Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection
Summary
Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.
Abstract
Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.
Introduction
Entamoeba histolytica is a single cell protozoan responsible of human amoebiasis, an intestinal infection causing inflammation and diarrhea. E. histolytica infects up to 50 million individuals yearly, but only about 10% of infected people develop the symptoms associated to amoebiasis1. Infection occurs upon ingestion of contaminated food or water containing E. histolytica cysts. In the intestine, cysts produce live trophozoites that adhere to colon mucin and proliferate2. Trophozoites usually form cysts that are excreted via stools. In other cases and for yet unknown reasons, trophozoites break the intestinal epithelial layer and invade underlying tissues. In worst cases, they enter the blood stream and affect other organs such as the liver3.
Breaking the epithelial barrier requires disruption of epithelial transmembranal structures that maintain cells joined. Epithelial cell contacts are formed by the apical junctional complex consisting of tight (TJ) and adherens junctions (AJ), and desmosomes4. The most apical junctions are TJ, and therefore, they are the first barrier affronted by E. histolytica and some other pathogens during host invasion. TJ are comprised of transmembranal adhesion receptors such as claudins, occludin and junctional adhesion molecules (JAM) that engage in homo- or heterophilic interactions with receptors of the neighboring cell. They are intracellularly bound by scaffold molecules of the zonula occludens (ZO) family that connect adhesion receptors to actin cytoskeleton to provide further mechanical strength to the epithelium. TJ are responsible for sealing intestinal tissue from the gut lumen, preventing excessive water and solute leakage. Thus, after TJ are disrupted by the parasite, tissues are invaded. E. histolytica secretes several molecules such as: (i) those involved in adhesion of amoebae to target cells5; (ii) membrane-active factors participating in killing of host cells by exocytosis, for example the ion channel-forming peptides termed amoebapores6,7; and (iii) proteinases that degrade extracellular matrix proteins and mediate tissue disintegration5,8,9.
The cysteine protease EhCP112 and the adhesion molecule EhADH112 that together form the EhCPADH112 complex are two E. histolytica virulence proteins that play a major role in the disassembly of TJ 10. Live trophozoites, their total lysates and secreted products induce molecular changes in the TJ complex and functional disturbance of the epithelial barrier. In this study, it is shown that EhCP112 and EhADH112 interact with occludin and claudin-1 proteins leading to internalization and degradation of cell proteins, thus facilitating E. histolytica entrance through the paracellular pathway.
Our data and those of other groups11-17strongly suggest the necessity of specific host-pathogen interactions that allow parasite invasion. Unraveling the molecular basis of these interactions is of utmost importance for a better understanding of amoebiasis pathogenesis. Selective disturbance of TJ by trophozoites, characterized by increased paracellular permeability, can be measured by a decrease in transepithelial electrical resistance (TER). The transference of parasitic proteins towards host epithelia can be determined by immunofluorescence staining and confocal laser microscopy, a method that can also reveal co-localization of amoeba virulence factors with epithelial junctional markers indicating possible direct interactions. In this article, we describe in detail how epithelial cells and trophozoites are cultivated, harvested and manipulated to examine host-pathogen interactions and their consequences.
Protocol
Representative Results
Discussion
In order to study in vitro host-pathogen interactions during epithelial infection by E. histolytica, it is crucial to work with well-established cultures of both epithelial cells and trophozoites. For example, formerly, E. histolytica cultures had usually been established in association with certain species of bacteria or trypanosomatids22,23. However, co-cultivation of E. histolytica cultures is counterproductive for the study of host-pathogen interactions because observed …
Declarações
The authors have nothing to disclose.
Acknowledgements
This work was supported by grants from the Institute of Science and Technology of the Federal District (ICyTDF, 64/2012 to EO) and the Mexican Council for Science and Technology (Conacyt, 179895 to MS).
Materials
| Entamoeba histolytica HM1:IMSS, Clone A | IMSS Hospital, Mexico | Without/number | Virulent trophozoites18 |
| TYI broth | Becton, Dickinson and Company Merck Merck Merck J.T. Baker Reproquifin SIGMA-Aldrich SIGMA-Aldrich |
211862 K35625437 626 21578 4873 3252-01 CAS 50-81-7 C7880 F-5879 |
3.45% BBL Biosate peptone 58 mM glucose 39 mM NaCl 5 mM KH2PO4 6.5 mM K2HPO4 16.3 mM ascorbic acid 8.1 mM L-cysteine 0.1 mM ferric ammonium citrate, adjust pH 6.819 |
| Bovine serum adult | Microlab , Labs., Mex. | SU146 | Use at 10% and inactivated to 56° C for 30 min |
| Diamond vitamin mixture- Tween 80 | In vitro | SR-07 | Use at 3% |
| Penicillin | Lakeside, Méx. | 34564SSA IV | 0.5 IU/mL |
| Streptomycin | Lakeside, Méx. | 75757SSA IV | 35 µg/ mL |
| Pyrex 15 mL screw cap culture tubes with PTFE lined phenolic caps | Corning-Pyrex | 9826-16X | 16×125 mm, capacity 15 mL and caps fabricated from special formula resistant to effects of temperature |
| Cell culture plates, 6 Well | Corning-Costar | 3516 | Sterile plates, well diameter 34.8 mm and growth area 9.5 cm2. Rings on lid prevent cross-contamination |
| 25cm2 cell culture flask | Corning-Costar | 430168 | Canted neck flasks |
| MDCK (Madin Darby canine kidney) type I | American Type Culture Collection | CCL34 | Kidney epithelial cells grown between the 60th and 90th passage |
| DMEM medium | Gibco | 12800-017 | Dulbecco's Modified Eagle Medium with high glucose. |
| Neonate Calf Serum | In vitro | S-02 | Use at 10%. |
| Penicillin/Streptomycin mixture | In vitro | A-01 | Stock solution 10,000 U/µg/mL |
| Insulin | AMSA | 398MJ94SSA IV | Stock solution 100 IU/mL |
| Trypsin solution | In vitro | EN-005 | 0.05% enzyme solution without calcium and magnesium |
| 75cm2 cell culture flask | Corning-Costar | 430720 | Canted neck flasks for trophozoite culture in TYI-S-33 medium |
| Transwell permeable supports | Corning-Costar | 3470 | 0.4. µm polyster membrane, 6.5 mm insert in 24 well plate, growth area 0.3 cm2 |
| 24 well cell culture dish | Corning-Costar | 3524 | Clear polystyrene, treated for optimal cell attachment, sterilized by gamma radiation and certified non-pyrogenic |
| Complete Mini | Roche | 11836 153 001 | Protease inhibitor cocktail inhibits a broad spectrum of serine, cysteine and metallo-proteases. Final concentration 1 mM |
| Trans-epoxysuccinyl-L-leucylamido (4-guanidino) butane (E-64) | SIGMA-Aldrich | E3132 | Cystein protease inhibitor, final concentration 40 µg/mL |
| pαZO-1 | Invitrogen | 402200 | IgG rabbit policlonal antibody against a synthetic peptide in the middle region of the ZO-1 human protein |
| mαEhCPADH112 | Homemade antibody | Without/ Number | IgM mouse monoclonal antibody against 444-601 epitope located at C-terminal of EhCPADH11221,27 |
| FITC-goat anti-mouse IgM | Zymed | 62-6811 | Fluorescein isotiocyanate (FITC)-labelled goat anti-mouse secondary antibody |
| TRITC- goat anti-rabbit IgG (H+L) | Zymed | 816114 | Tetramethyl-rhodamine isothiocyanate (TRITC)-labelled goat anti-rabbit IgG secondary antibody. |
| STX2 Electrode | World Precision Instrument | 102711 | Consists of a fixed pair of double electrodes, 4 mm wide and 1 mm thick. Each stick of the electrode pair contains a silver/silver-chloride pellet for measuring voltage and a silver electrode for passing current. For use with EVOM |
| EVOM epithelial voltohmmeter | World Precision Instrument | 12111 | Use in resistance mode and maintain unplugged during TER measurements |
| Neubauer chamber | MEARIENFELD | 610610 | Hemocytometer |
| Leica TCS_SP5_MO | Leica | Without/number | Laser confocal microscopy with Leica microsystems CMS Gmbh/leica Las af Lite/BIN software |
| Vectashield | Vector Laboratories, Inc. | H-1000 | Mounting medium for fluorescence |
| 4´, 6-diamino-2-phenylindole (Dapi) | SIGMA | D-9542 | 0.05 mM final concentration |
| Bovine serum albumin (BSA) | US Biological | A-1310 | 0.5% final concentration for blocking solution |
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