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.

Autophagy is now known to be an essential component of host innate and adaptive immunity. Several herpesviruses have developed various strategies to evade this antiviral host defense. Herpes simplex virus 1 (HSV-1) blocks autophagy in fibroblasts and in neurons, and the ICP34.5 protein is important for the resistance of HSV-1 to autophagy because of its interaction with the autophagy machinery protein Beclin 1. ICP34.5 also counteracts the shutoff of protein synthesis mediated by the doublestranded RNA (dsRNA)-dependent protein kinase PKR by inhibiting phosphorylation of the eukaryotic translation initiation factor 2␣ (eIF2␣) in the PKR/eIF2␣ signaling pathway. Us11 is a late gene product of HSV-1, which is also able to preclude the host shutoff by direct inhibition of PKR. In the present study, we unveil a previously uncharacterized function of Us11 by demonstrating its antiautophagic activity. We show that the expression of Us11 is able to block autophagy and autophagosome formation in both HeLa cells and fibroblasts. Furthermore, immediate-early expression of Us11 by an ICP34.5 deletion mutant virus is sufficient to render the cells resistant to PKR-induced and virus-induced autophagy. PKR expression and the PKR binding domain of Us11 are required for the antiautophagic activity of Us11. However, unlike ICP34.5, Us11 did not interact with Beclin 1. We suggest that the inhibition of autophagy observed in cells infected with HSV-1 results from the activity of not only ICP34.5 on Beclin 1 but also Us11 by direct interaction with PKR.

Afa/Dr diffusely adhering Escherichia coli strain IH11128 bacteria basolaterally entered polarized epithelial cells by a CD55-and CD66e-independent mechanism through interaction with the ␣ 5 ␤ 1 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-␤-cyclodextrin or the caveoladisrupting 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 Ca 2؉ -free medium supplemented with EGTA, a procedure that disrupts intercellular junctions and thus exposes the basolateral surface to bacteria. Basally infected fully differentiated polarized Caco-2/TC7 cells grown on inverted inserts mounted in chamber culture showed a highly significant level of intracellular IH11128 bacteria compared with cells subjected to the apical route of infection. No expression of CD55 and CD66e, the receptors for the Afa/Dr adhesins, was found at the basolateral domains of these cells. Consistent with the hypothesis that a cell-to-cell adhesion molecule acts as a receptor for polarized IH11128 entry, an antibody blockade using anti-␣ 5 ␤ 1 integrin polyclonal antibody completely abolished bacterial entry. Experiments conducted with the laboratory strain E. coli K-12 EC901 carrying the recombinant plasmid pBJN406, which expresses Dr hemagglutinin, demonstrated that the dra operon is involved in polarized entry of IH11128 bacteria. Examined as a function of cell differentiation, the number of internalized bacteria decreased dramatically beyond cell confluency. Surviving intracellular IH11128 bacteria residing intracellularly had no effect on the functional differentiation of Caco-2/TC7 cells.

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 strain survives and persists at a higher level in the feces of the untreated conventional mice than in those of the treated group. In L. casei GG germ-free mice, establishment of L. casei GG in the gut significantly delayed the occurrence of 100% mortality of the animals (15 days after C5 challenge versus 9 days in germ-free mice [P < 0.01]). Cecal colonization level and translocation rate of C5 to the mesenteric lymph nodes, spleen, and liver were significantly reduced during the first 2 days post-C5 challenge, although the L. casei GG population level in the gut dramatically decreased in these animals. Recently, Klaenhammer (21) proposed that Lactobacillus strains with well-defined properties should be selected and characterized for specific use. Among Lactobacillus strains used in fermented milks, Lactobacillus casei GG has been shown to promote clinical recovery from acute rotavirus diarrhea in infants (17-20, 24) and from either antibiotic-associated or traveller's diarrhea in adults (27, 36). It has been previously reported that L. casei GG adheres to enterocyte-like cells in culture (11). It exerts an antagonistic effect against several bacteria and produces an unknown antimicrobial substance against Escherichia coli (37). But the mechanism of action of L. casei GG in vivo remains unknown. We have recently reported that selected, adhering human L. acidophilus strains inhibit both cell association and cell entry of a variety of enterovirulent bacteria within human cultured enterocyte-like Caco-2 cells (5-8). In the present work, we have compared the antagonistic effect observed in vitro in a cellular model (Caco-2 cells) to that observed in an in vivo animal model (C3H/He/ Oujco mice). First, we studied whether L. casei GG can inhibit the invasion of cultured enterocyte-like Caco-2 cells by Salmonella typhimurium C5 (28). Second, the antagonistic activity of L. casei GG was examined in C3H/He/Oujco mice orally infected with S. typhimurium C5. Two experimental designs were used: (i) L. casei GG given daily to conventional animals as a ...

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 borderassociated 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 clustering of CD66e around adhering bacteria and (ii) Afa/Dr DAEC strains bound efficiently to stably transfected HeLa cells expressing CD66e, accompanied by CD66e clustering around adhering bacteria. Inhibition assay using monoclonal antibodies directed against CD55 SCR domains, and polyclonal anti-CD55 and anti-CD66e antibodies demonstrate that CD55 and CD66e function as a receptors for the C1845 and IH11128 bacteria. Moreover, using structural draE gene mutants, we found that a mutant in which cysteine replaced aspartic acid at position 54 displayed conserved binding capacity but failed to induce CD55 and CD66e clustering. Taken together, these data give new insights into the mechanisms by which Afa/Dr DAEC induces adhesin-dependent cross talk in the human polarized intestinal epithelial cells by mobilizing brush border-associated GPI-anchored proteins known to function as transducing molecules.

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 bifidobacteria. The LA1-SCS antibacterial activity was insensitive to proteases and independent of lactic acid production. MATERIALS AND METHODS Bacteria. L. acidophilus LA1 and LA10 were from the Nestec (Lausanne, Switzerland) collection. The bacteria were grown under anaerobic conditions (Gaspak H 2 ϩCO 2) in De Man-Rogosa-Sharpe (MRS) broth (Biokar Diagnostic, Beauvais, France) for 24 h at 37°C. Spent culture supernatant (LA1-SCS) was obtained by centrifugation at 10.000 ϫ g for 30 min at 4°C. Centrifuged LA1-SCS was passed through a sterile 0.22-m-pore-size Millex GS filter unit (Millipore, Molsheim, France). Filtered LA1-SCS was checked for the absence of LA1 bacteria by being plated on tryptic soy agar (TSA) to confirm the absence of bacterial colonies. Concentrated LA1-SCS were obtained by freeze-drying. Salmonella typhimurium SL1344 was a gift of B. A. D. Stocker (Stanford University, Stanford, Calif.) (12); S. typhimurium C5 was provided by M. Y. Popoff (Institut Pasteur, Paris, France) (40). Listeria monocytogenes EGD [HLY ϩ ] was provided by J. L. Gaillard (INSERM U411, Hôpital Necker, Paris, France). Enterobacter cloacae and Klebsiella pneumoniae were clinical isolates provided by A. Darfeuille-Michaud (Université Clermont I) (32). Shigella flexneri FL5M90T was provided by P. Sansonetti (Institut Pasteur, Paris, France). Staphylococcus aureus, Streptococcus group D, and Pseudomonas aeruginosa were stock clinical isolates from the microbiological laboratory of the Faculté de Pharmacie,

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, actin-depolymerizing factor, and tubulin was not modified. Using draE mutants, we found that a mutant in which cysteine replaces aspartic acid at position 54 conserved binding capacity but failed to induce F-actin disassembly. Accompanying the cytoskeleton injuries, we found that the distribution of brush border-associated functional proteins sucrase-isomaltase (SI), dipeptidylpeptidase IV (DPPIV), glucose transporter SGLT1, and fructose transporter GLUT5 was dramatically altered. In parallel, SI and DPPIV enzyme activity decreased.Diffusely adhering Escherichia coli (DAEC) strains are considered a heterogeneous group. It has been well established that some Afa/Dr DAEC expressing related adhesins adhere to host cells and cause symptomatic urinary tract and intestinal infections. Afa/Dr DAEC harboring the afimbrial adhesin I (AfaE-I) (34) and adhesin III (AfaE-III) (18, 35), the Dr hemagglutinin (51), and the adhesin DR-II (58) have been associated with 30% of cases of pyelonephritis in pregnant women. Afa/Dr DAEC strain C1845 harboring the fimbrial F1845 adhesin has been isolated from an infant with diarrhea (4). These virulent E. coli strains express a family of gene operons, including afa (19,20,30,34,35), dra (66), and daa (3, 4, 38). Moreover, a common system of adhesion involving the decay-accelerating-factor (DAF; CD55) as a receptor has been identified for Afa/Dr DAEC (48, 50).Yamamoto et al. (67) were first to report that adherence of DAEC induced elongated cellular projections in epithelial HeLa cells. Cookson and Nataro (10) observed that attachment of Afa/Dr DAEC onto epithelial Hep-2 cells is followed by induction of a long thin membrane extending from the cell surface. Moreover, Afa/Dr DAEC-induced cytoskeletal rearrangements in HeLa cells have been recently reported by Goluszko et al. (23). We have previously shown that Afa/Dr DAEC strains infect polarized human intestinal Caco-2 cells expressing a well-characterized brush border endowed with CD55 and forming a monolayer mimicking an epithelial barrier (32). When investi...

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 that recognized the short consensus repeat 3 domain of DAF (monoclonal antibody 1H4). Moreover, Afa/Dr DAEC strains induced tyrosine phosphorylation of several T84 proteins and activated the mitogen-activated protein kinases (ERK1/2 mitogen-activated protein, P38, and Jun-C kinases). These data demonstrated for the first time that, in vitro, Afa/Dr DAEC strains exert a proinflammatory signal in intestinal epithelial cells.