Invasion Of Intestinal Epithelial Cells Research Articles

A σ 28 -Regulated Nonflagella Gene Contributes to Virulence of Campylobacter jejuni 81-176

A Campylobacter jejuni 81-176 mutant in Cj0977 was fully motile but reduced >3 logs compared to the parent in invasion of intestinal epithelial cells in vitro. The mutant was also attenuated in a ferret diarrheal disease model. Expression of Cj0977 protein was dependent on a minimal flagella structure.

Role of SPI-1 in the interactions of Salmonella Typhimurium with porcine macrophages

Salmonella Pathogenicity Island 1 (SPI-1) genes are indispensable for virulence of Salmonella Typhimurium in mice after oral challenge. These genes mediate invasion in intestinal epithelial cells and induce cell death in murine macrophages. The role of SPI-1 in the pathogenesis of Salmonella Typhimurium infections in food producing animals is not known. It was the aim of the present study to characterize the interactions of a porcine Salmonella Typhimurium field strain and its isogenic mutants in the SPI-1 genes hilA, sipA and sipB with porcine macrophages. SPI-1 was found to be important in the invasion of porcine pulmonary alveolar macrophages (PAM) and the induction of the formation of spacious phagosomes. Both early and delayed cytotoxicity were seen in PAM, but only the early cytotoxicity was SPI-1 dependent. Exposure of PAM to Salmonella Typhimurium induced the production of reactive oxygen species (ROS) and interleukin-8, but no differences were noticed between the induction mediated by the wild type strain and its SPI-1 mutant strains. In conclusion, invasion of porcine macrophages and the induction of early, but not delayed, cytotoxicity by Salmonella Typhimurium is SPI-1 dependent. SPI-1 dependent invasion, however, is not a prerequisite to induce a pro-inflammatory response.

Lactobacillus caseiDN-114 001 Inhibits the Ability of Adherent-InvasiveEscherichia coliIsolated 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.

GRIM-19 Interacts with Nucleotide Oligomerization Domain 2 and Serves as Downstream Effector of Anti-bacterial Function in Intestinal Epithelial Cells

Nucleotide oligomerization domain 2 (NOD2) functions as a mammalian cytosolic pathogen recognition molecule, and variants have been associated with risk for Crohn disease. We recently demonstrated that NOD2 functions as an anti-bacterial factor limiting survival of intracellular invasive bacteria. To gain further insight into the mechanism of NOD2 activation and signal transduction, we performed yeast two-hybrid screening. We demonstrate that GRIM-19, a protein with homology to the NADPH dehydrogenase complex, interacts with endogenous NOD2 in HT29 cells. GRIM-19 is required for NF-kappaB activation following NOD2-mediated recognition of bacterial muramyl dipeptide. GRIM-19 also controls pathogen invasion of intestinal epithelial cells. GRIM-19 expression is decreased in inflamed mucosa of patients with inflammatory bowel diseases. GRIM-19 may be a key component in NOD2-mediated innate mucosal responses and serve to regulate intestinal epithelial cell responses to microbes.

N-linked protein glycosylation is required for full competence in Campylobacter jejuni 81-176.

The recent sequencing of the virulence plasmid of Campylobacter jejuni 81-176 revealed the presence of genes homologous to type IV secretion systems (TFSS) that have subsequently been found in Helicobacter pylori and Wolinella succinogenes. Mutational analyses of some of these genes have implicated their involvement in intestinal epithelial cell invasion and natural competence. In this report, we demonstrate that one of these type IV secretion homologs, Cjp3/VirB10, is a glycoprotein. Treatment with various glycosidases and binding to soybean agglutinin indicated that the structure of the glycan present on VirB10 contains a terminal GalNAc, consistent with previous reports of N-linked glycans in C. jejuni. Site-directed mutagenesis of five putative N-linked glycosylation sites indicated that VirB10 is glycosylated at two sites, N32 and N97. Mutants in the N-linked general protein glycosylation (pgl) system of C. jejuni are significantly reduced in natural transformation, which is likely due, in part, to lack of glycosylation of VirB10. The natural transformation defect in a virB10 mutant can be complemented in trans by using a plasmid expressing wild-type VirB10 or an N32A substitution but not by using a mutant expressing VirB10 with an N97A substitution. Taken together, these results suggest that glycosylation of VirB10 specifically at N97 is required for the function of the TFSS and for full competence in C. jejuni 81-176.

Molecular infection: the invasion complex between internalin ofListeria monocytogenesand human E-cadherin

The bacterium Listeria monocytogenes is a food-borne human pathogen. It infects humans by inducing its own uptake into epithelial cells of the intestine although these are normally non-phagocytic. Recognition, adhesion and invasion of intestinal epithelial cells is mediated by a single listerial surface protein, Internalin (InlA), through specific interaction with the host cell receptor E-cadherin. We have solved the crystal structure of the functional domain of InlA both uncomplexed and in complex with the extracellular, N-terminal domain of human E-cadherin (hEC1). In the complex between InlA and hEC1, the superhelically twisted leucine rich repeat (LRR) domain of InlA surrounds and specifically recognizes hEC1. Site-directed mutagenesis, analytical ultracentrifugation and Biacore experiments indicate that binding affinity is remarkably weak yet highly specific: Pro16 of hEC1, a major determinant for human susceptibility to L. Monocytogenes infection is essential for intermolecular recognition. Ca was found to stabilize the complex. Structurally, this is corroborated by a Ca-binding site bridging the two proteins. This indicates that complex formation in the intestine is favoured by high Ca-concentrations whereas low, intracellular concentrations induce dissociation, freeing the bacterium and allowing it to move through the eukaryotic cell. Our studies thus provide detailed insights into the molecular deception L. monocytogenes employs to exploit the host E-cadherin signal cascade through the expression of a single surface protein.

Protein Kinase C (PKC) βII Induces Cell Invasion through a Ras/Mek-, PKCι/Rac 1-dependent Signaling Pathway

Protein kinase C betaII (PKCbetaII) promotes colon carcinogenesis. Expression of PKCbetaII in the colon of transgenic mice induces hyperproliferation and increased susceptibility to colon cancer. To determine molecular mechanisms by which PKCbetaII promotes colon cancer, we established rat intestinal epithelial (RIE) cells stably expressing PKCbetaII. Here we show that RIE/PKCbetaII cells acquire an invasive phenotype that is blocked by the PKCbeta inhibitor LY379196. Invasion is not observed in RIE cells expressing a kinase-deficient PKCbetaII, indicating that PKCbetaII activity is required for the invasive phenotype. PKCbetaII induces activation of K-Ras and the Ras effector, Rac1, in RIE/PKCbetaII cells. PKCbetaII-mediated invasion is blocked by the Mek inhibitor, U0126, and by expression of either dominant negative Rac1 or kinase-deficient atypical PKCiota. Expression of constitutively active Rac1 induces Mek activation and invasion in RIE cells, indicating that Rac1 is the critical downstream effector of PKCbetaII-mediated invasion. Taken together, our results define a novel PKCbetaII --> Ras --> PKCiota /Rac1 --> Mek signaling pathway that induces invasion in intestinal epithelial cells. This pathway provides a plausible mechanism by which PKCbetaII promotes colon carcinogenesis.

Inhibitory effect of probiotic Escherichia coli strain Nissle 1917 on adhesion to and invasion of intestinal epithelial cells by adherent-invasive E. coli strains isolated from patients with Crohn's disease.

Pathogenic adherent-invasive Escherichia coli have been isolated from ileal lesions of Crohn's disease. : To investigate the non-pathogenic E. coli strain Nissle 1917 (Mutaflor) as possible maintenance therapy in inflammatory bowel disease by testing its ability to prevent adherent-invasive E. coli strains from adhering to and invading human intestinal epithelial cells in vitro. Bacterial adhesion to and invasion of intestinal epithelial cells (Intestine-407) were assessed by counting the colony-forming units. The inhibitory effect of E. coli Nissle 1917 was determined after co-incubation with adherent-invasive E. coli strains or after pre-incubation of the intestinal epithelial cells with this probiotic strain prior to infection with adherent-invasive E. coli strains. Strain Nissle 1917 exhibited dose- and time-dependent adherence to intestinal epithelial cells and inhibited the adhesion and invasion of various adherent-invasive E. coli strains. In co-infection experiments, the inhibitory effect on adherent-invasive E. coli adhesion reached 78-99.9%. Pre-incubation of intestinal epithelial cells with strain Nissle 1917 reduced adherent-invasive E. coli adhesion by 97.2-99.9%. The inhibitory effect on adherent-invasive E. coli invasion paralleled that on adhesion. As strong and significant inhibitory effects on adherent-invasive E. coli adhesion and invasion were observed in co-infection and pre-infection experiments, E. coli Nissle 1917 could be efficient for preventive or curative probiotic therapy in patients with Crohn's disease.

A Salmonella enterica serovar typhimurium translocated leucine-rich repeat effector protein inhibits NF-kappa B-dependent gene expression.

Nontyphoidal salmonellae are enteric pathogens that cause acute gastroenteritis and colonize the intestinal tract for prolonged periods. In the intestinal epithelia, these bacteria induce secretion of proinflammatory cytokines, such as interleukin-8 (IL-8), which leads to a profound inflammatory response through recruitment of polymorphonuclear leukocytes. Production of IL-8 induced by Salmonella spp. is due to the activation of the transcription factors nuclear factor kappa B (NF-kappa B) and activator protein-1 (AP-1). This work demonstrates that Salmonella enterica serovar Typhimurium can downmodulate IL-8 production after invasion of intestinal epithelial cells. The Salmonella translocated effector proteins SspH1 and SptP participate in this process. SspH1 is a member of the bacterial LPX repeat protein family that localizes to the mammalian nucleus and inhibits NF-kappa B-dependent gene expression. A Shigella flexneri translocated effector, IpaH9.8, which has a similar structure and subcellular localization in mammalian cells, also inhibits NF-kappaB-dependent gene expression. We propose that suppression of inflammatory responses by intracellular S. enterica serovar Typhimurium, and perhaps Shigella flexneri, contributes to bacterial colonization of host tissues and pathogenesis.

The role of Cryptosporidium parvum-derived phospholipase in intestinal epithelial cell invasion.

In the Cryptosporidium parvum-infected intestinal epithelial cell, the parasite occupies an unusual extracytoplasmic location at the luminal surface, but how the invading zoites interact with the host cell to achieve this niche is poorly understood. This study examined the role of secretory phospholipase A(2) (sPLA(2)), a known virulence factor for several pathogenic microorganisms, in establishing C. parvum intracellularly. Initially, it was established that there was sPLA(2) activity in homogenates of C. parvum oocysts. C. parvum reproduction in two human enterocyte cell lines was significantly reduced by a specific PLA inhibitor, p-bromophenacylbromide, and by sheep anti-sPLA(2) antibodies developed against PLA(2) of bee ( Apis mellifera) venom. Treatment of either C. parvum sporozoites or enterocytes with sPLA(2) derived from cobra ( Naja naja) venom before initiation of infection increased the numbers of intracellular parasites. Thus, C. parvum PLA(2 )may play an important part in establishing the parasite within the enterocyte.

DNA sequence and mutational analyses of the pVir plasmid of Campylobacter jejuni 81-176.

The circular pVir plasmid of Campylobacter jejuni strain 81-176 was determined to be 37,468 nucleotides in length with a G+C content of 26%. A total of 83% of the plasmid represented coding information, and all but 2 of the 54 predicted open reading frames were encoded on the same DNA strand. There were seven genes on the plasmid in a continguous region of 8.9 kb that encoded orthologs of type IV secretion proteins found in Helicobacter pylori, including four that have been described previously (D. J. Bacon, R. A. Alm, D. H. Burr, L. Hu, D. J. Kopecko, C. P. Ewing, T. J. Trust, and P. Guerry, Infect. Immun. 68:4384-4390, 2000). There were seven other pVir-encoded proteins that showed significant similarities to proteins encoded by the plasticity zones of either H. pylori J99 or 26695. Mutational analyses of 19 plasmid genes identified 5 additional genes that affect in vitro invasion of intestinal epithelial cells. These included one additional gene encoding a component of a type IV secretion system, an ortholog of Cj0041 from the chromosome of C. jejuni NCTC 11168, two Campylobacter plasmid-specific genes, and an ortholog of HP0996 from the plasticity zone of H. pylori 26695.

Transforming Growth Factor-β1 Promotes Invasiveness after Cellular Transformation with Activated Ras in Intestinal Epithelial Cells

Invasion is a defining event in carcinoma progression. In general, invasive carcinoma is characterized by an epithelial–fibroblastoid conversion associated with loss of cell–cell adhesion receptors such as E-cadherin and β-catenin. We report here that TGF-β1 promotes the invasiveness by modulating the alterations of cellular plasticity including a loss of cell–cell contact in Ras-transformed epithelial cells. In order to examine the role of TGF-β1 in the Ras-induced responses, intestinal epithelial cells expressing a conditionally activated Ha-RasVal12 (RIE-iRas cells) were used in this study. Induced expression of activated Ha-RasVal12 caused morphologic transformation of the RIE-iRas cells with an increase in vimentin expression and a decrease of E-cadherin levels. There was also redistribution of β-catenin from the cytoplasm to the nucleus after the induction of Ras. TGF-β1 treatment enhanced both the decrease in E-cadherin levels and the redistribution of β-catenin. Interestingly, the activation of Ras markedly decreased the level of TGF-β receptor type II (TβRII) in RIE-iRas cells. However, the expression of plasminogen activator inhibitor-1, which is known to be transcriptionally induced by TGF-β1, was strongly induced by TGF-β1 despite the marked downregulation of TβRII. The induction of Ha-RasVal12 markedly increased the invasiveness in RIE-iRas cells, as evaluated by a collagen type I-coated Boyden-chamber assay, and the Ras-mediated invasiveness was significantly enhanced by TGF-β1 treatment. Expression of a dominant-negative form of TβRII in the RIE-iRas cells abrogated both growth-inhibitory and invasion responses to TGF-β1. Collectively, these results suggest that TGF-β1 and oncogenic Ras collaborate in promoting cellular invasiveness in intestinal epithelial cells. The enhancement of invasiveness was correlated with decreased E-cadherin levels and subcellular distribution of β-catenin. The enhancement of oncogenic Ras-mediated cell transformation by TGF-β1 occurs via TβRII.

Escherichia coli strain nissie 1917 inhibits adhesion to an invasion of intestinal epithelial cells by adherent-invasive E. colil isolated from a Crohn's disease patient

Escherichia coli strain nissie 1917 inhibits adhesion to an invasion of intestinal epithelial cells by adherent-invasive E. colil isolated from a Crohn's disease patient

Escherichia coli strain nissie 1917 inhibits adhesion to an invasion of intestinal epithelial cells by adherent-invasive E. colil isolated from a Crohn's disease patient

Escherichia coli strain nissie 1917 inhibits adhesion to an invasion of intestinal epithelial cells by adherent-invasive E. colil isolated from a Crohn's disease patient

Type 1 pili-mediated adherence of Escherichia coli strain LF82 isolated from Crohn's disease is involved in bacterial invasion of intestinal epithelial cells

We previously characterized the invasive ability of Escherichia coli strain LF82, isolated from an ileal biopsy of a patient with Crohn's disease. In the present study, we performed TnphoA insertion mutagenesis to identify genes involved in LF82 invasion of intestinal epithelial cells. Most of the non-invasive mutants had an insertion mutation within the type 1 pili-encoding operon. Two non-invasive fim mutants, which harboured an insertion within the fimI and fimF genes, still adhered but had lost the ability to induce host cell membrane elongations at the sites of contact with the epithelial cells. Transcomplementation experiments with a fim operon cloned from E. coli K-12 restored both invasive ability and the ability to induce host cell membrane elongations. Expression of the cloned LF82 or K-12 fim operon into the non-invasive laboratory strain JM109 did not confer invasive properties. Thus, these findings showed that: (i) type 1 pili-mediated adherence is involved in LF82-induced perturbation of host cell signalling responsible for membrane elongations; (ii) native shafts are required for type 1 pilus-mediated induction of membrane elongations; (iii) this active phenomenon is a key step in the establishment of the invasive process; and (iv) type 1 pili alone are not sufficient to trigger bacterial internalization.

Type 1 pili-mediated adherence of Escherichia coli strain LF82 isolated from Crohn's disease is involved in bacterial invasion of intestinal epithelial cells.

We previously characterized the invasive ability of Escherichia coli strain LF82, isolated from an ileal biopsy of a patient with Crohn's disease. In the present study, we performed TnphoA insertion mutagenesis to identify genes involved in LF82 invasion of intestinal epithelial cells. Most of the non-invasive mutants had an insertion mutation within the type 1 pili-encoding operon. Two non-invasive fim mutants, which harboured an insertion within the fimI and fimF genes, still adhered but had lost the ability to induce host cell membrane elongations at the sites of contact with the epithelial cells. Transcomplementation experiments with a fim operon cloned from E. coli K-12 restored both invasive ability and the ability to induce host cell membrane elongations. Expression of the cloned LF82 or K-12 fim operon into the non-invasive laboratory strain JM109 did not confer invasive properties. Thus, these findings showed that: (i) type 1 pili-mediated adherence is involved in LF82-induced perturbation of host cell signalling responsible for membrane elongations; (ii) native shafts are required for type 1 pilus-mediated induction of membrane elongations; (iii) this active phenomenon is a key step in the establishment of the invasive process; and (iv) type 1 pili alone are not sufficient to trigger bacterial internalization.

Gamma interferon influences intestinal epithelial hyperplasia caused by Lawsonia intracellularis infection in mice.

Lawsonia intracellularis is a recently identified bacterial pathogen which causes disease in a broad range of animals. Invasion of intestinal epithelial cells and the resultant hyperplasia of infected cells are central processes in disease pathogenesis. In this study, we aimed to establish whether immunocompetent mice were susceptible to infection and whether gamma interferon (IFN-gamma) contributed to the pathogenesis of infection. Wild-type 129-Sv-Ev mice (129 mice) and IFN-gamma receptor knockout mice based on the 129 background (IFN-gammaR(-)) were challenged orally with approximately 5.5 x 10(7) L. intracellularis cells. Both 129 and IFN-gammaR(-) mice became infected, although the extent of infection (as determined by the proportion of infected crypts) was substantially lower in 129 mice than in IFN-gammaR(-) mice. Despite these differences, infected crypts showed characteristics typical of proliferative enteropathies of other animals, i.e., intracellular colonization of epithelial cells by L. intracellularis with resultant epithelial hyperplasia. Infection in 129 mice was cleared between days 21 and 28 postchallenge, whereas infection in IFN-gammaR(-) mice was evident in 100% of animals from day 21 onward. Additionally, in IFN-gammaR(-) mice the infection was so extensive that fatalities resulted. IFN-gamma therefore plays a significant role in limiting intracellular infection and increased cellular proliferation associated with L. intracellularis. L. intracellularis infection is generally associated with modest cellular infiltration; therefore, further comparative examinations will be necessary to determine pathogenicity factors and define the role of IFN-gamma in controlling this infection.

Invasion genes are not required for Salmonella enterica serovar typhimurium to breach the intestinal epithelium: evidence that salmonella pathogenicity island 1 has alternative functions during infection.

Salmonella enterica serovar Typhimurium invasion genes are necessary for bacterial invasion of intestinal epithelial cells and are thought to allow salmonellae to enter and cross the intestinal epithelium during infection. Many invasion genes are encoded on Salmonella pathogenicity island 1 (SPI1), and their expression is activated by HilA, a transcription factor also encoded on SPI1. We have studied the role of Salmonella invasion genes during infection of mice following intragastric inoculation. We have found that strains containing a mutation in hilA or invG were recovered from the intestinal contents, intestinal tissues, and systemic tissues at a lower frequency than their parental wild-type strain. In contrast, a strain in which SPI1 is deleted was recovered from infected mice at a frequency similar to that of its parental wild-type strain. The DeltaSPI1 phenotype indicates that S. enterica does not require invasion genes to cross the intestinal epithelium and infect systemic tissues. This result has forced us to reconsider the long-held belief that invasion genes directly mediate bacterial infection of the intestinal mucosa and traversion of the intestinal barrier during infection. Instead, our results suggest that hilA is required for bacterial colonization of the host intestine. The seemingly contradictory phenotype of the DeltaSPI1 mutant suggests that deletion of another gene(s) encoded on SPI1 suppresses the hilA mutant defect. We propose a model for S. enterica pathogenesis in which hilA and invasion genes are required for salmonellae to overcome a host clearance response elicited by another SPI1 gene product(s).

Blockade ofSalmonella enteritidisPassage across the Basolateral Barriers of Human Intestinal Epithelial Cells by Specific Antibody

Antibodies specific to Salmonella enteritidis (S.E.) were obtained from immunized egg yolk, and their protective effects against S.E. were studied by using monolayer-cultured human intestinal epithelial cells, Caco-2 and T84. The Salmonella adherence and entry to the cells were partially inhibited by the antibodies. The antibodies inhibited the decrease in transepithelial electrical resistance (TEER) of the intestinal epithelial monolayers and IL-8 secretion of the cells induced by S.E. invasion. Also, the antibodies blocked the penetration of bacteria through the cell layer although they did not inhibit the growth of bacteria in the cells. Confocal microscopic photographs revealed the bacteria in the infected monolayer cells were bound to antibodies. These results indicate that anti-S.E. antibodies may protect the cells from destruction induced by S.E. invasion in intestinal epithelial cells in addition to the partial inhibition of adhesion and invasion of S.E. at the cell surface. Passive antibodies against invasive bacteria would be useful to prevent the migration of S.E. to blood not only at the cell surface but also inside of intestinal epithelial cells.

TGF-B1 enhances ras-mediated invasiveness in intestinal epithelial cells

TGF-B1 enhances ras-mediated invasiveness in intestinal epithelial cells