Regulation of differentiation during the Giardia life cycle : An insight into the molecular control

Abstract: Giardia intestinalis (syn. G. lamblia, G. duodenalis) is an intestinal protozoan parasite that causes diarrheal infections worldwide. A critical step in maintaining its chain of transmission is the formation of infectious cysts from the disease causing trophozoites during a cell differentiation process called encystation. Giardia establishes noninvasive infection in the epithelial cells (IECs) of the upper small intestine of mammals, including humans, causing a diarrheal disease known as giardiasis. Giardiasis is a multifactorial disease in which some of the factors involved in pathogenesis and virulence have been identified. However, several questions of the effects of host-parasite interaction during cell differentiation remain unclear.This thesis is divided into three sections. The first part focused on producing a high-resolution gene expression map of encystation in Giardia (Paper I). For this purpose, we used deep RNAseq combined with a rich range of timepoints during encystation that showed a gradual change in gene expression along the time course of differentiation. Genes encoding proteins involved in forming the resistant cyst wall are induced early in encystation, and most genes are regulated by the transcription factor Myb2. However, the most extensive gene expression changes were observed during the late phase of encystation, mainly among cysteine-rich surface proteins (VSPs and HCMPs) and genes involved in regulation of chromatin structure.Secondly, we investigated how epigenetic regulation is related to processes controlling parasite virulence, host immune evasion, transmission, and cell differentiation. As a result, we produced the first molecular map of post-translational modifications in core histones (methylation, acetylation, and phosphorylation) in Giardia using mass spectrometry (Paper II).Finally, to study how cell differentiation affects the host and vice versa, we used a host-parasite interaction model combining IECs (differentiated Caco-2 cells) with different cell stages of Giardia (trophozoites, early encysting cells (7 h), and cysts). We analyzed these interactions using Dual RNA sequencing (Dual RNAseq) to identify differentially expressed genes (DEGs) in Giardia and the host cells. Our results showed that different life cycle stages of Giardia induce different gene expression responses in the host cells. Furthermore, the IECs affect the gene expression in trophozoites and early encysting cells (Paper III). This life cycle stage-specific host-parasite crosstalk stands as a crucial step to consider for future studies in the molecular pathogenesis of Giardia. In summary, this thesis has provided a deeper understanding of cell differentiation of Giardia intestinalis and its interaction with host cells. It shows that specific gene expression changes, regulated via transcription factors and epigenetic regulation, are involved in the host-parasite crosstalk during Giardia infections.