analysis-epithelial-damage-produced-entamoeba-histolytica

Analysis of the Epithelial Damage Produced by <em>Entamoeba histolytica</em> Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Center for Research and Advanced Studies of the National Polytechnic Institute

The purpose of this study was to compare human endothelial cells from Schlemm's canal (SCEs) and the trabecular meshwork (TMEs) in terms of ZO-1 isoform expression, hydraulic conductivity (HC) properties, and "giant" vacuole (GV) formation. The principal study methods were Western blot, RT-PCR, immunofluorescence, and perfusion chambers. Blot signals for alpha+ - and alpha- -isoforms were similar in SCEs but less intense for the alpha+ -relative to the alpha- -signal in TMEs. With the anti-alpha+ antibody used at 1/50 dilution, binding occurred at cell borders of both cell types, but only to SCEs when used at a >/=1/200 dilution in vitro and in vivo. SCEs were more resistive than TMEs (HC = 0.66 vs. 1.32 microl.min-1.mmHg-1.cm-2; P < 0.001) when perfused from apex to base. When perfused in the other direction, SCEs were again more resistive (5.23 vs. 9.04 microl.min-1.mmHg-1.cm-2; P < 0.01). GV formation occurred only in SCEs as a function of flow direction, perfusion pressure, and time. We conclude that SCEs and TMEs have distinctive phenotypic properties involving their content of ZO-1 isoforms, barrier function, and GV formation.

Endothelia of Schlemm's canal and trabecular meshwork: distinct molecular, functional, and anatomic features.

ZO-2 is a membrane-associated guanylate kinase (MAGUK) protein present at the tight junction (TJ) of epithelial cells. While confluent monolayers have ZO-2 at their cellular borders, sparse cultures conspicuously show ZO-2 at the nuclei. To study the role of nuclear ZO-2, we tested by pull-down assays and gel shift analysis the interaction between ZO-2 GST fusion proteins and different transcription factors. We identified the existence of a specific interaction of ZO-2 with Fos, Jun and C/EBP (CCAAT/enhancer binding protein). To analyze if this association is present "in vivo", we performed immunoprecipitation and immunolocalization experiments, which revealed an interaction of ZO-2 with Jun, Fos and C/EBP not only at the nucleus but also at the TJ region. To test if the association of ZO-2 with AP-1 (activator protein-1) modulates gene transcription, we performed reporter gene assays employing chloramphenicol acetyltransferase (CAT) constructs with promoters under the control of AP-1 sites. We observed that the co-transfected ZO-2 down-regulates CAT expression in a dose-dependent manner. Since ZO-2 is a multidomain protein, we proceeded to determine which region of the molecule is responsible for the modulation of gene expression, and observed that both the amino and the carboxyl domains are capable of inhibiting gene transcription.

The tight junction protein ZO-2 associates with Jun, Fos and C/EBP transcription factors in epithelial cells.

ZO-2 is a MAGUK protein that in confluent epithelial sheets localizes at tight junctions (TJ) whereas in sparse cultures accumulates in clusters at the nucleus. Here, we have characterized several nuclear properties of ZO-2. We observe that ZO-2 is present in the nuclear matrix and co-immunoprecipitates with lamin B(1) and actin from the nuclei of sparse cultures. We show that ZO-2 presents several NLS at its amino region, that when deleted, diminish the nuclear import of the ZO-2 amino segment and impair the ability of the region to regulate the transcriptional activity of promoters controlled by AP-1. Several RS repeats are detected in the ZO-2 amino segment, however, their deletion does not preclude the display of a speckled nuclear pattern. ZO-2 displays two putative NES. However, only the second one appears to be functional, as when conjugated to ovalbumin (OV), it is able to translocate this protein from the nucleus to the cytoplasm in a leptomycin B-sensitive way.

Characterization of the tight junction protein ZO-2 localized at the nucleus of epithelial cells.

JAM-C (junctional adhesion molecule C) has been implicated in the regulation of leukocyte migration, cell polarity, spermatogenesis, angiogenesis and nerve conduction. JAM-C has been also reported to concentrate at TJs (tight junctions) and desmosomes, although detailed localization studies remain incomplete.

Evidence for cross-reactivity of JAM-C antibodies: implications for cellular localization studies.

Phenol contamination of soil and water has raised concerns among people living near phenol-producing factories and hazardous waste sites containing the chemical. Phenol, particularly in high concentrations, is an irritating and corrosive substance, making mucosal membranes targets of toxicity in humans. However, few data on the effects of phenol after oral exposure exist. We used an in vitro model employing human intestinal epithelial cells (SK-CO15) cultured on permeable supports to examine effects of phenol on epithelial barrier function. We hypothesized that phenol disrupts epithelial barrier by altering tight junction (TJ) protein expression. The dose-response effect of phenol on epithelial barrier function was determined using transepithelial electrical resistance (TER) and FITC-dextran permeability measurements. We studied phenol-induced changes in cell morphology and expression of several tight junction proteins by immunofluorescence and Western blot analysis. Effects on cell viability were assessed by MTT, Trypan blue, propidium iodide and TUNEL staining. Exposure to phenol resulted in decreased TER and increased paracellular flux of FITC-dextran in a dose-dependent manner. Delocalization of claudin-1 and ZO-1 from TJs to cytosol correlated with the observed increase in permeability after phenol treatment. Additionally, the decrease in TER correlated with changes in the distribution of a membrane raft marker, suggesting phenol-mediated effects on membrane fluidity. Such observations were independent of effects of phenol on cell viability as enhanced permeability occurred at doses of phenol that did not cause cell death. Overall, these findings suggest that phenol may affect transiently the lipid bilayer of the cell membrane, thus destabilizing TJ-containing microdomains.

Effects of phenol on barrier function of a human intestinal epithelial cell line correlate with altered tight junction protein localization.

Entamoeba histolytica, the protozoan responsible for human amoebiasis, causes between 30,000 and 100,000 deaths per year worldwide. Amoebiasis is characterized by intestinal epithelial damage provoking severe diarrhea. However, the molecular mechanisms by which this protozoan causes epithelial damage are poorly understood. Here, we studied the initial molecular interactions between the E. histolytica EhCPADH112 virulence complex and epithelial MDCK and Caco-2 cells. By confocal microscopy, we discovered that after contact with trophozoites or trophozoite extracts (TE), EhCPADH112 and proteins forming this complex (EhCP112 and EhADH112) co-localize with occludin and claudin-1 at tight junctions (TJ). Immunoprecipitation assays revealed interaction between EhCPADH112 and occludin, claudin-1, ZO-1 and ZO-2. Overlay assays confirmed an interaction of EhCP112 and EhADH112 with occludin and claudin-1, whereas only EhADH112 interacted also with ZO-2. We observed degradation of all mentioned TJ proteins after incubation with TE. Importantly, inhibiting proteolytic activity or blocking the complex with a specific antibody not only prevented TJ protein degradation but also epithelial barrier disruption. Furthermore, we discovered that TE treatment induces autophagy and apoptosis in MDCK cells that could contribute to the observed barrier disruption. Our results suggest a model in which epithelial damage caused by E. histolytica is initiated by the interaction of EhCP112 and EhADH112 with TJ proteins followed by their degradation. Disruption of TJs then induces increased paracellular permeability, thus facilitating the entry of more proteases and other parasite molecules leading eventually to tissue destruction.

The EhCPADH112 complex of Entamoeba histolytica interacts with tight junction proteins occludin and claudin-1 to produce epithelial damage.

The intestinal epithelium forms a stable barrier protecting underlying tissues from pathogens in the gut lumen. This is achieved by specialized integral membrane structures such as tight and adherens junctions that connect neighboring cells and provide stabilizing links to the cytoskeleton. Junctions are constantly remodeled to respond to extracellular stimuli. Assembly and disassembly of junctions is regulated by interplay of actin remodeling, endocytotic recycling of junctional proteins, and various signaling pathways. Accumulating evidence implicate small G proteins of the Ras superfamily as important signaling molecules for the regulation of epithelial junctions. They function as molecular switches circling between an inactive GDP-bound and an active GTP-bound state. Once activated, they bind different effector molecules to control cellular processes required for correct junction assembly, maintenance and remodelling. Here, we review recent advances in understanding how GTPases of the Rho, Ras, Rab and Arf families contribute to intestinal epithelial homeostasis.

Article	Year
Endothelia of Schlemm's canal and trabecular meshwork: distinct molecular, functional, and anatomic features. American journal of physiology. Cell physiology\| PubMed ID: 14613887	2004
The tight junction protein ZO-2 associates with Jun, Fos and C/EBP transcription factors in epithelial cells. Experimental cell research\| PubMed ID: 14720506	2004
Characterization of the tight junction protein ZO-2 localized at the nucleus of epithelial cells. Experimental cell research\| PubMed ID: 15194440	2004
Evidence for cross-reactivity of JAM-C antibodies: implications for cellular localization studies. Biology of the cell / under the auspices of the European Cell Biology Organization\| PubMed ID: 19143587	2009
Effects of phenol on barrier function of a human intestinal epithelial cell line correlate with altered tight junction protein localization. Toxicology and applied pharmacology\| PubMed ID: 19679145	2009
The EhCPADH112 complex of Entamoeba histolytica interacts with tight junction proteins occludin and claudin-1 to produce epithelial damage. PloS one\| PubMed ID: 23762290	2013
Small GTPases of the Ras superfamily regulate intestinal epithelial homeostasis and barrier function via common and unique mechanisms. Tissue barriers\| PubMed ID: 24868497	2013

Abigail Betanzos

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