Transcriptomic profiling of high-density Giardia foci encysting in the murine proximal intestine

Abstract

Abstract. Giardia is a highly prevalent, understudied protistan parasite causing significant diarrheal 1 disease worldwide. Its life cycle consists of two stages: infectious cysts ingested from contaminated 2 food or water sources, and motile trophozoites that colonize and attach to the gut epithelium, later 3 encysting to form new cysts that are excreted into the environment. Current understanding of parasite 4 physiology in the host is largely inferred from transcriptomic studies using Giardia grown axenically or 5 in co-culture with mammalian cell lines. The dearth of information about the diversity of host-parasite 6 interactions occurring within distinct regions of the gastrointestinal tract has been exacerbated by a 7 lack of methods to directly and non-invasively interrogate disease progression and parasite physiology 8 in live animal hosts. By visualizing Giardia infections in the mouse gastrointestinal tract using 9 bioluminescent imaging (BLI) of tagged parasites, we recently showed that parasites colonize the gut in 10 high-density foci that may cause localized pathology to the epithelium. Encystation is also initiated in 11 these foci throughout the entire course of infection, yet how the physiology of parasites within high-12 density foci in the host gut differs from that of cells in laboratory culture is unclear. Here we use BLI to 13 precisely select parasite samples from high-density foci in the proximal intestine to interrogate in vivo 14 Giardia gene expression in the host. Relative to axenic culture, we noted significantly higher expression 15 (> 10-fold) of oxidative stress, membrane transporter, and metabolic and structural genes associated 16 with encystation in the high-density foci. These differences in gene expression within parasite foci in 17 the host may reflect physiological changes associated with high-density growth in localized regions of 18 the gut. We also identified and verified six novel cyst-specific proteins, including new components of 19 the cyst wall that were highly expressed in these foci. Our in vivo transcriptome data support an 20 emerging view that parasites encyst early in localized regions in the gut, possibly as a consequence of 21 nutrient limitation, and also impact local metabolism and physiology.

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

“…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 . 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 . Proteomic analyses showed that G. intestinalis cystatin is up‐regulated by secretions form human intestinal epithelial cells .…”

“…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].…”

“…Immunoblot analysis (Fig. 6) showed robust expression level of transgenic cystatin under endogenous promoter in trophozoites and encysting trophozoites as indicated earlier in transcriptome and proteome studies (https://giardiadb.org/giardiadb/app/record/gene/GL 50803_27918, [32,34,35]), thus suggesting a role of the protein throughout the parasite life cycle. Of note, both transgenic cell lines showed comparable growth rates identical to the parent strain and overexpression of cystatin by cwp1 promotor during encystation did not change the capacity of the trophozoites to form cysts in vitro (data not shown).…”

Methionyl-tRNA synthetase inhibitor has potentin vivoactivity in a novelGiardia lamblialuciferase murine infection model

“…To promote parasite colonization, mice were administered antibiotics (0.25 mg/mL ampicillin and neomycin) in drinking water for the duration of the experiment starting 6 days before infection. 17 A cocktail containing ampicillin, neomycin and metronidazole at concentrations of 50 mg/kg, 15 mg/kg and 50 mg/kg, respectively, was dosed by oral gavage 3 days before infection. A second antibiotic dose containing 50 mg/kg ampicillin and 15 mg/kg neomycin was given 1 day before infection.…”

Robust and stable transcriptional repression inGiardiausing CRISPRi

“…Over 40 percent of Giardia's 6000 genes encode "hypothetical" proteins that lack similarity to 19 proteins in the human host (Morrison et al, 2007). Many hypothetical proteins are highly 20 expressed during in vivo infections yet lack any known cellular function (Pham et al, 2017). 21…”