimicrobial molecules. The human intestinal microbiota is a source from which Lactobacillus probiotic strains have often been isolated. Only six probiotic Lactobacillus strains isolated from human intestinal microbiota, i.e., L. rhamnosus GG, L. casei Shirota YIT9029, L. casei DN-114 001, L. johnsonii NCC 533, L. acidophilus LB, and L. reuteri DSM 17938, have been well characterized with regard to their potential antimicrobial effects against the major gastric and enteric bacterial pathogens and rotavirus. In this review, we describe the current knowledge concerning the experimental antibacterial activities, including antibiotic-like and cell-regulating activities, and therapeutic effects demonstrated in well-conducted, placebo-controlled, randomized clinical trials of these probiotic Lactobacillus strains. What is known about the antimicrobial activities supported by the molecules secreted by such probiotic Lactobacillus strains suggests that they constitute a promising new source for the development of innovative anti-infectious agents that act luminally and intracellularly in the gastrointestinal tract.

Pathogenesis of human enterovirulent bacteria: lessons from cultured, fully differentiated human colon cancer cell lines.

Hosts are protected from attack by potentially harmful enteric microorganisms, viruses, and parasites by the polarized fully differentiated epithelial cells that make up the epithelium, providing a physical and functional barrier. Enterovirulent bacteria interact with the epithelial polarized cells lining the intestinal barrier, and some invade the cells. A better understanding of the cross talk between enterovirulent bacteria and the polarized intestinal cells has resulted in the identification of essential enterovirulent bacterial structures and virulence gene products playing pivotal roles in pathogenesis. Cultured animal cell lines and cultured human nonintestinal, undifferentiated epithelial cells have been extensively used for understanding the mechanisms by which some human enterovirulent bacteria induce intestinal disorders. Human colon carcinoma cell lines which are able to express in culture the functional and structural characteristics of mature enterocytes and goblet cells have been established, mimicking structurally and functionally an intestinal epithelial barrier. Moreover, Caco-2-derived M-like cells have been established, mimicking the bacterial capture property of M cells of Peyer's patches. This review intends to analyze the cellular and molecular mechanisms of pathogenesis of human enterovirulent bacteria observed in infected cultured human colon carcinoma enterocyte-like HT-29 subpopulations, enterocyte-like Caco-2 and clone cells, the colonic T84 cell line, HT-29 mucus-secreting cell subpopulations, and Caco-2-derived M-like cells, including cell association, cell entry, intracellular lifestyle, structural lesions at the brush border, functional lesions in enterocytes and goblet cells, functional and structural lesions at the junctional domain, and host cellular defense responses.

Note on the formulation of thermosensitive and mucoadhesive vaginal hydrogels containing the miniCD4 M48U1 as anti-HIV-1 microbicide.

The miniCD4 M48U1 was formulated into thermosensitive and mucoadhesive pluronic(®) hydrogels as anti-HIV-1 microbicide. The release kinetics of M48U1 from F127/HPMC (20/1 wt%) and F127/F68/HPMC (22.5/2.5/1 wt%) studied during 24h by using Franz diffusion cells showed that HEC hydrogel (1.5 wt%) used as control released 93% of the peptide, while about 25% of M48U1 remained in pluronic(®) hydrogels. The formulation of M48U1 in pluronic(®) hydrogels ensures a local delivery because no diffusion of the peptide was detected through vaginal Cynomolgus macaque mucosa using Ussing chamber. Finally, toxicological studies showed no significant difference in the HeLa cell viability of the pluronic(®) hydrogels in comparison with HEC and phosphate buffer saline.

Dysfunctions at human intestinal barrier by water-borne protozoan parasites: lessons from cultured human fully differentiated colon cancer cell lines.

Some water-borne protozoan parasites induce diseases through their membrane-associated functional structures and virulence factors that hijack the host cellular molecules and signalling pathways leading to structural and functional lesions in the intestinal barrier. In this Microreview we analyse the insights on the mechanisms of pathogenesis of Entamoeba intestinalis, Giardia and Cryptosporidium observed in the human colon carcinoma fully differentiated colon cancer cell lines, cell subpopulations and clones expressing the structural and functional characteristics of highly specialized fully differentiated epithelial cells lining the intestinal epithelium and mimicking structurally and functionally an intestinal barrier.

Afa/Dr diffusely adhering Escherichia coli strain C1845 induces neutrophil extracellular traps that kill bacteria and damage human enterocyte-like cells.

We recently documented the neutrophil response to enterovirulent diffusely adherent Escherichia coli expressing Afa/Dr fimbriae (Afa/Dr DAEC), using the human myeloid cell line PLB-985 differentiated into fully mature neutrophils. Upon activation, particularly during infections, neutrophils release neutrophil extracellular traps (NETs), composed of a nuclear DNA backbone associated with antimicrobial peptides, histones, and proteases, which entrap and kill pathogens. Here, using fluorescence microscopy and field emission scanning electron microscopy, we observed NET production by PLB-985 cells infected with the Afa/Dr wild-type (WT) E. coli strain C1845. We found that these NETs were able to capture, immobilize, and kill WT C1845 bacteria. We also developed a coculture model of human enterocyte-like Caco-2/TC7 cells and PLB-985 cells previously treated with WT C1845 and found, for the first time, that the F-actin cytoskeleton of enterocyte-like cells is damaged in the presence of bacterium-induced NETs and that this deleterious effect is prevented by inhibition of protease release. These findings provide new insights into the neutrophil response to bacterial infection via the production of bactericidal NETs and suggest that NETs may damage the intestinal epithelium, particularly in situations such as inflammatory bowel diseases.

Impairment of swimming motility by antidiarrheic Lactobacillus acidophilus strain LB retards internalization of Salmonella enterica serovar Typhimurium within human enterocyte-like cells.

We report that both culture and the cell-free culture supernatant (CFCS) of Lactobacillus acidophilus strain LB (Lactéol Boucard) have the ability (i) to delay the appearance of Salmonella enterica serovar Typhimurium strain SL1344-induced mobilization of F-actin and, subsequently, (ii) to retard cell entry by S. Typhimurium SL1344. Time-lapse imaging and Western immunoblotting showed that S. Typhimurium SL1344 swimming motility, as represented by cell tracks of various types, was rapidly but temporarily blocked without affecting the expression of FliC flagellar propeller protein. We show that the product(s) secreted by L. acidophilus LB that supports the inhibitory activity is heat stable and of low molecular weight. The product(s) caused rapid depolarization of the S. Typhimurium SL1344 cytoplasmic membrane without affecting bacterial viability. We identified inhibition of swimming motility as a newly discovered mechanism by which the secreted product(s) of L. acidophilus strain LB retards the internalization of the diarrhea-associated pathogen S. enterica serovar Typhimurium within cultured human enterocyte-like cells.

Lipid raft-dependent adhesion of Giardia intestinalis trophozoites to a cultured human enterocyte-like Caco-2/TC7 cell monolayer leads to cytoskeleton-dependent functional injuries.

Gardia intestinalis, the aetiological agent of giardiasis, one of the most common intestinal diseases in both developing and developed countries, induces a loss of epithelial barrier function and functional injuries of the enterocyte by mechanisms that remain unknown. Three possible mechanisms have been proposed: (i) Giardia may directly alter the epithelial barrier after a close interaction between the trophozoite and polarized intestinal cells, (ii) intestinal functions may be altered by factors secreted by Giardia including an 'enterotoxin', proteinases and lectins, and (iii) based on mouse studies, a mechanism involving the intervention of activated T lymphocytes. We used fully differentiated cultured human intestinal Caco-2/TC7 cells forming a monolayer and expressing several polarized functions of enterocytes of small intestine to investigate the mechanisms by which G. intestinalis induces structural and functional alterations in the host intestinal epithelium. We first report that adhesion of G. intestinalis at the brush border of enterocyte-like cells involves the lipid raft membrane microdomains of the trophozoite. We report an adhesion-dependent disorganization of the apical F-actin cytoskeleton that, in turn, results in a dramatic loss of distribution of functional brush border-associated proteins, including sucrase-isomaltase (SI), dipeptidylpeptidase IV (DPP IV) and fructose transporter, GLUT5, and a decrease in sucrose enzyme activity in G. intestinalis-infected enterocyte-like cells. We observed that the G. intestinalis trophozoite promotes an adhesion-dependent decrease in transepithelial electrical resistance (TER) accompanied by a rearrangement of functional tight junction (TJ)-associated occludin, and delocalization of claudin-1. Finally, we found that whereas the occludin rearrangement induced by G. intestinalis was related to apical F-actin disorganization, the delocalization of claudin-1 was not.

Secreted autotransporter toxin (Sat) triggers autophagy in epithelial cells that relies on cell detachment.

The secreted autotransporter toxin, Sat, which belongs to the subfamily of serine protease autotransporters of Enterobacteriaceae, acts as a virulence factor in extraintestinal and intestinal pathogenic strains of Escherichia coli. We observed that HeLa cells exposed to the cell-free culture supernatant of recombinant strain AAEC185p(Sat-IH11128) producing the Sat toxin (CFCS(Sat) ), displayed dramatic disorganization of the F-actin cytoskeleton before loosening cell-to-cell junctions and detachment. Examination of the effect of Sat on GFP-microtubule-associated protein light chain 3 (LC3) HeLa cells revealed that CFCS(Sat) -induced autophagy follows CFCS(Sat) -induced F-actin cytoskeleton rearrangement. The induced autophagy shows an acceleration of the autophagy flux soon after Sat treatment, followed later by a blockade of the flux leading to the accumulation of large GFP-LC3-positive vacuoles in the cell cytoplasm. CFCS(Sat) did not induce cell detachment in autophagy-deficient mouse embryonic fibroblasts in contrast with wild-type mouse embryonic fibroblasts. The CFCS(Sat) -induced large GFP-LC3 dots do not display the characteristics of autophagolysosomes including expression of cathepsin D and Lamp-1 and 2 proteins, and Lysotracker Red- and DQ-BSA-positive labelling. We provide evidences that CFCS(Sat) -induced autophagy is not a cell response intended to get rid of the intracellular toxin. By a pharmacological blockers approach, we found that the blockade of Erk1/2 and p38 MAPKs, but not JNK, inhibited the CFCS(Sat) -induced autophagy and cell detachment whereas phosphatidylinositol-3 kinase blockers inhibiting canonical autophagy were inactive. When attached CFCS(Sat) -treated cells start to detach they showed caspase-independent cell death and rearrangements of the focal adhesion-associated vinculin and paxillin. Collectively, our results support that Sat triggers autophagy in epithelial cells that relies on its cell-detachment effect.