Giardia duodenalis-induced alterations of commensal bacteria kill Caenorhabditis elegans: a new model to study microbial-microbial interactions in the gut

Abstract

Giardia duodenalis is the most common cause of parasitic diarrhea worldwide and a well-established risk factor for postinfectious irritable bowel syndrome. We hypothesized that Giardia-induced disruptions in host-microbiota interactions may play a role in the pathogenesis of giardiasis and in postgiardiasis disease. Functional changes induced by Giardia in commensal bacteria and the resulting effects on Caenorhabditis elegans were determined. Although Giardia or bacteria alone did not affect worm viability, combining commensal Escherichia coli bacteria with Giardia became lethal to C. elegans. Giardia also induced killing of C. elegans with attenuated Citrobacter rodentium espF and map mutant strains, human microbiota from a healthy donor, and microbiota from inflamed colonic sites of ulcerative colitis patient. In contrast, combinations of Giardia with microbiota from noninflamed sites of the same patient allowed for worm survival. The synergistic lethal effects of Giardia and E. coli required the presence of live bacteria and were associated with the facilitation of bacterial colonization in the C. elegans intestine. Exposure to C. elegans and/or Giardia altered the expression of 172 genes in E. coli. The genes affected by Giardia included hydrogen sulfide biosynthesis (HSB) genes, and deletion of a positive regulator of HSB genes, cysB, was sufficient to kill C. elegans even in the absence of Giardia. Our findings indicate that Giardia induces functional changes in commensal bacteria, possibly making them opportunistic pathogens, and alters host-microbe homeostatic interactions. This report describes the use of a novel in vivo model to assess the toxicity of human microbiota.

the protozoan Giardia duodenalis (syn. lamblia, intestinalis) is the most common cause of human parasitic diarrheal disease worldwide with an estimated 280 million infections every year (2, 37). Clinical symptoms range from nonsymptomatic infections to acute and chronic diarrheal diseases. During the acute phase of infection, Giardia trophozoites attach to the epithelium of the upper part of the small intestine and induce a series of events, including disruption of epithelial barrier function, diffuse shortening of brush border microvilli, small intestinal malabsorption and maldigestion, chloride hypersecretion, and increased rates of small intestinal transit, culminating with diarrhea (3, 8, 14). Although the mechanisms are incompletely understood, both host and parasite factors contribute to acute giardiasis (14).

Giardia has recently been linked to the development of postinfectious irritable bowel syndrome (IBS) long after clearance of the parasite. Follow-up studies of Giardia-infected individuals during an outbreak in Bergen, Norway attributes 69.7% of the local IBS cases to giardiasis (53). Giardia has also been implicated in various other bowel [inflammatory bowel disease (IBD), biliary tract dysfunction] and extraintestinal disorders (arthritis, uveitis, and retinal arteritis) (23). The mechanisms underlying postgiardiasis intestinal and extraintestinal disorders remain obscure. However, changes in luminal microbiota and metabolite composition are suspected to play a role (11, 43).

Metagenomic association studies have implicated gut microbiota dysbiosis in the pathophysiology of various intestinal and extraintestinal disorders. Identification of functional changes in microbiota by use of sequence analysis, however, is a challenge because of inherent functional redundancy between microbes (31). And the use of rodent model systems to understand mechanisms of functional changes in microbiota is limited because of the requirement for expensive germ-free facilities and their inapplicability for high-throughput functional studies. In contrast, the Caenorhabditis elegans model system is simple, genetically tractable, amenable for high-throughput applications, and readily used to assess effects of gut pathophysiology by monitoring reduced survival over short periods of time. C. elegans has been extensively used to model host-pathogen interactions and identify various pathogen specific virulence strategies and host responses (1). A number of human enteropathogens, including bacteria, have been shown to have pathogenic effects on the C. elegans intestine (42). Intestinal colonization and persistent infection, invasion, and biofilm formation are among the mechanisms by which pathogenic bacteria reduce survival of the infected worms (42). Virulence factors employed by pathogenic microbes include secreted toxins, exoproteins, type III effectors, virulence gene regulators, cell wall-associated factors, and proteins involved in intermediary metabolism (42). The C. elegans mechanisms of defense include pathogen and toxin avoidance, aversive behaviors, oxidative and xenobiotic stress responses, antimicrobial peptide production, and RNAi interference (55). These defense strategies are so far known to be orchestrated by recognition receptors (Toll-like receptor 1; Toll/interleukin-1 receptor), p38 MAPK pathways, DBL-1 transforming growth factor β-like pathways, DAF-2/DAF-16 insulin-like signaling pathways, and JNK-like MAPK pathways (49, 55). These observations further underscore that C. elegans is ideally suited to investigate mechanisms of host-microbial and microbial-microbial interactions in the gut.

In this study we sought to develop C. elegans as a model to study the effects of Giardia on microbiota and host interactions and to characterize novel Giardia-induced functional changes in commensal microbes. The findings reveal a novel, synergistic pathogenic effect of Giardia and commensal bacteria on the host, identify Giardia-induced changes in the expression of genes in commensal bacteria, and characterize alterations in host-commensal microbe interactions.

MATERIALS AND METHODS

Ethics Statement

Studies involving colon mucosal biopsy tissues were approved by the Conjoint Health Research Ethics Board (CHREB) at the University of Calgary and the Calgary Health Region. Patients and healthy individuals gave their written, informed consent as per the CHREB guidelines.

C. elegans, Bacteria, and Giardia Strains

Bristol N2 wild-type C. elegans, OP50 Escherichia coli, and HB101 E. coli strains were obtained from the Caenorhabditis Genetics Center, University of Minnesota, St. Paul, MN. Green fluorescent protein (GFP)-expressing E. coli HB101 was prepared by transforming competent E. coli HB101 with GFP-expressing pGFPuv plasmid (Clontech Laboratories). E. coli K12 strains were obtained from the Coli Genetic Stock Center, Yale University, New Haven, CT. Citrobacter rodentium wild-type, C. rodentium map mutant, and C. rodentium espF mutant strains were obtained from Dr. P. M. Sherman (Hospital for Sick Children, University of Toronto). C. rodentium is a rodent equivalent of human Enteropathogenic E. coli, and the attenuated virulence of the mutant strains has been well established in vivo, making this organism well suited for the purpose of the present study (13). The G. duodenalis isolate NF (Assemblage A) was originally obtained from an epidemic outbreak of human giardiasis in Newfoundland, Canada (51).

Bacteria Preparation

Bacteria were prepared as described previously (45). Briefly, bacteria were grown on Luria-Bertani (Miller) agar plates overnight, and a single colony of bacteria were transferred into Terrific Broth (TB) medium and grown overnight to saturation. TB medium was prepared as described previously (38). The resulting culture was used to inoculate TB medium 1 in 2000 and incubated at 37°C for 12 h. Bacterial cultures in exponential growth phase were washed three times with sterile distilled water (sdH₂O) to remove culture metabolites by centrifuging at 2,500 g for 10 min (44, 45). Then the bacterial pellets were resuspended in S-Basal complete medium (100 mg/ml), as described previously (45). S-basal complete medium is a buffered solution containing trace metals and cholesterol, prepared as described in Ref. 45.

Microbiota Preparation

Microbiota from human mucosal biopsy samples were prepared as described recently (47). Colon mucosal biopsies were collected from a patient with ulcerative colitis (UC) and from a healthy volunteer and placed in a transport medium (BBL Port-A-Cul tubes; BD Biosciences) to maintain viability of microbes, and placed immediately in a Bactron Anaerobic/Environmental Chamber (Sheldon Manufacturing, Cornelius, OR). Biopsies were homogenized and suspended in 10 ml supplemented tryptic soy with yeast (sTSY) and incubated overnight at 37°C in the anaerobic chamber. sTSY (47) is a tryptic soy broth (EMD Chemicals, Gibbstown, NJ) containing 4 g/l yeast extract (BD Biosciences) and supplemented with 0.05% l-cysteine-HCl (Sigma Aldrich) and 1% bovine hemin (Sigma Aldrich)-menadione (Sigma Aldrich) solutions. The overnight culture was used to inoculate fresh sTSY medium 1 in 300, placed in an anaerobic jar (Oxoid) with anaerobic atmosphere generator (Anaerogen, Oxoid), and incubated on a shaking incubator for 12 h at 37°C. The microbiota cultures were washed three times with sdH₂O (2,500 g, 10 min) and resuspended in S-Basal complete medium at 100 mg/ml.

Giardia Trophozoites Preparation

Giardia duodenalis trophozoites were maintained axenically at 37°C in Diamond's TY1-S-33 medium (16) supplemented with 2.5 mg/ml of piperacillin (Pipracil; Wyeth-Ayerst Canada, Montreal, Quebec, Canada) in 15-ml polystyrene centrifuge tubes (Falcon; Becton Dickinson, Franklin Lakes, NJ). Trophozoites were harvested by cold shock on ice for 20 min followed by centrifugation at 500 g for 10 min at 4°C, washed once with 4°C PBS and then resuspended in S-Basal complete medium. Giardia excreted/secreted factors were prepared by incubating 1 × 10⁸ Giardia trophozoites in 1.25 ml S-Basal complete medium for 6 h. The conditioned medium was centrifuged at 500 g and filtered with a 0.2-μm filter syringe to remove Giardia trophozoites.

Worm Preparation and Survival Assays

Worms were maintained on E. coli OP50 lawn and age-synchronized as previously described (6, 48). Adult-stage gravid worms incubated at 18°C on nematode growth medium plates were washed off the plates with sdH₂O and treated with sodium hypochlorite to isolate eggs. The eggs were then washed three times in S-Basal complete medium and allowed to hatch for 16 h in the same medium on a shaker at 20°C. Then E. coli OP50, 50 μg/ml carbenicillin (Sigma-Aldrich), 0.3 mM 5-fluoro-2′-deoxyuridine (FUDR; Sigma-Aldrich), and 0.1 μg/ml amphotericin B (Fungizone; Invitrogen) were added and incubated for 3 more days. After 3 days, worms were washed in sdH₂O (200 g, 2 min, three times) and resuspended in S-Basal complete medium. Survival assay experiments were set-up in 96-well untreated plates (VWR International) as described previously (45). Briefly, worms were transferred to a 96-well plate (∼15 worms/well) and bacteria or microbiota were added at final concentrations of 2.5 mg/ml (∼3 × 10⁸). Experiments were performed in a final volume of 200 μl S-Basal complete medium, 0.1 μg/ml amphotericin B, and 0.3 mM FUDR in the presence or absence of Giardia trophozoites (2 × 10⁶ trophozoites) or 25 μl Giardia-conditioned medium (prepared as described above). Half the concentrations of bacteria/microbiota and Giardia trophozoites were used for coincubations. Survival assays were carried out in a minimum of two independent experiments, run at least in triplicate wells each time, and in each of which ∼15 worms were assayed. Figures list N = total number of worms studied for each group. Live and dead worms (movement was used to determine whether worms were alive or dead) were counted before and after addition of bacteria or microbiota and/or Giardia samples at different time points.

Microarray Analyses

Experimental setup and RNA isolation.

E. coli HB101 (2.5 mg/ml final concentration, ∼10⁸ bacteria) was transferred to a 96-well plate and incubated at 25°C for 24 h alone or with C. elegans, Giardia-conditioned medium, or C. elegans and Giardia-conditioned medium in a total volume of 200 μl S-Basal complete medium. Samples were transferred to a microcentrifuge tube and centrifuged for 2 min at 100 g to remove C. elegans. The supernatants were centrifuged at 15,000 g for 10 min to collect E. coli HB101. Then bacteria was washed three times in S-Basal complete medium and treated with RNA Protect (Qiagen) to stabilize mRNA. Total RNA was purified by use of RNeasy Mini Kit (Qiagen). RNA quality was assessed by absorbance at 260 nm on spectrophotometer and by running samples on an agarose gel.

Target preparation, hybridization, washing, staining, and scanning.

cDNA synthesis, fragmentation, and terminal labeling were done by following prokaryotic target preparation protocols described in the GeneChip Expression Analysis Technical Manual (Affymetrix). The cDNA were cleaned with MinElute PCR Purification Kit (Qiagen) and quantified by absorbance at 260 nm on a spectrophotometer, and equal amounts were used for fragmentation. Gel shift assays were used to determine the efficiency of terminal labeling. Target hybridization, washing, staining, and scanning were performed at The Southern Alberta Microarray Facility (University of Calgary) by following the procedures described in the GeneChip Expression Analysis Technical Manual. Three independent microarray experiments were run using 12 arrays, three arrays per group. Microarray data for one of the arrays (belonging to E. coli HB101 group) was excluded from further analysis because of poor image quality. The microarray data have been deposited in the NCBI Gene Expression Omnibus database (accession number GSE61092).

Identification of differentially expressed genes.

The microarray data files were processed using statistical software R version 3.0.1 (36a) and Bioconductor packages (20). The Single-Channel Array Normalization (SCAN.UPC) package (35) was used to normalize the microarray data. Hierarchical clustering analysis was used to check for the presence of batch effects in the expression data, and batch effects were adjusted by using the Empirical Bayes method implemented in COMBAT software (26). Expression analysis of the adjusted data provided 5,532 probe set IDs of the 10,208 with adjusted P value < 0.05. Differential expression analysis of the data provided 194 probe set IDs with at least twofold changes in gene expression between groups and adjusted P value of < 0.05. Heatmap clustering of differentially expressed genes was done by the heatmap.2 function of the gplots package in R.

Quantitative RT-PCR

RNA and cDNA were prepared as described above. Primers were designed by using primer 3 software (http://bioinfo.ut.ee/primer3/). Lists of gene specific primers and primer sequences are given in Table 1. Quantitative RT-PCR (qRT-PCR) was performed using a Rotor-Gene Q thermal cycler (Qiagen) in duplicates by mixing equal amounts of cDNA, QuantiFast SYBR Green PCR master mix (Qiagen), and gene-specific forward and reverse primers. A PCR condition of 1 cycle of 5 min at 95°C and 40 cycles of 10 s at 95°C and 30 s at 60°C were used. Optimal PCR efficiency was determined for all genes tested, with the cysB gene used as an endogenous control. Fold changes in gene expression was determined by the comparative 2^−ΔΔCT method as previously described (39).

Table 1.

Primers used for quantitative RT-PCR

Genes	Primers	Sequences
zraP	Forward	5′-CAACTGGTGACGAAGCGTTA-3′
	Reverse	5′-ACGACTGACGCAAATTCTCC-3′
zraR	Forward	5′-ATCTTGTGCTTTGCGATGTG-3′
	Reverse	5′-CGCTGGAGTACGCAGTCATA-3′
cysC	Forward	5′-CTGGATGGCGACAATGTTC-3′
	Reverse	5′-ACCATCAAATTCGCCACTTC-3′
cysB	Forward	5′-GCTGATGCCGTCTCTAAAGG-3′
	Reverse	5′-ACAATAGCCCGATTCCAGTG-3′
cysG	Forward	5′-GAAAGCCTTCTCGACACCTG-3′
	Reverse	5′-CGTTACAGAAGATGCGACGA-3′
lepB	Forward	5′-GCGATTTCGTTCAGACCTTC-3′
	Reverse	5′-TTACGCTCGGAAAGACGAAT-3′
fliP	Forward	5′-AGCCATTCAGCGAAGAGAAA-3′
	Reverse	5′-TCAACTCGCTGGTCACGTAG-3′
fliL	Forward	5′-TTCTACGCGCTGGATACCTT-3′
	Reverse	5′-GGCAATCAGGTTTTTCTTGC-3′

Electron Microscopy

C. elegans adult worms were incubated alone or with E. coli HB101, Giardia trophozoites, or E. coli and Giardia trophozoites for 12 h. Worms were washed three times with sdH₂O (200 g, 2 min) and prepared for electron microscopy as previously described (22, 24). Worms were immersed in buffered 2.5% glutaraldehyde and 0.8% osmium tetroxide and cut to allow fixative penetration. Worms were then fixed in 0.8% glutaraldehyde and 2% osmium tetroxide and embedded in 1% agarose. Agarose embedded worms were dehydrated in ethanol and embedded in Spurr's low-viscosity resin (Electron Microscopy Sciences, Fort Washington, PA). Worms were thin-sectioned by using a diamond knife on a Reichert Ultracut E (Reichert Analytical Instruments, Depew, NY) and were stained with uranyl acetate and lead citrate (University of Calgary Microscopy and Imaging Facility). The stained sections were examined with a Hitachi H-7000 transmission electron microscope (Hitachi, Mississauga, ON, Canada) at 80 kV.

Statistical Analysis

All data were expressed as means ± SE, and statistical analyses of the data were performed with the Prism 5 software. All comparisons were made by two-way ANOVA with Bonferroni post hoc test with correction for multiple comparisons. P values <0.05 were considered statistically significant.

RESULTS

Giardia Synergizes with Commensal Bacteria to Kill C. elegans

To first investigate the effect of Giardia alone on C. elegans survival, worms were incubated in the presence or absence of Giardia trophozoites. Addition of Giardia alone had no effect on worm survival (Fig. 1A). In contrast, incubation of worms with Giardia trophozoites in the presence of E. coli OP50 decreased worm survival. Again, there was no detrimental effect on survival when worms were incubated with either Giardia trophozoites or E. coli OP50 alone (Fig. 1B). E. coli OP50 is a uracil auxotroph of an E. coli B strain and is normally used as a food source for C. elegans (48). E. coli B strains are commensal bacteria found in the gut of healthy individuals. Similar results were obtained when worms were incubated with Giardia and E. coli HB101, a commensal bacteria originally isolated from human gut (Fig. 1C). Taken together, the results indicate that Giardia and commensal bacteria synergize to kill C. elegans.

Fig. 1.

Giardia and commensal Escherichia coli bacteria synergize to kill Caenorhabditis elegans. Worms were incubated alone (untreated) or in the presence of E. coli OP50 (OP50), E. coli HB101 (HB101), Giardia trophozoites (Giardia), or Giardia trophozoites in combination with E. coli HB101. Giardia alone has no effect on C. elegans survival (A; untreated, N = 67 worms; Giardia, N = 94 worms). Giardia coincubation with E. coli OP50 (B; untreated, N = 87 worms; OP50, N = 114 worms; Giardia, N = 121 worms; OP50+Giardia, N = 122 worms) or with E. coli HB101 (C; untreated, N = 113 worms; HB101, N = 130 worms; HB101+Giardia, N = 102 worms) kills C. elegans. Error bars represent SE for 6–9 wells containing all the worms in each group. *P < 0.05 vs. untreated or Giardia, E. coli OP50, or E. coli HB101-treated groups.

Giardia Enhances Virulence of Attenuated C. rodentium Strains

To further investigate the synergistic effect of Giardia, we hypothesized that exposure to Giardia would be able to restore virulence in bacteria where virulence had been genetically attenuated. We coincubated Giardia with attenuated and wild-type C. rodentium strains (Fig. 2). C. rodentium map and espF mutant strains given alone were significantly less toxic to worms than the wild-type strain (Fig. 2A). Map and EspF are type III effector proteins responsible for C. rodentium infection-induced epithelial barrier dysfunction (21, 32). Coincubation with Giardia trophozoites restored the virulence of the mutant strains in C. elegans, bringing worm killing levels to those seen when worms were given the wild-type (Fig. 2B).

Fig. 2.

Giardia enhances virulence of C. rodentium map and espF mutant strains. Worms were incubated with C. rodentium mutant strains alone or in combination with Giardia trophozoites. C. rodentium map and espF mutant strains show attenuated virulence compared with the wild-type (WT) strain (A). In the presence of Giardia trophozoites, the virulence of the mutant strains is reversed to a WT level (B). WT, N = 121 worms; map, N = 105 worms; espF, N = 106 worms; map+Giardia, N = 143 worms; espF+Giardia, N = 155 worms. Error bars represent SE for 9 wells containing all the worms in each group. *P < 0.01 vs. espF and map treated groups.

Giardia Enhances Human Microbiota Toxicity in C. elegans

We then hypothesized that Giardia may similarly increase the virulence of human microbiota bacteria. Worms were incubated with microbiota obtained from colonic biopsies of a healthy individual and an UC patient, in the presence or absence of Giardia trophozoites. Microbiota bacteria obtained from the inflamed colonic site of an UC patient were significantly more toxic to C. elegans than the microbiota from the healthy individual; microbiota bacteria from the noninflamed site of the same UC patient were not toxic to the worms (Fig. 3A). Giardia enhanced the toxicity of microbiota bacteria from the healthy individual and that of microbiota from UC inflamed site (Fig. 3, B and C). The synergistic toxic effect to C. elegans was absent when worms are coincubated with Giardia and the noninflamed site microbiota bacteria (Fig. 3D). We monitored the effect of the noninflamed site microbiota in the presence or absence of Giardia for at least 4 days and found no effect on worm survival (data not shown).

Fig. 3.

Giardia trophozoites enhance the toxicity of human microbiota obtained from a healthy donor, or from an inflamed ulcerative colitis (UC) site. A: C. elegans incubated with E. coli OP50 (OP50, N = 114 worms), or with microbiota from the healthy donor (Healthy, N = 133 worms), or with microbiota from the inflamed UC site (Inflamed, N = 133 worms), or with microbiota from the noninflamed UC site (Noninflamed, N = 125 worms). B: worms incubated with human microbiota from a healthy donor and Giardia (Healthy+Giardia, N = 147 worms). C: worms incubated with microbiota from an inflamed UC site and Giardia (Inflamed+Giardia, N = 122 worms). D: worms incubated with microbiota from a noninflamed UC site and Giardia (Noninflamed+Giardia, N = 147 worms). Error bars represent SE for 9 wells containing all the worms in each group. A: *P < 0.05 vs. OP50 and Noninflamed; $P < 0.05 vs. Healthy. B: *P < 0.05 vs. OP50 and Inflamed. C: #P < 0.05 vs. OP50; &P < 0.05 vs. Healthy.

Giardia Secreted/Excreted Factors Are Sufficient to Induce Synergistic Killing, but Direct Contact of C. elegans With Live Bacteria Is Required

To understand how Giardia and E. coli synergize to kill C. elegans, we tested the effects of Giardia secretory/excretory factors. Coincubation of worms with Giardia-conditioned medium was sufficient to kill worms in the presence of E. coli (Fig. 4A). Heat treatment of Giardia-conditioned medium (boiling for 15 min) failed to abolish the effect of Giardia secreted/excreted factors (data not shown). We then tested whether Giardia interaction with E. coli results in the production of secretory-excretory factors toxic to C. elegans. We exposed worms to medium conditioned with Giardia and E. coli coincubations and found no effect on survival of worms, suggesting the requirement for physical presence of E. coli for this synergistic toxicity (Fig. 4A). Interestingly, Giardia coincubation with heat-killed bacteria had no effect on worm survival (Fig. 4B) indicating that the bacteria that had to be present also needed to be alive. We then investigated how exposure to Giardia may affect interactions between E. coli and the host. Although E. coli HB101 given alone failed to colonize the C. elegans gut, the coincubation with Giardia induced E. coli HB101 to colonize the C. elegans intestine (Fig. 5A). Corroborating these observations, in the intestines of these colonized worms, intact bacteria could be seen by transmission electron microscopy, and the intestines appeared distended (Fig. 5, D and E). Together, the findings suggest that Giardia may promote intestinal colonization by commensal bacteria.

Fig. 4.

Giardia secreted/excreted factors are sufficient to cause synergistic toxicity, but direct contact with live E. coli is required for the synergistic effects of Giardia and E. coli. A: worms were incubated with E. coli OP50 (OP50, N = 191 worms), E. coli OP50 in combination with Giardia-conditioned medium (SC^Giardia, N = 189 worms), or medium conditioned with both E. coli OP50 and Giardia (SC^OP50+Giardia, N = 237 worms). All S-Basal complete media were conditioned for 6 h. B: worms were incubated with live (OP50, N = 124 worms) or dead (DOP50, N = 137 worms) E. coli OP50 in the presence or absence of Giardia trophozoites (Giardia). OP50+Giardia, N = 160 worms; DOP50+Giardia, N = 190 worms. *P < 0.05 vs. OP50, SC^{(OP50+Giardia)}. #P < 0.05 vs. OP50, DOP50, DOP50+Giardia. Error bars represent SE for 9 wells containing all the worms in each group.

Fig. 5.

Giardia promotes colonization by E. coli in C. elegans. A: C. elegans intestine colonization by E. coli. Worms were incubated in a 96-well plate for 3 h with GFP-expressing E. coli HB101 in the presence (HB101GFP+Giardia) or absence (HB101GFP) of Giardia trophozoites, transferred onto nematode growth medium agar plates, and monitored for colonization. B–E: electron microscopy analysis of worms incubated with E. coli HB101 (B), Giardia trophozoites (C), and E. coli HB101 and Giardia trophozoites (D and E) for 12 h. E is higher magnification of rectangle area in D. Scale bars: 500 nm in B–D; 100 nm in E.

Giardia-Induced Changes in E. coli HB101 Gene Expression

To gain insights into the effects of Giardia on the virulence of commensal bacteria, we compared microarray gene expression profiles of E. coli HB101 controls with that of E. coli HB101 exposed to Giardia-conditioned medium alone, or to C. elegans alone, or to both Giardia-conditioned medium and C. elegans. We identified 172 genes differentially expressed by at least twofold, with P values less than 0.05 between groups (Fig. 6; Supplemental Table S1; Supplemental Material for this article is available on the Journal website). The microarray data were validated by qRT-PCR for eight genes (Fig. 7) showing a significant correlation of 0.97 (Spearman's ρ, P < 0.0001, N = 42) and 0.82 (Pearson's ρ, P < 0.0001, N = 42). Enrichment analysis indicated that most of these genes are involved in gene expression, gene translation, stress response, and hydrogen sulfide metabolism (Fig. 6B). Expression of pspB, activator of the PspB-PspC toxin-antitoxin stress response systems, and taurine ABC transport genes (tauA, tauB, and tauD), responsible for taurine uptake, was decreased, whereas expression of oxalate metabolic/catabolic genes (oxc, frc) and hydrogen sulfide biosynthesis genes was increased in E. coli HB101 upon exposure to C. elegans (Supplemental Table S1). More than 50% of the genes were only differentially expressed between any two of the groups. There were 44 and 57 genes showing differential expression only upon exposure to Giardia and C. elegans, respectively (Fig. 6C). Genes whose expression was affected only upon exposure to Giardia include those involved in taurine metabolism and transport (tauD, Taurin ABC transporter genes) and in translation (ribosomal protein genes; rpsS, rpsR, rpsP, rpsN, rpsM, rpsJ, rpsF, rpsA, rpmI, rpmG, rpmB, rplW, rplV, rplM, rplK, rplD, rplC, rplB, rplA). These 47 genes are preferentially enriched for expression regulation functions. The expression of taurine metabolism and transport genes was decreased, whereas expression of the ribosomal protein genes was increased upon exposure to Giardia-conditioned medium (Fig. 8A). Genes whose expression was affected only upon exposure to C. elegans include those involved in biofilm formation regulation and in flagella biosynthesis (Fig. 8B). Upon exposure to C. elegans, the expression of E. coli biofilm formation inhibitory genes (bssR, bssS) was increased, whereas the expression of flagella biosynthesis genes (fliL, fliP) was decreased (Supplemental Table S1, Fig. 8B). Preferentially enriched functions were not detected in the 57 genes affected only upon exposure to C. elegans reflecting the complexity of homeostatic interactions between commensal E. coli and C. elegans. There were 12 genes differentially expressed only between E. coli HB101 exposed to C. elegans and E. coli HB101 exposed to both C. elegans and Giardia-conditioned medium (Fig. 6C). The expression of these genes was increased in the E. coli group exposed to C. elegans (Fig. 9). The genes involved were stress response genes, such as phage shock protein operon (pspB, pspC, pspE), osmoregulatory trehalose synthesis (otsA, otsB), and superoxide dismutase (sodC). Most of the hydrogen sulfide biosynthesis genes expression was increased upon exposure to C. elegans but decreased upon exposure to Giardia-conditioned medium, both in the presence or absence of C. elegans (Figs. 6A and 10A). Since these genes are positively regulated by CysB, and since cysB gene expression remained unaffected in the different groups, we sought to test whether CysB mutant E. coli has a detrimental effect on C. elegans. Indeed, deletion of the cysB gene in E. coli was sufficient to kill worms in the absence of Giardia (Fig. 10B).

Fig. 6.

Giardia alters expression of E. coli stress response, hydrogen sulfide metabolism, and ribosomal protein genes. A: expression pattern of differentially expressed genes in E. coli HB101 incubated alone (HB101) or in the presence of Giardia-conditioned medium (HB101+GI), C. elegans (HB101+CE), or both Giardia-conditioned medium and C. elegans (HB101+CE+GI). The figure illustrates the expression patterns for top 33 most differentially expressed genes. B: functional category analysis of the differentially expressed genes. C: Venn diagram showing total number of genes differentially expressed between the different groups. Total number of genes differentially expressed only between any 2 of the groups are underlined.

Fig. 7.

Microarray data validation. Fold changes in expression of differentially expressed (fliP, cysC, zraR, fliL, and zraP) and nonaffected (cysG, lepB) genes were determined by quantitative RT-PCR (qRT-PCR) and compared with fold changes in the expression of the genes in the microarray data. The qRT-PCR and microarray data show significant correlation of 0.97 (Spearman's ρ, P < 0.0001, N = 42) and 0.82 (Pearson's ρ, P < 0.0001, N = 42).

Fig. 8.

Giardia alters expression of taurine metabolism and ribosomal protein genes whereas C. elegans alters expression of biofilm regulation and flagella biosynthesis genes. A: heat map of genes differentially expressed only upon exposure to Giardia-conditioned medium. Taurine metabolism and export genes are marked with red bar and ribosomal protein genes are marked with green bars. B: heat map of genes differentially expressed only upon exposure to C. elegans. Biofilm regulation genes are marked with green bars and flagella biosynthesis genes are marked with red bar. Average gene expressions from 3 independent experiments are shown.

Fig. 9.

Giardia counters C. elegans-increased expression of E. coli stress response genes. Heatmap shows genes differentially expressed between the C. elegans exposed and the C. elegans and Giardia-conditioned medium exposed E. coli. Average gene expressions from 3 independent experiments are shown.

Fig. 10.

Hydrogen sulfide biosynthesis genes are important in E. coli and C. elegans homeostatic interactions. A: heat map shows genes differentially expressed in all the E. coli groups. Hydrogen sulfide biosynthesis genes are marked with red bars. Average gene expressions from 3 independent experiments are shown. B: survival of C. elegans incubated with E. coli K12 wild-type (WT, N = 103 worms) and cysB-null mutant bacteria (cysB, N = 133 worms). *P < 0.005 vs. WT. Error bars represent SE for 7 wells containing all the worms in each group.

DISCUSSION

More research on the consequences of enteropathogen-induced microbiota disruptions is sorely needed to help uncover novel mechanisms of intestinal pathophysiology. The present study used C. elegans as a model system to investigate interactions between Giardia, commensal gut bacteria, and the host. Incubation of worms with Giardia trophozoites in the presence of E. coli was lethal to the worms, whereas exposure of C. elegans to Giardia or E. coli alone allowed for worm survival. Giardia was able to induce worm killing when coadministered with attenuated pathogenic strains of C. rodentium or with microbiota from a healthy individual. Giardia also enhanced worm killing by microbiota of inflamed colonic sites from an UC patient. Intriguingly, microbiota from noninflamed UC sites were not toxic and even conferred protection against the synergistic effect of Giardia coexposure, perhaps reflecting hitherto unknown protective benefits of the microbiota at intestinal sites not affected by inflammation in IBD. The purpose of using human donors for the present study was not to elaborate on patient-based findings but instead was simply to generate various microbiota samples. Clearly, large-scale functional characterization of microbiota from healthy donors and from patients with IBD by use of such a model should be carried out to help unravel new mechanistic insights into the pathogenic role of microbiota dysbiosis. Indeed, although mucosa-associated microbiota from inflamed UC sites are less diverse than those from noninflamed sites (41), it is not known whether the observed differences are a cause or an effect in IBD pathophysiology.

Together, the data suggest that Giardia may induce virulence in commensal gut bacteria. Gene expression analyses confirmed that indeed Giardia is able to alter expression of genes in E. coli, which has the potential to make commensal bacteria opportunistic pathogens. The physical presence of live E. coli was required for worm killing, suggesting that the synergistic toxicity is executed, at least in part, by the altered bacteria. Indeed, Giardia induced colonization of the C. elegans intestine with an otherwise noncolonizing E. coli bacteria.

Heat-insensitive secretory-excretory products from Giardia were sufficient to synergize with bacteria and kill C. elegans. Giardia secretory-excretory products, which contain heat-stable lipids and glycoproteins (28), modulate the host immune response and are implicated in pathogenesis via unknown mechanisms (15, 25, 28). Protein contents of the Giardia spent medium were not assessed in the present study, and further investigations are needed to identify the active component(s) of the effects of Giardia secretory-excretory products. Giardia exists as eight genotypically distinct Assemblages, of which only two, A and B, are infectious to humans (26). Future investigations will assess whether the synergistic toxicity of Giardia with commensal bacteria is Assemblage specific. No intact trophozoites were detected in the C. elegans intestine under electron microscopy. Whether the trophozoites were too big to fit inside the worm pharynx, did not survive grinding by the pharynx, or were unable to colonize the intestine requires further investigation. Whether and how worms may be able to extract nutrients from parasite trophozoites also represent an area for future research.

The analyses of gene expression reported herein shed light on the mechanisms whereby an enteropathogen like Giardia may synergize with commensal E. coli to induce pathogenic effects in a host. Exposure to Giardia preferentially enriched ribosomal proteins in E. coli HB101 genes. These findings raise the intriguing possibility that enteric pathogens may target gene expression in microbiota bacteria, which in turn may contribute to intestinal disease. Further studies are needed to determine whether the changes in ribosomal proteins reported here are at the core of alterations in virulence, or may instead, for example, simply reflect stress responses. Indeed, changes in ribosomal proteins modulate the degree and specificity of protein synthesis, and link posttranscriptional regulation of genes to specific cues such as stress (9, 33). Increased expression of ribosomal proteins represents a signature gene regulation in biofilm bacteria, with repression of flagellar genes, and increased expression of adhesion genes (30). Taurine transport genes (tauA, B, and C), important for taurine uptake in E. coli (48), were suppressed upon exposure to Giardia-conditioned medium. Taurine has been shown to ameliorate experimental colitis in rats (27, 46). These findings pave the way toward novel research into the mechanisms whereby enteropathogen-induced alterations in gut microbiota may contribute to postinfectious intestinal disease.

The present study revealed different gene expression patterns in E. coli HB101 upon exposure to C. elegans and Giardia-conditioned medium, which sheds further light on mechanisms of host-commensal interaction. The absence of preferential enrichment of functions in genes upon exposure to C. elegans alone may reflect the complexity of host-microbial interactions in homeostasis. Several genes coding for virulence are present but silenced in commensal E. coli during homeostasis (17), and the present findings indicate that host-microbiota interactions may help keep resident microbial virulence in check. For example, the expression of flagella biosynthesis genes (fliL and fliP) was decreased during E. coli HB101 interaction with C. elegans. The fliL gene loss has different effects in different bacteria, including loss of flagella (40), and, in Proteus mirabilis, it is involved in the ability of the bacteria to regulate expression of virulence genes (5). FliP also contributes to the production of virulence factors in Yersinia enterocolitica (54) and in Campylobacter jejuni (7, 12, 19, 29, 34, 36, 56). Similarly, expression of the citrate lyase gene (citE), the homolog of which is associated with virulence in Salmonella enterica (10), is suppressed during interaction with C. elegans. Genes negatively regulating biofilm formation (bssR and bssS) (18) were increased in E. coli interacting with C. elegans alone. More research is needed to characterize the role of the bacterial biofilm phenotype in host-microbiota homeostatic interactions.

E. coli stress response genes appear to be important in homeostatic host-microbial interactions as their expression was increased upon exposure to C. elegans, whereas the presence of Giardia-conditioned medium countered these host-induced changes (Fig. 9). The role of hydrogen sulfide biosynthesis genes of commensal bacteria in homeostatic interactions with the host remains poorly understood. However, the significance of hydrogen sulfide in protecting the gut against inflammation is now well established (52). It is interesting to find that these genes were increased upon exposure to C. elegans, but decreased upon exposure to Giardia-conditioned medium in the presence or absence of C. elegans (Fig. 6A). Finally, E. coli bacteria lacking the cysB gene, a positive regulator of hydrogen sulfide biosynthesis, directly reduced the survival of C. elegans (Fig. 10B). This further supports the hypothesis that the microbial hydrogen sulfide biosynthesis system plays an important role in host-commensal homeostatic interactions.

In conclusion, using this novel C. elegans model system, findings from this study establish new paradigms of host microbial interactions in the gut. The results demonstrate that Giardia secretes/excretes factors that synergize with commensal bacteria to disrupt homeostatic host-microbial interactions. The mechanisms involve enteropathogen-induced alterations of gene expression in commensal bacteria. These findings are consistent with recent observations that Giardia-induced microbiota dysbiosis is able to cause intestinal inflammation in germ-free mice (4). Since microbial-microbial synergies can be readily modeled in C. elegans by monitoring worm survival in a 96-well plate, our study presents a high-throughput model system for mechanistic investigations of host-microbial interactions.

GRANTS

This work was supported by a discovery grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to A. G. Buret, by a grant from Crohn's and Colitis Canada Foundation, and by the Eli and Edythe Broad Foundation. T. K. Gerbaba's fellowship was supported by the NSERC CREATE Host-Parasite Interactions program at the University of Calgary.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

T.K.G. and A.G.B. conception and design of research; T.K.G. and P.G. performed experiments; T.K.G., P.G., K.R., D.H., and A.G.B. analyzed data; T.K.G. and A.G.B. interpreted results of experiments; T.K.G. prepared figures; T.K.G. drafted manuscript; T.K.G., K.R., D.H., and A.G.B. edited and revised manuscript; T.K.G. and A.G.B. approved final version of manuscript.