High Cysteine Membrane Proteins (HCMPs) Are Up-Regulated During Giardia-Host Cell Interactions

Abstract

Giardia intestinalis colonizes the upper small intestine of humans and animals, causing the diarrheal disease giardiasis. This unicellular eukaryotic parasite is not invasive but it attaches to the surface of small intestinal epithelial cells (IECs), disrupting the epithelial barrier. Here, we used an in vitro model of the parasite's interaction with host IECs (differentiated Caco-2 cells) and RNA sequencing (RNAseq) to identify differentially expressed genes (DEGs) in Giardia, which might relate to the establishment of infection and disease induction. Giardia trophozoites interacted with differentiated Caco-2 cells for 1.5, 3, and 4.5 h and at each time point, 61, 89, and 148 parasite genes were up-regulated more than twofold, whereas 209, 265, and 313 parasite genes were down-regulated more than twofold. The most abundant DEGs encode hypothetical proteins and members of the High Cysteine Membrane Protein (HCMP) family. Among the up-regulated genes we also observed proteins associated with proteolysis, cellular redox balance, as well as lipid and nucleic acid metabolic pathways. In contrast, genes encoding kinases, regulators of the cell cycle and arginine metabolism and cytoskeletal proteins were down-regulated. Immunofluorescence imaging of selected, up-regulated HCMPs, using C-terminal HA-tagging, showed localization to the plasma membrane and peripheral vesicles (PVs). The expression of the HCMPs was affected by histone acetylation and free iron-levels. In fact, the latter was shown to regulate the expression of many putative giardial virulence factors in subsequent RNAseq experiments. We suggest that the plasma membrane localized and differentially expressed HCMPs play important roles during Giardia-host cell interactions.

Keywords: RNAseq; chromatin; diarrhea; protozoa; small intestine.

FIGURE 1

(A) Venn diagram showing the number of differentially expressed genes (DEGs) in Giardia intestinalis WB trophozoites incubated with differentiated Caco-2 cells in vitro for 1.5, 3, and 4.5 h. (B) Gene Ontology (GO) analysis of the DEGs at the three time points represented by a heat map. The map shows the up-regulated DEGs enriched for biological functions. (C) Gene Ontology (GO) analysis of DEGs at the three time points represented by a heat map. The map shows the down-regulated DEGs enriched for biological functions. Note that up-regulated (red) and down-regulated (blue) heat maps are color-coded.

FIGURE 2

Fluorescent microscopy analyses of three C-terminally HA-tagged, episomally expressed HCMPs. Panels (A–C) (A-7715, B-91707, and C-115066) show Giardia trophozoites after axenic growth to 80% confluence in TYDK. The 3 transfectants were also co-incubated with human IECs (D–I) and epitope tagged HCMP localization was studied at the start of interaction (T = 0 h, D,F,H) and after 3 h interaction with differentiated Caco-2 cells (T = 3 h, E,G,I).

FIGURE 3

(A) Gene expression analyses of five HCMP genes (HCMP 9620, 7715, 11309, 91707, and 115066) during growth of trophozoites (10⁴ starting trophozoites) for 1 and 3 days in the presence of three HDAC inhibitors [Trichostatin A (TSA), Sodium butyrate (NaB) and Nicotinamide (Nt)]. The experiment was repeated four times. (B) Expression of HCMPs 9620, 7715, 11309, 91707, and 115066 in medium containing different levels of iron; TYDK medium with extra iron (TYDK+ Iron), TYDK medium without extra iron (TYDK– Iron) and TYDK without extra iron and addition of 50 μM of the metal ion chelator 2,2′-Bipyridyl (TYDK-Iron + Chelator). The experiment was repeated three times. Levels of expression were measured by qPCR in both panels. Significant results are indicated by ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001.