Strategies To Discover the Structural Components of Cyst and Oocyst Walls

Abstract

bCysts of Giardia lamblia and Entamoeba histolytica and oocysts of Toxoplasma gondii and Cryptosporidium parvum are the infectious and sometimes diagnostic forms of these parasites. To discover the structural components of cyst and oocyst walls, we have developed strategies based upon a few simple assumptions. Briefly, the most abundant wall proteins are identified by monoclonal antibodies or mass spectrometry. Structural components include a sugar polysaccharide (chitin for Entamoeba, ␤-1,3-linked glucose for Toxoplasma, and ␤-1,3-linked GalNAc for Giardia) and/or acid-fast lipids (Toxoplasma and Cryptosporidium). Because Entamoeba cysts and Toxoplasma oocysts are difficult to obtain, studies of walls of nonhuman pathogens (E. invadens and Eimeria, respectively) accelerate discovery. Biochemical methods to dissect fungal walls work well for cyst and oocyst walls, although the results are often unexpected. For example, echinocandins, which inhibit glucan synthases and kill fungi, arrest the development of oocyst walls and block their release into the intestinal lumen. Candida walls are coated with mannans, while Entamoeba cysts are coated in a dextran-like glucose polymer. Models for cyst and oocyst walls derive from their structural components and organization within the wall. Cyst walls are composed of chitin fibrils and lectins that bind chitin (Entamoeba) or fibrils of the ␤-1,3-GalNAc polymer and lectins that bind the polymer (Giardia). Oocyst walls of Toxoplasma have two distinct layers that resemble those of fungi (␤-1,3-glucan in the inner layer) or mycobacteria (acid-fast lipids in the outer layer). Oocyst walls of Cryptosporidium have a rigid bilayer of acid-fast lipids and inner layer of oocyst wall proteins. Acid-fast lipids, Fibrils of beta-glucan, Dityrosine glows.-Anonymous, Haiku of the oocyst wall I n this minireview, we discuss strategies that have worked well to discover structural components of protist walls that allow them to travel by the fecal-oral route from one person to the next (1-5). Proteins and sugars are the major components of cyst walls of Entamoeba and Giardia, which cause amebic dysentery and diarrhea, respectively (6-8). In addition to proteins and carbohydrates, lipids are important structural components of oocyst walls of Cryptosporidium and Toxoplasma, which cause diarrhea and disseminated infections, respectively (9-11).To characterize the structural components of walls of so many different organisms, we have had to focus on what is likely to matter most. Many of these wall-oriented strategies are based upon a set of assumptions that may not be obvious to investigators working on stages of the parasites that are motile and dividing (12). Therefore, we review here the strategies and assumptions most useful to determine the protein, carbohydrate, and lipid compositions of cyst and oocyst walls. The compositions of these cyst and oocyst walls are then used to make simple, if incomplete, models for their structure and assembly.

Toxoplasma gondii merozoite gene expression analysis with comparison to the life cycle discloses a unique expression state during enteric development

“…In an opposite manner to FAB, FAD was largely downregulated in the invasive stages, the tachyzoite and merozoite, and upregulated in the encysted stages, the oocysts and bradyzoite. The regulation of the FAD pathway through life cycle development may provide the encysted stages a poised state to utilize fatty acid stores that are present, for example, in the oocyst [31]. Overall, the GO and KEGG analysis demonstrated that the merozoite has an elevated gene expression state for cell growth and metabolic related genes, indicating unique growth requirements of this stage.

Figure 3 Clustering of significantly regulated life cycle gene groups.

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The aromatic amino acid hydroxylase genes AAH1 and AAH2 in Toxoplasma gondii contribute to transmission in the cat

“…Dityrosine has a strong blue auto fluorescence under UV light, a fluorescence observed in the oocysts of both Eimeria and T . gondii [29]. Furthermore, microarray data indicates that the AAH genes are upregulated during oocyst development in T .…”

The single epsin homolog in Giardia lamblia localizes to the ventral disk of trophozoites and is not associated with clathrin membrane coats

“…Encystation entails synthesis and regulated secretion of a composite biopolymer matrix composed of three cyst wall proteins (CWPs) and glycan [14]. Cyst wall material (CWM) is selectively accumulated and matured in encystation-specific vesicles (ESVs) [10].…”