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Peptidylarginine Deiminase Inhibition Abolishes the Production of Large Extracellular Vesicles From Giardia intestinalis, Affecting Host-Pathogen Interactions by Hindering Adhesion to Host Cells.

ABSTRACT: Giardia intestinalis is a microaerophilic protozoan that is an important etiologic agent of diarrhea worldwide. There is evidence that under diverse conditions, the parasite is capable of shedding extracellular vesicles (EVs) which modulate the physiopathology of giardiasis. Here we describe new features of G. intestinalis EV production, revealing its capacity to shed two different enriched EV populations: large (LEV) and small extracellular vesicles (SEV) and identified relevant adhesion functions associated with the larger population. Proteomic analysis revealed differences in proteins relevant for virulence and host-pathogen interactions between the two EV subsets, such as cytoskeletal and anti-oxidative stress response proteins in LEVS. We assessed the effect of two recently identified inhibitors of EV release in mammalian cells, namely peptidylarginine deiminase (PAD) inhibitor and cannabidiol (CBD), on EV release from Giardia. The compounds were both able to effectively reduce EV shedding, the PAD-inhibitor specifically affecting the release of LEVs and reducing parasite attachment to host cells in vitro. Our results suggest that LEVs and SEVs have a different role in host-pathogen interaction, and that treatment with EV-inhibitors may be a novel treatment strategy for recurrent giardiasis.

SUBMITTER: Gavinho B

PROVIDER: S-EPMC7539837 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Peptidylarginine Deiminase Inhibition Abolishes the Production of Large Extracellular Vesicles From Giardia intestinalis, Affecting Host-Pathogen Interactions by Hindering Adhesion to Host Cells.

Gavinho Bruno B Sabatke Bruna B Feijoli Veronica V Rossi Izadora Volpato IV da Silva Janaina Macedo JM Evans-Osses Ingrid I Palmisano Giuseppe G Lange Sigrun S Ramirez Marcel Ivan MI

Frontiers in cellular and infection microbiology 20200923

Giardia intestinalis is a microaerophilic protozoan that is an important etiologic agent of diarrhea worldwide. There is evidence that under diverse conditions, the parasite is capable of shedding extracellular vesicles (EVs) which modulate the physiopathology of giardiasis. Here we describe new features of G. intestinalis EV production, revealing its capacity to shed two different enriched EV populations: large (LEV) and small extracellular vesicles (SEV) and identified relevant a ...[more]

PMID: 33072615

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Project description:BackgroundThe unicellular protozoan parasite Giardia intestinalis, which primarily infects humans and animals such as cattle and sheep, is having a major negative impact on public health. Giardia is able to evade the recognition and elimination of the host immune system because of the trophozoite surface and extracellular vesicles (EVs) covered by variant-specific surface proteins (VSPs). As key proteins for immune evasion, whether VSPs can regulate Giardia-induced pyroptosis and promote Giardia evasion of host immune responses has not been reported.MethodsTo examine the role of Giardia VSPAS7 on Giardia-induced activation of the signaling pathway, secretion of pro-inflammatory cytokines, pyroptosis and the mechanism involved, we constructed the pcDNA3.1-vspas7 expression plasmid and transfected this plasmid into mouse macrophages. Key proteins for pyroptosis, IL-1β secretion and LDH release were detected in pcDNA3.1-vspas7-transfected wild-type (WT) cells and NLRP3-deficient cells by western blot, ELISA and LDH assays, respectively. The interactions of Giardia VSPAS7 and mouse NLRP3 were examined using immunofluorescence assays (IFA), co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays.ResultsVSPAS7 could decrease the levels of phosphorylated-p65 (P-p65), P-IκBα and P-ERK caused by Giardia and reduce the production levels of Giardia-induced pro-inflammatory cytokine IL-6, IL-12 p40 and TNF-α. The results showed that VSPAS7 inhibited Giardia-mediated activation of NF-κB, ERK/MAPK signaling and secretion of pro-inflammatory cytokines. Furthermore, VSPAS7 suppressed Giardia-induced macrophage pyroptosis by reducing GSDMD cleavage, caspase-1 activation, IL-1β secretion and LDH release. We further found that VSPAS7 could interact with mouse NLRP3 directly, and in NLRP3-deficient cells the suppression of Giardia-induced macrophage pyroptosis by VSPAS7 was significantly attenuated.ConclusionsOverall, VSPAS7 could inhibit Giardia-induced activation of signaling pathways and pyroptosis in host macrophages, allowing Giardia evasion of host immune responses. Studies on Giardia VSP-mediated immune evasion provide an important theoretical basis for in-depth studies on Giardia pathogenicity.

Project description:The alveolates (Superphylum Alveolata) comprise a group of primarily single-celled eukaryotes that have adopted extremely diverse modes of nutrition, such as predation, photoautotrophy and parasitism. The alveolates consists of several major phyla including the apicomplexans, a large group of unicellular, spore forming obligate intracellular parasites, and chromerids, which are believed to be the phototrophic ancestors of the parasitic apicomplexans. Molecular pathways involved in Alveolata host-pathogen interactions, epigenetic regulation and metabolism in parasite development remain to be fully understood. Peptidylarginine deiminases (PADs) are a phylogenetically conserved enzyme family which causes post-translational protein deimination, affecting protein function through the conversion of arginine to citrulline in a wide range of target proteins, contributing to protein moonlighting in physiological and pathological processes. The identification of deiminated protein targets in alveolate parasites may therefore provide novel insight into pathogen survival and host-pathogen interactions. The current study assessed PAD homologues and deiminated protein profiles of two alveolate parasites, Piridium sociabile (Chromerida) and Merocystis kathae (Apicomplexa). Histological analysis verified strong cytoplasmic PAD expression in both Alveolates, detected deiminated proteins in nuclear and cytoplasmic compartments of the alveolate parasites and verified the presence of citrullinated histone H3 in Alveolata nucleus, indicating roles in epigenetic regulation. Histone H3 citrullination was also found significantly elevated in the host tissue, indicative of neutrophil extracellular trap formation, a host-defence mechanism against a range of pathogens, particularly those that are too large for phagocytosis. Proteomic analysis of deiminated proteins from both Alveolata identified GO and KEGG pathways strongly relating to metabolic and genetic regulation, with some species-specific differences between the apicomplexan and the chromerid. Our findings provide novel insights into roles for the conserved PAD/ADI enzyme family in the regulation of metabolic and epigenetic pathways in alveolate parasites, possibly also relating to their life cycle and host-pathogen interactions.

Dataset Information

Peptidylarginine Deiminase Inhibition Abolishes the Production of Large Extracellular Vesicles From Giardia intestinalis, Affecting Host-Pathogen Interactions by Hindering Adhesion to Host Cells.

Publications

Peptidylarginine Deiminase Inhibition Abolishes the Production of Large Extracellular Vesicles From <i>Giardia intestinalis</i>, Affecting Host-Pathogen Interactions by Hindering Adhesion to Host Cells.

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