Inhibition of adhesion of enteroinvasive pathogens to human intestinal Caco-2 cells by Lactobacillus acidophilus strain LB decreases bacterial invasion

Abstract Salmonella typhimurium and enteropathogenic Escherichia coli (EPEC) were found to adhere to the brush border of differentiated human intestinal epithelial Caco-2 cells in culture, whereas Yersinia pseudotuberculosis and Listeria monocytogenes adhered to the periphery of undifferentiated Caco-2 cells. All these enterovirulent strains invaded the Caco-2 cells. Using a heat-killed human Lactobacillus acidophilus (strain LB) which strongly adheres both to undifferentiated and differentiated Caco-2 cells, we have studied inhibition of cell association with and invasion within Caco-2 cells by enterovirulent bacteria. Living and heat-killed Lactobacillus acidophilus strain LB inhibited both cell association and invasion of Caco-2 cells by enterovirulent bacteria in a concentration-dependent manner. The mechanism of inhibition of both adhesion and invasion appears to be due to steric hindrance of human enterocytic pathogen receptors by whole-cell lactobacilli rather than to a specific blockade of receptors.     

Lactobacillus casei DN-114 001 inhibits the ability of adherent-invasive Escherichia coli isolated from Crohn's disease patients to adhere to and to invade intestinal epithelial cells

Ileal lesions in 36.4% of patients with Crohn's disease are colonized by pathogenic adherent-invasive Escherichia coli. The aim of this study was to determine the in vitro inhibitory effects of the probiotic strain, Lactobacillus casei DN-114 001, on adhesion to and invasion of human intestinal epithelial cells by adherent-invasive E. coli isolated from Crohn's disease patients. The experiments were performed with undifferentiated Intestine-407 cells and with undifferentiated or differentiated Caco-2 intestinal epithelial cells. Bacterial adhesion to and invasion of intestinal epithelial cells were assessed by counting CFU. The inhibitory effects of L. casei were determined after coincubation with adherent-invasive E. coli or after preincubation of intestinal cells with L. casei prior to infection with adherent-invasive E. coli. Inhibitory effects of L. casei on adherent-invasive E. coli adhesion to differentiated and undifferentiated intestinal epithelial cells reached 75% to 84% in coincubation and 43% to 62% in preincubation experiments, according to the cell lines used. Addition of L. casei culture supernatant to the incubation medium increased L. casei adhesion to intestinal epithelial cells and enhanced the inhibitory effects of L. casei. The inhibitory effects on E. coli invasion paralleled those on adhesion. This effect was not due to a bactericidal effect on adherent-invasive E. coli or to a cytotoxic effect on epithelial intestinal cells. As Lactobacillus casei DN-114 001 strongly inhibits interaction of adherent-invasive E. coli with intestinal epithelial cells, this finding suggests that the probiotic strain could be of therapeutic value in Crohn's disease.     

Invasion of tissue culture cells by diarrhoeagenic strains of Escherichia coli which lack the enteroinvasive inv gene

Abstract Invasive Escherichia coli strains of certain serotypes invade by the same mechanism as the Shigella sp. It has been proposed that invasion of epithelial cells by EPEC strains may also occur; this is a previously overlooked property. In the present study E. coli strains isolated from patients with diarrhoea or ulcerative colitis, lacking the inv plasmid mediating classical invasion, but hybridizing with probes for different adhesins, were analyzed for their ability to invade HeLa and Caco-2 cells. The majority of strains invaded Caco-2 cells to a higher extent than HeLa cells. Adhesion to Caco-2 cells was a prerequisite for subsequent invasion of the cells but EAF, eae , EAgg and other known virulence factors were not sufficient to mediate invasion. In 8/9 E. coli strains invasion was enhanced after growth under iron restriction. Growth during anaerobic conditions did not influence subsequent invasion by E. coli strains whereas 6/9 strains had their invasive ability significantly decreased after growth in the presence of 1% glucose. The invasive process was inhibited by mannose but not by lactose, fucose or galactose. Our data indicate that strains of E. coli may invade Caco-2 cells by novel mechanisms which require adhesion to the cells but which differ from those of Salmonella sp., Yersinia sp., Shigella sp. and classical enteroinvasive E. coli .     

Ability of acceptance for bacteriophages during verotoxin production by bacilli of the Enterobacteriaceae family]

Lysogenised verotoxigenic strains are the source of structural genes of verocytotoxins (stx-1 and stx-2) for the others intestinal bacili. The aim of the study was to estimate the ability of transfer of bacteriophages induced with UV irradiation from reference verotoxigenic strains of E. coli O157:H7 (CB571 and EDL933) into 125 wild-strains of bacili of Enterobacteriaceae family. None of tested recipient strains showed the production of cytotoxin on Vero and HeLa cell lines, what was acknowledged as the lack of six genes. Contrary to the laboratory strain of E. coli C600 none of 125 tested recipient strains accepted the phages. Obtained lysogenised laboratory strains of E. coli C600/CB571 and E. coli C600/EDL933, besides of the ability to produce verotoxins (with the presence of stx-1 and stx-2 genes), did not differ phenotypically and genotypically from parent strain of E. coli C600. The estimation of the ability to transfer of phages carried stx-1 and/or stx-2 genes was impossible because of too small number of tested wild strain of bacili or because of really low frequency of acceptation of phages by wild strains of intestinal bacili.     

Fermentative degradation of acetone by an enrichment culture in membrane-separated culture devices and in cell suspensions

Abstract We have recently demonstrated that cultured human intestinal HT-29 and Caco-2 cell lines express receptors for the F1845 fimbrial adhesin harbored by the diarrheagenic C1845 Escherichia coli (Kernéis et al., Infect. Immun. 59 (1991) 4013–4018). This adhesinn belongs to a family of adhesins including the Dr hemagglutinin and the afimbrial adhesin AFA-1 harbored by uropathogenic E. coli . Here we investigated the cell association of laboratory E. coli strains expressing the Dr hemagglutinin and the afimbrial adhesin AFA-I with human cultured enterocyte-like or mucosecreting cells. We observed that the E. coli strains bearing these adhesins adhere both to human intestinal undifferentiated and differentiated fluid-transporting cells, and to mucus-secreting cells. This result strongly suggests a high capacity of intestinal colonization for the uropathogenic E. coli harboring adhesive factors belonging to the Dr adhesin family. These results further corroborate the intestinal colonization by uropathogenic E. coli of the Dr family related to the fecal-perineal-urethral hypothesis of urinary tract infection pathogenesis.     

Rotavirus infection induces cytoskeleton disorganization in human intestinal epithelial cells: implication of an increase in intracellular calcium concentration

Rotavirus infection is the most common cause of severe infantile gastroenteritis worldwide. In vivo, rotavirus exhibits a marked tropism for the differentiated enterocytes of the intestinal epithelium. In vitro, differentiated and undifferentiated intestinal cells can be infected. We observed that rotavirus infection of the human intestinal epithelial Caco-2 cells induces cytoskeleton alterations as a function of cell differentiation. The vimentin network disorganization detected in undifferentiated Caco-2 cells was not found in fully differentiated cells. In contrast, differentiated Caco-2 cells presented Ca(2+)-dependent microtubule disassembly and Ca(2+)-independent cytokeratin 18 rearrangement, which both require viral replication. We propose that these structural alterations could represent the first manifestations of rotavirus-infected enterocyte injury leading to functional perturbations and then to diarrhea.     

Prevalence of enterohaemorrhagic Escherichia coli from serotype O157 and other attaching and effacing Escherichia coli on bovine carcasses in Algeria

AIMS: Bovine meat is the principal source of human contamination of attaching and effacing Escherichia coli, including enterohaemorrhagic E. coli O157. The aim was to study the prevalence of these strains on bovine carcasses in Algeria. METHODS AND RESULTS: Two-hundred and thirty carcasses were swabbed and analysed by classical microbiological methods for total E. coli counts and for the presence of pathogenic E. coli. The E. coli counts were high, with a 75th percentile of 444.75 CFUs cm(-2). For pathogenic E. coli, more than 7% of the tested carcasses were positive for E. coli O157. Eighteen E. coli O157 strains were isolated and typed by multiplex PCR. The main isolated pathotype (78%) was eae+ stx2+ ehxA+. In addition to E. coli O157, other attaching and effacing E. coli (AEEC) were also detected from carcasses by colony hybridization after pre-enrichment and plating on sorbitol MacConkey agar using eae, stx1 and stx2 probes. Thirty carcasses (13%) on the 230 analysed harboured at least one colony positive for one of the tested probes. These positive carcasses were different from those positive for E. coli O157. Sixty-six colonies (2.9%) positive by colony hybridization were isolated. The majority (60.6%) of the positive strains harboured an enteropathogenic E. coli-like pathotype (eae+ stx-). Only three enterohaemorrhagic E. coli (EHEC)-like (eae+ stx1+) colonies were isolated from the same carcass. These strains did not belong to classical EHEC serotypes. CONCLUSIONS: In this study, the global hygiene of the slaughterhouse was low, as indicated by the high level of E. coli count. The prevalence of both E. coli O157 and other AEEC was also high, representing a real hazard for consumers. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study of this type in Algeria, which indicates that the general hygiene of the slaughterhouse must be improved.     

Generation of Escherichia coli intimin derivatives with differing biological activities using site-directed mutagenesis of the intimin C-terminus domain

Intimins, encoded by eae genes, are outer membrane proteins involved in attaching–effacing (A/E) lesion formation and host cell invasion by pathogenic bacteria, including enteropathogenic Escherichia coli (EPEC) and Citrobacter rodentium . A series of intimins, harbouring specific mutations close to the C-terminus, were constructed using pCVD438, which encodes the eae gene from EPEC strain E2348/69. These mutant plasmids were introduced into EPEC strain CVD206 and C. rodentium strain DBS255, which both contain deletion mutations in their eae genes. CVD206, CVD206(pCVD438) and CVD206(pCVD438) derivatives were assessed for their ability to promote A/E lesion formation or invasion of HEp-2 cells and to induce A/E lesions on fresh human intestinal in vitro organ cultures (IVOC). The pathogenicity of C. rodentium DBS255 harbouring these plasmid derivatives was also studied in mice. Here, we report that intimin-mediated A/E lesion formation can be segregated from intimin-mediated HEp-2 cell invasion. Moreover, adherence to IVOC, EPEC-induced microvillus elongation and colonization of the murine intestine by C. rodentium were also modulated by the modified intimins.     

Lactobacillus casei DN-114 001 Inhibits the Ability of Adherent-Invasive Escherichia coli Isolated from Crohn's Disease Patients To Adhere to and To Invade Intestinal Epithelial Cells

Ileal lesions in 36.4% of patients with Crohn's disease are colonized by pathogenic adherent-invasive Escherichia coli. The aim of this study was to determine the in vitro inhibitory effects of the probiotic strain, Lactobacillus casei DN-114 001, on adhesion to and invasion of human intestinal epithelial cells by adherent-invasive E. coli isolated from Crohn's disease patients. The experiments were performed with undifferentiated Intestine-407 cells and with undifferentiated or differentiated Caco-2 intestinal epithelial cells. Bacterial adhesion to and invasion of intestinal epithelial cells were assessed by counting CFU. The inhibitory effects of L. casei were determined after coincubation with adherent-invasive E. coli or after preincubation of intestinal cells with L. casei prior to infection with adherent-invasive E. coli. Inhibitory effects of L. casei on adherent-invasive E. coli adhesion to differentiated and undifferentiated intestinal epithelial cells reached 75% to 84% in coincubation and 43% to 62% in preincubation experiments, according to the cell lines used. Addition of L. casei culture supernatant to the incubation medium increased L. casei adhesion to intestinal epithelial cells and enhanced the inhibitory effects of L. casei. The inhibitory effects on E. coli invasion paralleled those on adhesion. This effect was not due to a bactericidal effect on adherent-invasive E. coli or to a cytotoxic effect on epithelial intestinal cells. As Lactobacillus casei DN-114 001 strongly inhibits interaction of adherent-invasive E. coli with intestinal epithelial cells, this finding suggests that the probiotic strain could be of therapeutic value in Crohn's disease.     

Adhesion of human bifidobacterial strains to cultured human intestinal epithelial cells and inhibition of enteropathogen-cell interactions.

Thirteen human bifidobacterial strains were tested for their abilities to adhere to human enterocyte-like Caco-2 cells in culture. The adhering strains were also tested for binding to the mucus produced by the human mucus-secreting HT29-MTX cell line in culture. A high level of calcium-independent adherence was observed for Bifidobacterium breve 4, for Bifidobacterium infantis 1, and for three fresh human isolates from adults. As observed by scanning electron microscopy, adhesion occurs to the apical brush border of the enterocytic Caco-2 cells and to the mucus secreted by the HT29-MTX mucus-secreting cells. The bacteria interacted with the well-defined apical microvilli of Caco-2 cells without cell damage. The adhesion to Caco-2 cells of bifidobacteria did not require calcium and was mediated by a proteinaceous adhesion-promoting factor which was present both in the bacterial whole cells and in the spent supernatant of bifidobacterium culture. This adhesion-promoting factor appeared species specific, as are the adhesion-promoting factors of lactobacilli. We investigated the inhibitory effect of adhering human bifidobacterial strains against intestinal cell monolayer colonization by a variety of diarrheagenic bacteria. B. breve 4, B. infantis 1, and fresh human isolates were shown to inhibit cell association of enterotoxigenic, enteropathogenic, diffusely adhering Escherichia coli and Salmonella typhimurium strains to enterocytic Caco-2 cells in a concentration-dependent manner. Moreover, B. breve 4 and B. infantis 1 strains inhibited, dose dependently, Caco-2 cell invasion by enteropathogenic E. coli, Yersinia pseudotuberculosis, and S. typhimurium strains.     

Interactions of clinical and environmental Aeromonas isolates with Caco-2 and HT29 intestinal epithelial cells

AIM: Evaluation of adherence and invasion of Aeromonas spp. to human colon carcinoma cell lines Caco-2 and HT29 and assessment of cytotoxic activity. METHODS AND RESULTS: A number of 27 strains of Aeromonas caviae and 23 strains of Aeromonas hydrophila was analysed. All strains were capable to adhere to sub-confluent monolayers of Caco-2 and HT29 cell types, presenting aggregative and diffuse adherence patterns cells, respectively. In the cytotoxic assays all strains showed cytopathic and/or cytotoxic activities to Vero cells. The evaluation of the tetrazolium salt (MTT test) reduction capability was carried out in Vero, Caco-2, and HT29 cells. MTT test showed that Vero cell line was the most sensitive cell type. In the invasion test, 13 strains were analysed on Caco-2 and HT29 monolayers. Only two (15%) of the 13 strains, A. hydrophila and A. caviae species, both isolated from vegetables were invasive to Caco-2 cells. No strains were able to invade the HT29 cells. CONCLUSIONS: A. hydrophila and A. caviae isolated from human diarrhoeic faeces, vegetables, and water, were able to adhere to and produce cytotoxic/cytopathic effects in intestinal epithelial cell lines. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of Aeromonas spp. in food and water samples expressing virulence factors suggest that these sources may act as dissemination vehicles of human pathogen with implication in the public health.     

Increased production of the ether-lipid platelet-activating factor in intestinal epithelial cells infected by Salmonella enteritidis

When exposed to enteric pathogens intestinal epithelial cells produce several cytokines and other proinflammatory mediators. To date there is no evidence that the ether-lipid platelet-activating factor (PAF) is one of these mediators. Our results revealed a significant increase in PAF production by human colonic tissue 4 h after infection by enterohemorrhagic Escherichia coli (EHEC) or Salmonella enteritidis. PAF is produced in the gut by cells of the immune system in response to bacterial infection. To determine whether the epithelial cells of colonic mucosa might also modulate PAF levels, we carried out PAF quantification and analysis of the enzymes involved in PAF synthesis in 5-day-old (undifferentiated) or 28-day-old (differentiated) Caco-2 cell cultures. Infection of undifferentiated Caco-2 cells with either bacterium had no effect on PAF levels, whereas in differentiated cells, infection by S. enteritidis increased PAF levels. Following infection by S. enteritidis, there were no changes in the activity of dithiothreitol-insensitive choline phosphotransferase. However, the enzymes of the remodeling pathway cytosolic phospholipase A(2), which catalyzes the formation of the PAF precursor lysoPAF, and lysoPAF acetyltransferase, are activated in the infected epithelial cells. This response is Ca(2+)-dependent.     

Characterization of the vitamin D receptor from the Caco-2 human colon carcinoma cell line: effect of cellular differentiation.

The human colon carcinoma cell line, Caco-2, is the only intestinal cell line to spontaneously differentiate in culture to a population exhibiting structural and biochemical characteristics of mature enterocytes. We conducted studies to establish the presence of the vitamin D receptor (VDR), determine changes in VDR concentration and affinity with differentiation and determine whether 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) mediates a functional response in this cell line. We found that Caco-2 cells possess a specific 1,25(OH)2D3 binding protein similar to the mammalian VDR. It has an equilibrium dissociation constant (Kd) of 0.72 nM, binds vitamin D analogues in order of their biological activities in vivo (1,25(OH)2D3 greater than 25(OH)D3 greater than 24,25(OH)2D3), sediments as a single peak on sucrose density gradients at 3.7 S, and is eluted from a DNA-cellulose column by 0.16 M KCl. The maximum number of binding sites was 2.6-fold greater in the differentiated cell (Day 15) compared to the preconfluent, undifferentiated (Day 4) cell (23 fmol/mg protein vs 56 fmol/mg protein). Cell growth was reduced 59% when exposed to 10(-7) M 1,25(OH)2D3 for 8 days. Alkaline phosphatase activity significantly increased in cultures incubated with 10(-8) M 1,25(OH)2D3 for up to 4 days when treatment was started in both undifferentiated cells (Day 5) and differentiated cells (Day 11). These findings suggest that the VDR present in undifferentiated and differentiated Caco-2 cells is functional. Caco-2 cells provide a unique in vitro model to study vitamin D-regulated functions in differentiated mammalian enterocytes.     

Adherens junction-dependent PI3K/Akt activation induces resistance to genotoxin-induced cell death in differentiated intestinal epithelial cells

The crypt-villi axis of intestinal mucosa maintains homeostasis by renewal of epithelia, and also exhibits different properties from undifferentiated to terminally differentiated cells. We investigated differential susceptibility to genotoxin-induced cell death, based on the degree of differentiation of epithelial cells, and its mechanism. Differentiation was induced by post-confluence culture in Caco-2 cells. Methyl methanesulfonate (MMS), a direct-acting DNA alkylating agent, was used for genotoxin-induced cell death. Compared to subconfluent Caco-2 cells, 7 days post-confluent cells showed resistance to MMS-induced cell death. With increasing expression of adherens junction components of E-cadherin and β-catenin, E-cadherin and p-Akt expression increased in 7 days post-confluent Caco-2 cells, and in human intestinal tissue, expression of E-cadherin and p-Akt also increased in the upper portion of villi, compared to the crypt. Inhibition of cell-cell adhesion using EGTA decreased Akt phosphorylation, which was reversed by calcium restoration. Akt phosphorylation by calcium-mediated cell-cell adhesion was more prominent in differentiated cells. In addition, treatment of a PI3K inhibitor, LY294002, inhibited Akt phosphorylation by calcium-mediated cell-cell adhesion. Finally, the differential sensitivity to MMS-induced cell death between subconfluent and 7 days post-confluent Caco-2 cells was eliminated by inhibiting cell-cell adhesion or PI3K. Our data demonstrated that cell adhesion-mediated PI3K/Akt activation could be one of the important mechanisms of resistance to genotoxin-induced cell death in differentiated epithelial cells.     

Enteropathogenic Escherichia coli inhibits butyrate uptake in Caco-2 cells by altering the apical membrane MCT1 level

Enteropathogenic Escherichia coli (EPEC), a food-borne human pathogen, is responsible for infantile diarrhea, especially in developing countries. The pathophysiology of EPEC-induced diarrhea, however, is not completely understood. Our recent studies showed modulation of Na+/H+ and Cl-/HCO3- exchange activities in Caco-2 cells in response to EPEC infection. We hypothesized that intestinal short-chain fatty acid absorption mediated by monocarboxylate transporter 1 (MCT1) might also be altered by EPEC infection. The aim of the current studies was to examine the effect of EPEC infection on butyrate uptake. Caco-2 cells were infected with wild-type EPEC, various mutant strains, or nonpathogenic E. coli HS4, and [14C]butyrate uptake was determined. EPEC, but not nonpathogenic E. coli, significantly decreased butyrate uptake. Infection of cells with strains harboring mutations in escN, which encodes a putative ATPase for the EPEC type III secretion system (TTSS), or in the espA, espB, or espD genes encoding structural components of the TTSS, had no effect on butyrate uptake, indicating the TTSS dependence. On the other hand, strains with mutations in the effector protein genes espF, espG, espH, and map inhibited butyrate uptake, similar to the wild-type EPEC. Surface expression of MCT1 decreased considerably after EPEC but not after nonpathogenic E. coli infection. In conclusion, our studies demonstrate inhibition of MCT1-mediated butyrate uptake in Caco-2 cells in response to EPEC infection. This inhibition was dependent on a functional TTSS and the structural proteins EspA, -B, and -D of the translocation apparatus.     

蒙脱石对细菌黏附Caco-2细胞的影响

采用Caco-2细胞培养模型,观察两歧双歧杆菌、嗜酸乳杆菌、嗜水气单胞菌、副溶血弧菌、大肠杆菌、鼠伤寒沙门菌的黏附率,并在培养液中加入蒙脱石,计算蒙脱石对细菌黏附的阻断率,探讨蒙脱石对上述细菌黏附作用的影响。结果表明:所试菌与Caco-2细胞均有不同程度的黏附作用;蒙脱石对细菌黏附Caco-2细胞均有不同程度的阻断作用,对病原菌黏附Caco-2细胞的阻断作用要明显大于其对益生菌的阻断效果,其中对大肠杆菌、鼠伤寒沙门菌、嗜水气单胞菌、副溶血弧菌黏附的阻断率分别为54.22%、48.41%、60.53%、50.64%,而对两歧双歧杆菌、嗜酸乳杆菌黏附的阻断率分别为25.64%和21.49%。结果提示蒙脱石可有效阻断病原菌黏附,从而防治肠道细菌感染和细菌移位。     

Ultrastructural and immunocharacterization of undifferentiated myocardial cells in the developing mouse heart

The recent discovery of several myogenic cardiac progenitor cells in the post-natal heart suggests that some myocardial cells may remain undifferentiated during embryonic development. In this study, we examined the subcellular characteristics of the embryonic (E) mouse ventricular myocardial cells using transmission electron microscopy (TEM). At the ultrastructural level, we identified three different cell populations within the myocardial layer of the E11.5 heart. These cells were designated as undifferentiated cells (43 +/- 6%), moderately differentiated cells (43 +/- 2%) and mature cardiomyocytes (14 +/- 4%). Undifferentiated cells contained a large nucleus and sparse cytoplasm with no myofibrillar bundles. Moderately differentiated cells contained randomly arranged myofilaments in the cytoplasm. In contrast, mature cardiomyocytes contained well-developed sarcomere structures. We also confirmed the presence of similar undifferentiated cells albeit at low levels in the E16.5 ( approximately 20%) and E18.5 ( approximately 7%) myocardium. Further we used immunogold labeling technique to test whether these distinct cell populations were also positive for markers such as Nkx2.5, ISL1 and ANF. A preponderance of anti-Nkx2.5 label was found in the undifferentiated and moderately differentiated cell types. Anti-ANF label was found only in the cytoplasmic compartment of moderately differentiated and mature myocardial cells. All of the undifferentiated cells were negative for anti-ANF labeling. We did not find immuno-gold labeling with ISL1 in any of the three myocardial cell types. Based on these results, we suggest that embryonic myocardial cell differentiation is a gradual process and undifferentiated cells expressing Nkx2.5 in post-chamber myocardium may represent a progenitor cell population while cells expressing Nkx2.5 and ANF represent differentiating myocytes.