HT29-MTX Cells Research Articles

Transport of lipophilic drug molecules in a new mucus-secreting cell culture model based on HT29-MTX cells.

A new mucus-secreting in vitro drug absorption model based on monolayers of goblet-cell like sub-clones of the human colon carcinoma cell line HT29 obtained by methotrexate (MTX) treatment was investigated. Twelve sub-clones were isolated and characterized by light microscopy (LM), transelectron microscopy (TEM), confocal laser scanning microscopy (CLSM), transepithelial electrical resistance (TEER) and the transport of a paracellular marker FITC-Dextran (Mw 4400) (FD-4). Significant differences of microscopical appearance, TEER-values and permeability of FD-4 between the sub-clones were evident. However, two of them, namely MTX-D1 and MTX-E12. formed tight confluent monolayers with a thick mucus-layer on the apical surface. They were used to compare the apparent permeability coefficient (Papp) of a series of lipophilic drugs, which should be affected by the mucus-layer, namely barbiturates (barbituric acid, barbital, phenobarbital, methylphenobarbital and heptabarbital) and testosterone, as a reference, to mucus-free Caco-2 cells. The permeability of drugs with a partition coefficient (log P) > 1 was decreased in the mucus-producing cell lines. Testosterone, the most lipophilic compound, showed a decrease of up to 43%. We demonstrated that the mucus layer is a significant barrier to drug absorption for lipophilic drugs. In conclusion, our model may serve as a suitable in-vitro cell culture model to study the influence of the mucus layer on drug diffusion.

Listeriolysin O-induced stimulation of mucin exocytosis in polarized intestinal mucin-secreting cells: evidence for toxin recognition of membrane-associated lipids and subsequent toxin internalization through caveolae

Lysteriolysin O (LLO) induces a microtubule-dependent activation of mucin exocytosis in the human mucin-secreting HT29-MTX. Cholesterol inhibits the LLO-induced mucin exocytosis, whereas the oxidized form of cholesterol had no inhibitory effect. LLO-induced mucin exocytosis inhibited by cholesterol can be restored by enzymatic treatment with cholesterol oxidase. Inhibition of cholesterol synthesis in HT29-MTX cells results in a decrease in the LLO-induced mucin exocytosis. Other lipids such as gangliosides are able to inhibit the LLO-induced mucin exocytosis, suggesting that the binding of the toxin occurs at a multiplicity of membrane-associated lipids acting as receptors. Incubation of the toxin with lipids such as cholesterol or gangliosides does not decrease binding of LLO to target membranes. The present work also provides evidence that the LLO-induced mucin exocytosis develops independently of the pore-forming activity of the toxin. Finally, we demonstrated that the toxin associates with detergent-insoluble glycolipid microdomains (DIGs) containing VIP/21 caveolin, allowing internalization of the toxin and subsequent activation of the mucin exocytosis.

The influence of prednisolone on the secretion of MUC5AC from TH29-MTX cell culture

INTRODUCTION: Glucocorticoids have been used in the treatment of otitis media with effusion with promising but inconsistent resluts. The HT29-MTX cell line is a completely differentiated and almost exclusively mucous secreting cell line, which therefore has the potential to act as an ideal model for the study of mucus-secreting epithelia. To assess this potential of steroids in suppressing mucin secretion we have studied the response of the cell culture to prednisolone. METHODS: Confluent cell cultures were trypsinized, subcultured in six-well plates and incubated with four doses of prednisolone from 10-5 M to 10-11 M and over varying time courses from 12 to 36 h. ELISA was performed using a monoclonal mouse antibody to human gastric mucin by dot-blot ELISA. RESULTS: Prednisolone caused a reduction in mucin production from the cell line consistently. Media control values ranged from 3495 to 3559 compared with untreated controls of 18 998-27 176 and steroid treatment values of 12 244-27 792. Increasing concentrations of prednisolone result in increasing suppression of MUC5AC (P = 0.005). There was no independent time-related effect. CONCLUSION: There does appear to be a genuine suppression of mucus production by prednisolone and this appears to be dose-dependent but not time-dependent at the times studied. The effect size was small.

Involvement of activator protein 1 complexes in the epithelium-specific activation of the laminin γ2-chain gene promoter by hepatocyte growth factor (scatter factor)

Laminin-5 is a trimer of laminin α3, β3 and γ2 chains that is found in the intestinal basement membrane. Deposition of the laminin γ2 chain at the basement membrane is of great interest because it undergoes a developmental shift in its cellular expression. Here we study the regulatory elements that control basal and cytokine-activated transcriptional expression of the LAMC2 gene, which encodes the laminin γ2 chain. By using transient transfection experiments we demonstrated the presence of constitutive and cytokine-responsive cis-elements. Comparison of the transcriptional activity of the LAMC2 promoter in the epithelial HT29mtx cells with that in small-intestinal fibroblastic cells (C20 cells) led us to conclude that two regions with constitutive epithelium-specific activity are present between positions -1.2 and -0.12 kb. This was further validated by transfections of primary foetal intestinal endoderm and mesenchyme. A 2.5 kb portion of the LAMC2 5ʹ flanking region was equally responsive to PMA and hepatocyte growth factor (HGF), whereas it was less responsive to transforming growth factor β1. A minimal promoter limited to the initial 120 bp upstream of the transcriptional start site maintained inducibility by PMA and HGF. This short promoter fragment contains two activator protein 1 (AP-1) elements and the 5ʹ-most of these is a composite AP-1/Sp1 element. The 5ʹAP-1 element is crucial to the HGF-mediated activity of the promoter; analysis of interacting nuclear proteins demonstrated that AP-1 proteins containing JunD mediate the response to HGF.

Involvement of activator protein 1 complexes in the epithelium-specific activation of the laminin gamma2-chain gene promoter by hepatocyte growth factor (scatter factor).

Laminin-5 is a trimer of laminin alpha3, beta3 and gamma2 chains that is found in the intestinal basement membrane. Deposition of the laminin gamma2 chain at the basement membrane is of great interest because it undergoes a developmental shift in its cellular expression. Here we study the regulatory elements that control basal and cytokine-activated transcriptional expression of the LAMC2 gene, which encodes the laminin gamma2 chain. By using transient transfection experiments we demonstrated the presence of constitutive and cytokine-responsive cis-elements. Comparison of the transcriptional activity of the LAMC2 promoter in the epithelial HT29mtx cells with that in small-intestinal fibroblastic cells (C20 cells) led us to conclude that two regions with constitutive epithelium-specific activity are present between positions -1.2 and -0.12 kb. This was further validated by transfections of primary foetal intestinal endoderm and mesenchyme. A 2.5 kb portion of the LAMC2 5' flanking region was equally responsive to PMA and hepatocyte growth factor (HGF), whereas it was less responsive to transforming growth factor beta1. A minimal promoter limited to the initial 120 bp upstream of the transcriptional start site maintained inducibility by PMA and HGF. This short promoter fragment contains two activator protein 1 (AP-1) elements and the 5'-most of these is a composite AP-1/Sp1 element. The 5'AP-1 element is crucial to the HGF-mediated activity of the promoter; analysis of interacting nuclear proteins demonstrated that AP-1 proteins containing JunD mediate the response to HGF.

Antagonistic activity against Helicobacter infection in vitro and in vivo by the human Lactobacillus acidophilus strain LB.

The purpose of the present study was to examine the activity of the human Lactobacillus acidophilus strain LB, which secretes an antibacterial substance(s) against Helicobacter pylori in vitro and in vivo. The spent culture supernatant (SCS) of the strain LB (LB-SCS) dramatically decreased the viability of H. pylori in vitro independent of pH and lactic acid levels. Adhesion of H. pylori to the cultured human mucosecreting HT29-MTX cells decreased in parallel with the viability of H. pylori. In conventional mice, oral treatment with the LB-SCS protected against infection with Helicobacter felis. Indeed, at both 8 and 49 days post-LB-SCS treatment (29 and 70 days postinfection), inhibition of stomach colonization by H. felis was observed, and no evidence of gastric histopathological lesions was found. LB-SCS treatment inhibits the H. pylori urease activity in vitro and in H. pylori that remained associated with the cultured human mucosecreting HT29-MTX cells. Moreover, a decrease in urease activity was detected in the stomach of the mice infected with H. felis and treated with LB-SCS.

Listeria monocytogenes stimulates mucus exocytosis in cultured human polarized mucosecreting intestinal cells through action of listeriolysin O.

When the intracellular pathogen Listeria monocytogenes infects cultured human mucosecreting polarized HT29-MTX cells apically, it induces the stimulation of mucus exocytosis without cell entry. Using a set of isogenic mutants and purified listeriolysin O (LLO), we identified the L. monocytogenes thiol-activated exotoxin LLO as the agonist of mucus secretion. We demonstrated that the LLO-induced mucus exocytosis did not result from the LLO membrane-damaging activity. We found that LLO-induced mucus exocytosis is an event requiring the binding of LLO to a brush border-associated receptor and membrane oligomerization of the exotoxin. By a pharmacological approach, we demonstrated that no regulatory system or intracellular transducing signal known to be involved in control of mucin exocytosis was activated by LLO. Based on the present data, the stimulatory action of LLO on mucin exocytosis could be accounted for either by an unknown signaling system which remains to be determined or by direct action of LLO with the membrane vesicle components involved in the intracellular vesicular transport of mucins.

Changes in glycosaminoglycan structure and composition of the main heparan sulphate proteoglycan from human colon carcinoma cells (perlecan) during cell differentiation

Colon carcinoma cells provide a useful model to study the biochemical processes associated with cell differentiation. Undifferentiated HT29, differentiated HT29MTX(-3) and HT29MTX(-6), and Caco2 human colon carcinoma cells have been used to study the production of proteoglycans and to characterize the glycosaminoglycan structure of the heparan sulphate chains. All the cell lines produce mainly a heparan sulphate proteoglycan that is found partly in the extracellular medium and associated to the cell membrane. The heparan sulphate proteoglycans from the media were purified by ion-exchange chromatography and subjected to structural analysis. The heparan sulphate proteoglycan from differentiated cells is larger and more homogeneous in size than the heparan sulphate proteoglycan from undifferentiated HT29 cells. No differences in protein core structure were observed when cells were labeled with [35S]methionine and the protein cores visualized by gel electrophoresis. Nevertheless, differences in glycosaminoglycan composition were found correlated with the degree of differentiation. The heparan sulphate chains from differentiated HT29MTX(-3) and HT29MTX(-6) cells have a higher sulphation degree than those from undifferentiated HT29 cells. The heparan sulphate from Caco2 cells is the most highly sulphated species. The differences are mainly attributed to O-sulphate groups. The increase in O-sulphation was more pronounced for D-glucosamine 6-O-sulphate than for L-iduronic acid 2-O-sulphate groups.

O-Glycosylation and Cellular Differentiation in a Subpopulation of Mucin-Secreting HT-29 Cell Line

Malignant transformation of epithelial cells is associated with abnormal glycosylation of mucins. The aim of this work was to evaluate the changes in the O-glycosylation processes during differentiation of tumor cells by performingin vitroreactions using crude microsomal preparations obtained from a subpopulation of HT-29 cells capable of differentiating into mucin-secreting cells (HT-29 MTX cells). The reactions of O-glycosylation were carried out at different times of culture: before confluence (Day 5), when cells are still undifferentiated, and after confluence (Day 21), when cells display a mucin-secreting phenotype. As acceptor for the UDP-N-acetylgalactosamine:polypeptideNacetylgalactosaminyltransferase (GalNAc transferase), the peptide motif TTSAPTTS (tandem repeat deduced from MUC5AC human gastric gene, expressed in HT-29 MTX cells) was used. A higher rate of enzyme activity was observed in preconfluent cells, and analysis by capillary electrophoresis and electrospray mass spectrometry showed a different pattern of galactosaminylation in pre- and postconfluent cells. Core 1 UDP-galactose:N-acetyl-α-galactosaminyl-R 3-β-galactosyltransferase (3-β-galactosyltransferase) activityalso decreased with the differentiation, whereas CMP-neuraminic acid:galactose–β-1, 3-N-acetyl-α-galac- tosaminyl-R 3-α-sialyltransferase activity increased. In comparison, the evolving process of mucin biosynthesis was tested by the analysis of purified mucins of HT-29 MTX cells, in amino acid and carbohydrate composition, and immunoreactivity assays using several antibodies and lectins. The results suggested that (i) no mucins were detected at Day 5, while the GalNAc transferase and 3-β-galactosyltransferase activities were already at high rates; (ii) the mucins purified from postconfluent cells showed a high content of sialic acid in an α-2,3-linkage to galactose residues; and (iii) cellular differentiation seemed to be accompanied by more regulated processes of glycosylation. This study of the O-glycosylation in HT-29 MTX cells is thus an interesting approach to analyzing the regulation of mucin biosynthesis during cellular differentiation.

MRNA Expression of HNF-4 Isoforms and of HNF-1α/HNF-1β Variants and Differentiation of Human Cell Lines That Mimic Highly Specialized Phenotypes of Intestinal Epithelium

The mRNA expression of HNF-4 isoforms and the ratio of HNF-1α/HNF-1β variants in cell lines representing highly specialized phenotypes of human intestinal epithelium were studied by RT-PCR. A strong rise in expression of HNF-4 isoforms α2, α4 and γ correlates with commitment into highly differentiated enterocyte-like phenotype of Caco-2 cells which best mimic enterocytes, whereas only isoform α4 expression is high in the less differentiated HT-29 G−cells. These increased expressions are not encountered in the highly differentiated mucous-secreting HT-29 MTX cells. Differentiation into highly specialized enterocyte-like Caco-2 cells and mucous-secreting HT-29 MTX cells is accompanied by a moderate rise in HNF-1 without change in the ratio of its variants. Our data corroborate those of Späthet al.(Mol. Cell. Biol.,1997, 17, 1913) in hepatoma cells and suggest that HNF-4 isoforms α2, α4 and γ play a major role in the differentiation of enterocytes.

Regulation of cathepsin D dependent on the phenotype of colon carcinoma cells

We have studied the intracellular trafficking of cathepsin D in different colon carcinoma cell populations: the HT-29 cell line, composed of >95% undifferentiated cells; 2 subpopulations derived from this cell line, containing cells committed to differentiation into mucin-secreting cells (HT-29 MTX) or enterocyte-like cells (HT-29 G−) after confluence; and the Caco-2 cell line, which spontaneously differentiates into enterocyte-like cells after confluence. Post-confluent undifferentiated HT-29 cells and differentiated enterocyte-like HT-29 G− and Caco-2 cells secrete significant levels of cathepsin D in culture medium, in contrast to post-confluent differentiated mucin-secreting HT-29 MTX cells, which secrete this enzyme at a very low level. The intracellular content and the mRNA level of cathepsin D increase after confluence in the different cell types, particularly in Caco-2 cells, which intensify the secretion of cathepsin D along with the differentiation process post-confluence. Membrane-associated mature cathepsin D was detected in HT-29 cells but not in Caco-2 cells. In the different types of cell, pro-cathepsin D associates with the membrane concomitantly to its binding to an M, 72,000 protein. Membrane association persists after dissociation of the complex in HT-29 cells but not in Caco-2 cells. In the mucin-secreting HT-29 MTX cells, cathepsin D was immunolocalised to the membrane of mucin vacuoles localised under the brush border. Our results show that cathepsin D can be regulated differently in colon carcinoma cells, and this finding might have specific functional implications for each cell type. © 1996 Wiley-Liss, Inc.

Regulation of cathepsin D dependent on the phenotype of colon carcinoma cells.

We have studied the intracellular trafficking of cathepsin D in different colon carcinoma cell populations: the HT-29 cell line, composed of >95% undifferentiated cells; 2 subpopulations derived from this cell line, containing cells committed to differentiation into mucin-secreting cells (HT-29 MTX) or enterocyte-like cells (HT-29 G-) after confluence; and the Caco-2 cell line, which spontaneously differentiates into enterocyte-like cells after confluence. Post-confluent undifferentiated HT-29 cells and differentiated enterocyte-like HT-29 G- and Caco-2 cells secrete significant levels of cathepsin D in culture medium, in contrast to post-confluent differentiated mucin-secreting HT-29 MTX cells, which secrete this enzyme at a very low level. The intracellular content and the mRNA level of cathepsin D increase after confluence in the different cell types, particularly in Caco-2 cells, which intensify the secretion of cathepsin D along with the differentiation process post-confluence. Membrane-associated mature cathepsin D was detected in HT-29 cells but not in Caco-2 cells. In the different types of cell, pro-cathepsin D associates with the membrane concomitantly to its binding to an Mr 72,000 protein. Membrane association persists after dissociation of the complex in HT-29 cells but not in Caco-2 cells. In the mucin-secreting HT-29 MTX cells, cathepsin D was immunolocalised to the membrane of mucin vacuoles localised under the brush border. Our results show that cathepsin D can be regulated differently in colon carcinoma cells, and this finding might have specific functional implications for each cell type.

Benzyl-N-acetyl-alpha-D-galactosaminide inhibits the sialylation and the secretion of mucins by a mucin secreting HT-29 cell subpopulation.

We have analysed the mucins synthesized by the HT-29 MTX cell subpopulation, derived from the HT-29 human colon carcinoma cells through a selective pressure with methotrexate (Lesuffleur et al., 1990, Cancer Res 50: 6334-43), in the presence of benzyl-N-acetyl-alpha-galactosaminide (GalNAc alpha-O-benzyl), which is a potential competitive inhibitor of the beta 1,3-galactosyltransferase that synthesizes the T-antigen. The main observation was a 13-fold decrease in the sialic acid content of mucins after 24 h of exposure to 5 mM GalNAc alpha-O-benzyl. This effect was accompanied by an increased reactivity of these mucins to peanut lectin, testifying to the higher amount of T-antigen. The second observation was a decrease in the secretion of the mucins by GalNAc alpha-O-benzyl treated cells. The decrease in mucin sialylation was achieved through the in situ beta-galactosylation of GalNAc alpha-O-benzyl into Gal beta 1-3GalNAc alpha-O-benzyl, which acts as a competitive substrate of Gal beta 1-3GalNAc alpha 2,3-sialyltransferase, as shown by the intracellular accumulation of NeuAc alpha 2-3Gal beta 1-3GalNAc alpha-O-benzyl in treated cells.

HT29-MTX/Caco-2 Cocultures as an in Vitro Model for the Intestinal Epithelium: In Vitro–in Vivo Correlation with Permeability Data from Rats and Humans

The diverse secretory and absorptive functions of the intestinal epithelium are conducted by a mixed population of absorptive cells and mucus-producing goblet cells as the major cell types. In order to approach the main characteristics in an in vitro model, a coculture system of absorptive Caco-2 cells and mucus-secreting HT29-MTX cells was developed and the permeability of a range of different drugs was tested. Variable goblet cell frequency can be achieved, preserving a significant barrier to drug transport and maintaining the differentiated features of both cell types. Absorption rates for actively transported drugs are rather underestimated in the cell culture model when compared to in vivo data. However, a good correlation with fraction absorbed in humans was attained separating the range of passively transported drugs into two groups of well-absorbable compounds with Peff ≥ 10 × 10−6 cm/s and drugs that are absorbed 40–70% with Peff=0.1–1 ×10−6 cm/s. A permeability of Peff<0.1 × 10−6 cm/s is suggested for low absorbable drugs.

Identification of a 42-kDa Nuclear Factor (NF1-MUC5B) from HT-29 MTX Cells That Binds to the 3′ Region of Human Mucin GeneMUC5B

MUC5B gene is one of the four human mucin genes mapped to chromosome 11p15. The identification of three potential Sp1 binding sites located between the tandem repeat and the 3′ end ofMUC5Bsuggests a possible regulatory role for this region. In this report we show by electrophoretic mobility shift assay that only one potential Sp1 binding site (NAU62) leads to a specific interaction with a nuclear factor from HT-29 MTX cells which does not exist in parental HT-29 cells. By using mutated versions of NAU62, an 18 mer sequence within this later was shown to be directly involved in the interaction. The nuclear factor called NF1-MUC5B which binds to this element has a Mrof 42000 and is not Sp1. These results suggest thatMUC5Bcontains a sequence in its 3′ region that might act as a cis-element. This report opens the field of transcriptional regulation of human mucin genes encoding secreted mucins.

Characterization of mucins and proteoglycans synthesized by a mucin-secreting HT-29 cell subpopulation.

HT-29 cells selected by adaptation to 10(-5) M methotrexate (HT-29 MTX) are a homogeneous cell population producing high amounts of mucin. Intracellular mucins and proteoglycans were isolated from these cells by ultracentrifugation of cell lysates on a cesium bromide gradient and further separated by anion-exchange high performance liquid chromatography. The major mucin fraction isolated was characterized by a high hydroxy amino acid content (40%), a Thr/Ser ratio of 1.52, a high sialic acid content, and a low sulfate content. When the same procedure was applied to undifferentiated HT-29 cells, a minor mucin fraction was isolated which appeared less sialylated and more sulfated. The major proteoglycan species identified in HT-29 MTX cells showed less acidic behavior than the proteoglycan isolated from HT-29 cells. The effect of brefeldin A and the sugar analog GalNAc-alpha-O-benzyl on the synthesis and biochemical properties of mucins synthesized by HT-29 MTX cells was examined. Brefeldin A induced the synthesis of more-sulfated mucins. GalNAc-alpha-O-benzyl treatment resulted in mucins with an increased content of T antigen and a 13-fold lower sialic acid content. We show that GalNAc-alpha-O-benzyl was metabolized by the cells to Gal beta 1-3GalNAc-alpha-O-benzyl, which, in turn, was a potent competitive inhibitor of the O-glycan alpha-2,3-sialyltransferase. These results illustrate the suitability of HT-29 MTX cells as a model to analyse mucin synthesis and sialylation.

Expression of cytochrome P-450 3A in HT29-MTX cells and Caco-2 clone TC7

HT-29 sublines and Caco-2 clones were analyzed for the expression of cytochrome P-450 3A. The enzyme was found to be expressed in differentiated HT-29 cells selected by resistance to methotrexate and in one of seven Caco-2 clones, TC7. Its expression parallels the differentiation process, with highest levels being observed at late confluency. P-450 3A mRNA and protein patterns, as well as subcellular distribution, are intermediate between those observed in human adult intestine and fetal liver.

Adhesion of human enterotoxigenic Escherichia coli to human mucus secreting HT-29 cell subpopulations in culture.

Enterotoxigenic Escherichia coli (ETEC) bearing the fimbrial colonisation factor antigens CFA/I, CFA/II, CFA/III, and the non-fimbrial antigen 2230 were tested for their ability to adhere to two cultured human intestinal HT-29 mucus secreting cell subpopulations. These populations are referred to as HT29-MTX and HT29-FU, which differ in the amount of secreted mucins and in their gastric or colonic mucin immunoreactivity respectively. Adherence of radiolabelled bacteria to cell monolayers infected apically was assessed. All ETEC strains adhered to the mucus secreting HT29-FU subpopulation, which secretes mucins of colonic immunoreactivity. Visualisation of bacteria by scanning electron microscopy showed that ETEC bound to the HT29-FU cells possessing a brush border, but not to the mucus and that ETEC binding developed as a function of cell differentiation. The adhesion of ETEC to cells possessing a brush border and to mucus secreting cells was also analysed by indirect immunofluorescence in HT29-MTX cells, which secrete mucins of gastric immunoreactivity. Fluorescein isothiocyanate labelling using specific anti-CFA/I antibody was used to show ETEC; rhodamine isothiocyanate labelling using a monoclonal antibody (designated M1) against purified human gastric mucus was used to detect secreted mucins, and rhodamine isothiocyanate labelling using a monoclonal antibody (designated 4H3) against human dipeptidylpeptidase IV was used to show cells possessing a brush border. Binding of bacteria colocalised with dipeptidylpeptidase IV of enterocytes and not with mucins.

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.

Identification and characterization of adhesive factors of Clostridium difficile involved in adhesion to human colonic enterocyte‐like Caco‐2 and mucus‐secreting HT29 cells in culture

Experiments reported in this communication showed that the highly toxinogenic Cd 79685, Cd 4784, and Wilkins Clostridium difficile strains and the moderately toxinogenic FD strain grown in the presence of blood adhere to polarized monolayers of two cultured human intestinal cell lines: the human colonic epithelial Caco-2 cells and the human mucus-secreting HT29-MTX cells. Scanning electron microscopy revealed that the bacteria interacted with well-defined apical microvilli of differentiated Caco-2 cells and that the bacteria strongly bind to the mucus layer that entirely covers the surface of the HT29-MTX cells. The binding of C. difficile to Caco-2 cells developed in parallel with the differentiation features of the Caco-2 cells, suggesting that the protein(s) which constitute C. difficile-binding sites are differentiation-related brush border protein(s). To better define this interaction, we tentatively characterized the mechanism(s) of adhesion of C. difficile with adherence assays. It was shown that heating of C. difficile grown in the presence of blood enhanced the bacterial interaction with the brush border of the enterocyte-like Caco-2 cells and the human mucus-secreting HT29-MTX cells. A labile surface-associated component was involved in C. difficile adhesion since washes of C. difficile grown in the presence of blood without heat shock decreased adhesion. After heating, washes of C. difficile grown in the presence of blood did not modify adhesion. Analysis of surface-associated proteins of C. difficile subjected to different culture conditions was conducted. After growth of C. difficile Cd 79685, Cd 4784, FD and Wilkins strains in the presence of blood and heating, two predominant SDS-extractable proteins with molecular masses of 12 and 27 kDa were observed and two other proteins with masses of 48 and 31 kDa disappeared. Direct involvement of the 12 and 27 kDa surface-associated proteins in the adhesion of C. difficile strains was demonstrated by using rat polycolonal antibodies pAb 12 and pAb 27 directed against the 12 and 27 kDa proteins. Indeed, adhesion to Caco-2 cell monolayers of C. difficile strains grown in the presence of blood, without or with heat-shock, was blocked. Taken together, our results suggest that C. difficile may utilize blood components as adhesins to adhere to human intestinal cultured cells.