Abstract
Giardia lamblia is an intestinal protozoan parasite required to survive in the environment in order to be transmitted to a new host. To ensure parasite survival, flagellated trophozoites colonizing the small intestine differentiate into non-motile environmentally-resistant cysts which are then shed in the environment. This cell differentiation process called encystation is characterized by significant morphological remodeling which includes secretion of large amounts of cyst wall material. Although much is known about the transcriptional regulation of encystation and the synthesis and trafficking of cyst wall material, the investigation of global changes in protein content and abundance during G. lamblia encystation is still unaddressed.In this study, we report on the quantitative analysis of the G. lamblia proteome during encystation using tandem mass spectrometry. Quantification of more than 1000 proteins revealed major changes in protein abundance in early, mid and late encystation, notably in constitutive secretory protein trafficking. Early stages of encystation were marked by a striking decrease of endoplasmic reticulum-targeted variant-specific surface proteins and significant increases in cytoskeleton regulatory components, NEK protein kinases and proteins involved in protein folding and glycolysis. This was in stark contrast to cells in the later stages of encystation which presented a surprisingly similar proteome composition to non-encysting trophozoites. Altogether these data constitute the first quantitative atlas of the Giardia proteome covering the whole process of encystation and point towards an important role for post-transcriptional control of gene expression in Giardia differentiation. Furthermore, our data provide a valuable resource for the community-based annotation effort of the G. lamblia genome, where almost 70% of all predicted gene models remains “hypothetical”.
Highlights
Some of the most widespread protozoan parasites rely on the development of an environmentally resistant infectious form (ERIF)
To ensure correct induction of cyst wall material (CWM) biosynthesis and trafficking to encystation specific vesicle (ESV), we used immunofluorescence assays to monitor CWP1 accumulation and translocation in aliquots of non-encysting and encysting cells harvested during the timecourse; CWP1 is commonly used as a marker for CWM trafficking and ESV neogenesis [27]
Wide-field microscopy observation of fixed cells labeled with anti-CWP1 monoclonal antibody conjugated to the Texas-Red fluorophore demonstrated the timely induction of CWP1 expression in 70–80% of the cells (Figure 1A) and the progressive development of ESVs during their previously documented stages of neogenesis and maturation [15]
Summary
Some of the most widespread protozoan parasites rely on the development of an environmentally resistant infectious form (ERIF). Environmental shedding of mature ERIFs allows for per oral parasite transmission to a new host, achieving completion of the infectious cycle. To species such as Toxoplasma gondii [1], Eimeria tenella [2], Entamoeba invadens [3] and most recently Dientamoeba fragilis [4], the diplomonad Giardia lamblia Giardial cysts are shed in fecal matter which may contaminate water sources Following ingestion, they differentiate into flagellated excyzoites after passage through the stomach. They differentiate into flagellated excyzoites after passage through the stomach These intermediate cell stages rapidly undergo 2 rounds of cell division, giving rise to 4 fully developed trophozoites. This infection usually develops into a full-fledged parasitic disease known as giardiasis which accounts for the majority of nonbacterial diarrheal waterborne illness [6]
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