Search

This study focuses on the impact of actin on adhesion and translocation of Enterococcus (E.) faecalis OG1RF, E. faecalis Symbioflor(®), and E. faecalis V583. Insight into the role of actin aggregation in the mediation of bacterial adhesion and translocation was provided by a two-chamber translocation assay, which employed Ptk6 cells. Determination of translocation rates, cytochalasin D treatment, and laser scanning confocal microscopic observation revealed actin as a predominant brace for enterococci to pass through the epithelial cell layer. As the three enterococci had moderate adhesion ability to actin, actin-binding proteins were isolated and characterized by LC-MS/MS. The isolated proteins were identified as pyruvate formate lyase, enolase, glyceraldehyde-3-phosphate dehydrogenase, and GroEL. All these proteins belong to two major groups of moonlighting proteins, i.e., proteins, which display additional functions other than their described major biochemical catalysis. Both groups of moonlight proteins were determined to be associated with epithelial cell binding.

Background & Aims Matrix metalloproteases (MMPs) mediate pathogenesis of chronic intestinal inflammation. We characterized the role of the gelatinase (GelE), a metalloprotease from Enterococcus faecalis , in the development of colitis in mice. Methods Germ-free, interleukin-10–deficient (IL-10 −/− ) mice were monoassociated with the colitogenic E faecalis strain OG1RF and isogenic, GelE-mutant strains. Barrier function was determined by measuring E-cadherin expression, transepithelial electrical resistance (TER), and translocation of permeability markers in colonic epithelial cells and colon segments from IL-10 −/− and TNF ΔARE/Wt mice. GelE specificity was shown with the MMP inhibitor marimastat. Results Histologic analysis (score 0–4) of E faecalis monoassociated IL-10 −/− mice revealed a significant reduction in colonic tissue inflammation in the absence of bacteria-derived GelE. We identified cleavage sites for GelE in the sequence of recombinant mouse E-cadherin, indicating that it might be degraded by GelE. Experiments with Ussing chambers and purified GelE revealed the loss of barrier function and extracellular E-cadherin in mice susceptible to intestinal inflammation (IL-10 −/− and TNF ΔARE/Wt mice) before inflammation developed. Colonic epithelial cells had reduced TER and increased translocation of permeability markers after stimulation with GelE from OG1RF or strains of E faecalis isolated from patients with Crohn's disease and ulcerative colitis. Conclusions The metalloprotease GelE, produced by commensal strains of E faecalis, contributes to development of chronic intestinal inflammation in mice that are susceptible to intestinal inflammation (IL-10 −/− and TNF ΔARE/Wt mice) by impairing epithelial barrier integrity.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with poor patient outcome often resulting from late diagnosis in advanced stages. To date methods to diagnose early-stage PDAC are limited and in vivo detection of pancreatic intraepithelial neoplasia (PanIN), a preinvasive precursor of PDAC, is impossible. Using a cathepsin-activatable near-infrared probe in combination with flexible confocal fluorescence lasermicroscopy (CFL) in a genetically defined mouse model of PDAC we were able to detect and grade murine PanIN lesions in real time in vivo. Our diagnostic approach is highly sensitive and specific and proved superior to clinically established fluorescein-enhanced imaging. Translation of this endoscopic technique into the clinic should tremendously improve detection of pancreatic neoplasia, thus reforming management of patients at risk for PDAC.

The association of Helicobacter pylori with gastric cancer is the best-studied relationship between a bacterial infection and cancer. Other bacterial pathogens in humans and rodents are now being recognized as potentially having a direct role in carcinogenesis. Thus, it might be possible to understand the pathogenesis and prevention of certain cancers by studying the bacterial infections associated with them, and their effects on the host. However, the mechanisms by which bacteria contribute to cancer formation are complex, and recent investigations show that they involve the interplay between chronic inflammation, direct microbial effects on host cell physiology and, ultimately, changes in tissue stem cell homeostasis.

The transforming growth factor-beta (TGFbeta) signaling pathway is one important player in the regulation of extracellular matrix turnover and cell proliferation in epithelial regeneration. We used cerulein-induced pancreatitis in rats as a model to investigate the regulation of TGFbeta receptor type I and type II expression on protein and messenger RNA level during regeneration. In the regenerating pancreas, mRNA levels of TGFbeta receptor I and II were significantly increased with a maximum after 2 days. On protein level, expression of TGFbeta receptor II was significantly increased after three to 3-5 days. This elevated expression could be inhibited by neutralizing the endogenous biological activity of TGFbeta1 with a specific antibody. In cultured pancreatic epithelial cells, TGFbeta1 reduced cell proliferation as measured by [3H]thymidine incorporation. Furthermore the transcript levels of TGFbeta1 as well as mRNA and protein concentrations of type I and type II receptor increased during TGFbeta stimulation in vitro. These results indicate that epithelial pancreatic cells contribute to the enhanced TGFbeta1 synthesis during pancreatic regeneration by an autocrine mechanism. TGFbeta1, furthermore, upregulates the expression of its own receptors during the regenerative process, thereby contributing to the increase of the TGFbeta-induced cellular responses.

Caveolin-1 (Cav1) is a scaffold protein and pathogen receptor in the mucosa of the gastrointestinal tract. Chronic infection of gastric epithelial cells by Helicobacter pylori (H. pylori) is a major risk factor for human gastric cancer (GC) where Cav1 is frequently down-regulated. However, the function of Cav1 in H. pylori infection and pathogenesis of GC remained unknown. We show here that Cav1-deficient mice, infected for 11 months with the CagA-delivery deficient H. pylori strain SS1, developed more severe gastritis and tissue damage, including loss of parietal cells and foveolar hyperplasia, and displayed lower colonisation of the gastric mucosa than wild-type B6129 littermates. Cav1-null mice showed enhanced infiltration of macrophages and B-cells and secretion of chemokines (RANTES) but had reduced levels of CD25+ regulatory T-cells. Cav1-deficient human GC cells (AGS), infected with the CagA-delivery proficient H. pylori strain G27, were more sensitive to CagA-related cytoskeletal stress morphologies (“humming bird”) compared to AGS cells stably transfected with Cav1 (AGS/Cav1). Infection of AGS/Cav1 cells triggered the recruitment of p120 RhoGTPase-activating protein/deleted in liver cancer-1 (p120RhoGAP/DLC1) to Cav1 and counteracted CagA-induced cytoskeletal rearrangements. In human GC cell lines (MKN45, N87) and mouse stomach tissue, H. pylori down-regulated endogenous expression of Cav1 independently of CagA. Mechanistically, H. pylori activated sterol-responsive element-binding protein-1 (SREBP1) to repress transcription of the human Cav1 gene from sterol-responsive elements (SREs) in the proximal Cav1 promoter. These data suggested a protective role of Cav1 against H. pylori-induced inflammation and tissue damage. We propose that H. pylori exploits down-regulation of Cav1 to subvert the host's immune response and to promote signalling of its virulence factors in host cells., Author Summary Infection with the bacterium Helicobacter pylori (H. pylori) mainly affects children in the developing countries who are at risk to progress to gastric cancer (GC) as adults after many years of persistent infection, especially with strains which are positive for the oncogenic virulence factor CagA. Eradication of H. pylori by antibiotics is a treatment of choice but may also alter the susceptibility to allergies and other tumor types. Thus, novel diagnostic or prognostic markers are needed which detect early molecular changes in the stomach mucosa during the transition of chronic inflammation to cancer. In our study, we found that the tumor suppressor caveolin-1 (Cav1) is reduced upon infection with H. pylori, and CagA was sufficient but not necessary for this down-regulation. Loss of Cav1 was caused by H. pylori-dependent activation of sterol-responsive element-binding protein-1 (SREBP1), and this event abolished the interaction of Cav1 with p120 RhoGTPase-activating protein/deleted in liver cancer-1 (p120RhoGAP/DLC1), a second bona fide tumor suppressor in gastric tissue. Conclusively, Cav1 and DLC1 may constitute novel molecular markers in the H. pylori-infected gastric mucosa before neoplastic transformation of the epithelium.

Bacillus cereus causes food poisoning and serious non-gastrointestinal-tract infections. Non-hemolytic enterotoxin (Nhe), which is present in most B. cereus strains, is considered to be one of the main virulence factors. However, a B. cereus ΔnheBC mutant strain lacking Nhe is still cytotoxic to intestinal epithelial cells. In a screen for additional cytotoxic factors using an in vitro model for polarized colon epithelial cells we identified B. cereus sphingomyelinase (SMase) as a strong inducer of epithelial cell death. Using single and double deletion mutants of sph, the gene encoding for SMase, and nheBC in B. cereus we demonstrated that SMase is an important factor for B. cereus cytotoxicity in vitro and pathogenicity in vivo. SMase substantially complemented Nhe induced cytotoxicity in vitro. In addition, SMase but not Nhe contributed significantly to the mortality rate of larvae in vivo in the insect model Galleria mellonella. Our study suggests that the role of B. cereus SMase as a secreted virulence factor for in vivo pathogenesis has been underestimated and that Nhe and SMase complement each other significantly to cause full B. cereus virulence hence disease formation.

Helicobacter pylori infection is the most common bacterial infection worldwide and is strongly associated with gastric oncogenesis. Recently, we discovered that the H. pylori protein CagA, a risk factor for carcinogenesis, consists of two distinct membrane-targeting domains. The C-terminal membrane-binding domain induces host cell responses associated with a high oncogenic potential. The N-terminal membrane-targeting domain, however, localizes to a different membrane substructure at the site of newly formed cell-cell contacts thereby diminishing the effects of C-terminal signaling motifs on host cell physiology. This inhibitory function may allow H. pylori to establish a colonization niche in the host by maintaining the host epithelial architecture and thus decreasing the oncogenic potential as a side effect. From a bacterial standpoint, however, its main purpose maybe is to translocate the CagA protein via the type IV secretion apparatus into host epithelial cells.

The Helicobacter pylori protein CagA (cytotoxin-associated gene A) is associated with an increased risk for gastric cancer formation. After attachment to epithelial cells, the bacteria inject CagA via a type IV secretion apparatus into host cells, where it exerts its biological activity. Host cell responses to intracellular CagA have been linked exclusively to signaling motifs in the C terminus of the CagA protein. Little is known about the functional role of the remaining CagA protein. Using transgenic expression of CagA mutants in epithelial cells, we were able to identify a novel CagA inhibitory domain at the N terminus consisting of the first 200 amino acids. This domain localizes to cell-cell contacts and increases the rate and strength of cell-cell adhesion in epithelial cells. Thus, it compensates for the loss of cell-cell adhesion induced by the C terminus of the CagA protein. Consistent with its stabilizing role on cell-cell adhesion, the CagA N terminus domain reduces the CagA-induced β-catenin transcriptional activity in the nucleus. Furthermore, it inhibits apical surface constriction and cell elongations, host cell phenotypes induced by the C terminus in polarized epithelia. Therefore, our study suggests that CagA contains an intrinsic inhibitory domain that reduces host cell responses to CagA, which have been associated with the formation of cancer.

Whereas the final differentiation of conventional dendritic cells (CDCs) from committed precursors occurs locally in secondary lymphoid or peripheral tissues, plasmacytoid dendritic cells (PDCs) are thought to fully develop in the bone marrow from common DC progenitors before migrating to the periphery. In our study, we define, for the first time, a subpopulation of CCR9− major histocompatibility complex class IIlow PDCs in murine bone marrow, which express E2-2 and are immediate precursors of CCR9+ fully differentiated PDCs. However, CCR9− PDCs have the plasticity to acquire the phenotype and function of CD11b+ CD8α− major histocompatibility complex class IIhigh CDC-like cells under the influence of soluble factors produced by intestinal epithelial cells or recombinant GM-CSF. This deviation from the PDC lineage commitment is regulated on the level of transcription factors reflected by down-regulation of E2-2 and up-regulation of ID2, PU.1, and BATF3. Thus, CCR9− PDCs are immediate PDC precursors that can be reprogrammed to differentiate into CDC-like cells with higher antigen-presenting and cytokine-producing capacity under the influence of the local tissue microenvironment.

The number of proteins found associated with cell-cell adhesion substructures is growing rapidly. Based on potential protein-protein interactions, complex protein networks at cell-cell contacts can be modeled. Traditional studies to examine protein-protein interactions include co-immunoprecipitation or pull-down experiments of tagged proteins. These studies provide valuable information that proteins can associate directly or indirectly through other proteins in a complex. However, they do not clarify if a given protein is part of other protein complexes or inform about the specificity of those interactions in the context of adhesion substructures. Thus, it is not clear if models compiled from these types of studies reflect the combination of protein interactions in the adhesion complex in vivo for a specific cell type. Therefore, we present here a method to separate cell-cell contact membrane substructures with their associated protein complexes based on their buoyant behavior in iodixanol density gradients. Analysis of 16 proteins of the apical junctional complex (AJC) in epithelial Madin-Darby canine kidney cells revealed a more simple organization of the AJC adhesion complex than that predicted from the combination of all possible protein-protein interactions defined from co-immunoprecipitation and pull-down experiments.

The epithelial apical junctional complex (AJC) is an important regulator of cell structure and function. The AJC is compartmentalized into substructures comprising the tight and adherens junctions, and other membrane complexes containing the membrane proteins nectin, junctional adhesion molecule, and crumbs. In addition, many peripheral membrane proteins localize to the AJC. Studies of isolated proteins indicate a complex map of potential binding partners in which there is extensive overlap in the interactions between proteins in different AJC substructures. As an alternative to a direct search for specific protein-protein interactions, we sought to separate membrane substructures of the AJC in iodixanol density gradients and define their protein constituents. Results show that the AJC can be fractured into membrane substructures that contain specific membrane and peripheral membrane proteins. The composition of each substructure reveals a more limited overlap in common proteins than predicted from the inventory of potential interactions; some of the overlapping proteins may be involved in stepwise recruitment and assembly of AJC substructures.

The epithelial apical–junctional complex is a key regulator of cellular functions. In addition, it is an important target for microbial pathogens that manipulate the cell to survive, proliferate and sometimes persist within a host. Out of a myriad of potential molecular targets, some bacterial and viral pathogens have selected a subset of protein targets at the apical–junctional complex of epithelial cells. Studying how microbes use these targets also teaches us about the inherent physiological properties of host molecules in the context of normal junctional structure and function. Thus, we have learned that three recently uncovered components of the apical–junctional complex of the Ig superfamily — junctional adhesion molecule, Nectin and the coxsackievirus and adenovirus receptor — are important regulators of junction structure and function and represent critical targets of microbial virulence gene products.

Helicobacter pylori translocates the protein CagA into gastric epithelial cells and has been linked to peptic ulcer disease and gastric carcinoma. We show that injected CagA associates with the epithelial tight-junction scaffolding protein ZO-1 and the transmembrane protein junctional adhesion molecule, causing an ectopic assembly of tight-junction components at sites of bacterial attachment, and altering the composition and function of the apical-junctional complex. Long-term CagA delivery to polarized epithelia caused a disruption of the epithelial barrier function and dysplastic alterations in epithelial cell morphology. CagA appears to target H. pylori to host cell intercellular junctions and to disrupt junction-mediated functions.

BackgroundE-cadherin is a major component of adherens junctions. Impaired expression of E-cadherin in the small intestine and colon has been linked to a disturbed intestinal homeostasis and barrier function. Down-regulation of E-cadherin is associated with the pathogenesis of infections with enteropathogenic bacteria and Crohn's disease.Methods and findingsTo genetically clarify the function of E-cadherin in intestinal homeostasis and maintenance of the epithelial defense line, the Cdh1 gene was conditionally inactivated in the mouse intestinal epithelium. Inactivation of the Cdh1 gene in the small intestine and colon resulted in bloody diarrhea associated with enhanced apoptosis and cell shedding, causing life-threatening disease within 6 days. Loss of E-cadherin led cells migrate faster along the crypt-villus axis and perturbed cellular differentiation. Maturation and positioning of goblet cells and Paneth cells, the main cell lineage of the intestinal innate immune system, was severely disturbed. The expression of anti-bacterial cryptidins was reduced and mice showed a deficiency in clearing enteropathogenic bacteria from the intestinal lumen.ConclusionThese results highlight the central function of E-cadherin in the maintenance of two components of the intestinal epithelial defense: E-cadherin is required for the proper function of the intestinal epithelial lining by providing mechanical integrity and is a prerequisite for the proper maturation of Paneth and goblet cells.