Ethics statement and chemicals

The cell lines used in this study were all obtained from American Type Culture Collection (ATCC): Giardia intestinalis isolates WB clone C6 (ATCC30957; assemblage A) and GS, clone H7 (ATCC50581; assemblage B) were used in this study in combination with the human colonic adenocarcinoma cell line, Caco-2 clone TC7 (ATCC HTB-37). All the experiments were approved by the Programme Review Board at Department of Cell and Molecular Biology, Uppsala University, Sweden. All chemicals were purchased from Sigma-Aldrich (MO, USA), unless otherwise stated.

Parasites and intestinal epithelial cells (IECs)

Giardia trophozoites were cultured in 10 ml and 50 ml tubes filled with TYDK medium (37°C) supplemented with 10% heat inactivated bovine serum (Sigma-Aldrich) as described in [27]. The Caco-2 clone TC7 (passage numbers 7–12) cell line was grown and differentiated as previously described [16].

Medium for collection of excretory/secretory products (ESPs) and trophozoite viability

To collect Giardia ESPs from axenic cultures we used a modified RPMI-1640 medium containing 55.5 mM glucose, 22.8 mM L-arginine, 11.4 mM L-cysteine hydrochloride monohydrate, 11.4 mM ascorbic acid, 2mM Glutamax (Gibco), 1 mM sodium pyruvate (Gibco) and 1x MEM essential amino acids. The medium was filter-sterilized and placed in a water bath (final pH is 6.85 at 37°C) until used.

Confluent Giardia culture tubes, prepared as described above, were used for trophozoite viability assessment. Briefly, the TYDK medium was decanted from each tube followed by three washes in Hank’s Balanced Salt Solution with glucose (HBSS-G) supplemented with 11.4 mM L-cysteine (pH 6.8, 37°C). Culture tubes were replenished with the modified medium, closed tightly and incubated for 2h or 6h (37°C). At the end of each incubation, tubes were placed on ice to detach trophozoites (10 min), followed by counting using the expulsion of Trypan Blue and flagellar motility as viability criteria.

Collection and processing of ESPs from axenic cultures and host parasite interactions

Tubes with Giardia trophozoites in modified RPMI-1640 medium (50 ml, two tubes of each isolate) were used to collect Giardia ESPs in axenic cultures. The tubes and media, following counting and viability assessment, were centrifuged (930 xg, 10 min, 4°C) to pellet trophozoites. Collected media were filter-sterilized (0.22 μM) and treated with protease inhibitor cocktail tablets (cOmplete, Mini EDTA-free, Roche, Sigma-Aldrich). Media were concentrated down to 200–300 μl using the Amicon Ultra 15 mL centrifugal filters with 3kDa cutoff (Merck-Millipore, Darmstadt, Germany). To further eliminate any traces of residual serum, concentrates were processed through the AlbuminOUT kit (G-Biosciences, MO, USA) according to the manufacturer’s instructions. Three biological replicates (2h and 6h) were prepared and stored at -80°C until used for MS analysis. The whole experimental setup is described in Fig 1A.

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Fig 1. Experimental setup for proteomic analyses and characterization of Giardia ESPs.

(A) The collection of Giardia intestinalis, isolates WB and GS, excretory-secretory products (ESPs) in a serum-free medium from axenic culture and co-culture with differentiated intestinal epithelial cells (IECs), Caco-2, for 2h and 6h. Samples were processed as described in Materials and Methods followed by liquid chromatography/mass spectrometry to identify secreted proteins. (B) Studies of IECs exposed to G. intestinalis ESPs using differential gene expression analyses by RNA sequencing, quantitative real time PCR, Western blots, ELISA and confocal microscopy. The panel illustrates the experimental setup (left to right) showing IECs exposed to ESPs of either Giardia isolates through a Transwell insert (direct exposure) or different amounts of ESPs collected from axenic culture (i.e. the absence of IECs, 1 μg, 5 μg or 10 μg per ml of medium). Abbreviation: Dulbecco’s Modified Eagles Medium (DMEM).

https://doi.org/10.1371/journal.pntd.0006120.g001

Differentiated Caco-2 cells (75 cm² flasks) were washed with warm PBS (3x) and incubated with 6 x 10⁷ trophozoites resuspended in 25 ml of DMEM (no FBS) for 2h or 6h. Trophozoites were washed (3x, HBSS-G plus 11.4 mM L-cysteine) prior addition to cells. Caco-2 cells treated as above and incubated alone in DMEM (2h and 6h) served as controls (Fig 1A). All incubations were performed in 10% CO₂ humidified tissue culture incubator at 37°C. At the end of each interaction, flasks were placed on ice (10 min) to detach trophozoites for counting and viability assessment. Media were then collected, processed as described earlier and stored at -80°C until used for MS analysis. Three biological replicates were prepared (2h or 6h including controls).

Isolation of Giardia extracellular vesicles (EVs) and electron microscopy

The ESP enrichment experiment for axenic cultures described earlier was repeated to collect Giardia EVs. Samples were processed with the ExoQuick TC Kit (SBI System Biosciences, CA, USA) according to the manufacturer’s instructions. Giardia EVs were pelleted by centrifugation (1500 xg, 30 min) and resuspended in 100 μl PBS buffer and processed immediately for electron microscopy (EM). Samples (5μl) were adsorbed onto a Formvar-coated 200 mesh grid (15 seconds), followed by treatment with 2% molybdenum (10 seconds) and contrasting using 2% uranyl acetate (10 seconds). Grids were dried and visualized using a transmission electron microscope at the BioVis Core Facility, Uppsala University, Uppsala, Sweden. A negative control (media alone) was included and processed similarly.

Liquid chromatography and mass spectrometry (LC/MS) analysis

Before digestion, the buffer in the collected samples was exchanged to a urea-containing lysis buffer (20 mM HEPES, 9 M urea, Complete Mini EDTA-free protease inhibitor cocktail (Roche) using 3 kDa molecular weight cut off filters (Thermo Fisher Scientific, MA, USA). Proteins were then subjected to dithiothreitol (DTT, 12.5 mM) reduction and iodoacetamide (IAA, 6.9 mM) alkylation before digested in-solution with trypsin (1 μg added per sample). The samples were desalted using Pierce C₁₈ Spin Columns (Thermo Fisher Scientific). Eluates were dried and resolved in 15 μL 0.1% formic acid of which 5 μL were injected. The nanoLC-MS/MS experiments were performed using a Q Exactive Orbitrap mass spectrometer (ThermoFisher Scientific, Bremen, Germany) equipped with a nano-electrospray ion source. The peptides were separated by C₁₈ reversed phase liquid chromatography using an EASY-nLC 1000 system (Thermo Fisher Scientific). A set-up of pre-column and analytical column was used. The pre-column was a 2 cm EASYcolumn (ID 100 μm, 5 μm particles) (Thermo Fisher Scientific) while the analytical column was a 10 cm EASY-column (ID 75 μm, 3 μm particles, Thermo Fisher Scientific). Peptides were eluted with a 150-min linear gradient from 4% to 100% acetonitrile at 250 nL min-1. The mass spectrometer was operated in positive ion mode acquiring a survey mass spectrum with resolving power 70,000 (full width half maximum), m/z 400–1750 using an automatic gain control (AGC) target of 3×10⁶. The 10 most intense ions were selected for higher-energy collisional dissociation (HCD) fragmentation (25% normalized collision energy) and MS/MS spectra were generated with an AGC target of 5×10⁵ at a resolution of 17,500. Mass spectrometer worked in data-dependent mode.

Database searches were performed using the Sequest algorithm, embedded in Proteome Discoverer 1.4 (Thermo Fisher Scientific). Proteins in axenic cultures were identified by performing separate searches against the fasta files of the Giardia proteome for each isolate (GS clone containing 4470 entries and WB clone 9667 entries) (available at www.giardiadb.org; giardiadb.org/common/downloads/release5.0/). The fasta file for human proteins was downloaded from Uniprot 2015–05 and contained 20192 entries. Proteins in co-cultures were searched against a combined database of Giardia and human proteins in a MudPIT search combining all three replicates. For both searches, the following parameters were used for data processing: Maximum 10 ppm and 0.02 Da error tolerances for the survey scan and MS/MS analysis, respectively, trypsin as digesting enzyme, carbamidomethylation of cysteins as fixed modifications, oxidation of methionine and deamidation of asparagine and glutamine as variable modifications, maximum of two miss cleavages sites. The target decoy PSM validator was used to calculate false discovery rate. The search criteria for protein identification were set to at least two matching peptides of 95% confidence level per protein.

Identified proteins were subject to gene ontology (GO) enrichment analysis to identify their biological as well as proteins class. The analysis was performed using the PANTHER algorithms provided within the Gene Ontology Consortium website (http://geneontology.org/page/go-enrichment-analysis). The results were processed through the GO slim option for biological GO terms, with Bonferroni correction applied for multiple testing (P < 0.05). In addition, the STRING Database (http://string-db.org/) was also used to search for InterPro domains, applying Bonferroni correction at P < 0.05 as above. Identified proteins were also analyzed for the presence of secretion signal peptides (SSPs) using the SignalP program embedded within the GiardiaDB (http://giardiadb.org/giardiadb/queries_tools.jsp). The search criteria were set to include any of the following; a SignalP-NN conclusion score of 3, SignalP-NN D-score of 0.5 and SignalP-HMM signal probability of 0.5.

Differential gene transcription of differentiated Caco-2 cells in response to Giardia ESPs

We used RNA sequencing (RNA Seq) or quantitative PCR (qPCR) to identify differential gene expression on the RNA level in IECs (i.e. differentiated Caco-2 cells) exposed to Giardia ESPs in two different experimental setups (Fig 1B). First, IECs (6-well plate) were exposed to parasite ESPs via a Transwell insert (0.4 μm pore polyester membrane, Corning, Sigma Aldrich). Here, IECs were physically separated from parasite but directly exposed to their ESPs. For this experiment, we used RNA Seq to identify global changes in gene expression. Second, IECs were exposed to 1, 5 or 10 μg/ml of ESPs from axenic cultures. This aimed at assessing changes in pro-inflammatory gene expression in IECs exposed to ESPs released in axenic culture (i.e. no IECs) and assess how IECs respond to different amounts of Giardia ESPs. Changes of gene expression were studied using qPCR. An illustration of the experiments above is depicted in Fig 1B.

The experiments below were performed using complete DMEM with 10% HI-FBS and all incubations were performed in a 10% CO₂ humidified incubator at 37°C. Before the beginning of all experiments, tissue culture flasks or well-plates were washed twice with warm PBS and incubated with fresh media for 2h. Trophozoite cultures (50 ml) were washed once with warm PBS then cold PBS, incubated on ice (10 min), counted and pelleted by centrifugation (930 xg, 10 min, 4°C). Media collected in the insert experiment were centrifuged (930 xg, 10 min, 4°C), filter-sterilized (0.45 μM) and stored at -20°C to be used later for cytokines measurement. Three biological replicates were analyzed with RNA Seq for the insert experiment, whereas four biological replicates were used for qPCR in the other experiment. Each biological replicate had a control (IECs alone in media) and IECs exposed to ESPs of either WB or GS isolate. For RNA collection, IECs were lyzed directly in the tissue culture flasks or well-plates using Trizol reagent (Ambion, Thermo Fisher Scientific) and stored at -80°C until processed later.

In the insert experiment, IECs were washed and replenished with 4 ml of fresh DMEM added directly into the well. Trophozoites were processed as stated above and resuspended in DMEM, from which 2 ml (1 x 10⁷ trophozoites) were added into the Transwell inserts and incubated for 2h or 6h. Media were collected at the end of each time point. In the second experiment, IECs (25 cm² flasks) were washed and replenished with 10 ml of fresh DMEM. The amounts of protein in ESPs from either the WB and GS isolates were quantified using the Qubit protein kit (Thermo Fisher Scientific) and added to flasks at a concentration of 1, 5 or 10 μg/ml followed by incubation for 2 h or 6h.

Enzyme linked immunosorbent assay (ELISA) for cytokines in collected media

Media collected from Caco-2-trophozoite interactions were analyzed by ELISA to measure the concentration of cytokines released by IECs in response to parasite ESPs. The Human CXCL8/IL-8 Quantikine ELISA Kit, Human CCL20/MIP-3 alpha Quantikine ELISA Kit and Human CXCL1/GRO alpha Quantikine ELISA Kit were purchased from R&D Signalling and the samples were processed for cytokine measurements following the manufacturer’s recommendations. The results were plotted and analyzed using a four Parameter Logistic ELISA curve fitting provided at http://www.elisaanalysis.com.

RNA extraction, library preparation and RNA sequencing

Samples collected as described earlier were processed using the PureLink RNA Mini Kit (Ambion, Thermo Fisher scientific) according to the manufacturer’s instruction. During RNA extraction, a DNaseI treatment step (PureLink DNase Set, Ambion, Thermo Fisher Scientific) was performed on the spin columns to remove genomic DNA. The quality of extracted RNA was assessed by checking the 260/280 and 260/230 ratios using a NanoDrop 1000 Spectrometer (Thermo Fisher Scientific) and by running samples (500 ng) on a 1.5% Tris-Borate-EDTA (TBE) agarose gel prepared with 20 mM of guanidium isothiocyanate (GITC). For cDNA library preparation and RNA Sequencing, RNA (50 ng each) from each sample was reverse transcribed according to the instructions provided in the Ion AmpliSeq Transcriptome Human Gene Expression kit (Revision A.0, Thermo Fisher Scientific) and the generated cDNA was amplified using the Ion AmpliSeq Transcriptome Human Gene Expression core panel (Thermo Fisher Scientific). Primer sequences were partially digested followed by adaptors ligation (Ion P1 Adapter and Ion Xpress Barcode Adapter, Thermo Fisher Scientific) and purification using the Agencourt AMPure XP reagent (Beckman Coulter Inc., CA, USA). Adaptor-ligated amplicons were eluted into the amplification mix (Platinum PCR SuperMix High Fidelity and Library Amplification Primer Mix, Thermo Fisher Scientific) and amplified. Amplicons were subject to size-selection and purification using Agencourt AMPure XP reagent (Beckman Coulter) and were quantified using the Fragment Analyzer instrument (Advanced Analytical Technologies Inc., Ankeny, IA, USA) with DNF-474 High Sensitivity NGS Fragment Analysis Kit (Advanced Analytical Technologies Inc.). Samples were then pooled, followed by emulsion PCR on the Ion OneTouch 2 system with Ion PI Template OT2 200 Kit v3 (Thermo Fisher Scientific) and enrichment using the Ion OneTouch ES (Thermo Fisher Scientific). Samples were loaded on an Ion PI chip Kit v3 and sequenced in the Ion Proton System using the Ion PI^™ Sequencing 200 Kit v3 (Thermo Fisher Scientific).

Bioinformatic analysis of RNA sequencing data

Acquired reads from RNA Seq were analyzed using the hg19 AmpliSeq RNA plugin in the Torrent Suite Server with default settings. This analysis tool produces tables with raw read counts as well as normalized expression values for all genes in the Ion AmpliSeq Human Transcriptome panel. For each experiment, the RPKM values were normalized across samples using the moose2 software (https://github.com/grabherr/moose2). Differential expression was estimated based on Laplace/Normal distributions calculated from replicate samples, computing individual significance values as with r_i and r_j being the RPKM values, for up-and down-regulation respectively for all given pairwise comparisons i and j, applying a correction term c based on a normal distribution of read counts. The expectations and for comparing all replicates from condition k with l testing for up- or down-regulation in time point k were estimated as for all and n being the number of values, and Bonferroni-corrected to account for multiple testing. For reporting significance, a threshold of E < 0.00001 was applied.

Genes with significant change in the level of transcription were also subject to GO Terms analysis (http://geneontology.org/page/go-enrichment-analysis) to identify biological or molecular functions as well as protein class. We used the GO slim option with Bonferroni correction applied for multiple testing (P < 0.05).

Quantitative polymerase chain reaction (qPCR)

For qPCR experiments, RNA was extracted as above and cDNA was synthesized from 1μg RNA using the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. qPCR was performed on the genes, il-8, cxcl1-3, ccl2 and ccl20 with gapdh used as an endogenous control. The Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific) was used in the qPCR reactions with 250 nM of each primer and 10 ng template. Cycling conditions included an initial polymerase heat activation step (10 min, 95°C), and 40 cycles of 15 sec denaturation (95°C), 30 sec annealing (60°C) and 30 sec extension (72°C). The change in gene transcription was calculated using the ΔΔCt method.

Activator protein 1 (AP-1) reporter plasmid luciferase assay

AP-1 is a transcription factor involved in inducing the transcription of inflammatory genes [28,29]. It is activated by MAPKs and could work in conjunction with NFκB to activate gene transcription [28,29]. To detect AP-1 activation in response to Giardia ESPs, Caco-2 cells were transfected with a plasmid pGL4.44[luc2P/AP1 RE/Hygro] expressing the luciferase reporter gene luc2P (Photinus pyralis) under the control of a promoter that has the transcription response element of AP-1. The pGL4.75 Vector (encoding Renilla luciferase) was used as a normalisation control and was co-transfected into Caco-2 cells with the pGL4.44 plasmid. Both plasmids were purchased from Promega (WI, USA). Transfections were performed in a 24-wells plate format according to the supplier’s protocol, using 1.1μg of plasmid DNA/10⁵ cells and the FuGENE HD reagent (Promega). In the beginning of experiment, both parasite and Caco-2 cells were washed and processed as described earlier and the wells were replenished with 500 μl DMEM (no phenol red as it interferes with luminescence reading). Trophozoites were re-suspended in DMEM (no phenol red) and 10⁶ trophozoites were added into the Transwell insert in a total volume of 100 μl and incubated for 2h or 6h (37°C, 10% CO₂). Three wells were used for each time point and the controls. At the end of each incubation, the inserts were removed and the cells were washed (3x) with 200 μl PBS at RT, and lyzed with an equal amount of the Dual-Glo Luciferase Assay reagent. A 100 μl was dispensed into the well of 96-well plate in triplicates and luminescence was read from both plasmids following the instructions of Dual-Glo Luciferase Assay reagent and using an Infinite M200 Pro Tecan machine (Tecan Group Ltd., Männedorf, Switzerland). Cells with no plasmid (background fluorescence), cells with plasmid alone (negative control), and cells incubated with Phorbol 12-myristate 13-acetate (PMA) (positive control) served as controls. Three biological replicates were performed in this experiment.

Western blot analyses

To correlate RNA Seq results with those at protein level, we studied the effects of Giardia ESPs on differentiated Caco-2 cells using Western blotting. We tested for the activation/inhibition of mitogen activated protein kinases (MAPKs) and the nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), both of which are involved in inflammatory gene transcription [30,31]. Briefly, the insert experiment was repeated as described earlier with the inclusion of three washes with warm PBS (37°C) at the end of each incubation. In the last wash, cells were scraped off and pelleted by centrifugation (930 xg, 10 min, 4°C) and the pellet was lyzed using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) according to supplier’s instructions. Both Halt Protease and Phosphatase Inhibitor Cocktails (Thermo Fisher Scientific) were added to lysis buffers during extractions. Cytoplasmic and nuclear fractions were aliquoted and stored at -80°C until later use. To confirm that Giardia ESPs can modulate/inhibit immune signaling in IECs, we repeated the experiment above but IECs were previously treated with lipopolysaccharide (LPS, Sigma-Aldrich) (10 μg/ml) or tumour necrosis factor-alpha (TNF-α, Sigma-Aldrich) (100 ng/ml) for 2h prior adding trophozoites into the inserts. Trophozoites in inserts and IECs, in the presence of inflammatory signal, were left to interact for 2h and 6h, upon which IECs were processed as above for protein collection. IECs treated with LPS and TNF-α were tested for MAPKs activation/inhibition and the nuclear translocation of NF-κB.

Protein samples collected as described above were used for SDS-PAGE and Western blots. Briefly, 30 μg of total proteins were electrophoresed on AnykD-stain-free gels (Bio-Rad) (100 v, 4°C) and transferred onto a PVDF membrane (100 v, 90 min, 4°C). Membranes were washed with Tris-Buffered Saline with 0.1% Tween-20 (TBST) and blocked with 5% non-fat dry milk in TBST for 1h at RT, followed by incubation with primary antibodies overnight (4°C). Blots were then washed with TBST (3x) and incubated for 1h at RT with a secondary anti-mouse HRP conjugated or anti-rabbit HRP conjugated antibodies (1:5000 dilution, Cell Signaling Technology, MA, USA). Blots were developed using the Clarity ECL Western Blotting Substrate (Bio-Rad). For re-probing, blots were stripped using a mild (200 mM glycine, 0.1% w/v SDS, 1% Tween 20, pH 2.2) or harsh stripping buffer (2% SDS, 62.5 mM Tris, pH 6.8, and 114.4 mM beta-mercaptoethanol) and probed for the loading control, which is either an anti-human GAPDH (cat no. MAB5718, R&D Systems, Inc., MN, USA), anti α-tubulin antibody (Cell Signaling Technology) or TATA box binding protein (TBP) for nuclear samples (MA5-14739, Thermo Fisher Scientific). To detect the activation of MAPKs (ERK1/2, P38 and JNK), the following antibodies were used against the phosphorylated and non-phosphorylated forms of kinases: phospho-p44/42 MAPK (Erk1/2) (cat no. 8544) and p44/42 MAPK (Erk1/2) (cat no. 4348), Phospho-p38 MAPK (cat no. 4511) and p38 MAPK (cat no. 8690), Phospho-SAPK/JNK (cat no. 4671) and SAPK/JNK (cat no. 9258), and NF-κB1 p105/p50 (cat no. 13586). Antibodies were purchased from Cell Signaling Technology and used at the dilution recommended by the supplier.

Immunofluorescence of labelled ESPs and confocal microscopy

For ESPs labelling with Alexa Fluor 488, proteins collected from axenic culture were washed (3x) with cold PBS during sample concentration to remove free amino acids and antioxidants that might interfere with protein labelling (recommended in the kit). ESPs (45μg) were incubated with 11.3 nmol/μl of Alexa Fluor-488 and excess dye was removed by gel filtration following the instructions in the Alexa Fluor 488 Microscale Protein Labelling Kit (Thermo Fisher Scientific).

To visualise ESPs interaction with host cells, chamber slides were washed and replenished with fresh DMEM as above, followed by adding labelled ESPs to the wells at 1 or 5 μg/ml and incubation for 2 h or 6 h (37°C, 10% CO₂). The amounts of labelled proteins were quantified using a NanoDrop 1000 Spectrometer (Thermo Fisher Scientific). Caco-2 cells incubated in DMEM only (no Labelled ESPs added) served as a control. Slides were washed, fixed (4% paraformaldehyde in PBS) and mounted (Vectashield anti-fade reagent with DAPI, Vector Laboratories, CA, USA), excluding the blocking step. The fluorescent label on IECs was visualised using a Zeiss LSM700 confocal microscope (Zeiss, Oberkochen, Germany) at the BioVis Core Facility, SciLifeLab, Uppsala, Sweden.

Statistical analyses

The results from the cytokine measurements and differential gene expression for qPCR results were analysed using a one-way analysis of variance (ANOVA) to assess overall significance among the tested groups at α < 0.05. When significant, the values from the replicates were processed through the Bonferroni-Holm’s and significance was marked at P < 0.05.

The secretome of Giardia trophozoites in the absence and presence of host cells

In order to identify proteins in the Giardia secretome, we collected ESPs from Giardia trophozoites grown axenically or in the presence of IECs (Fig 1A). The secretomes of axenic WB (assemblage A) and GS (assemblage B) Giardia trophozoites were analyzed in a modified serum-free medium (see Materials and methods) that could support the full viability of trophozoites for 2h and estimated viabilities of 98.74% ± 1.03 (WB, n = 5) and 98.32% ± 0.81 (GS, n = 5) under 6h of incubation. In total, 196 proteins were identified in the secretome of the WB isolate with 149 proteins detected in 2h culture and 180 in 6h culture; 133 proteins overlapped in both time points (Fig 2A). For the GS isolate, 155 proteins were identified including 141 proteins from the 2h culture and 83 proteins from 6h culture; 69 proteins overlapped in both time points (Fig 2A). The reproducibility of the secretome profiles was conserved in the three biological replicates. Full lists of identified proteins, including tryptic peptides, score, coverage and number of peptide spectrum matches (PSM) are presented in S1 Table (WB) and S2 Table (GS). The secretomes of both isolates were also compared leading to the identification of 122 common secreted proteins (S3 Table).

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Fig 2. Secretomes of Giardia trophozoites and differentiated Caco-2 cells during interactions.

(A) Number of secreted WB and GS proteins in axenic culture. (B) Number of secreted WB and GS proteins during host cell interactions. (C) Number of secreted Caco-2 cell proteins during interaction with Giardia WB and GS trophozoites. (D) and (E) GO term analyses of WB and GS secretomes during interaction with host cells. (F) and (G) GO term analyses of secreted Caco-2 cell proteins during interactions with Giardia WB and GS trophozoites.

https://doi.org/10.1371/journal.pntd.0006120.g002

We also collected and analyzed the medium from Giardia trophozoites incubated with differentiated Caco-2 cells (Fig 1A). In total, 248 WB proteins were identified in the co-culture medium, 156 at 2h, 240 at 6h and 148 in both time points (Fig 2B, S4 Table). For the GS isolate, 152 proteins in total were identified, 122 at 2h, 128 at 6h and 98 at both time points (Fig 2B, S5 Table). The top 50 proteins, based on their peptide score, are listed in Table 1. These proteins overlap with those in axenic culture (S1 and S2 Tables). A comparison was performed between the secretomes of WB trophozoites incubated with (S4 Table) and without IECs (S1 Table) through which 161 common proteins and 87 interaction-specific proteins were identified (Table 2). Similarly, an analysis of the secreted proteins from GS isolate with (S5 Table) and without IECs (S2 Table) identified 111 common proteins and 41 proteins specific to the interaction (Table 2). Amongst the proteins specific to interactions, 13 proteins overlap between the two isolates (denoted in Table 2) whereas 74 WB trophozoite proteins and 28 GS trophozoite proteins are exclusive to the interaction process and represent an isolate-specific response to IECs. This overall shows that most of the secreted Giardia proteins during interactions with IECs are conserved between the WB and GS isolates but there are assemblage-specific differences.

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Table 1. Top 50 identified proteins of Giardia intestinalis isolates, WB and GS, secreted into the medium of interaction with intestinal epithelial cells in vitro.

The proteins are ranked based on their peptide score. Note that all listed proteins were also detected in axenic cultures.

https://doi.org/10.1371/journal.pntd.0006120.t001

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Table 2. Proteins specifically released by Giardia intestinalis isolates, WB and GS, during interaction with differentiated Caco-2 cells.

https://doi.org/10.1371/journal.pntd.0006120.t002

Secreted Giardia proteins in co-culture were analyzed for the enrichment of certain biological functions and for protein class. For the GS isolate, since the current online algorithms do not support GO analysis, orthologous genes in WB were used to perform the quest. The results are presented in Fig 2D and 2E. The largest number of detected proteins have functions associated with metabolism (85 proteins in WB and 64 in GS) with different subgroups in the child linages associated with protein metabolism, amino acid catabolic and metabolic processes, glycolysis, monosaccharide metabolic process and the generation of precursor metabolites and energy (Fig 2D). These metabolic groups were also seen in the secretome of axenic cultures (S1 Fig). Other metabolic functions such as the hexose monophosphate shunt and arginine metabolism could also be identified when secreted proteins were manually delineated into different metabolic pathways (S1 Fig). Furthermore, a nuclease with endonuclease I activity (Table 1), possibly involved in nucleic acids salvaging, and proteins involved in pyrimidine salvage (cytidine deiminase and uridine phosphorylase (UPL-1)) as well as in lipid metabolism were identified in secretome of co-culture (S1 Fig). The lipid metabolic proteins included enzymes involved in phospholipids (PLs) remodelling and salvaging fatty acids (phospholipase B, PLB), producing monoacylglycerol (lysophospholipid phosphatase) and generating inositol-3 phosphate (I3P, I3P synthase), the precursor for all inositol containing compounds including PLs [32]. These result show that the major part of Giardia secretome is involved in different metabolic functions.

The majority of proteins in the two isolates metabolo-secretome fell under four major categories: the hydrolase, dehydrogenase, oxidoreductase and protease groups. The oxidoreductase group contained 25 proteins in WB and 14 proteins in GS (Fig 2E). Compared with axenic culture (14 proteins) (S2A Fig), the WB isolate released 11 additional proteins with oxidoreductase activity including peroxidases when incubated with IECs (e.g. PFOR, NADH oxidase lateral transfer candidate, peroxiredoxin 1, FixW protein alcohol dehydrogenases (ALDs), glutamate dehydrogenase and glutamate synthase and a HP GL50803_9355, Table 2). This suggests a response to counteract IECs oxidative defences such as reactive oxygen species. It has been suggested that oxidative stress provides the trigger to initiate encystation [33] and proteins involved in cyst wall synthesis (e.g. glucosamine 6-phosphate deaminase and N-acetylglucosamine 6-phosphate mutase) were released by WB isolate interacting with IECs, corroborating this notion. For GS, on the other hand, peroxidases and antioxidant proteins were already secreted by trophozoites without exposure to IECs (S2 Table) and thus the results above show that the two isolates exhibit different anti-oxidative responses during interaction with IECs.

The protease group contained 13 secreted proteins in both isolates (Fig 2E) and harboured the cysteine- (cathepsins B and L), serine- (Alanyl dipeptidyl peptidases) and metallo-type (Aminoacyl-histidine dipeptidases and Xaa-Pro dipeptidases) of proteases. For WB isolate, two secreted metalloproteases (GL50803_8407 and GL50803_3822) were released in response to IECs whereas the rest of proteases were the same as those in axenic culture. For GS isolate, the same proteases were detected in both co-culture and axenic culture with two serine proteases (GL50581_3181 and GL50581_2704) being non-orthologous to any WB isolate proteins.

Many of the proteins detected in our analysis have been previously reported as immunoreactive proteins during human giardiasis (S6 Table) [21]. Amongst these proteins, the variable surface proteins (VSPs) are immunodominant during infection [2] and together with the high cysteine membrane proteins (HCMPs) they occupied a large portion of the WB isolate secretome (23 and 12, respectively, S1 Table). In the GS isolate secretome, lower numbers of VSPs and HCMPs were detected (4 and 5, respectively, S2 Table), more likely due to the fragmented genome used in protein identification. VSPs and HCMPs are cysteine-rich proteins and those detected in our analysis have an epidermal growth factor (EGF)-like domain in common (InterPro:IPR000742, 39 in WB and 10 in GS) which is also found in other secreted cysteine-rich proteins (e.g. tenascins, high cysteine proteins (HCPs) and neurogenic locus Notch protein precursor, S1 and S2 Tables). EGF-like domains are often present in secreted protein [34] and thus, this promoted us to look for proteins with secretion signal peptides (SSPs). A genome-wide screen in the Giardia database showed that 1361 proteins of the WB isolate have SSPs, amongst which 82 proteins were detected in our co-culture secretome analysis (~29%, S7 Table) with 41 of those being N-glycosylated [35] (S7 Table). For the GS isolate, 422 proteins in Giardia DB contain SSPs, 44 were detected in our analysis (~29%, S7 Table). The majority of proteins with SSPs (S7 Table) contained either an EGF-like domain (38 proteins for WB and 16 for GS) or a peptidase C1A domain (8 proteins for WB and 8 proteins for GS). The detection of a relatively small number of proteins with SSPs in Giardia secretomes was not surprising as this has been previously reported in other protozoan parasites like Trypanosoma. brucei (~20%) and Leishmania donovani (~9%) [36,37], suggesting the presence of alternative secretory pathways. Many protozoan parasites produce extracellular vesicles (MVs and exosomes), which can release proteins without SPPs [38]. MVs have recently been shown to be released from Giardia trophozoites [26] and we could detect two types of vesicles (100–250 nm and 100 nm, S2B Fig) in the supernatants of axenic trophozoite culture. These vesicles might explain the release of proteins without SSPs but further experiments are needed to show how these vesicles are formed and what proteins they contain.

The secretome of differentiated Caco-2 cells during parasite interactions

The secretome of differentiated Caco-2 cells incubated with WB trophozoites was analyzed for proteins released in co-culture. Overall, 384 Caco-2 cell proteins were identified in the medium of interaction with 21 proteins secreted at 2h, 136 at 6h and 227 at both time points (Fig 2C). The identified proteins, together with the number of peptides, score, coverage and PSMs are presented in S4 Table. A comparison between the secretomes of Caco-2 cells incubated alone (i.e. control, S8 Table) and with WB trophozoites (S4 Table) identified 308 overlapping proteins (S9 Table) and 76 proteins released in response to WB trophozoites (Table 3). The secretome of Caco-2 cells incubated with GS trophozoites was also analyzed for the proteins released by host cells in response to interaction. In total, 355 proteins were secreted in co-culture, 60 proteins were secreted at 2h, 78 at 6h and 217 at both time points (Fig 2C). The proteins identified, together with the number of peptides, score, coverage and PSMs are presented in S5 Table. The top 50 proteins, based on their peptide scores, are presented in Table 4. A comparison between the secretomes of co-culture (S5 Table) and control (S8 Table) identified 310 common proteins (S10 Table) and 45 proteins that were specifically released in response to GS trophozoites (Table 3). Amongst the proteins specific to interaction (45 proteins) 31 proteins overlapped in the response to both isolates.

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Table 3. Proteins secreted by differentiated Caco-2 cells specific to interaction with Giardia intestinalis isolates, WB and GS.

https://doi.org/10.1371/journal.pntd.0006120.t003

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Table 4. Top 50 identified proteins of secreted by differentiated Caco-2 cells co-incubated with Giardia intestinalis isolates, WB and GS in vitro.

The proteins are ranked based on peptide score. Note that all listed proteins were also detected in Caco-2 cells culture without Giardia.

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GO enrichment analyses of parasitized Caco-2 cell secretomes (both isolates, Fig 2F and 2G) revealed that the majority of proteins have metabolic functions (152 protein in response to WB and 135 in response to GS) such as protein metabolism (including proteolysis), carbohydrate metabolism (including monosaccharides and polysaccharide metabolism as well as glycolysis), nucleobase-containing compounds metabolic process and lipid metabolism (only WB). The same metabolic groups were also seen in the control Caco-2 cell secretome (S2C Fig). One of the big groups that emerged in our analysis was the cytoskeletal proteins (41 proteins in response to WB isolate and 32 proteins in response to GS). This group included the intermediate filament and the actin family cytoskeletal proteins (including non-motor actin binding protein in the child lineage). Except for the actin family cytoskeletal protein group which was not enriched in control Caco-2 cell secretome, the rest of cytoskeletal protein groups contained more proteins compared to the control secretome. This clearly indicates the effect of Giardia colonization on IECs actin cytoskeleton. An interesting finding is that seven microvilli proteins were detected in the secretome of Caco-2 cells incubated with WB trophozoites (GO:0005903, intestinal epithelial brush border) (e.g. villin-1, intelectin-1, myosin heavy chain-9 and -14, beta 1,4- galactosyltransferase polypeptide 1, plectin and LIM domain and actin binding 1). These proteins might have been cleaved off/dissociated from damaged microvilli as a result of trophozoite attachment.

Two of the proteins that we identified in the parasitized Caco-2 cell secretome are associated with immunological functions. The first protein is a cytokine called macrophage migration inhibitory factor (MIF) (Table 3). MIF is a pleiotropic cytokine that regulates the expression of inflammatory genes (e.g. TNF-α, cyclooxygenase 2 and inducible NO synthase (iNOS)) [39], indicating its involvement in innate immune responses. The second protein is complement c3 identified in the secretome of Caco-2 cell incubated with GS only (Table 3), suggesting differences in Giardia isolates abilities to activate the complement system.

In conclusion, we have identified the major secreted proteins of host and parasite during interaction. Our analysis showed that both, the host and parasite, release proteins with similar functions (sugar, protein, lipid and nucleic acid metabolism). Both assemblages/isolates (A and B) induced similar responses but there are assemblage-specific differences that might explain observed differences in infectivity and symptoms.

Transcriptional response of IECs to Giardia ESPs

Interaction between Giardia trophozoites and IECs in vitro induces the transcription of a large number of genes including the chemokines ccl2, ccl20, and cxcl1-3 that are up-regulated up to 100-fold at the RNA level [40]. To test whether Giardia ESPs are capable of inducing similar effects, differentiated Caco-2 cells were exposed to Giardia ESPs through Transwell inserts (Fig 1B) for 2h and 6h. Differentially expressed genes (DEGs) were identified by RNA sequencing (RNA Seq) (S11 Table). Overall, 120 and 87 genes were differentially expressed (DE) in IECs exposed to ESPs of WB and GS trophozoites, respectively, with a global gene expression profile showing differences between the two time points and between the two isolates (S11 Table, Fig 3A).

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Fig 3. Gene expression changes in IECs after ESP exposure.

(A) Heat-map of differentially expressed genes in the differentiated human colon carcinoma cell line Caco-2 exposed to Giardia intestinalis excretory secretory products. (B) Induction of AP-1 activity using a luciferase reporter plasmid transfected into Caco-2 cells. The cells were exposed to ESPs of Giardia intestinalis isolate WB or GS for 2h or 6h through a Transwell insert placed in the tissue culture plate (Mean ± SD, n = 4). (C) Relative RNA levels (Mean ± SD, n = 4) of inflammatory genes in differentiated Caco-2 cells incubated with different amounts of ESPs (5μg/ml and 10μg/ml) collected from Giardia intestinalis isolate WB or GS. Significant results in the panels are indicated by (*) for P < 0.05 or (**) for P < 0.01. (D) ELISA measurement (Mean ± SD, n = 3) of interleukin-8 (IL-8), chemokine (C-X-C motif) ligand 1 (CXCL1) and chemokine (C-C motif) ligand 20 (CCL20) in the medium of differentiated human colon carcinoma cell line, Caco-2, exposed to ESPs of G. intestinalis WB or GS isolate through a Transwell insert. No statistical significance was found between controls and treatments (P > 0.05).

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At 2h, seven genes were DE in IECs exposed to ESPs of the WB isolate, six of which were up-regulated and one gene (il8) was down-regulated (Table 5). The up-regulated genes encode immediate early genes [41], including the protein constituents of AP-1 transcription factor (FBJ murine osteosarcoma viral oncogene homolog, fos and jun B proto-oncogene, junb). To verify AP-1 activation, Caco-2 cells we transfected with a plasmid expressing a luciferase gene under the control of AP-1 transcription response element and luminescence was measured upon cell exposure to Giardia ESPs. At 2h, no significant change was observed in the AP-1 promoter activity with ESPs of either isolates (1.07 for ESPs of WB isolate and 1.12 for ESPs of GS isolate, P > 0.05) (Fig 3B). At 6h, on the other hand, despite the slight increase in AP-1 promoter activity (1.17 for ESPs of WB isolate and 1.15 for ESPs of GS isolate), the average fold change was significant (n = 4, P < 0.05) (Fig 3B). These findings are in line with the RNA Seq results and indicate that AP-1 is activated in response to Giardia ESPs. The gene encoding DUSP1 was the highest up-regulated gene at 2h and the only up-regulated gene in IECs exposed to ESPs of GS trophozoites (Table 5). Its transcriptional level increased at 6h together with dual specificity phosphatases 4 and 5 (dusp4 and dusp5), and tribbles pseudokinase 1 (trib1) (Table 5). The products of these genes play important roles in the regulation of MAPKs’ activity [42–44].

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Table 5. Selected groups of differentially expressed genes in differentiated human colon carcinoma Caco-2 cells exposed to excretory-secretory products of Giardia intestinalis isolates WB and GS via Transwell insert.

Bold fonts indicate a significantly up-regulated gene whereas italic fonts indicate a significantly down-regulated gene.

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The majority of DEGs (120 in WB and 87 in GS) were up-regulated at 6h (S11 Table) with a higher fold change for DEGs compared to 2h (Table 5, S11 Table). One exception was that the increase in RNA levels il8 in of IECs exposed to GS trophozoite (1.95 fold) was not significant. Insulin induced gene 1 (insig1) was the highest up-regulated gene in IECs in response to ESPs of both isolates at 6h (Table 5) and this gene encodes a protein that regulates cholesterol metabolism, lipogenesis, and glucose homeostasis [45–47]. More genes associated with glucose uptake and response to glucose starvation (Table 5) were DE at 6h, indicating that Giardia releases ESPs that interfere with glucose homeostasis. Nuclear receptor subfamily 4 group A member 1 (nr4a1) was the second top up-regulated gene (ESPs of both isolates, Table 5) and this gene has functions associated with cell cycle, inflammation, and induction of apoptosis [48–50]. Another two cell cycle genes associated with response to DNA damage, were amongst the top ten up-regulated genes and these include BTG family member 2 (btg2) [51] and growth arrest and DNA damage inducible alpha (gadd45a) [52] (ESPs of both isolates, Table 5). This suggests that Giardia ESPs induce damaging effects on host DNA and arrests IECs in the cell cycle. The tight junction proteins, claudin 3 and 4 (cldn 3 and cldn 4), were also up-regulated (Table 5) and these proteins play essential roles in the maintenance of the intestinal epithelial barrier (IEB) [53].

To obtain a general overview on the function of DEGs, they were processed through the GO Database for biological and molecular functions. For IECs exposed to ESPs of the WB isolate, cell proliferation (GO:0008283) and MAPK cascade (GO:0000165) constituted the biggest biological groups encompassing 11 genes each. Both chemokines (e.g. CXCL1-3 and IL-8) and growth factors (e.g. amphiregulin, proepiregulin, Proheparin-binding EGF-like growth factor) were contained within the cell proliferation group. All chemokine genes, including ccl2 and ccl20, were listed under chemokine activity (GO:0008009) and cytokine receptor binding (GO:0005126) by molecular function. In addition to these cytokines, the gene clcf1 encoding cardiotrophin-like_cytokine_factor_1 was DE in IECs exposed to ESPs of WB trophozoites. CLCF1 is an IL-6 family cytokine capable of activating NFκB and exerts stimulatory functions on B cells [54]. For MAPK cascade, it was interesting to see genes encoding RNA binding proteins (zfp36 and zfp36l2) within this group, both of which are associated with anti-inflammatory functions [55]. Another DE gene is Regnase 1 (ZC3H12A), an RNase that controls inflammatory responses by inducing mRNA decay of specific cytokine transcripts [56]. The rest of functional groups in this analysis included response to external stimulus (GO:0009605), locomotion (GO:0040011), localization (GO:0051179), behaviour (GO:0007610) and cell death (GO:0008219). For IECs exposed to ESPs of the GS isolate, the groups cell proliferation, MAPK cascade, cell death and endoderm development (GO:0007492) emerged by biological functions and no specific groups emerged by molecular function analysis. Only cxcl2, cxcl3 and clcf1 were significantly induced in IECs exposed to ESPs of the GS trophozoites. Overall, based on the transcriptomic profile we could identify a general response in IECs exposed to Giardia ESPs, which involves inducing and regulating inflammatory responses, glucose homeostasis, apoptosis, cell cycle, and maintenance of IEB.

Next, we examined which amount of ESPs could induce the transcription of pro-inflammatory genes in IECs. ESPs collected from axenic culture (1, 5 and 10 μg/ml) were incubated with IECs for 2h or 6h (Fig 3C) and RNA levels were assessed with qPCR. At 2h, 5 μg/ml of WB isolate ESPs induced a 2 to 2.9-fold increase in RNA levels of il8, ccl20, and cxcl1-3 but not ccl2, scoring the highest for cxcl3 (Fig 3C). The fold change, however, was statistically insignificant for all the genes tested (P > 0.05). The fold change in RNA levels remained below 2 for 1 μg/ml (not shown) and 10 μg/ml of ESPs (Fig 3C). At 6h, all genes were significantly up-regulated (2.84–8.46 fold) in IECs incubated with 5 μg/ml WB isolate ESPs, scoring the highest for ccl20 (P < 0.05) and the genes ccl2, ccl20, cxcl1 and cxcl3 remained significantly up-regulated (2.84–4.76 fold) when IECs were incubated with 10 μg/ml of ESPs (Fig 3C). For ESPs of the GS isolate, the fold change at RNA level for all the genes tested at the three concentrations of ESPs was below 2 (P > 0.05) (Fig 3D). Nevertheless, at 6h, il8, ccl20, cxcl1 and cxcl3 were significantly up-regulated (4.23–4.71 fold) with il8 being significantly up-regulated at all ESPs concentrations tested and ccl20 and cxcl1 at 10 μg/ml (Fig 3C). The results show that ESPs of both isolates induce variable transcriptional responses of inflammatory genes in IECs and these responses differ with the amounts of ESPs IECs are exposed to. However, direct host-cell interactions have a much stronger effect on the induction of chemokine gene transcription [40].

To assess whether the up-regulated chemokine genes were translated into their protein products, we selected three chemokines (IL-8, CXCL1 and CCL20) and measured their levels in the collected media from the insert experiment (Fig 3D). The amounts of IL8, CXCL1 and CCL20 were very low and their concentration was similar or lower than their controls at 6h (50.2, 42.1 and 57.9 pg/ml, respectively). A slight increase in the amounts of CXCL1 and CCL20 amounts could be seen at 2h upon IECs exposure to ESPs of the WB trophozoites (12.9 pg/ml in control versus 18.2 pg/ml) and GS trophozoites (24.7 pg/ml in control versus 27.1 pg/ml), respectively (P > 0.05) (Fig 3D). Overall, the levels of measured chemokines were either close to control or slightly lower and no significant differences could be seen between IECs exposed or unexposed to ESPs (P > 0.05) (Fig 3D). The low levels of measured chemokines indicate that their mRNA levels are post-transcriptionally regulated or the chemokines being degraded upon release from IECs.

The effect of ESPs on cell signaling

Giardia ESPs from axenic cultures were labelled with Alexa Fluor488 and added to the medium to visualize their interaction with IECs. Confocal images taken upon 2h and 6h of incubation with IECs showed that the labelled ESPs of WB and GS trophozoites bind to the IECs surface as well as the intercellular space (i.e. intercellular junctions) and some are internalized into differentiated Caco-2 cells (Fig 4). This indicates the presence of ligands or receptors on IECs surface and a potential of the internalized ESPs to modulate cellular processes and signaling.

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Fig 4. The binding and internalization of Giardia intestinalis excreted-secreted products (ESPs) to intestinal epithelial cells (IECs).

Confocal microscopy images of differentiated human colon carcinoma cell line, Caco-2, incubated with Alexa Fluor488-labelled ESPs of G. intestinalis isolate WB or GS. Note that the colour of nucleus, stained with DAPI, was changed to red for better contrast.

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We consequently examined MAPK signaling in IECs exposed to ESPs via inserts because this pathway emerged in the functional analysis of RNA Seq data. Western blot analyses showed a slight decrease in the phosphorylation levels of ERK1/2 and P38 at 2h but an increased level of nuclear translocation of NFκB compared to control (Fig 5A). Despite the slight decrease in ERK1/2 and P38 phosphorylation, their roles in inducing the nuclear recruitment of NFκB cannot be excluded and it indicates a possibility of other factors contributing to NFκB activation (e.g. growth factors, stress, hypoxia or nutrient depletion). At 6h, we could see a marked reduction in the phosphorylation levels of ERK1/2 and P38 compared to control and this coincided with a decrease in the levels of NFκB nuclear translocation (Fig 5A). The nuclear translocation of NFκB, however, was not completely abolished. The phosphorylated form of JNK could not be detected (S3A Fig). This shows that Giardia ESPs actively modulate MAPK signaling to avert the induction of strong inflammatory responses and this effect is built up upon prolonged exposure of IECs to ESPs. To test whether Giardia ESPs can also inhibit inflammatory signaling, we incubated IECs with LPS or TNF-α for 2h, then added Giardia trophozoites into Transwell inserts in the persistence of inflammatory stimuli for 2h or 6h. While the controls exhibited increased phosphorylation of ERK1/2 and P38 as well as nuclear recruitment of NFκB, these levels were reduced in inflamed tissue upon exposure to Giardia ESPs at both time points (Fig 5B). These results show that Giardia ESPs can attenuate inflammatory signaling mediated by MAPK phosphorylation and NFκB recruitment. It is possible that the parasite senses IECs inflammatory signals and release specific protein factors to subvert such effects.

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Fig 5. The effects of ESPs on IEC cell signaling.

(A) Western blot analyses of differentiated Caco-2 cells exposed to G. intestinalis ESPs through a Transwell insert for 2h or 6h in the absence of inflammatory stimuli (e.g. lipopolysaccharides or tumour necrosis factor-alpha). Blots were probed for total proteins (phosphorylated/non-phosphorylated forms) of mitogen activated proteins kinases (MAPKs, ERK1/2 and P38) and nuclear factor kappa beta (NFκB). (B) Western blot analyses after the induction of inflammation with LPS and TNF-alpha.

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