giardia disrupts the arrangement of tight, adherens and...

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Parasitology International xxx (2011) xxx–xxx

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PARINT-00951; No of Pages 8

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Parasitology International

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Giardia disrupts the arrangement of tight, adherens and desmosomal junctionproteins of intestinal cells

C. Maia-Brigagão a,d, J.A. Morgado-Díaz b, W. De Souza a,c,⁎a Laboratório de Ultraestrutura Celular Hertha Meyer, Universidade Federal do Rio de Janeiro, Brazilb Grupo de Biologia Estrutural, Instituto Nacional de Câncer, Brazilc Diretoria de Programas, Instituto Nacional de Metrologia, Normalização e Qualidade Industrial, Brazild Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Brazil

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⁎ Corresponding author at: Av. Carlos Chagas Filho, CCiências da Saúde, CEP 21941-599, Brazil.

E-mail address: [email protected] (W. De Souza).

1383-5769/$ – see front matter © 2011 Elsevier Irelanddoi:10.1016/j.parint.2011.11.002

Please cite this article as: Maia-Brigagão C,intestinal cells, Parasitol Int (2011), doi:10

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Article history:Received 11 March 2011Received in revised form 20 October 2011Accepted 1 November 2011Available online xxxx

Keywords:Giardia duodenalisIntestinal epitheliumJunctional complexHost cell interaction

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Giardia duodenalis is a parasitic protozoan that causes diarrhea and other symptoms which together consti-tute a disease known as giardiasis. Although the disease has been well defined, the mechanisms involvingthe establishment of the infection have not yet been fully elucidated. In this study, we show that after 24 hof interaction between parasites and intestinal Caco-2 cells, there was an alteration of the paracellular per-meability, as observed by an approximate 42% of reduction in the transepithelial electrical resistance andpermeation to ruthenium red, which was concomitant with ultrastructural changes. Nevertheless, epitheliumviability was not affected. We also demonstrate that there was no change in expression of junctional proteins(tight and adherens) but that the distribution of these proteins in Caco-2 cells after parasite adhesion wassignificantly altered, as observed via laser scanning confocal microscopy 3D reconstruction. The presentwork shows that adhesion of Giardia duodenalis trophozoites to intestinal cells in vitro induces disturbancesof the tight, adherens and desmosomal junctions.

© 2011 Elsevier Ireland Ltd. All rights reserved.

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RRE1. Introduction

Giardia duodenalis is a widespread etiologic agent of thewaterbornedisease, giardiasis. This disease causes acute or chronic diarrhoea,dehydration, abdominal discomfort, and weight loss [1, 2]. The pro-liferative form, named trophozoite, colonises the small intestine byadhering to the apical surface of the enterocyte [2]. However, themechanisms involved in the induction of diarrhea during giardiasishave not been fully elucidated.

In the intestinal epithelium, the junctional complex is responsiblefor the maintenance of the epithelium organisation and the integrityof intercellular contacts. It is formed by three main structures: (a)the tight junction (TJ), which is an occluding junction that regulatesthe passage of molecules between epithelial cells; (b) the adherensjunction, which is associated with actin filaments; and (c) desmo-somes, which are associated with intermediate filaments. Tightjunctions are localised in the most apical portion of the epithelialcells and claudins and occludins are the main protein components.Claudins are 21–28 kDa transmembrane proteins and are the keycomponents for the structure and function of TJs [3, 4]. On the cyto-plasmic side of TJ strands, the membrane-associated guanylate kinasefamily proteins ZO-1 and ZO-2 bind to the C-terminal cytoplasmic

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Ltd. All rights reserved.

et al, Giardia disrupts the ar.1016/j.parint.2011.11.002

domain of claudins [5, 6]. Among the tight junction-associated proteins,ZO-1 and ZO-2 have been shown to be indispensable for TJ formationin epithelial cells [3]. Found inmost epithelial cells, the TJ forms beltsthat link the cells into a continuous sheet and separate the apical andbasolateral membranes of each highly polarised cell.

Adherens junctions (AJs) are cell–cell adhesion complexes thathave important contributions for tissue homeostasis [7–10]. Cadherinadhesion molecules are transmembrane proteins that are the core ofAJs. Cadherins, including E-cadherin, additionally have a highly con-served cytoplasmic tail that interacts with a defined set of cytoplasmicproteins, the catenins. Among them, β-catenin binds to the cyto-plasmic tail of cadherins, and together with α-catenin, it forms thecadherin–catenin complex [8, 11, 12]. Desmosomes are intercellularadhering junctions characterised by the presence of intermediatefilaments associated to the junctional area. They are made of trans-membrane glycoproteins that are the adhesive interface of this an-choring structure [13].

Interference at the cell-to-cell adhesion region, which can be eval-uated by the integrity of tight junctions, is one of the first indicationsof epithelium injury. Some recent studies have shown that G. duode-nalis increases the permeability of epithelial monolayers [14–18].However, a less recent article showed the opposite to be true [19].These contrasting results may be due to the use of different experi-mental conditions including (a) strains of Giardia used, (b) time ofinteraction, (c) parasite–cell ratio used, and (d) cell lines used. Otherprotozoa that interact with epithelial cells, such as Trichomonas and

rangement of tight, adherens and desmosomal junction proteins of

http://dx.doi.org/10.1016/j.parint.2011.11.002

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Entamoeba, also have the ability to increase paracellular permeabilityfollowing adhesion [20, 21].

The present study addresses the hypothesis of whether epithelialfunctional disturbance caused by G. duodenalis is associated with al-terations at the junctional complex of Caco-2 cells, a cell line derivedfrom an epithelial human colonic adenocarcinoma. This lineage waschosen because it is widely used to analyse the structure and phys-iology of the epithelium in vitro and because when in confluence,the cells are highly differentiated, and they express the junctionalproteins involved in the maintenance of epithelium integrity.

2. Materials and methods

2.1. Antibodies and reagents

Primary antibodies used include rabbit polyclonal anti-claudin-1(Zymed Laboratories, San Francisco, CA, USA); mouse monoclonalanti-ZO-1 and anti-ZO-2 (Zymed Laboratories San Francisco, CA, USA);mouse monoclonal anti-desmocollin-2/3 (Zymed Laboratories SanFrancisco, CA, USA); mouse monoclonal anti-E-cadherin clone 36(BD Biosciences – San Diego, CA, USA); and rabbit polyclonal anti-β-catenin (Sigma Chemical Co. – St. Louis, MO, USA).

Secondary antibodies used for immunofluorescence include anti-mouse and anti-rabbit Alexa Fluor 488 and 546 (Molecular Probes,Invitrogen Life Sciences, California, USA). Secondary antibodies usedfor western blotting include goat anti-mouse and anti-rabbit andconjugated to peroxidase (Sigma Chemical Co. – St. Louis, MO, USA).

MTT [3-(4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bro-mide) was purchased from Sigma (Sigma Chemical Co. – St. Louis,MO, USA), and DMSO was purchased from Merck (Merck KGaA,Darmstadt, Germany). Tetramethylrhodamine B isothiocyanate(TRITC)-conjugated phalloidin was purchased from Sigma–Aldrich(St. Louis, MO).

2.2. Parasite cultures

Trophozoites of G. duodenalis WB strain (clone C6, ATCC No.30957) were cultivated in TYI-S-33 medium (pH 7.2) that wassupplemented with 0.1% bovine bile and 10% foetal calf serum(Gibco BRL, Gaithersburg, MD) [22]. Cultures were maintained in10 ml tubes at 37 °C for 72 h. After this time, the tubes were placedat 4 °C for approximately 10 min to detach the cells. They were gentlyshaken, and a parasite's aliquot was taken to make new subcultures.Tubes containing end log phase cells were used in the experiments.

2.3. Intestinal cell cultures

Caco-2 cells were obtained from the American Type Culture Col-lection (ATCC, # HTB-37, Manassas, VA, USA) and were grown inculture flasks (TPP®, Switzerland) using high glucose Dulbecco'smodified Eagle's medium (DMEM) supplemented with 10% foetalcalf serum (Gibco BRL, Gaithersburg, MD), 0.1 g/L streptomycin,and 100 U/ml penicillin. Cultures were maintained at 37 °C in ahumidified atmosphere of 5% CO2 and passaged weekly with 0.05%trypsin/0.02% EDTA in PBS solution. The culture medium was changedevery other day to avoid nutrient depletion. All experiments wereperformed when cultures had achieved about 15 days of confluence.

2.4. Parasite/host interaction conditions

All of the interaction analyses were performed when the Caco-2cells achieved confluence and when the parasites were at the finalgrowth phase (72 h post passage). Before starting the interactionassays, the Caco-2 cells were washed once with growth medium, andnew medium replaced the old medium. Tubes containing the parasiteswere chilled at 4 °C for 10 min to detach cells and centrifuged at 1050 g

Please cite this article as: Maia-Brigagão C, et al, Giardia disrupts the arintestinal cells, Parasitol Int (2011), doi:10.1016/j.parint.2011.11.002

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for 5 min. The supernatant was discarded, and the pellet was resus-pended in supplemented DMEM medium (the same supplementsused in the Caco-2 cultures). The proportion of parasites per intestinalcells was 2:1 for all assays. During all experiments, the interactionsamples were held at 37 °C and 5% CO2. Depending on the experiment,the infection was terminated in different ways as described below.

2.5. Transepithelial electrical resistance (TER)

Caco-2 cells were grown in Transwell polyester membranes(Corning Incorporated, Corning, NY, USA). The membranes had anarea of 0.33 cm2 with pores of 0.4 μm. Upon reaching confluence,the TER was measured using a Millicell-ERS voltmeter (Millipore),and a current of 20 μA was applied to verify if the cultures had reachedcomplete confluence. An electrodewas inserted into the upper chamberto access the monolayer, and a second electrode was inserted in thelower chamber. The passage of ions through the paracellular pathwaywas quantified by measuring the difference in electrical potentialbetween the two chambers.G. duodenalis trophozoiteswere introducedinto the system at a ratio of 2:1 and allowed to interact for 24 h. Afterincubation, monolayers were washed with PBS at 4 °C to remove para-sites. Thereafter, TER was monitored until 24 h post interaction, com-paring results between control and infected samples. The interactiontests did not begin before reaching total confluence of monolayers anda minimum TER value of 300Ω/cm2 had been reached. All TER valueswere normalized for the area of the filter and these values wereobtained after the substraction of the blank Transwell filter and bathsolution that had been cultured in parallel. Data obtained from threeindependent TER measurements was expressed as percentages in rela-tion to the control group (100%). Statistical analysis was performedusing one-way analysis of variance with a post hoc Bonferroni's test.

2.6. MTT cell viability assay

Analyses of cell viability in control samples and in cells allowedto interact with the protozoa were performed using the MTT assay(MTT [3-(4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bro-mide) as previously described [23]. Briefly, cells (approximately8×104 Caco-2/well) were grown to confluence in 96-well plates(TPP®, 0.335 cm2

flat bottom) at 37 °C in an atmosphere of 5%CO2. Parasites (1.6×105/well) were seeded into the medium, andinteraction was performed for 24 h. This quantity of parasites isnot sufficient to be detected by the MTT method, so our readingswere restricted to Caco-2 monolayers [24]. After this interval, MTTwas added in an amount equal to 10% of the culture medium volumeand the incubation with the reagent lasted for 2 h. Next, dimethylsulfoxide (DMSO) was used to dissolve the formazan crystals, andthe resulting purple solutions were spectrophotometrically mea-sured at 570 nm in a SpectraMax 190 Gemini XS spectrophotometer(Molecular Devices, Sunnyvale, CA, USA). Data were analysed usingSoftMax PRO 4.3 LS software. Statistical analysis was completedusing a one-way ANOVA, considering Pb0.05 as significant.

2.7. Immunofluorescence microscopy

For this analysis, Caco-2 cells were grown on glass coverslipswithin 24-well plates and maintained until complete confluence, atwhich point trophozoites were added. After the interaction period,monolayers were washed with cold PBS at 4 °C to remove theadhered parasites. To identify TJ proteins, samples were fixed with80% methanol and 20% acetone for 20 min at −20°C. Next, cellswere incubated in blockage solution (3% BSA in PBS, pH 8.0) for1 h. Subsequently, samples were incubated overnight at 4°C withprimary antibodies against TJ proteins (dilution 1:20 for ZO-1 andclaudin-1), washed 3 times with blockage solution and followed by1 h with the respective secondary antibodies (dilution 1:500). The



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Fig. 1. TER of Caco-2 cells decreased over time during interaction with G. duodenalis,reaching a reduction of approximately 42%.

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coverslips were washed and mounted using n-propyl-gallate. Toidentify AJ proteins (1:250, E-cadherin; 1:400, β-catenin) anddesmocollin-2/3 (1:20), samples were fixed with 4% formaldehydein PBS (pH 7.1) for 10 min at room temperature, permeabilisedwith 0.1% Triton X-100 at room temperature for 10 min, and blockedwith 50 mM ammonium chloride for 10 min at room temperature.Monolayers were then blocked again in a 3% BSA solution and pro-cessed as described above for TJ proteins. All samples were analysedunder a Leica TCS SP5 laser scanning confocal microscope with anargon laser (Ar 488). 3D images were reconstructed from confocalsections using LAS 2.2.0 software. In some of the reconstructions acolour coded resource from the software was used to facilitate theunderstanding of 3D reconstructions.

2.8. F-actin detection

For visualisation of F-actin distribution, cell monolayers werefixed and permeabilised as described above and incubated with500 ng/ml TRITC-phalloidin for 30 min at room temperature. Afterwashing, stained monolayers were analysed using a Leica TCS SP5laser scanning confocal microscope with a 543-nm excitation laser.Individual images through the cell volume of similar confluent re-gions were collected and optical sections near the apical, medium(5 μm) and basal (9 μm) planes from monolayers (x–y plane) wereobtained. Images shown are representative of at least three indepen-dent experiments.

2.9. High resolution (field emission – FESEM) and conventional scanningelectron microscopies

After parasite–cell interactions, as described above, the sampleswere fixed in Karnovsky solution (2.5% glutaraldehyde, 4% formalde-hyde and 5 mM calcium chloride in 0.1 M sodium cacodylate buffer,pH 7.2), washed twice in cacodylate buffer for 10 min and post-fixed for 1 h in 1% OsO4, 0.8% potassium ferrocyanide and 2.5 mMCaCl2 in 0.1 M cacodylate buffer. Samples were washed twice in 0.1 Mcacodylate buffer for 10 min, dehydrated in increasing concentrationsof ethanol (50%, 70%, 90% and 3 times 100%) and critical point-driedwith CO2. The samples were covered with a thin layer of gold andobserved using a Jeol 6340 field emission scanning electronmicroscope(FESEM).

2.10. Transmission Electron Microscopy (TEM)

Control cell monolayers, as well as those allowed to interact withprotozoa, were fixed in a solution containing 0.6% ruthenium red,2.5% glutaraldehyde, 1% formaldehyde and 2 mM CaCl2 in 0.1 Msodium cacodylate buffer (pH 7.2) for 1 h under stirring in thedark. Subsequently, the samples were washed in 0.1 M cacodylatebuffer containing ruthenium red dye 0.6% and incubated in a solu-tion of 2% osmium tetroxide, also with ruthenium at the sameconcentration for 45 min, stirring in the dark. Next, the sampleswere dehydrated in increasing concentrations of acetone andembedded in Epon resin. Ultrathin sections were obtained and con-trasted with lead citrate only, for 2 min; they were then analysedusing a Zeiss CEM-900 and Jeol 1200 EX transmission electronmicroscopes.

2.11. Western blotting and densitometric analysis

Following the interaction experiments, samples were washed withPBS and homogenised in lysis buffer that was composed of 1% TritonX-100, 0.5% sodium deoxycholate, 0.2% SDS, 150 mM sodium chloride,2 mM EDTA, 10 mM Hepes (pH 7.4), 20 mM sodium fluoride, 1 mMsodium orthovanadate and a cocktail of protease inhibitors (1 mMPMSF, 1 mg/ml aprotinin, 2.5 mM phenanthroline, 10 mM Leupeptin


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and 5 mg/mL pepstatin – Sigma Chemical Co.) at 4 °C. After centrifuga-tion at 10,000 g for 10 min, the supernatant was collected and stored at−20 °C. To analyse the expression of proteins, 30 μg of the respectiveproteins were separated by SDS-PAGE using gels of 6–12% and furthertransferred to nitrocellulose sheets using a semidry transfer cell(BioRad, Hercules, CA, USA) at 10 V for 60 min. The membranes wereblocked for 10 min in a solution of 10% non-fat dried milk in TBS-T(20 mM Tris, pH 7.6, 137 mm NaCl, 0.1% Tween-20). After, they wereincubated overnight at 4 °C with antibodies recognising: (a) the tightjunction proteins claudin-1 and ZO-1 (both 1:20), (b) the adherensjunction protein E-cadherin (1:2,500) and β-catenin (1:4,000), (c) thedesmosomal protein desmocollin-2/3 (1:20), and (d) the housekeepingprotein α-tubulin (diluted 1:200). After five washes with TBS-T, themembraneswere incubatedwith secondarymouse IgG or rabbit IgG an-tibodies conjugated to peroxidase, diluted 1:30,000 in TBS-T containing5% non-fat dried milk for 60 min. Finally, the membranes were washed3 times in TBS-T and the reactivity to target proteins was determinedusing a commercial chemiluminescence kit (ECL, Amersham Biosci-ences GE Healthcare, Buckinghamshire, UK). Band images were quanti-fied by optical density using LabWorks 4.6 software (Bio-Rad, Hercules,CA). Densitometric analysis is presented as the means+/−S.D. Com-parison between control and interaction samples in three independentexperiments was performed using GraphPad Prism 4.02 software(GraphPad Software Inc., San Diego, CA, USA). Results were consideredstatistically significantwhen the P valuewas b0.05 by One-way ANOVAanalysis.

ED3. Results

3.1. Giardia duodenalis attachment to the epithelial cell surface alters theparacellular permeability but not the cell viability

Transepithelial electrical resistance (TER) of polarised cell mono-layers is considered to be an excellent indicator of tight junctionintegrity. We used this approach to analyse the potential interferenceof G. duodenalis attachment to the epithelial cell surface on the TER ofconfluent Caco-2 cells. As shown in Fig. 1, TER significantly decreasedfollowing the interaction. This effect was observed even after 2 h ofinteraction, when a reduction of approximately 20% was observed,reaching a decrease of approximately 40% after 24 h (Pb0.001). Weadditionally used ruthenium red dye and transmission electron mi-croscopy analysis to monitor the paracellular permeability of epithe-lial cells after parasite interaction. Fig. 2A illustrates that in controlcells a light electron-dense material can be seen covering the micro-villi, but not the lateral surface between neighbouring cells. Afterinteraction with the parasites, the plasma membrane of the epithelialcell surfaces was labelled, and a thin permeation of the dye throughthe junctional region was observed (Fig. 2B). Despite a marked



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Fig. 2. In control cells (A), ruthenium red permeation is blocked (arrow) at the TJ level. After interaction (B), the dye flowed through paracellular space, and the microvilli werereduced. M=Microvilli, G=Giardia, arrow=cell–cell contacts. Bars: 1 μm.


alteration of the microvilli, as they were considerably shorter or evenabsent, we could not see a drastic disruption of cell–cell contacts.

We also examined the viability of Caco-2 cells following incuba-tion in the presence of the protozoa. In these experiments we usedthe MTT formazan technique, which has been proven to be an excel-lent method to assay cell viability. As shown in Fig. 3, the resultsafter 24 h of interaction illustrate that the cell viability of controlcells and those allowed to interact with G. duodenalis were verysimilar. The culture medium used in these assays (DMEM) did notalter the colorimetric analysis (data not shown).

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3.2. Expression and spatial reorganisation of junctional proteins after theinteraction process

Another approach to verify the integrity of the epithelial cell junc-tional complex is to analyse the expression and distribution of themajor proteins involved in the maintenance of the junctions that areestablished between the cells. Immunoblotting assays using antibodiesrecognising claudin-1, ZO-1, β-catenin and E-cadherin showed nosignificant differences of expression among these proteins betweencontrol cells and those that had interacted with the protozoa for 2or 24 h (Fig. 4). Although we tested various protocols for immuno-blotting, we could not identify the desmocollin-2/3 protein. This isprobably because the protein is at a low concentration in thesecells. However, by using immunofluorescence microscopy to ana-lyse the distribution of these proteins, we observed that in controlcells the tight junction proteins (ZO-1 and claudin-1), adherensjunction proteins (E-cadherin and β-catenin) and desmosomal

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Fig. 3. MTT assays showing the viability of the control and Caco-2 interaction sam-ples. In addition, the culture medium used in the assays did not alter the MTT color-imetric reaction. Statistical analysis showed no significant differences among thedata obtained.


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the cell–cell contact regions (Figs. 5, 6, 7 and supplementaryvideos). However, after parasite adhesion, both TJ proteins wereinternalised into host cells (Fig. 5, and supplementary videos).This change in protein distribution could be seen more clearlyusing a three-dimensional reconstruction via confocal laser scan-ning microscopy (Fig. 5). Images obtained from cells labelled forthe ZO-1 protein showed that there was a branch-like distributionin the presence of the parasites (Fig. 5). Unlike the TJ proteins, AJproteins remained at the cell–cell contacts even after the interactionwith Giardia (Fig. 6). Nevertheless, the distribution of E-cadherin wasaltered, with an extra punctual pattern observed (Fig. 6), while β-catenin showed a more uniform distribution than what is typically ob-served (Fig. 6). The same pattern of change observed for β-cateninalso occurred during analysis of the desmosomal protein desmocollin-2/3, as clearly shown when the 3D reconstruction was completed(Fig. 7 and supplementary video).

3.3. Giardia modulates actin filaments in host cells

Due to the fact that in epithelial cells the apical perijunctional F-actin belt affiliates with the apical junction complex (AJC) and con-trols assembly and barrier properties of this complex, we investigated

Fig. 4. Western blots of junctional proteins. Column numbers refer to samples: 1,Caco-2 after 2 h of experiment; 2, interaction 2 h; 3, Caco-2 after 24 h of experiment;4, interaction 24 h. No differences in protein regulation could be noticed, both in TJ(claudin-1 and ZO-1, 22 kDa and 225 kDa, respectively) and in AJ proteins (β-cateninand E-cadherin, 94 kDa and 12 kDa, respectively). Statistical analysis, one-wayANOVA, Pb0.05, showed no significant differences among data obtained. Beta tubulinwas used as the protein loading control.



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383Fig. 5. The distribution of tight junction proteins was affected after 24 h of interactionwith the parasites. ZO-1 changed to a branch-like pattern, and claudin-1 was interna-lised. This may be seen more clearly in the colour coded reconstructions of total slicesof ZO-1 labelling, and with a rotation angle of 7°, which correspond to control and in-teraction images, respectively. Bars: 10 μm.

Fig. 6. Adherens junction protein labelling in control and infected monolayers. The dis-tribution pattern of proteins was changed after the contact with parasites. Bars: 25 μm.

Fig. 7. Desmocollin-2/3 protein is also rearranged after G. duodenalis adhesion, betterviewed in 3D reconstruction (C: control; D: interaction; Rotation angle of 7° for a bettervisualisation). Bars: 25 μm.


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Rthe role of the actin cytoskeleton in the AJC disassembly after interac-tion with parasites. We evaluated the disposition of actin microfila-ments in Caco-2 cells using confocal laser scanning microscopy ofcells labelled with rhodamine-phalloidin. Fig. 8 shows the arrange-ment of actin filaments in different regions of the epithelial cell. Inthe apical section of normal monolayers a punctuated distributionof actin labelling can be observed due to its presence in microvilli aswell as in the cell–cell contact regions (Fig. 8). At the medial level,labelling for actin predominated at cell junctions (Fig. 8), while atthe basal side, stress fibres were observed (Fig. 8). Interaction withGiardia caused changes in actin cytoskeleton organisation in bothapical (Fig. 8) and basal (Fig. 8) regions of Caco-2 cells. In the firstcase, the actin distribution loses the punctual pattern and becomesconcentrated in areas near cellular contacts, thus indicating a disorga-nisation of microvilli (Fig. 8). In the basal portion, a reduced labellingof stress fibres was observed (Fig. 8). Furthermore, in the medialregion the punctuated pattern slightly decreased, while the appear-ance of a more continuous labelling pattern was observed (Fig. 8).

3.4. Ultrastructural changes observed during the cell interaction model

Scanning electron microscopy was used to analyse the surfaceof the epithelial cells and the pattern of adhesion of G. duodenalistrophozoites to the epithelial cells. Fig. 9A shows the appearance


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of control cells and their general shape and the distribution of themicrovilli can be seen. After interaction, contacts between cellswere disrupted (Fig. 9B). In addition, there was a clear retractionof the microvilli surrounding the areas where the parasites wereattached (Fig. 9B, C). Instead of typical microvilli, a large numberof globous structures were observed (Fig. 9C).

4. Discussion

Most parasitic protozoa living in the gastrointestinal tract ofmammals do not penetrate into the epithelial cells but rather exert



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Fig. 8. Confocal slices of actin staining, showing its affected pattern at apical, middleand basal regions before and after Giardia infection. Bars: 10 μm.

Fig. 9. Changes on the surface of Caco-2 monolayers caused by the interaction with G.duodenalis under SEM. A: Control monolayer; B: Breakdown of cell contacts caused bythe parasite (arrows); C: Retraction of microvilli at parasite adhesion site (arrow).Bars: 2 μm.


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their parasitic action through adhesion to the cell surface. This is thecase of G. duodenalis, where the trophozoites adhere intensely to themicrovilli of the intestinal cells [1]. The current article presents newobservations regarding the interaction between Giardia andintestinal cells, focusing on the cell surface as well as on tight andadherens junctions and how this interaction interferes with intercel-lular epithelial permeability. Furthermore, we also clearly show thatthis parasite is able to disturb the epithelium at the desmosomeslevel.

Our present observations also show the existence of dramaticchanges in the organisation of the intestinal cell surface due to theinteraction of the protozoan with the microvilli. There was a signifi-cant retraction of the microvilli leading to the observation of only afew short projections. This change may also explain the new patternof the actin filament distribution seen in cells interacting with theprotozoan, as will be discussed below. Because the microvilli playan important role in the absorption of nutrients, their disorganisationmay explain some of the physiological changes that take place inanimals and humans infected with G. duodenalis [26].

In the present study, we focused on the junctional properties ofthe epithelium during interaction with the protozoan. In vitro studiesusing epithelial cell monolayers have shown that alterations of theexpression or localisation of claudin-1 [3, 27–31] dramatically in-crease the paracellular permeability concomitantly with a decrease ofthe transepithelial resistance (TER), a fundamental property relatedwith the functional integrity of the epithelium and the transport ofsubstances through the intercellular space. In our model, we found


that TER of the intestinal epithelium significantly decreased after inter-action with trophozoites, an observation that is in agreement withprevious results from other research groups [14–18]. Disturbances atthe cell–cell contacts were also observed by transmission electronmicroscopy with the use of the ruthenium red marker. It is knownthat claudin C terminus appears to influence protein stability, alteringprotein turnover and consequently the paracellular permeability [33].Also, the C terminus portion of the protein still binds cytoplasmic TJproteins, such as zonula occludens (ZO proteins) [4, 34, 35]. In theinteraction assays we observed, by immunofluorescence microsco-py, a rearrangement of the ZO-1 protein. Together, these observa-tions can explain the increased epithelium permeability as well asthe variation of the TER in our experiments.

It has been demonstrated that claudin-1 is downregulated duringchronic giardiasis [14]. Nevertheless, our results using epithelialmonolayers of Caco-2 cells showed that there is no change in expressionof any junctional proteins, but only a reallocation of them. Another study



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[32] showed that a large number of genes displayed changed expressionpatterns during interactions between Giardia and Caco-2 cells, but theauthors did not find any differences around the junctional proteins. Wepropose that the adhesion of trophozoites on the surface of epithelialcells might be enough to destabilise the cell junctions, leading to effectsevidenced in the present work.

As mentioned above, we observed a clear rearrangement of sev-eral junctional proteins, but we did not observe a decrease in theirexpression. The distribution pattern seen in our study for someproteins differs from those published previously [15, 17, 36]. In thecase of the tight junction protein ZO-1, we saw that its redistribu-tion led to the appearance of a branch-like form that had a tendencyto migrate into the cytoplasm. Buret and colleagues have alreadyreported that G. duodenalis acts at the level of ZO-1 with focaldisruptions, punctuates concentrations of this protein along thepericellular junctions and promotes cytoplasmic accumulation [15].The same group later described that ZO-1 migrates from the cellularmargins into the cytoplasm and towards the cell nucleus after incuba-tion with Giardia sonicates [17]. Spatial rearrangement of ZO-1 wasalso demonstrated during infection with other intestinal parasites[36], including the anaerobic T. vaginalis [20].

We additionally observed changes in the distribution pattern ofE-cadherin and desmocollin, proteins that are part of the adherensjunction and desmosomes, respectively. These changes becamemore evident when a 3-dimensional reconstruction of labelled cellswas completed using confocal laser scanning microscopy. In polarisedepithelial cells, the TJs and AJs are physically linked to apical F-actinfilaments that are organised in a perijunctional belt-like structure[37]. The perijunctional actin belt is critical for the formation of theapical junctional complex (AJC) [38–40] and for the regulation of para-cellular permeability [41, 42]. In our study, we described that after cellinteraction with the parasite the actin filaments in the apical region ofCaco-2 only surrounded the cells, with concomitant loss of microvillistaining for actin. We suggest that actin retraction provides mechanicalforces that disassemble the AJC (tight and adherens junctions), but fur-ther studies are necessary to support this hypothesis. A mode of actionsimilar to that proposed here has been previously suggested based onexperiments in which calcium was depleted from the culture medium[27, 43, 44].

In conclusion, our present observations show the disturbancecaused by the parasite Giardia duodenalis in intestinal cells goesbeyond the tight junction, as it also reaches the adherens and desmo-somal junctions. We also show that Giardia trophozoites do notpromote downregulation of junctional proteins, but only rearrangethem, which would be enough to cause any intestinal imbalanceobserved in vivo. The adhesion of the parasites to the host cells maybe activating or deactivating a signalling cascade, leading to thedestabilisation of the junctional proteins with a loss of their func-tionality. These effects may be due to the release of toxins by theparasite or by the movements of the trophozoites during adhesionon epithelial cells. This destabilisation applies both to the apicaljunctions (tight and adherens) as well as the desmosomal junctionproteins. However, further studies will be necessary to supportthese hypotheses.

Supplementary materials related to this article can be found onlineat doi:10.1016/j.parint.2011.11.002

5. Uncited reference

[25].

Acknowledgements

We thank the Structural Biology group (INCa) for the discussionsand technical support. We are also grateful to other members fromour laboratory and to the Laboratório de Biotecnologia (INMETRO)


for the technical assistance and helpful discussions. The manuscriptwas revised by the American Journal Experts and Mr. David Straker.This work was supported by the CAPES, CNPq and FAPERJ fundingagencies.

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