Induction of virulence factors in Giardia duodenalis independent of host attachment

Abstract

Giardia duodenalis is responsible for the majority of parasitic gastroenteritis in humans worldwide. Host-parasite interaction models in vitro provide insights into disease and virulence and help us to understand pathogenesis. Using HT-29 intestinal epithelial cells (IEC) as a model we have demonstrated that initial sensitisation by host secretions reduces proclivity for trophozoite attachment, while inducing virulence factors. Host soluble factors triggered up-regulation of membrane and secreted proteins, including Tenascins, Cathepsin-B precursor, cystatin, and numerous Variant-specific Surface Proteins (VSPs). By comparison, host-cell attached trophozoites up-regulated intracellular pathways for ubiquitination, reactive oxygen species (ROS) detoxification and production of pyridoxal phosphate (PLP). We reason that these results demonstrate early pathogenesis in Giardia involves two independent host-parasite interactions. Motile trophozoites respond to soluble secreted signals, which deter attachment and induce expression of virulence factors. Trophozoites attached to host cells, in contrast, respond by up-regulating intracellular pathways involved in clearance of ROS, thus anticipating the host defence response.

Giardia secretome highlights secreted tenascins as a key component of pathogenesis

“…Here instead, we chose to provide a detailed baseline from cultured Giardia trophozoites secreting proteins under a steady state in vitro . Nevertheless, our results are strongly supportive of a recent proteomic study looking at the effect of host attachment on the profile of Giardia -secreted proteins [15]. Prior to that study, several metabolic enzymes had been proposed to be released by Giardia trophozoites upon interaction with intestinal epithelial cells (IECs) [13], such as arginine deiminase (ADI), enolase, and ornithine carbamoyltransferase (OCT), which we were also able to identify from the culture supernatants of both assemblages.…”

“…Interestingly, when GS was resequenced, GL50803_15564 was found to comprise 3 recently diverged orthologs (GSB_153537, GSB_155477, GSB_150353), and it may be that the positive selection pressure observed has been generated as a result of recent gene duplications in the assemblage B strain. GL50803_16779, an assemblage A (WB) GCATB, has previously been shown to be upregulated and involved in trophozoite motility in the early pathogenesis of Giardia [15]. In this study, this protein was found to be in WB’s top 5 secreted proteins (Table S4); its GS ortholog (GL50581_78) was also present but at a considerably lower level, suggesting that for this GCATB may play a more significant role in assemblage A than assemblage B.…”

“…They appear to have evolved from a group of proteins specific to vertebrates, presumably co-evolving with the EGF receptor, and so the presence of homologous proteins in Giardia evolving independently from HCMPs is a clear example of the kind of convergent evolution best described as molecular mimicry. Interestingly, a Giardia tenascin (WB-GL50803_8687/GS-GL50581_4316), secreted by both strains, and Giardia tenascin (WB-GL50803_14573/GS-GL50581_1475), secreted only by the WB strain (Table 1; Table S4), were found to be induced by host soluble factors and implicated in the regulation of trophozoite attachment [15], supporting the case for secreted tenascins acting as virulence factors in Giardia pathogenesis.…”

“…Just 2 of these were of unknown function, and 2 groups dominated the annotated genes encoding the rest of these proteins; 5 were annotated as tenascins and 3 as cathepsin B cysteine proteases. The most abundant enriched protein was found to be pyridoxamine 5'-phosphate oxidase (PNPO), a flavin mononucleotide–dependent enzyme capable of fixing molecular oxygen that lacks a signal peptide and which was also recently identified as a secreted Giardia trophozoite protein upregulated during interaction with epithelial cells [15]. An extracellular nuclease was also present, along with a high-cysteine membrane protein, as well as a protein product of a gene misannotated as a variant surface protein (VSP; as it was well conserved between assemblages).…”

Giardia: a pathogen or commensal for children in high-prevalence settings?

“…Indeed, Giardia contains an impressive repertoire of immunoevasive products [36*] that are upregulated upon sensing secreted host factors even prior to parasite attachment [37] and could have implications for modulating host responses to co-pathogens or resident microbiota. For example, cathepsin B-producing Giardia strains are capable of cleaving IL-8 and attenuating neutrophil chemotaxis to inflammatory stimuli [38, 39].…”