Life Cycle Giardia cysts can contaminate food, water, and surfaces, and they can cause giardiasis when swallowed in this infective stage of their life cycle. Infection occurs when a person swallows Giardia cysts from contaminated water, food, hands, surfaces, or objects. When Giardia cysts are swallowed, they pass through the mouth, esophagus, and stomach into the small intestine where each cyst releases two trophozoites through a process called excystation.
The Giardia trophozoites then feed off and absorb nutrients from the infected person. Giardia trophozoites multiply by splitting in two in a process called longitudinal binary fission, remaining in the small intestine where they can be free or attached to the inside lining of the small intestine.
The Giardia trophozoites then move toward the colon and transform back into cyst form through a process called encystation. The Giardia cyst is the stage found most commonly in stool.
Both Giardia cysts and trophozoites can be found in the stool of someone who has giardiasis and may be observed microscopically to diagnose giardiasis. Giardia cysts are immediately infectious when passed in the stool or shortly afterward, and the cysts can survive several months in cold water or soil. Life cycle image and information courtesy of DPDx. Isolation and characterization of two distinct EV populations produced from Giardia intestinalis. B Quantification of vesicle numbers from nanoparticle tracking analysis NTA.
C Time-course for culture confluence of trophozoites induced to produce EVs for 1, 3, and 6 h, respectively. In addition, we assessed the ability to block host-pathogen interactions following treatment with the EV-inhibitors. Indeed, both compounds were capable of decreasing trophozoite adhesion to the Caco-2 cell monolayer Figure 2B. Figure 2. E Cytotoxic effects of the EV-inhibitors on Caco-2 cell monolayers. Cell viability was determined by the crystal violet method.
EVs were exposed to heat inactivation before treatment of co-cultures; trophozoites treated with Cl-am and EVs showed a higher attachment to Caco-2 cells when compared to parasites treated with Cl-am and heat-inactivated EVs Supplementary Figure 2.
We performed a toxicity test using mammalian cells, and PAD-inhibitor or CBD had no cytotoxic effects at 48 h post-treatment, compared with albenzadole, one of the common drugs used for giardiasis treatment Figure 2E.
Trophozoites exposed to the EV-inhibitors for 1 h in the vesiculation protocol were resuspended in culture tubes and did not show any significant difference in growth-curves, compared with the control culture Supplementary Figure 3.
These compounds did not affect parasite adhesion Figure 2F. LEVs derived from the parasite were capable of restoring the adherence phenotype following treatment with Cl-amidine, in a dose-dependent manner Figure 3A. In contrast, no effect was observed in the SEVs treated groups. These results suggest that physical properties related to adherence can be found in the larger Giardia EVs and therefore, EVs produced by the parasite may selectively influence its phenotype.
Figure 3. Parasite derived EVs are selectively involved with Giardia adhesion to host cells. B Host-pathogen assay after treatment of Caco-2 cell monolayer with Cl-amidine and incubation with mammalian cell derived EVs. We next assessed if the EVs from the host contribute to the adherence process to host cells. Confluent Caco-2 cell monolayers were washed and thereafter treated with Cl-amidine, and trophozoites were added to the wells, followed by treatment with Caco-2 cell-derived EVs Figure 3B.
Moreover, we sought to investigate the influence of cysteine peptidase inhibitors on parasite adhesion to Caco-2 cells, treated with IAA. The cysteine protease inhibitor caused a significant reduction in trophozoite adhesion to host cells Supplementary Figure 4 but this effect was reduced when the parasites were incubated with LEVs from the parasites, indicating that protease activity is not related to the effect on adhesion, observed for EVs.
Important products related to the Giardia genre were found common to both EVs, including antigenic Variable Surface Proteins VSPs , giardins, cathepsin B, and other virulence factors arginine deiminase, ornithine carbamoyltransferase.
There were 77 proteins exclusively found in LEVs related to cytoskeleton composition and protein binding Figures 4A,B. Products identified in LEV are associated with the cytoskeleton, as well as oxidative stress responses, such as Peroxiredoxin-1 Supplementary Table 2. SEVs exclusively contained 19 proteins of which some related to ribosome metabolism. Bioinformatic analysis of the Giardia EVs proteome suggests the enrichment of enzymatic and cytoskeletal products, metabolic processes, as well as stress response to oxygen Figure 5.
Analysis of LEV and SEV specific proteins revealed a distinct cellular sub-localization, such as cytoplasm, nucleus, and plasma membrane, while SEVs proteins were mostly cytoplasmic. Protein-protein interaction and gene ontology analysis of LEV and SEV unique proteins revealed different biological processes and different structural domains Figures 6A—C.
Figure 4. EV subtypes derived from G. Figure 5. A Protein-protein interaction networks. B Gene ontology. C Enriched metabolic pathways. Figure 6. B,C Biological processes and Pfam enriched domains are shown, respectively.
Confocal microscopy revealed punctuated patterns of fluorescence distributed intracellularly in Caco-2 cells Figure 7A. Both populations appeared to be taken up by the host cells in a dose-dependent manner. Despite LEV intensity internalization being observed to be higher overall, which may relate to larger vesicle size Figure 7B , final intracellular destiny of the EVs was not determined.
Figure 7. The two G. B Internalized EVs were quantified by means of fluorescence intensity. A role for one of the identified EV populations in host-pathogen interactions was demonstrated, as treatment of G. EV modulatory strategies have been highlighted as an increasingly important approach in a range of pathologies Jorfi et al.
Therefore, our current findings highlight that such strategies in giardiasis may be of considerable importance. The pan-PAD-inhibitor Cl-amidine has previously been described as a potent EV-inhibitor, compared to a range of other compounds, in various cancer cells Kosgodage et al.
Studies have also shown that PAD-inhibitors can be strategically used to sensitize cancer cells to chemotherapy Kholia et al. The effect of PAD-inhibitors on EV release furthermore seems to be a phylogenetically conserved pathway as PAD-inhibitors were also found to reduce EV release from bacteria and accordingly, to effectively sensitize bacteria to antibiotics Kosgodage et al. The EV-modulatory functions of CBD were also recently revealed, and CBD has even been found to be a more potent EV inhibitor than Cl-amidine in some cancer cell types, also to have chemosensitizing effects and showing selective inhibition on smaller or larger EVs according to cancer type Kosgodage et al.
Furthermore, CBD was recently revealed to reduce bacterial EV release, modify proteomic content of bacterial EVs, and to sensitize certain bacteria to antibiotic treatment via this pathway Kosgodage et al. Such EV-modulatory functions, as also observed in our current study in Giardia , may correlate to the reported effects of cannabinoids as anti-parasitic agents, where inhibitory effects on parasite invasion and the immunosuppression of trypanosomiasis has been reported Nok et al.
Cannabinoids have furthermore been shown to be effective anti-helmitics Roulette et al. Our current findings may therefore be of considerable interest for putative use of CBD in giardiasis. The field of EV research is still rapidly growing, with characterization of functions of subpopulations gaining increased attention. The complex function of LEVs revealed here in Giardia , suggests that their influence on phenotypes could be even more diverse than those of SEVs Tkach et al.
No biomarkers were considered in the present study, since both EV populations are enriched mixtures of vesicles that fail to contain any unique marker Kalra et al. Properties related to different functions of LEV have been studied in non-infectious models. LEVs microvesicles derived from platelets were also associated with polymorphonuclear leucocytes increase in adhesion Fujimi et al. On the other hand, properties related to cellular adhesion for SEVs isolated from two cancer cell lines have also been identified while the same was not detected for LEVs Jimenez et al.
LEVs identified in Fasciola hepatica contained protein cargo related to digestion cathepsin L1 zymogen , while proteomic and functional analyses identified membrane structure components and immunomodulation factors in SEVs Cwiklinski et al. EVs from Giardia have previously been studied in host-pathogen interactions. Evans-Osses et al. Moyano et al. According to Saha et al. Wampfler et al. The latter appears to assume a functional role similar to the endoplasmic reticulum, such as recruitment of ribosomes to organelle membranes Wampfler et al.
Benchimol studied the release of ESVs on giardial cell surface. They detected large granules docking in the plasma membrane. In addition, Midlej et al. Using electron microscopy techniques, they demonstrated the exocytosis of those vesicles and recovery in the supernatant.
In another study, a proteomic analysis of excretory-secretory products ESP , was conducted, including EVs of axenic cultures and cultures of trophozoite interacting with mammalian cells, identifying proteins related to metabolism, without signal peptides on EVs Ma'ayeh et al. Our proteomic analysis of Giardia EVs detected relevant virulence factors and immunogenic molecules such as cathepsin-B, arginine-metabolizing enzymes, and VSPs in both EV subpopulations.
Many of the proteins found are associated with parasite cytoskeleton, such as giardins, katanin, and ankyrin repeat proteins. Due to the prominent role of proteins from cytoskeletons in parasite adhesion and virulence, we propose that Giardia adhesion to epithelial cells could be related to surface molecules and Disk-associated proteins, identified here to be contained in LEVs. These proteins are rich in ankyrin repeats and may contribute to attachment, protein-protein interactions, and stability Weiland et al.
Some of the giardins detected in LEVs, such as the alpha-1 giardin, are capable of binding to glycosaminoglycans present in the intestinal epithelial monolayer, and hence can play a role in the early host—parasite interplay Weiland et al.
Giardia has to survive an unfriendly environment in the small intestine, while it lacks mitochondria and a conventional ROS-scavenging enzyme, such as catalase, superoxide dismutase, and glutathione GSH peroxidase. These products were also identified in the work of Ansell et al. Ma'ayeh et al. Isolate GS revealed higher levels of Peroxiredoxin-1 and other antioxidative products.
In another work from the same authors Ma'ayeh et al. Our evidence of dose-dependent internalization of EVs, together with proteomic data on cytoskeleton protein enrichment, suggests that EVs may be associated with the recovery of trophozoite adhesion capacity altered with the PAD-inhibitor.
Experiments with overexpression of genes in trophozoites, to be released by vesicles as blockage with monoclonal antibodies, could give an idea of whether the type of interaction is specific and assess downstream effect on molecular EV-cargo.
Understanding the internalization and intracellular destiny of EVs and EV subpopulations is a future challenge for further in-depth studies. While the majority of Giardia infected individuals are asymptomatic, giardiasis is a major contributor to malnutrition and growth impairment in children from developing countries Fink and Singer, Additionally, the disease may also last for a long term chronic infection.
Therefore, it is important to identify and study novel clinical strategies that can lead to host recovery. PAD-inhibitor Cl-amidine and CBD were here shown to effectively decrease parasitic EV release, which contributes to parasite adherence into intestinal epithelial cells Caco-2 cells. They may therefore pose as novel therapeutic candidate agents for cases of chronic giardiasis.
Our results suggest that the two EV populations identified in G. Since adhesion in the epithelial intestine is fundamental to parasite fitness and invasion, and LEVs clearly aid this process, the use of targeted EV—inhibitors, such as Cl-amidine identified here, can be used to selectively interfere with EV secretion, allowing novel treatment strategies in the control of giardiasis. Figure 8. The original contributions presented in the study are publicly available. All authors planned experiments.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We would like to thank Dr. Thanks to Prof. VF is supported by CNPq. Supplementary Figure 1. EV protein estimation from parasite and mammalian cells Caco Supplementary Figure 2. Supplementary Figure 3. Supplementary Figure 4. Host-pathogen assay treated with a protease inhibitor and LEVs. Supplementary Table 1.
Supplementary Table 2. Products associated with cytoskeleton microtubules , ventral disk, median body and flagella, and oxidative stress responses found in LEVs. Andersson, J. A genomic survey of the fish parasite Spironucleus salmonicida indicates genomic plasticity among diplomonads and significant lateral gene transfer in eukaryote genome evolution. BMC Genomics Ankarklev, J. Behind the smile: cell biology and disease mechanisms of Giardia species. To overcome the parasite survival barrier, most in vitro studies have utilized high multiplicities of infection in addition to the short incubation times.
These conditions do not reflect a typical Giardia infection where a low infectious dose leads to an active infection that can span several days to weeks. Therefore, establishing an in vitro model that allows for the protracted co-culture of host epithelial and immune cells with Giardia trophozoites would greatly contribute to the understanding of late infection interactions.
Our model utilizes transwell inserts to co-culture a human intestinal epithelial cell line Caco-2 and a murine peritoneal macrophage cell line IC in a manner that represents the apical-basolateral orientation of the small intestine Figure 1. IC murine peritoneal macrophages were selected for their similarity to human macrophages, including their typical macrophage morphology in culture, their expression of macrophage-specific antigens, their activation by lipopolysaccharide, their phagocytic ability [31] , and their expression of IgG receptors [68].
Caco-2 cells, derived from a human colonic adenocarcinoma retain both morphologic and phenotypic characteristics of intestinal epithelial cells when fully differentiated, including polarized morphology, microvilli on the apical surface, expression of brush border enzymes, and adjacent cell tight junctions reviewed in [69] , [58].
The in vitro differentiation of Caco-2 cells into a phenotype similar to small intestinal epithelial cells is a time-mediated event that is dependent on many factors, including passage number, seeding density, media composition, and substrate support reviewed in [58]. In our study, the use of Caco-2 cells at three days post plating allows for the assessment of how the parasite affects the proliferation and differentiation process of enterocytes in the intestine.
The epithelial barrier of the intestine is replenished every 4—5 days; therefore, enterocyte renewal through stem cell differentiation is critical for normal functioning of the human gut reviewed in [70] , [71]. Caco-2 cells have been used to model the differentiation process of enterocytes in the small intestine [72]. Although immature proliferating Caco-2 cells show differences in gene expression [73] , the protein expression profiles are remarkably similar [74] when compared to fully differentiated Caco-2 cells.
Thus far, Caco-2 cells are the best described enterocyte cell line and the most common epithelial cell line used in in vitro Giardia —host interactions. Therefore, using Caco-2 cells to characterize our co-culture model allows us to compare our results with those published in the literature. Following establishment of cell-cell communication and epithelial monolayer formation, Giardia trophozoites were added to the system using a lower starting density than what has previously been reported.
This model allows the parasite to proliferate in culture, more accurately reflects the infection in vivo , and allows for the characterization of host- Giardia interactions from the start of an infection through its termination, including the role if any of immune cells in limiting the infection. Using transwell inserts filled with medium, we were able to limit the oxygen exposure of the parasites, while allowing the epithelial cells to exchange oxygen and nutrients through their basolateral surface.
The drop in parasite density after 5 days is attributed to daily feeding of the insert cultures. Increased metabolism in co-cultures modeling infectious disease can drastically alter the media composition, pH, and by-product accumulation in the system [28] ; therefore, a feeding strategy must be employed to offset these effects.
We elected to only remove half the culture medium every other day until day five so that the host-parasite interface remained undisturbed until the infection was established. After five days, parasite density peaked and unattached parasites had to be removed daily. This approach mimics in vivo conditions where unattached parasites are pushed down the intestine and are ultimately excreted from the host.
Therefore, further optimization of the insert model, such as altering parasite inoculation density or media composition, could allow parasite survival in the absence of host cells. This would make assessment of host-induced changes in Giardia gene expression feasible using our developed model.
Our experimental design recapitulates the architecture of the gastrointestinal tract where Giardia trophozoites attach apically to the epithelium and epithelial cells interact basolaterally with macrophages in the lamina propria. Using human differentiated macrophages isolated from buffy coats for the cytokine array further illustrates the versatility of the model.
However, due to low isolation numbers, lack of proliferation, and difficulty in maintaining the human monocyte-derived macrophages in culture, IC cells were used in all other experiments to characterize our model. Epithelial cell apoptosis as a mechanism of barrier dysfunction during giardiasis has been well documented in vitro [19] , [18] , in human biopsies [17] , and in mouse models with G.
However, the results of those experiments have been contradictory with regards to the degree of apoptosis observed as well as the Giardia assemblage s capable of inducing epithelial cell death. Studies using sonicated Giardia lamblia strain WBC6 assemblage A , failed to elicit epithelial cell apoptosis [19] ; a finding inconsistent with other work using live WBC6 trophozoites [18]. To assess apoptosis in our model, we compared caspase-3 activation in Caco-2 cells on inserts to the long-established monoculture plate environment.
Our results indicate that live WBC6 trophozoites can induce apoptosis in a time-dependent manner. Since sonicated Giardia WBC6 parasites fail to produce the same response [19] , this may indicate this particular Giardia strain mediates host cell death through a direct parasite-epithelial cell interaction.
The difference in apoptosis observed between plate and insert cultures at 5 days in our studies is likely due to parasite density in the different culture conditions. Significantly more parasites are observed in the insert environment even though the plate and insert cultures received the same starting density of parasites.
We speculate that the reduced parasite proliferation in the plate is due to high oxygen tension, which is deleterious to Giardia trophozoites [76]. Together, these data suggest that our model is a better representation of giardiasis as parasites can reach higher densities in the insert environment and remain viable over many days.
Indeed, the contradictory data on apoptosis during giardiasis is likely due to many factors including Giardia strain utilized, parasite density, and type of epithelial cell line used. The cytokine array illustrated the importance of co-culture in modulating cell phenotype.
Caco-2 cells exhibit a different cytokine profile in the presence of human differentiated macrophages, which has been previously reported [77]. Secretion of chemotatic cytokines, such as GRO isoforms and MCP-1, by intestinal epithelial cells incubated with Giardia has been reported [15]. However, Giardia failed to elicit secretion of these cytokines from Caco-2 cells cultured alone and, in fact, suppressed the cytokine expression of Caco-2 cells cultured with macrophages by abolishing IL-8 and GRO secretion.
Immuno-regulation of host defenses has been observed in Giardia [78] and other parasitic infections Reviewed in [79] , [59]. Differences in experimental design and Caco-2 cell differentiation state between the two studies as well as parasite density could explain some of the disparities. Furthermore, differences in the cytokine profile of intestinal epithelial cells during Giardia infection could be attributed to Giardia assemblage.
Differences in virulence between assemblage A and B have been previously suggested [10] — [12]. The role of macrophages in human giardiasis has yet to be fully resolved. Macrophages can actively phagocytose trophozoites in vitro [23] and in infected mice [21].
However, only low numbers of macrophages are observed in the lumen of Giardia infected animals [81] ; therefore, phagocytosis alone probably does not contribute greatly to parasite control as Giardia is mostly non-invasive and does not cross the epithelial barrier. Using the co-culture model, we assessed if macrophages play a role in Giardia pathology by secreting cytokines or stimulating epithelial cell proliferation.
In other tissues, macrophage cytokine secretion can activate epithelial cell proliferation as a means to repair damage or maintain homeostasis [82] , [55]. In our studies, macrophages did not induce proliferation of Caco-2 cells exposed to Giardia parasites. The decrease in Caco-2 cell number in the presence of Giardia is likely due to epithelial cell apoptosis.
The increase in caspase-3 activity in Caco-2 cells incubated with Giardia was quite substantial as it surpassed the levels induced by the camptothecin, a strong inhibitor of DNA synthesis. The dissociation between macrophages and epithelial cell proliferation in the gastrointestinal tract could be in part due to the unresponsive, anergic nature of intestinal macrophages reviewed in [83] , [84]. Additionally, macrophages do not control Giardia infection through cytokine secretion as the cytokine profile of the macrophages did not change in the presence of parasites.
If this is due to regulation of epithelial cell cytokine secretion by the parasite is yet to be determined. As monolayer integrity is compromised at later time points, this model can investigate disease physiology, such as altered transport function leading to malabsorption, until about 13 days post-infection.
Incubations spanning longer than 13 days can be utilized for pathological studies. This model can be adapted to define culture conditions for the long-term culture of other Giardia strains, which will allow for the identification of strain-specific effects on host cells that may contribute to the wide spectrum of disease symptoms and infection duration.
In addition, using the co-culture model for additional characterization of cytokine profiles unique to Giardia infections will provide insights into the underlying mechanisms of host immune suppression by the parasite. Overall, this model can help identity mechanisms of disease in giardiasis that can then be used as targets of therapeutic intervention. Immunofluorescence microscopy analysis of parasites in the media mixes. Parasites were incubated with a cyst specific antibody green.
Nuclei were stained with DAPI blue. Pictures represent the merged images of DAPI and cyst antibody. IC macrophages in the co-culture. A macrophage is identified with an arrow. Video of co-culture at day 0. The co-culture consisting of Caco-2 cells and IC macrophages was assembled as described and incubated for 3 days.
Giardia trophozoites were then added at , total parasites and a video was taken immediately. The authors would like to thank Dr. Randal Buddington Professor, University of Memphis for helpful suggestions on experimental design. Performed the experiments: BSF. Wrote the paper: BSF. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract Globally, there are greater than , deaths per year associated with diarrheal disease. Introduction Giardia lamblia , also known as G.
Co-culture model IC cells were plated on the bottom of a 0. Download: PPT. Effect of formononetin treatment on signaling in Caco-2 cells Lauwaet et al [33] used the isoflavone formononetin to rapidly detach Giardia from intestinal cells. Microscopy Live images of the co-culture were obtained for day 1, 5, 13, and 21 using a Nikon Eclipse TS microscope with the Nikon Digital Sight camera.
Cell number assay Co-cultures were plated as previously described. Cytokine array Human macrophages were differentiated from blood monocytes isolated from donated buffy coats Key Biologics, Memphis, TN using Percoll density gradients as described [36]. Results Defining the co-culture requirements for Giardia and Caco-2 cells The life cycle of the Giardia parasite has two distinct stages, the infective cyst and the metabolically active trophozoite found in the intestine.
Figure 2. Morphology and viability of Caco-2 cells in media mixes. Figure 3. Giardia trophozoite proliferation and attachment in co-culture media G. Figure 4. Detachment of trophozoites using formononetin has no effect on MAPK signaling in Caco-2 cells Traditionally, Giardia trophozoites are removed from surfaces, including epithelial cells, by cold shock [32].
Figure 5. Effect of formononetin on stress activated kinases in Caco-2 cells. Giardia decreases epithelial cell number Previous in vitro monolayer co-culture studies have shown decreased epithelial cell proliferation in response to Giardia interaction [15] , [54] ; however, co-culture studies with lung epithelial cells and activated macrophages have revealed that cytokine production by macrophages can induce epithelial cell proliferation as a mechanism to repair tissue damaged during the inflammation process [55].
Giardia increases caspase-3 activity in Caco-2 cells To investigate the mechanisms involved in epithelial cell number decrease during Giardia interaction, we measured the impact of Giardia on Caco-2 cell apoptosis.
Figure 7. Caspase-3 activity in Caco-2 cells incubated with Giardia. Modulation of host cytokines by Giardia Cytokines are important in determining the type of adaptive immune response initiated while also advancing the innate response [59] ; however, little is known about these processes in giardiasis. Figure 8. Cytokine profile of Caco-2 cells and macrophages in the co-culture system.
Long-term viability of cells and parasites in the co-culture model Since Giardia infections can span several days to weeks in both mice [64] and humans [65] , [14] , constructing a model that allows for long-term incubation of parasites with epithelial cells is essential to understanding long-term infection interactions on the cellular level. Figure 9. Giardia trophozoites in the day co-culture. Figure Morphology of Giardia and Caco-2 cells over 21 days.
Discussion Several co-culture models of the human intestine have been developed [37] , [66] ; however, no experimental design has been adapted for prolonged Giardia -host interactions. Supporting Information.
Figure S1. Figure S2. Figure S3. Live image of co-culture model at 1 day. Figure S4. Live image of co-culture model at 5 days. Figure S5.
Live image of co-culture model at 13 days. Figure S6. Live image of co-culture model at 21 days. Figure S7. Video S1. Acknowledgments The authors would like to thank Dr. References 1. Infectious Disorders - Drug Targets —
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