Microvesicles released from Giardia intestinalis disturb host-pathogen response in vitro

Abstract

a b s t r a c tGiardia intestinalis (G.I), is an anaerobic protozoan and the aetiological agent of giardiasis, a diarrhoea present worldwide and associated with poverty. G.I has a simple life cycle alternating between cyst and trophozoite. Cysts are transmitted orally to the stomach and transform to trophozoites in the intestine by a multifactorial process. Recently, microvesicles (MVs) have been found to be released from a wide range of eukaryotic cells. We have observed a release of MVs during the life cycle of G.I., identifying MVs from active trophozoites and from trophozoites differentiating to the cyst form. The aim of the current work was to investigate the role of MVs from G.I in the pathogenesis of giardiasis. MVs from log phase were able to increase the attachment of G. intestinalis trophozoites to Caco-2 cells. Moreover, MVs from G. intestinalis could be captured by human immature dendritic cells, resulting in increased activation and allostimulation of human dendritic cells. Lipid rafts participate in the MV biogenesis and in the attachment to Caco-2 cells. Nevertheless, proteomic analysis from two types of MVs has shown slight differences at the protein levels. An understanding of biogenesis and content of MVs derived from trophozoites might have important implications in the pathogenesis of the disease.

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

“…In the current study, both PAD-inhibitor and CBD were able to decrease adhesion of Giardia to mammalian cells, similar to as our group previously observed with the cholesterol-chelating agent methyl-β-cyclodextrin treatment (Evans-Osses et al, 2017). For our protozoa model in the current study, Cl-amidine regulated LEVs specifically.…”

“…The first objective was to obtain different EV populations from G. intestinalis. The previous protocol (Evans-Osses et al, 2017) was slightly modified to separate putative large extracellular (LEV) and small extracellular vesicles (SEV) from the total extracellular vesicles described before (Fig.1A). Two different EV populations were recovered from this method: LEVs at 15,000xg and SEVs at 100,000xg.…”

“…Parasites from confluent cultures were decanted by chilling for 15 min in ice-cold, centrifuged twice (600xg/5min) and the pellets suspended with fresh TYI-S-33 without adult bovine serum (FBS). Parasites were then counted using a hemocytometer, and diluted to 1×10 6 per sample according to Evans-Osses et al(2017). 1mM of CaCl 2 were added to the parasite culture for EV induction and incubated for 1 hour at 37°C.…”

“…Groups were as follows: medium only (control), 100 or 50 μM PAD-inhibitor Cl-amidine (a kind gift from Prof Paul Thompson, UMASS), or with 10 or 5 μM CBD (90899_SIAL, Sigma-Aldrich™). After 60 minutes of incubation (37°C), samples were processed according to Evans-Osses et al (2017).…”

“…EVs are found in most biological fluids and are 30-1000 nm lipid-bilayer vesicles, which are shed from cells and transport a range of biomolecules, participating in cell communication in physiological and pathophysiological processes (Coakley et al, 2015; Maas et al, 2017; van Niel et al, 2018; Ramirez et al, 2018; Ryu et al, 2018). Our group has previously described EV release of G. intestinalis and established that protozoa EVs are involved in pathogen interactions via immunomodulation and trophozoite persistence (Evans-Osses et al, 2017). In recent years, there has been a growing interest to improve current knowledge of the nature, constitution and biogenesis of all secreted EVs.…”