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The infectious and diagnostic stage of Giardia lamblia (also known as G. intestinalis or G. duodenalis) is the cyst. The Giardia cyst wall contains fibrils of a unique β-1,3-linked N-acetylgalactosamine (GalNAc) homopolymer and at least three cyst wall proteins (CWPs) composed of Leu-rich repeats (CWPLRR) and a C-terminal conserved Cys-rich region (CWPCRR). Our goals were to dissect the structure of the cyst wall and determine how it is disrupted during excystation. The intact Giardia cyst wall is thin (∼400 nm), easily fractured by sonication, and impermeable to small molecules. Curled fibrils of the GalNAc homopolymer are restricted to a narrow plane and are coated with linear arrays of oval-shaped protein complex. In contrast, cyst walls of Giardia treated with hot alkali to deproteinate fibrils of the GalNAc homopolymer are thick (∼1.2 µm), resistant to sonication, and permeable. The deproteinated GalNAc homopolymer, which forms a loose lattice of curled fibrils, is bound by native CWP1 and CWP2, as well as by maltose-binding protein (MBP)-fusions containing the full-length CWP1 or CWP1LRR. In contrast, neither MBP alone nor MBP fused to CWP1CRR bind to the GalNAc homopolymer. Recombinant CWP1 binds to the GalNAc homopolymer within secretory vesicles of Giardia encysting in vitro. Fibrils of the GalNAc homopolymer are exposed during excystation or by treatment of heat-killed cysts with chymotrypsin, while deproteinated fibrils of the GalNAc homopolymer are degraded by extracts of Giardia cysts but not trophozoites. These results show the Leu-rich repeat domain of CWP1 is a lectin that binds to curled fibrils of the GalNAc homopolymer. During excystation, host and Giardia proteases appear to degrade bound CWPs, exposing fibrils of the GalNAc homopolymer that are digested by a stage-specific glycohydrolase.

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Multiple system atrophy (MSA) is a fatal late-onset neurodegenerative disease. Although presenting with distinct pathological hallmarks, which in MSA consist of glial cytoplasmic inclusions (GCIs) containing fibrillar α-synuclein in oligodendrocytes, both MSA and Parkinson's disease are α-synucleinopathies. Pathologically, MSA can be categorized into striatonigral degeneration (SND), olivopontocerebellar atrophy (OPCA) or mixed subtypes. Despite extensive research, the regional vulnerability of the brain to MSA pathology remains poorly understood. Genetic, epigenetic and environmental factors have been proposed to explain which brain regions are affected by MSA, and to what extent. Here, we explored for the first time epigenetic changes in post-mortem brain tissue from MSA cases. We conducted a case-control study, and profiled DNA methylation in white mater from three brain regions characterized by severe-to-mild GCIs burden in the MSA mixed subtype (cerebellum, frontal lobe and occipital lobe). Our genome-wide approach using Illumina MethylationEPIC arrays and a powerful cross-region analysis identified 157 CpG sites and 79 genomic regions where DNA methylation was significantly altered in the MSA mixed-subtype cases. HIP1, LMAN2 and MOBP were amongst the most differentially methylated loci. We replicated these findings in an independent cohort and further demonstrated that DNA methylation profiles were perturbed in MSA mixed subtype, and also to variable degrees in the other pathological subtypes (OPCA and SND). Finally, our comethylation network analysis revealed several molecular signatures (modules) significantly associated with MSA (disease status and pathological subtypes), and with neurodegeneration in the cerebellum. Importantly, the co-methylation module having the strongest association with MSA included a CpG in SNCA, the gene encoding α-synuclein. Altogether, our results provide the first evidence for DNA methylation changes contributing to the molecular processes altered in MSA, some of which are shared with other neurodegenerative diseases, and highlight potential novel routes for diagnosis and therapeutic interventions.

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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.