Dual RNA Sequencing Reveals Key Events When Different Giardia Life Cycle Stages Interact With Human Intestinal Epithelial Cells In Vitro

Abstract

Giardia intestinalis is a protozoan parasite causing diarrheal disease, giardiasis, after extracellular infection of humans and other mammals’ intestinal epithelial cells (IECs) of the upper small intestine. The parasite has two main life cycle stages: replicative trophozoites and transmissive cysts. Differentiating parasites (encysting cells) and trophozoites have recently been shown to be present in the same regions of the upper small intestine, whereas most mature cysts are found further down in the intestinal system. To learn more about host-parasite interactions during Giardia infections, we used an in vitro model of the parasite’s interaction with host IECs (differentiated Caco-2 cells) and Giardia WB trophozoites, early encysting cells (7 h), and cysts. Dual RNA sequencing (Dual RNAseq) was used to identify differentially expressed genes (DEGs) in both Giardia and the IECs, which might relate to establishing infection and disease induction. In the human cells, the largest gene expression changes were found in immune and MAPK signaling, transcriptional regulation, apoptosis, cholesterol metabolism and oxidative stress. The different life cycle stages of Giardia induced a core of similar DEGs but at different levels and there are many life cycle stage-specific DEGs. The metabolic protein PCK1, the transcription factors HES7, HEY1 and JUN, the peptide hormone CCK and the mucins MUC2 and MUC5A are up-regulated in the IECs by trophozoites but not cysts. Cysts specifically induce the chemokines CCL4L2, CCL5 and CXCL5, the signaling protein TRKA and the anti-bacterial protein WFDC12. The parasite, in turn, up-regulated a large number of hypothetical genes, high cysteine membrane proteins (HCMPs) and oxidative stress response genes. Early encysting cells have unique DEGs compared to trophozoites (e.g. several uniquely up-regulated HCMPs) and interaction of these cells with IECs affected the encystation process. Our data show that different life cycle stages of Giardia induce different gene expression responses in the host cells and that the IECs in turn differentially affect the gene expression in trophozoites and early encysting cells. This life cycle stage-specific host-parasite cross-talk is an important aspect to consider during further studies of Giardia’s molecular pathogenesis.

Trophozoite fitness dictates the intestinal epithelial cell response to Giardia intestinalis infection

“…intestinalis infection remains the subject of only a single recent study [ 32 ], which revealed IEC barrier breakdown linked to cyclic AMP/protein kinase A signalling during mature stages of the infection. Notably, the authors could by RNA sequencing (RNA-seq) not detect any IEC immune response during the early infection stage (first ~1.5 h), which stands in contrast to previous works in colorectal adenocarcinoma cell line cultures [ 20 , 22 ]. The reasons for these discrepancies are unclear.…”

Functional Differentiation of Cyclins and Cyclin-Dependent Kinases in Giardia lamblia

“…The transcript of glcyclin 13874 increased in Giardia cells at the G1/S phase (1.5-fold). Interestingly, dual RNA sequencing analysis of Giardia interacting with human intestinal epithelial cells indicated that expression of 2 GlCDKs and 11 Glcyclins was decreased ( 45 ). The exact mechanisms of how the number of Glcyclins is varied and which Glcyclins exert their differential function in the Giardia cell cycle are important issues that need to be addressed in the future.…”

Spliceosomal introns in the diplomonad parasite Giardia duodenalis revisited

“…A summary of samples and their metadata can be found in Table S1, available in the online version of this article. The compiled data were published in six separate studies all using comparable library preparation protocols and Illumina sequencing chemistry [6,15,[18][19][20][21]. From this dataset and our described detection strategy, we identified a total of 42 candidate cis-splicing introns, including all eight introns previously characterized using laboratory methods, plus 34 novel ones.…”

“…In this work, we gathered a collection of mRNA sequence data from six independent studies published between 2013 and 2022 [6,[16][17][18][19][20][21] and used these as the basis of a novel bioinformatics investigation of the gene models currently annotated in the G. duodenalis reference genome. Thus, our objectives in this study were to identify and characterize novel splicing activity to improve the gene models used for reference genome annotation.…”