Characterization of the Giardia intestinalis secretome during interaction with human intestinal epithelial cells: The impact on host cells

Abstract

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.

Giardia intestinalis cystatin is a potent inhibitor of papain, parasite cysteine proteases and, to a lesser extent, human cathepsin B

“…The stefins are mainly intracellular inhibitors but they were found in body fluids or in the excretory-secretory products (ESPs) of trematodes [48][49][50]. We confirmed robust expression of G. intestinalis cystatin in trophozoites and encysting trophozoites, as previously indicated by separate studies under different stress conditions, differentiation and host-parasite interactions [3,32,34,35,51]. In one of the studies equally robust expression of cystatin comparable to trophozoites cultures was shown in the mouse intestine, further highlighting the potentially important role of cystatin during infection [35].…”

“…B). We further tested the inhibition capacity of G. intestinalis cystatin to human cathepsin B, which is highly expressed in human intestine and abundant in the secretome of intestinal cell lines during interaction with Giardia trophozoites (https://www.proteinatlas.org/ENSG00000164733-CTSB/tissue, ), and the inhibiting activity was weaker ( K i of 79.88 ± 16.76 n m ) as compared to papain (Fig. C).…”

“…In the gut, professional APC collect antigens via M cells in the Peyer's patches and epithelial cells also express high levels of MHC class II molecules [58]. Cathepsins are also released upon contact with Giardia parasites [3]. We therefore speculate that Giardia cystatin might contribute to the overall immune modulating effect mediated by Giardia during infection, however, its potential role in Giardia immunomodulatory function requires further investigation [1].…”

Peptidylarginine Deiminase inhibition abolishes the production of large extracellular vesicles fromGiardia intestinalis, affecting host-pathogen interactions by hindering adhesion to host cells

“…EVs from Giardia have previously been studied in host-pathogen interactions through proteomic analysis of excretory-secretory products (ESP), including EVs of axenic cultures and cultures of trophozoite interacting with mammalian cells, where proteins related to metabolism were found, without signal peptides on EVs (Ma’ayeh et al 2017). In addition, ESP containing cleavage activity trough cysteine proteases have been identified (de la Mora-de la Mora et al, 2019).…”

The compact genome ofGiardia murisreveals important steps in the evolution of intestinal protozoan parasites

“…Its G. intestinalis homolog is up-regulated and secreted during interactions with human 480 intestinal epithelial cells 8 and it cleaves chemokines, tight junction proteins and 481 defensins 12,75 . Thus, this is most likely also an important virulence factor in G. muris.…”