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BackgroundGiardia intestinalis is a non-invasive protozoan parasite that causes giardiasis in humans, the most common form of parasite-induced diarrhea. Disease mechanisms are not completely defined and very few virulence factors are known.MethodologyTo identify putative virulence factors and elucidate mechanistic pathways leading to disease, we have used proteomics to identify the major excretory-secretory products (ESPs) when Giardia trophozoites of WB and GS isolates (assemblages A and B, respectively) interact with intestinal epithelial cells (IECs) in vitro.FindingsThe main parts of the IEC and parasite secretomes are constitutively released proteins, the majority of which are associated with metabolism but several proteins are released in response to their interaction (87 and 41 WB and GS proteins, respectively, 76 and 45 human proteins in response to the respective isolates). In parasitized IECs, the secretome profile indicated effects on the cell actin cytoskeleton and the induction of immune responses whereas that of Giardia showed anti-oxidation, proteolysis (protease-associated) and induction of encystation responses. The Giardia secretome also contained immunodominant and glycosylated proteins as well as new candidate virulence factors and assemblage-specific differences were identified. A minor part of Giardia ESPs had signal peptides (29% for both isolates) and extracellular vesicles were detected in the ESPs fractions, suggesting alternative secretory pathways. Microscopic analyses showed ESPs binding to IECs and partial internalization. Parasite ESPs reduced ERK1/2 and P38 phosphorylation and NF-κB nuclear translocation. Giardia ESPs altered gene expression in IECs, with a transcriptional profile indicating recruitment of immune cells via chemokines, disturbances in glucose homeostasis, cholesterol and lipid metabolism, cell cycle and induction of apoptosis.ConclusionsThis is the first study identifying Giardia ESPs and evaluating their effects on IECs. It highlights the importance of host and parasite ESPs during interactions and reveals the intricate cellular responses that can explain disease mechanisms and attenuated inflammatory responses during giardiasis.

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Giardiasis is a common diarrheal disease caused by the protozoan parasite Giardia intestinalis. Cysteine proteases (CPs) are acknowledged as virulence factors in Giardia but their specific role in the molecular pathogenesis of disease is not known. Herein, we aimed to characterize the three main secreted CPs (CP14019, CP16160 and CP16779), which were identified by mass spectrometry in the medium during interaction with intestinal epithelial cells (IECs) in vitro. First, the CPs were epitope-tagged and localized to the endoplasmic reticulum and cytoplasmic vesicle-like structures. Second, we showed that recombinant CPs, expressed in Pichia pastoris, are more active in acidic environment (pH 5.5-6) and we determined the kinetic parameters using fluorogenic substrates. Third, excretory-secretory proteins (ESPs) from Giardia trophozoites affect the localization of apical junctional complex (AJC) proteins and recombinant CPs cleave or re-localize the AJC proteins (claudin-1 and -4, occludin, JAM-1, β-catenin and E-cadherin) of IECs. Finally, we showed that the ESPs and recombinant CPs can degrade several chemokines, including CXCL1, CXCL2, CXCL3, IL-8, CCL2, and CCL20, which are up-regulated in IECs during Giardia-host cell interactions. This is the first study that characterizes the role of specific CPs secreted from Giardia and our results collectively indicate their roles in the disruption of the intestinal epithelial barrier and modulating immune responses during Giardia infections.

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