Transcriptomic Profiling of High-Density Giardia Foci Encysting in the Murine Proximal Intestine

Abstract

Giardia is a highly prevalent, understudied protistan parasite causing significant diarrheal disease worldwide. Its life cycle consists of two stages: infectious cysts ingested from contaminated food or water sources, and motile trophozoites that colonize and attach to the gut epithelium, later encysting to form new cysts that are excreted into the environment. Current understanding of parasite physiology in the host is largely inferred from transcriptomic studies using Giardia grown axenically or in co-culture with mammalian cell lines. The dearth of information about the diversity of host-parasite interactions occurring within distinct regions of the gastrointestinal tract has been exacerbated by a lack of methods to directly and non-invasively interrogate disease progression and parasite physiology in live animal hosts. By visualizing Giardia infections in the mouse gastrointestinal tract using bioluminescent imaging (BLI) of tagged parasites, we recently showed that parasites colonize the gut in high-density foci. Encystation is initiated in these foci throughout the entire course of infection, yet how the physiology of parasites within high-density foci in the host gut differs from that of cells in laboratory culture is unclear. Here we use BLI to precisely select parasite samples from high-density foci in the proximal intestine to interrogate in vivo Giardia gene expression in the host. Relative to axenic culture, we noted significantly higher expression (>10-fold) of oxidative stress, membrane transporter, and metabolic and structural genes associated with encystation in the high-density foci. These differences in gene expression within parasite foci in the host may reflect physiological changes associated with high-density growth in localized regions of the gut. We also identified and verified six novel cyst-specific proteins, including new components of the cyst wall that were highly expressed in these foci. Our in vivo transcriptome data support an emerging view that parasites encyst early in localized regions in the gut, possibly as a consequence of nutrient limitation, and also impact local metabolism and physiology.

Responses of the Differentiated Intestinal Epithelial Cell Line Caco-2 to Infection With the Giardia intestinalis GS Isolate

“…Experiments in vivo further showed a reduced uptake of L-phenyl-alanine, L-lysine and L-aspartic acid in the intestines of infected swiss albino mice and a significant drop in the transport of L-alanine and glycine in mice (Anand et al, 1982 ). Giardia also scavenges the gut lumen for lipids including cholesterol (Pham et al, 2017 ), and secretes enzymes involved in phospholipids remodeling and scavenging fatty acids (Ma'ayeh et al, 2017 ). Cholesterol, specifically, is required for lipid raft formation and cholesterol starvation induces encystation (Yichoy et al, 2011 ; Mendez et al, 2015 ).…”

The compact genome of Giardia muris reveals important steps in the evolution of intestinal protozoan parasites

“…We also noted that the encystation-related genes are among the most highly expressed genes in G. muris trophozoites in vivo (Fig. S3c, Table S1b), similar to G. intestinalis infection in mice [42, 43].…”

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

“…In response to IECs, both Giardia isolates released proteins involved in cyst wall formation ( Fig 6 ) and these proteins were not detected in axenic culture indicating that host cells provide the signal to initiate encystation. Interestingly, in a recent study that investigated transcriptional changes in bioluminescent WB isolate trophozoites from high density foci in the mice intestines, the authors reported the up-regulation of encystation, oxidative stress and metabolic genes (carbohydrate, protein, lipid and nucleoside base metabolism) [ 83 ]. Herein, we show that the protein products for many of up-regulated genes in that study are being released into the interaction medium.…”

“…So far, most studies of Giardia -host cell interactions have been performed with simplified models using a small selection of human cell lines but it will be important to follow up these results using more complex in vitro host systems like enteroids [ 119 ]. This can be complemented by in vivo experiments of secreted proteins in mice where trophozoites are mixed with early encysting cells [ 83 ]. Thus, Giardia interaction with IECs is an intricate process and further understanding of this cross-talk could be the key for understanding giardiasis.…”