The adhering human Lactobacillus acidophilus strain LA1 inhibits the cell association and cell invasion of enteropathogens in cultured human intestinal Caco-2 cells (M. F. Bernet, D. Brassard, J. R. Neeser, and A. L. Servin, Gut... more
The adhering human Lactobacillus acidophilus strain LA1 inhibits the cell association and cell invasion of enteropathogens in cultured human intestinal Caco-2 cells (M. F. Bernet, D. Brassard, J. R. Neeser, and A. L. Servin, Gut 35:483-489, 1994). Here, we demonstrate that strain LA1 developed its antibacterial activity in conventional or germ-free mouse models orally infected by Salmonella typhimurium. We present evidence that the spent culture supernatant of strain LA1 (LA1-SCS) contained antibacterial components active against S. typhimurium infecting the cultured human intestinal Caco-2 cells. The LA1-SCS antibacterial activity was observed in vitro against a wide range of gram-negative and gram-positive pathogens, such as Staphylococcus aureus, Listeria monocytogenes, S. typhimurium, Shigella flexneri, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Enterobacter cloacae. By contrast, no activity was observed against species of the normal gut flora, such as lactobacilli and b...
Research Interests: Electron Microscopy, Biology, Medicine, Lactic Acid Metabolism, Humans, and 15 moreMice, Applied, Female, Animals, Listeria monocytogenes, Applied Environmental Microbiology, Electron, Gram Positive Bacteria, Antibacterial activity, Cells, Lactic Acid, Lactobacillus Acidophilus, Enterobacter cloacae, Antibiosis, and Cultured
Research Interests: Biology, Calcium, Cell Biology, Medicine, Cytoskeleton, and 15 moreSignal Transduction, Biological Sciences, Infection and immunity, Cell line, Humans, Escherichia coli, Actin, Protein Tyrosine Kinase, Intestines, Isoenzymes, Bacterial Adhesion, Protein Kinase C, Phosphatidylinositol 3-KINASE, Protein tyrosine kinases, and Medical and Health Sciences
The association of enterotoxigenic Escherichia coli expressing colonization factor antigen I (CFA/I) with the cultured human colon adenocarcinoma cell, a model of the mature enterocyte of the small intestine, is dependent on the binding... more
The association of enterotoxigenic Escherichia coli expressing colonization factor antigen I (CFA/I) with the cultured human colon adenocarcinoma cell, a model of the mature enterocyte of the small intestine, is dependent on the binding of CFA/I to a brush border-associated component. Binding of the purified radiolabeled [125I]CFA/I- and 14C-labeled CFA/I-positive bacteria could be displaced by an increasing concentration of unlabeled CFA/I. Moreover, we showed that expression of the specific CFA/I binding developed as a function of cell differentiation in Caco-2 cells, whereas expression of the nonspecific binding did not. Expression of the brush border differentiation-associated component acting as a binding site for CFA/I was up-regulated by glucose. Indeed, the enterocyte-like HT-29 glc- cell subpopulation not expressing the CFA/I binding site when cultured in dialyzed serum and hexose-free medium regained the ability to bind CFA/I when the cells were returned to culture medium ...
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The aim of this study was to compare the antagonistic properties of Lactobacillus casei GG exerted in vitro against Salmonella typhimurium C5 in a cellular model, cultured enterocyte-like Caco-2 cells, to those exerted in vivo in an... more
The aim of this study was to compare the antagonistic properties of Lactobacillus casei GG exerted in vitro against Salmonella typhimurium C5 in a cellular model, cultured enterocyte-like Caco-2 cells, to those exerted in vivo in an animal model, C3H/He/Oujco mice. Our results show that a 1-h contact between the invading strain C5 and either the culture or the supernatant of L. casei GG impeded the invasion by the Salmonella strain in Caco-2 cells, without modifying the viability of the strain. After neutralization at pH 7, no inhibition of the invasion by C5 was observed. The antagonistic activity of L. casei GG was examined in C3H/He/Oujco mice orally infected with C5 as follows: (i) L. casei GG was given daily to conventional animals as a probiotic, and (ii) it was given once to germ-free animals in order to study the effect of the population of L. casei GG established in the different segments of the gut. In vivo experiments show that after a single challenge with C5, this strai...
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The spent culture supernatant of the human Lactobacillus acidophilus strain LB produces an antibacterial activity against a wide range of gram-negative and gram-positive pathogens. It decreased the in vitro viability of Staphylococcus... more
The spent culture supernatant of the human Lactobacillus acidophilus strain LB produces an antibacterial activity against a wide range of gram-negative and gram-positive pathogens. It decreased the in vitro viability of Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, Shigella flexneri, Escherichia coli, Klebsiella pneumoniae, Bacillus cereus, Pseudomonas aeruginosa, and Enterobacter spp. In contrast, it did not inhibit lactobacilli and bifidobacteria. The activity was heat stable and relatively sensitive to enzymatic treatments and developed under acidic conditions. The antimicrobial activity was independent of lactic acid production. Activity against S. typhimurium SL1344 infecting human cultured intestinal Caco-2 cells was observed as it was in the conventional C3H/He/oujco mouse model with S. typhimurium C5 infection and oral treatment with the LB spent culture supernatant.
Research Interests: Microbiology, Medical Microbiology, Biology, Medicine, Humans, and 15 moreEscherichia coli, Gram Positive, Mice, Animals, Listeria monocytogenes, Antimicrobial activity, Antimicrobial, Antibacterial activity, Mouse Model, Antimicrobial agents, Microbial Sensitivity Tests, Bacillus Cereus, Lactic Acid, Cell Survival, and Lactobacillus Acidophilus
First extensive reformulation of clofazimine (CLZ) in nanoporous silica particles (NSPs) for tackling antibiotic-resistant tuberculosis (TB) infections. Solid-state characterization of several CLZ-encapsulated NSP formulations was... more
First extensive reformulation of clofazimine (CLZ) in nanoporous silica particles (NSPs) for tackling antibiotic-resistant tuberculosis (TB) infections. Solid-state characterization of several CLZ-encapsulated NSP formulations was followed by in vitro drug solubility, Caco-2 intestinal cells drug permeability and TB antibacterial activity. NSPs stabilize the amorphous state of CLZ (shelf stability >6 months) and dramatically increase the drug solubility in simulated gastric fluid (up to 20-fold) with different dissolution kinetics depending on the NSPs used. CLZ encapsulation in NSP substantially enhances the permeation through model intestinal cell layer, achieving effective antimicrobial concentrations in TB-infected macrophages. Promising results toward refurbishment of an approved marketed drug for a different indication suitable for oral anti-TB formulation.
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Afa/Dr diffusely adhering Escherichia coli (Afa/Dr DAEC) strains cause symptomatic urinary tract and intestinal infections. The proinflammatory effects of Afa/Dr DAEC strains in vitro have been not investigated to date. In the present... more
Afa/Dr diffusely adhering Escherichia coli (Afa/Dr DAEC) strains cause symptomatic urinary tract and intestinal infections. The proinflammatory effects of Afa/Dr DAEC strains in vitro have been not investigated to date. In the present study, we used confluent polarized monolayers of intestinal cell line T84 to evaluate the consequences of epithelial infection by Afa/Dr DAEC strains in terms of proinflammatory response. Polymorphonuclear leukocyte (PMNL) migration across the epithelial barrier was induced after incubation of the T84 monolayers with the wild-type Afa/Dr DAEC strain C1845 harboring the fimbrial F1845 adhesin and strain IH11128 harboring the Dr hemagglutinin, and the E. coli laboratory strain HB101 was transformed with the pSSS1 plasmid, producing Afa/Dr F1845 adhesin. PMNL migrations were correlated with a basolateral secretion of interleukin-8 by T84 cells and were abolished after incubation of epithelial cells with an anti-decay accelerating factor (DAF) antibody tha...
Research Interests: Microbiology, Biology, Medicine, Signal Transduction, Biological Sciences, and 15 moreInfection and immunity, Cell line, Humans, Intestinal Mucosa, Escherichia coli, Cell Polarity, Phosphorylation, Mitogen Activated Protein Kinase, Neutrophils, Monoclonal Antibody, Bacterial Adhesion, Secretion, Tyrosine, Interleukin, and Medical and Health Sciences
Diffusely adhering Escherichia coli strains harboring Afa/Dr adhesins (Afa/Dr DAEC) have been associated with diarrhea and urinary tract infections (UTIs). The present work is the first extensive molecular study of a Afa/Dr DAEC strain... more
Diffusely adhering Escherichia coli strains harboring Afa/Dr adhesins (Afa/Dr DAEC) have been associated with diarrhea and urinary tract infections (UTIs). The present work is the first extensive molecular study of a Afa/Dr DAEC strain using the representational difference analysis technique. We have searched for DNA sequences present in strain C1845, recovered from a diarrheagenic child, but absent from a nonpathogenic K-12 strain. Strain C1845 harbors part of a pathogenicity island (PAI CFT073 ) and several iron transport systems found in other E. coli pathovars. We did not find genes encoding factors known to subvert host cell proteins, such as type III secretion system or effector proteins. Several C1845-specific sequences are homologous to putative virulence genes or show no homology with known sequences, and we have analyzed their distribution among Afa/Dr and non-Afa/Dr clinical isolates and among strains from the E. coli Reference Collection. Three C1845-specific sequences (...
Research Interests: Genetics, Microbiology, Biology, Intelligent Transport System, Medicine, and 15 moreBiological Sciences, Infection and immunity, Humans, Escherichia coli, Infection, DNA sequence design, Iron, Enterobacteriaceae, Species Specificity, Base Sequence, Bacterial Adhesion, Escherichia Coli Infections, DNA sequence, Molecular Sequence Data, and Medical and Health Sciences
We undertook a study of the mechanism by which Dr-positive bacteria invade epithelial cells. Our findings show that Dr-positive bacteria enter via a zipper-like mechanism that is independent of the Dr-induced mobilization of F-actin and... more
We undertook a study of the mechanism by which Dr-positive bacteria invade epithelial cells. Our findings show that Dr-positive bacteria enter via a zipper-like mechanism that is independent of the Dr-induced mobilization of F-actin and of the signaling molecules that control Dr-induced F-actin rearrangements. We also observed that Dr-positive IH11128 bacteria entered cells that were positive for the caveola marker VIP21/caveolin (HeLa and Caco-2/Cav-1 cells) to the same extent as those that were not (parental Caco-2 cells). Using fluorescence labeling and confocal laser scanning microscopy, we provide evidence that during the adhesion step, the α5β1 integrin, which plays a pivotal role in Afa/Dr diffusely adhering Escherichia coli bacterial entry, is mobilized around adhering Dr-positive bacteria. We show that the receptor for Afa/Dr adhesins, glycosylphosphatidylinositol-anchored CD55; the raft marker, ganglioside GM1; and VIP21/caveolin are all recruited around adhering Dr-positi...
Research Interests: Biology, Cell Biology, Medicine, Biological Sciences, Infection and immunity, and 14 moreInternalization, Humans, Escherichia coli, Cholesterol, Infection, Lipid Raft, Caco-2 cells, Epithelium, HeLa cells, Confocal Laser Scanning Microscopy, Caco 2 Cell, Bacterial Adhesion, Cell Membrane, and Medical and Health Sciences
Afa/Dr diffusely adhering Escherichia colistrain IH11128 bacteria basolaterally entered polarized epithelial cells by a CD55- and CD66e-independent mechanism through interaction with the α5β1 integrin and a pathway involving caveolae and... more
Afa/Dr diffusely adhering Escherichia colistrain IH11128 bacteria basolaterally entered polarized epithelial cells by a CD55- and CD66e-independent mechanism through interaction with the α5β1 integrin and a pathway involving caveolae and dynamic microtubules (MTs). IH11128 invasion within HeLa cells was dramatically decreased after the cells were treated with the cholesterol-extracting drug methyl-β-cyclodextrin or the caveola-disrupting drug filipin. Disassembly of the dynamically unstable MT network by the compound 201-F resulted in a total abolition of IH11128 entry. In apically infected polarized fully differentiated Caco-2/TC7 cells, no IH11128 entry was observed. The entry of bacteria into apically IH11128-infected fully differentiated Caco-2/TC7 cells was greatly enhanced by treating cells with Ca2+-free medium supplemented with EGTA, a procedure that disrupts intercellular junctions and thus exposes the basolateral surface to bacteria. Basally infected fully differentiated p...
Research Interests: Biology, Cell Biology, Medicine, Biological Sciences, Infection and immunity, and 15 moreInternalization, Cell Differentiation, Humans, Intestinal Mucosa, Escherichia coli, Endocytosis, Cell Polarity, Urinary Tract Infections, Microtubules, Epithelial cells, HeLa cells, Caveolae, Bacterial Adhesion, Cell Adhesion Molecules, and Medical and Health Sciences
Diffusely adhering Escherichia coli (DAEC) strains expressing adhesins of the Afa/Dr family bind to epithelial cells in a diffuse adherence pattern by recognizing a common receptor, the decay-accelerating factor (CD55). Recently, a novel... more
Diffusely adhering Escherichia coli (DAEC) strains expressing adhesins of the Afa/Dr family bind to epithelial cells in a diffuse adherence pattern by recognizing a common receptor, the decay-accelerating factor (CD55). Recently, a novel CD55-binding adhesin, named Dr-II, was identified from the pyelonephritogenic strain EC7372. In this report, we show that despite the low level of sequence identity between Dr-II and other members of the Afa/Dr family, EC7372 induces pathophysiological effects similar to those induced by other Afa/Dr DAEC strains on the polarized epithelial cell line Caco-2/TC7. Specifically, the Dr-II adhesin was sufficient to promote CD55 and CD66e clustering around adhering bacteria and apical cytoskeleton rearrangements. Unlike other Afa/Dr DAEC strains, EC7372 expresses a functional hemolysin that promotes a rapid cellular lysis. In addition, cell death by apoptosis or necrosis was observed in EC7372-infected Caco-2/TC7 cells, depending on infection time. Our r...
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Diffusely adhering Escherichia coli (DAEC) strains expressing F1845 fimbrial adhesin or Dr hemagglutinin belonging to the Afa/Dr family of adhesins infect cultured polarized human intestinal cells through recognition of the brush... more
Diffusely adhering Escherichia coli (DAEC) strains expressing F1845 fimbrial adhesin or Dr hemagglutinin belonging to the Afa/Dr family of adhesins infect cultured polarized human intestinal cells through recognition of the brush border-associated decay-accelerating factor (DAF; CD55) as a receptor. The wild-type Afa/Dr DAEC strain C1845 has been shown to induce brush border lesions by an adhesin-dependent mechanism triggering apical F-actin rearrangements. In the present study, we undertook to further characterize cell injuries following the interaction of wild-type Afa/Dr DAEC strains C1845 and IH11128 expressing fimbrial F1845 adhesin and Dr hemagglutinin, respectively, with polarized, fully differentiated Caco-2/TC7 cells. In both cases, bacterium-cell interaction was followed by rearrangement of the major brush border-associated cytoskeletal proteins F-actin, villin, and fimbrin, proteins which play a pivotal role in brush border assembly. In contrast, distribution of G-actin, ...
Research Interests: Electron Microscopy, Biology, Calcium, Transmission Electron Microscopy, Medicine, and 15 moreCytoskeleton, Biological Sciences, Infection and immunity, Protein Structure and Function, Virulence, Humans, Intestinal Mucosa, Escherichia coli, Cell Polarity, Villin, Brush Border, Caco-2 cells, Bacterial Adhesion, microvilli, and Medical and Health Sciences
The Afa/Dr family of diffusely adhering Escherichia coli (Afa/Dr DAEC) includes bacteria expressing afimbrial adhesins (AFA), Dr hemagglutinin, and fimbrial F1845 adhesin. We show that infection of human intestinal Caco-2/TC7 cells by the... more
The Afa/Dr family of diffusely adhering Escherichia coli (Afa/Dr DAEC) includes bacteria expressing afimbrial adhesins (AFA), Dr hemagglutinin, and fimbrial F1845 adhesin. We show that infection of human intestinal Caco-2/TC7 cells by the Afa/Dr DAEC strains C1845 and IH11128 is followed by clustering of CD55 around adhering bacteria. Mapping of CD55 epitopes involved in CD55 clustering by Afa/Dr DAEC was conducted using CD55 deletion mutants expressed by stable transfection in CHO cells. Deletion in the short consensus repeat 1 (SCR1) domain abolished Afa/Dr DAEC-induced CD55 clustering. In contrast, deletion in the SCR4 domain does not modify Afa/Dr DAEC-induced CD55 clustering. We show that the brush border-associated glycosylphosphatidylinositol (GPI)-anchored protein CD66e (carcinoembryonic antigen) is recruited by the Afa/Dr DAEC strains C1845 and IH11128. This conclusion is based on the observations that (i) infection of Caco-2/TC7 cells by Afa/Dr DAEC strains is followed by ...
Research Interests: Biology, Medicine, Biological Sciences, Infection and immunity, Humans, and 15 moreEscherichia coli, Animals, Epitope mapping, Cell Polarity, Brush Border, Intestines, HeLa cells, Monoclonal Antibody, Carcinoembryonic Antigen, Bacterial Adhesion, Cell Adhesion Molecules, microvilli, CHO cells, Cricetinae, and Medical and Health Sciences
The Afa/Dr diffusely adhering Escherichia coli (DAEC) C1845 strain harboring the F1845 fimbrial adhesin interacts with the brush border-associated CD55 molecule and promotes elongation of brush border microvilli resulting from... more
The Afa/Dr diffusely adhering Escherichia coli (DAEC) C1845 strain harboring the F1845 fimbrial adhesin interacts with the brush border-associated CD55 molecule and promotes elongation of brush border microvilli resulting from rearrangement of the F-actin network. This phenomenon involves the activation of a cascade of signaling coupled to the glycosylphosphatidylinositol-anchored receptor of the F1845 adhesin. We provide evidence that infection of the polarized human intestinal cell line Caco-2/TC7 by strain C1845 is followed by an increase in the paracellular permeability for [ 3 H]mannitol without a decrease of the transepithelial resistance of the monolayers. Alterations in the distribution of tight-junction (TJ)-associated occludin and ZO-1 protein are observed, whereas the distribution of the zonula adherens-associated E-cadherin is not affected. Using the recombinant E. coli strains HB101(pSSS1) and -(pSSS1C) expressing the F1845 fimbrial adhesin, we demonstrate that the adhe...
Research Interests: Biology, Membrane Proteins, Cell Biology, Medicine, Cytoskeleton, and 15 moreBiological Sciences, Infection and immunity, Cell line, Humans, Intestinal Mucosa, Escherichia coli, Permeability, Cell Polarity, Brush Border, Occludin, Bacterial Adhesion, Actin Filaments, Cadherins, Adherens Junction, and Medical and Health Sciences
Research Interests: Electron Microscopy, Biology, Cell Culture, Cell line, Cell Differentiation, and 15 moreEscherichia coli, Animals, Cellular differentiation, Cell, Bacteria, Differentiation, Cellular Automaton Model, Digestive System, Carcinoma, Epithelium, Epithelial cells, Bacterial Adhesion, Biochemistry and cell biology, Cell invasion, and Colonic Neoplasms
Research Interests: Biology, Cell Biology, Medicine, Cell Culture, Cell line, and 15 moreCell Differentiation, Humans, Animals, Cellular differentiation, Cell, Monoclonal Antibodies, Differentiation, Integrins, Intestines, Epithelium, Epithelial cells, Colon Carcinoma, Biochemistry and cell biology, Paediatrics and reproductive medicine, and Colonic Neoplasms
Wild-type diffusely adhering Escherichia coli (DAEC) harbouring afimbrial adhesin (Afa) or fimbrial Dr and F1845 adhesins (Afa/Dr DAEC) apically infecting the human intestinal epithelial cells promote injuries in the brush border of the... more
Wild-type diffusely adhering Escherichia coli (DAEC) harbouring afimbrial adhesin (Afa) or fimbrial Dr and F1845 adhesins (Afa/Dr DAEC) apically infecting the human intestinal epithelial cells promote injuries in the brush border of the cells. We report here that infection by Afa/Dr DAEC wild-type strains C1845 and IH11128 in polarized human fully differentiated Caco-2/TC7 cells dramatically impaired the enzyme activity of functional brush border-associated proteins sucrase-isomaltase (SI) and dipeptidylpeptidase IV (DPP IV). Blockers of the transduction signal molecules, previously found to be active against the Afa/Dr DAEC-induced cytoskeleton injury, were inactive against the Afa/Dr-induced decrease in sucrase enzyme activity. In parallel, Afa/Dr DAEC infection promotes the blockade of the biosynthesis of SI and DPP IV without affection enzyme stability. The observation that no changes occurred in mRNA levels of SI and DPP IV upon infection suggested that the decrease in biosynthesis probably resulted from a decrease in the translation rate. When the cells were infected with recombinant E. coli strains expressing homologous adhesins of the wild-type strains, neither a decrease in sucrase and DPP IV enzyme activities nor an inhibition of enzyme biosynthesis were observed. In conclusion, taken together, these data give new insights into the mechanisms by which the wild-type Afa/Dr DAEC strains induce functional injuries in polarized fully differentiated human intestinal cells. Moreover, the results revealed that other pathogenic factor(s) distinct from the Afa/Dr adhesins may play(s) a crucial role in this mechanism of pathogenicity.