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 amoebapores^6,7; and (iii) proteinases that degrade extracellular matrix proteins and mediate tissue disintegration^5,8,9.

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 groups^11-17strongly 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

1. Establishment and Maintenance of E. histolytica Cultures

1. Grow axenically (entirely free of all other contaminating organisms) 1 x 10⁵ trophozoites of Entamoeba histolytica strain HMl:IMSS clone A¹⁸ in 16 x 125 mm culture tubes with Teflon liner screw caps (or 1 x 10⁶ trophozoites in a disposable T-25 flask) and 15 ml (or 50 ml in T-25 flask) of TYI-S-33 medium (TYI broth supplemented with 3% Diamond vitamin mixture, 10% heat inactivated adult bovine serum, 0.5 IU/ml penicillin and 35 µg/ml streptomycin)¹⁹ in an incubator at 37 °C.
2. Harvest trophozoites during the logarithmic growth phase usually at 48-96 hr intervals (Figure 1) by chilling the culture tubes for 5-10 min in an ice-water bath to release trophozoites attached to the glass culture tube.
3. Transfer the culture into a conical tube and invert it several times to disperse the cells. Determine cell number using a hemocytometer (Neubauer chamber), and transfer an inoculum into a culture tube containing fresh TYI-S-33 medium.
   1. Use low numbers of amoebas for longer incubation periods (~3 x 10⁵ cells for ~5 days) and a higher number for shorter periods (~1 x 10⁶ cells for ~1 day). While counted inocula are desirable, established cultures become predictable so that estimated volumes of inocula are feasible.
   2. Titrate the amount of trophozoites to optimize cell numbers for each experiment.
   3. Maintain a parallel duplicate culture to have a back-up in case of inadvertent contamination or tube breakage.
4. Cap tubes tightly and incubate them at 37 °C in a 5° tilted horizontal angle.
5. Check cultures by visual inspection regularly, since an excessive growth of the culture may contain many lysed cells.

2. Establishment and Maintenance of MDCK Culture

1. Grow MDCK (Madin Darby Canine Kidney) cells type I (high resistance) in disposable T-75 flasks with 10 ml of DMEM media supplemented with penicillin (100 IU/ml), streptomycin (100 mg/ml), insulin (0.08 U/ml) and 10% neonate calf serum in a humidified atmosphere of 5% CO₂ and 95% air at 37 °C.
2. To split the cells, check them on an inverted microscope. Split cells when they are ~ 85-100% confluent.
3. Use a vacuum aspirator in a sterile hood and a sterile glass Pasteur pipette to remove media from the culture. To avoid scraping cells, turn the flask upside down and aspirate media from the bottom corner.
4. Dispense 10 ml of prewarmed PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4) into a T-75 flask (5 ml in the case of a T-25 flask) and gently rock the flask to wash the cells. Remove PBS by vacuum aspiration. Extensive washing is required since serum inhibits trypsin.
5. Dispense 1.5 ml of 0.05% trypsin into a T-75 flask (for T-25 flask use 0.5 ml) and gently rock the flask to cover the cell layer with trypsin.
6. Incubate for 5-10 min in the incubator (exact time depends on trypsin activity).
7. Check for cell detachment by holding flask against the light and look for flowing cells. Cell detachment (rounded cells) can also be checked using a microscope. Often, cells release faster with a quick, gentle slap to the side of the flask.
8. Add 15 ml of supplemented DMEM to a T-75 flask (5 ml for a T-25 flask). Resuspend cells by pipetting up and down 4 or 5 times.
9. To propagate cells, dilute cell suspension 1:5 with fresh supplemented DMEM. Cells number can also be determined at this point using a hemocytometer but this is usually only necessary if splitting cells into special formats for certain assays.

3. Preparation of Trophozoite Total Lysates

1. Detach trophozoites from culture tubes by chilling the tube for 5-10 min in an ice-water bath. Transfer the culture aseptically into a conical tube and centrifuge at 360 x g for 10 min at 4 °C. Carefully discard the supernatant by decantation, add ice-cold PBS to the pellet, invert the tube several times to disperse the cells and centrifuge again at 360 x g for 10 min. Repeat this step twice to remove all traces of TYI-S-33 medium.
2. Determine trophozoites number using a hemocytometer.
3. Lyse trophozoites by freeze-thaw cycles. Snap-freeze a measured inoculum of trophozoites (600,000 trophozoites/ml) diluted in ice-cold PBS, in liquid nitrogen for 1 min. Unfreeze the sample at 4 °C and vortex vigorously. Repeat freezing and thawing 3 times to complete cell lysis.
4. Activate proteases present in the lysates with 0.02% β-mercaptoethanol.

4. Preparation of Trophozoite Secreted Products

1. Perform steps 3.1 to 3.2.
2. Determine cells number and viability at this point using a hemocytometer and applying a trypan blue exclusion test²⁰:
   1. Dilute 9 x 10⁶ trophozoites in 3 ml ice-cold PBS and transfer cells into a 50 ml conical tube. Take 10 µl of this suspension and add 1 µl of 0.4% trypan blue stock solution pH 7.2.
   2. Use a Neubauer chamber to count cells at low magnification.
   3. Count all cells and separate the number of blue cells, since blue cells have taken up the dye and must be considered dead.
   4. Calculate the percentage of viable cells by dividing the number of viable cells by the total number of cells and multiplying by 100.
3. Transfer the conical tube containing the trophozoite suspension into the incubator at 37 °C for 2 hr in a 5° tilted horizontal angle.
4. To collect the secreted products, centrifuge tubes at 360 x g for 10 min. Use a disposable and sterile syringe and carefully collect the supernatant, avoiding contamination of samples with trophozoites from the pellet. Eliminate any transferred cells by passing the supernatant through a 0.22 μm cellulose acetate membrane. Activate proteases with 0.02% β-mercaptoethanol.
5. To discard putative unwanted contamination with molecules released from dead cells, reapply the trypan blue exclusion test for cell viability. Viable and total number of cells should be the same as obtained in step 4.2. If this is not the case, the collected supernatant may not only contain secreted proteins and should be discarded.

5. Interaction of MDCK Cells with Trophozoites, Trophozoite Lysates or Secreted Products

1. Incubate confluent MDCK cell monolayers with live trophozoites (an MDCK-to-amoeba ratio of 1:1), trophozoite lysates (an MDCK-to-amoeba ratio of 1:2) or secreted products (an MDCK-to-amoeba ratio of 1:10) at 37 °C for 2 and 30 min (Figure 2A).
2. Wash epithelial cells five times with ice-cold PBS to eliminate unbound molecules or trophozoites.

6. Preparation of Samples for Immunofluorescence

1. Culture MDCK cells on sterile glass coverslips placed inside a 24-multiwell cell culture dish. Inspect the cells on the inverted microscope until they reach 100% of confluence. Then, replace medium with 1 ml of fresh supplemented DMEM medium and transfer the plate to a 37 °C incubator for 24 hr more.
2. Remove the medium using a vacuum aspirator and a sterile glass Pasteur pipette and add 1 ml of warm PBS to each well. Remove PBS and repeat the wash with fresh PBS. Take care not to scrape cells from the bottom of the well with the glass pipette.
3. Add different conditions of trophozoites diluted in 1 ml of warm PBS: live trophozoites (T), trophozoite total lysates (TL) and secreted products (SP).
   1. Put the culture plate in the incubator for 2 or 30 min.
   2. Prepare two control conditions: one named time 0 min, where MDCK cells should be not incubated with E. histolytica but only with 1 ml of warm PBS; and another condition as secondary antibodies control, in this case incubate MDCK with T, TL or SP for 2 or 30 min and omit incubation with primary antibodies.
4. Wash epithelial cells five times with cold PBS to eliminate unbound molecules or trophozoites.
5. Fix and permeabilize the cells with 1 ml of 96% ethanol for 30 min at -20 °C.
6. To remove ethanol, perform three quick washes with 1 ml of PBS at room temperature.
7. Perform the following steps in a humid chamber to avoid drying of samples:
   1. Use any available flat box that can be closed and filled with a wet paper towel on which samples can be safely placed. For example, an empty pipette tip box serves this purpose well.
   2. Inside the chamber, evenly place a Parafilm layer and pipette 25 μl of blocking solution (0.5% BSA) for each coverslip.
   3. Take coverslips out of the wells with fine forceps, carefully remove excess liquid from the edge with a filter paper and put coverslip into the drop containing blocking solution (0.5% BSA)with the cells facing the blocking solution. Incubate the samples for 1 hr at room temperature.
8. Prepare a mixture of primary antibodies in PBS: IgM mouse anti-EhCPADH112 (mα-EhCPADH112; 1:10 dilution) and IgG rabbit anti-ZO-1 (pα-ZO-1; 1:100 dilution).
9. Place a fresh sheet of Parafilm in the humid box and pipette 25 μl of antibodies solution for each coverslip.
10. Lift the coverslips from the blocking solution, dry any excess liquid at the edges using a filter paper, and place them into the drops with the cells facing the antibody solution. Incubate the samples overnight at 4 °C.
11. Put each coverslip into a well of a 24-multiwell (cell side on top) and add 1 ml of PBS.
    1. Wash five times with 1 ml of PBS to remove unbound molecules.
12. Prepare a mixture of fluorescent secondary antibodies in PBS: goat anti-IgM mouse coupled to FITC (1:100 dilution) and goat anti-IgG rabbit coupled to TRITC (1:50 dilution).
13. Place a fresh sheet of Parafilm into the humid box and pipette 25 µl of the secondary antibodies solution for each coverslip.
14. Transfer the samples as in 6.10 and incubate for 1 hr at room temperature in the dark to avoid bleaching of the fluorescent dyes. Repeat step 6.11.1.
15. For nuclear staining, incubate for 3 min with 200 μl of 0.05 mM DAPI solution at room temperature and protect from light. Repeat step 6.11.1.
16. To conserve the fluorescent dyes, place the coverslips (cells facing down) into 5 μl Vecta Shield antifade mounting solution on glass microscope slides. Seal the cover slips using nail polish to avoid sample drying.
17. For long term storage, keep slides in a microscope slide box at -20 °C.
18. Analyze preparations by confocal microscopy through Z-stack sections and xz-planes (Figure 2A).

7. Incubation of Trophozoites with Protease Inhibitors or Specific Antibody

1. Repeat steps 3.1 to 3.2. For each experimental condition, incubate 1 x 10⁵ trophozoites (resuspended in 50 μl PBS) with protease inhibitors (1 mM Complete and 40 µg/ml E-64) or 30 µg monoclonal antibody against EhCPADH112 (mαEhCPADH112)²¹ for 20 min at 4 °C (Figure 2B). Use these preparations for co-incubation assays as described in step 8.5.

8. Measurement of Transepithelial Electrical Resistance (TER)

1. Culture MDCK cells on sterile transwell permeable supports (0.4 μm pore size). In the upper compartment, place 100 μl of cellular suspension and in the lower compartment place 600 μl of supplemented DMEM medium.
   1. Check the medium level periodically. Fresh medium can be added as required.
   2. Inspect the cells on an inverted microscope until they reach 100% of confluence. Change medium every 2-3 days.
   3. To improve cell attachment (if necessary), equilibrate the transwell filters overnight at 37 °C in DMEM before adding the cell suspension.
2. Sterilize STX2 electrodes in the hood by immersion in ethanol and then in sterile PBS for 15 min each, under UV light. Connect electrodes to an EVOM epithelial voltohmmeter and select resistance mode.
3. Collect medium from the upper compartment of each transwell and pool them. Take 50 μl of this media mixture and combine it with 50 μl of trophozoites suspension (1 x 10⁵ trophozoites in PBS) or with only PBS (control). Use a similar mixture for each transwell.
4. Add mixtures to the upper chamber of each transwell containing the MDCK cells. Experimental conditions include MDCK cells without trophozoites (control), a co-culture with trophozoites or with trophozoites pre-incubated with protease inhibitors or mαEhCPADH112. To eliminate the resistance provided by filters, use transwells incubated with medium only. For each experimental condition use at least three transwells.
5. Immediately, measure TER by dipping the STX2 electrodes into each transwell.
   1. Ensure that the longer electrode touches the bottom of the lower chamber and that the shorter electrode is covered by medium in the upper compartment (see Figure 2B).
   2. Make sure to hold the electrodes perpendicularly each time. This will improve reproducibility, significantly. Avoid moving or tilting the electrode during the measurements as this will lead to variation of data.
6. This measurement should be considered the initial TER value. Then, monitor the TER during the following 30 min.
7. To calculate the final TER value, subtract the TER value of filters only. To compare results from different experiments normalize each data point to the initial values, taking these as 100% (Figure 2B).

Results

For a successful E. histolytica culture, two important conditions must be fulfilled: growth in axenic conditions and harvest in logarithmic phase. Previously, cultures of E. histolytica were readily established in association with certain species of bacteria or trypanosomatids²². However, nowadays it is common to have axenic cultivation of this parasite meaning an indefinite subcultivation of amoebae in an environment free of metabolizing bacteria, fungi, protozoa, or metazoan cells. Addition...

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 trypanosomatids^22,23. However, co-cultivation of E. histolytica cultures is counterproductive for the study of host-pathogen interactions because observed ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 x 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
25 cm2 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
75 cm2 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