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.Fil: Humen, Martin Andres. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; ArgentinaFil: Perez, Pablo Fernando. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; ArgentinaFil: Liévin Le Moal, Vanessa. Université Paris Sud; Francia. Institut National de la Santé et de la Recherche Médicale; Franci

Up-Regulation of Intestinal Vascular Endothelial Growth Factor by Afa/Dr Diffusely Adhering Escherichia coli

BACKGROUND: Angiogenesis has been recently described as a novel component of inflammatory bowel disease pathogenesis. The level of vascular endothelial growth factor (VEGF) has been found increased in Crohn's disease and ulcerative colitis mucosa. To question whether a pro-inflammatory Escherichia coli could regulate the expression of VEGF in human intestinal epithelial cells, we examine the response of cultured human colonic T84 cells to infection by E. coli strain C1845 that belongs to the typical Afa/Dr diffusely adhering E. coli family (Afa/Dr DAEC). METHODOLOGY: VEGF mRNA expression was examined by Northern blotting and q-PCR. VEGF protein levels were assayed by ELISA and its bioactivity was analysed in endothelial cells. The bacterial factor involved in VEGF induction was identified using recombinant E. coli expressing Dr adhesin, purified Dr adhesin and lipopolysaccharide. The signaling pathway activated for the up-regulation of VEGF was identified using a blocking monoclonal anti-DAF antibody, Western blot analysis and specific pharmacological inhibitors. PRINCIPAL FINDINGS: C1845 bacteria induce the production of VEGF protein which is bioactive. VEGF is induced by adhering C1845 in both a time- and bacteria concentration-dependent manner. This phenomenon is not cell line dependent since we reproduced this observation in intestinal LS174, Caco2/TC7 and INT407 cells. Up-regulation of VEGF production requires: (1) the interaction of the bacterial F1845 adhesin with the brush border-associated decay accelerating factor (DAF, CD55) acting as a bacterial receptor, and (2) the activation of a Src protein kinase upstream of the activation of the Erk and Akt signaling pathways. CONCLUSIONS: Results demonstrate that a Afa/Dr DAEC strain induces an adhesin-dependent activation of DAF signaling that leads to the up-regulation of bioactive VEGF in cultured human intestinal cells. Thus, these results suggest a link between an entero-adherent, pro-inflammatory E. coli strain and angiogenesis which appeared recently as a novel component of IBD pathogenesis

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

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 enterohemorrhagic 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

Lactobacillus Adhesion to Mucus

Mucus provides protective functions in the gastrointestinal tract and plays an important role in the adhesion of microorganisms to host surfaces. Mucin glycoproteins polymerize, forming a framework to which certain microbial populations can adhere, including probiotic Lactobacillus species. Numerous mechanisms for adhesion to mucus have been discovered in lactobacilli, including partially characterized mucus binding proteins. These mechanisms vary in importance with the in vitro models studied, which could significantly affect the perceived probiotic potential of the organisms. Understanding the nature of mucus-microbe interactions could be the key to elucidating the mechanisms of probiotic adhesion within the host

Distinct Pathogenesis and Host Responses during Infection of C. elegans by P. aeruginosa and S. aureus

The genetically tractable model host Caenorhabditis elegans provides a valuable tool to dissect host-microbe interactions in vivo. Pseudomonas aeruginosa and Staphylococcus aureus utilize virulence factors involved in human disease to infect and kill C. elegans. Despite much progress, virtually nothing is known regarding the cytopathology of infection and the proximate causes of nematode death. Using light and electron microscopy, we found that P. aeruginosa infection entails intestinal distention, accumulation of an unidentified extracellular matrix and P. aeruginosa-synthesized outer membrane vesicles in the gut lumen and on the apical surface of intestinal cells, the appearance of abnormal autophagosomes inside intestinal cells, and P. aeruginosa intracellular invasion of C. elegans. Importantly, heat-killed P. aeruginosa fails to elicit a significant host response, suggesting that the C. elegans response to P. aeruginosa is activated either by heat-labile signals or pathogen-induced damage. In contrast, S. aureus infection causes enterocyte effacement, intestinal epithelium destruction, and complete degradation of internal organs. S. aureus activates a strong transcriptional response in C. elegans intestinal epithelial cells, which aids host survival during infection and shares elements with human innate responses. The C. elegans genes induced in response to S. aureus are mostly distinct from those induced by P. aeruginosa. In contrast to P. aeruginosa, heat-killed S. aureus activates a similar response as live S. aureus, which appears to be independent of the single C. elegans Toll-Like Receptor (TLR) protein. These data suggest that the host response to S. aureus is possibly mediated by pathogen-associated molecular patterns (PAMPs). Because our data suggest that neither the P. aeruginosa nor the S. aureus–triggered response requires canonical TLR signaling, they imply the existence of unidentified mechanisms for pathogen detection in C. elegans, with potentially conserved roles also in mammals

A gastrointestinal anti-infectious biotherapeutic agent: the heat-treated 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

The Front Line of Enteric Host Defense against Unwelcome Intrusion of Harmful Microorganisms: Mucins, Antimicrobial Peptides, and Microbiota

The intestinal tract is a complex ecosystem that combines resident microbiota and the cells of various phenotypes with complex metabolic activities that line the epithelial wall. The intestinal cells that make up the epithelium provide physical and chemical barriers that protect the host against the unwanted intrusion of microorganisms that hijack the cellular molecules and signaling pathways of the host and become pathogenic. Some of the organisms making up the intestinal microbiota also have microbicidal effects that contribute to the barrier against enteric pathogens. This review describes the two cell lineages present in the intestinal epithelium: the goblet cells and the Paneth cells, both of which play a pivotal role in the first line of enteric defense by producing mucus and antimicrobial peptides, respectively. We also analyze recent insights into the intestinal microbiota and the mechanisms by which some resident species act as a barrier to enteric pathogens. Moreover, this review examines whether the cells producing mucins or antimicrobial peptides and the resident microbiota act in partnership and whether they function individually and/or synergistically to provide the host with an effective front line of defense against harmful enteric pathogens