The Most Abundant Glycoprotein of Amebic Cyst Walls (Jacob) Is a Lectin with Five Cys-Rich, Chitin-Binding Domains

Frisardi, Marta; Ghosh, Sudip Kumar; Field, Jessica; Dellen, Katrina Van; Rogers, Rick A.; Robbins, Phillips W.; Samuelson, John

doi:10.1128/iai.68.7.4217-4224.2000

Cited by 73 publications

(104 citation statements)

References 52 publications

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“…Such staining is consistent with processing of the EhNucI-HA and EhNucII-HA proteins through the rudimentary endoplasmic reticulum present in these parasites for entry into the secretory system. Importantly, very similar immunofluorescence staining patterns have been reported for other secretory proteins in these parasites (44,45). In contrast to the above, no fluorescence was detected in cells transfected with the ExEhNeo control plasmid (data not shown).…”

Section: -180 Min)supporting

confidence: 48%

Identification and Biochemical Characterization of Unique Secretory Nucleases of the Human Enteric Pathogen, Entamoeba histolytica

McGugan

Joshi

Dwyer

2007

Journal of Biological Chemistry

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The ancient eukaryotic human pathogen, Entamoeba histolytica, is a nucleo-base auxotroph (i.e. lacks the ability to synthesize purines or pyrimidines de novo) and therefore is totally dependent upon its host for the supply of these essential nutrients. In this study, we identified two unique 28-kDa, dithiothreitol-sensitive nucleases and showed that they are constitutively released/secreted by parasites during axenic culture. Using several different molecular approaches, we identified and characterized the structure of EhNucI and EhNucII, genes that encode ribonuclease T2 family proteins. Homologous episomal expression of epitope-tagged EhNucI and EhNucII chimeric constructs was used to define the functional and biochemical properties of these released/secreted enzymes. Results of coupled immunoprecipitation-enzyme activity analyses demonstrated that these "secretory" enzymes could hydrolyze a variety of synthetic polynucleotides, as well as the natural nucleic acid substrate RNA. Furthermore, our results demonstrated that sera from acutely infected amebiasis patients recognized and immunoprecipitated these parasite secretory enzymes. Based on these observations, we hypothesize that within its host, these secretory nucleases could function, at a distance away from the parasite, to harness (i.e. hydrolyze/access) host-derived nucleic acids to satisfy the essential purine and pyrimidine requirements of these organisms. Thus, these enzymes might play an important role in facilitating the survival, growth, and development of this important human pathogen.The ancient eukaryotic human parasite, Entamoeba histolytica, is the causative agent of invasive amebiasis in humans infecting 10% of the world population and resulting in over 100,000 deaths annually, chiefly from amebic liver abscess (1). Infection is acquired through the ingestion of the quadranucleate cyst form of the organism from fecally contaminated food or water supplies. Excystation occurs in the small intestine, releasing immature trophozoites, which move into and colonize the nutrient-poor large intestine.As ancient eukaryotes, Entamoeba parasites contain only rudimentary endoplasmic reticulum and Golgi apparatus (2, 3). Despite this, vesicle trafficking pathways appear to be essential to the pathogenesis of these parasites as (i) uptake and digestions of nutrients by mature trophozoites in the large intestine, (ii) invasion of the intestinal epithelium, (iii) delivery of cyst wall components during encystation, and (iv) dissemination and establishment of extra-intestinal infections all rely on endocytosis and secretion. In addition, these parasites lack many traditional eukaryotic biochemical pathways, including the de novo biosynthesis of purine and pyrimidine nucleobases. As a result, these parasites are completely dependent upon their host for the supply of these essential preformed nutrients. In that regard, it has been reported previously that Entamoeba are capable of salvaging both purines and pyrimidines from the extracellular milieu during in vit...

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Section: -180 Min)supporting

confidence: 48%

Identification and Biochemical Characterization of Unique Secretory Nucleases of the Human Enteric Pathogen, Entamoeba histolytica

McGugan

Joshi

Dwyer

2007

Journal of Biological Chemistry

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“…2E) (24). Similarly, the walls of Saccharomyces and the sheath of nematodes are a single layer of uniform thickness and staining.…”

Section: First Strategy: Assume That At Least Some Of the Important Cmentioning

confidence: 99%

Strategies To Discover the Structural Components of Cyst and Oocyst Walls

et al. 2013

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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.

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“…In turn, FITC-lectin avidity of Acanthamoeba and Naegleria strains differed between morphological forms, species and pathogenic or nonpathogenic strains of the same species (Bose et al 1989). More sophisticated Entamoeba studies have not only demonstrated further the experimental potential of fluorescent lectins in elucidating protozoan cell surface nature and dynamics (Ribeiro et al 1997), but also the natural role of lectins as encystation-specific glycoproteins in cyst-wall maturation (Frisardi et al 2000). The studies outlined below employ the techniques of flow cytometry and a panel of FITC-lectins and concepts developed from ligand-receptor studies, to characterize Acanthamoeba polyphaga cyst and trophozoite lectin avidity, hence surface carbohydrate nature.…”

Section: Introductionmentioning

confidence: 99%

Analysis of Acanthamoeba polyphaga surface carbohydrate exposure by FITC-lectin binding and fluorescence evaluation

et al. 2004

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Aims: Characterization of the representative protozoan Acanthamoeba polyphaga surface carbohydrate exposure by a novel combination of flow cytometry and ligand-receptor analysis. Methods and Results: Trophozoite and cyst morphological forms were exposed to a panel of FITC-lectins. Population fluorescence associated with FITC-lectin binding to acanthamoebal surface moieties was ascertained by flow cytometry. Increasing concentrations of representative FITC-lectins, saturation binding and determination of K d and relative B max values were employed to characterize carbohydrate residue exposure. FITC-lectins specific for N-acetylglucosamine, N-acetylgalactosamine and mannose/glucose were readily bound by trophozoite and cyst surfaces. Minor incremental increases in FITC-lectin concentration resulted in significant differences in surface fluorescence intensity and supported the calculation of ligand-binding determinants, K d and relative B max , which gave a trophozoite and cyst rank order of lectin affinity and surface receptor presence. Conclusions: Trophozoites and cysts expose similar surface carbohydrate residues, foremost amongst which is N-acetylglucosamine, in varying orientation and availability. Significance and Impact of the Study: The outlined versatile combination of flow cytometry and ligandreceptor analysis allowed the characterization of surface carbohydrate exposure by protozoan morphological forms and in turn will support a valid comparison of carbohydrate exposure by other single-cell protozoa and eucaryotic microbes analysed in the same manner.

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The Most Abundant Glycoprotein of Amebic Cyst Walls (Jacob) Is a Lectin with Five Cys-Rich, Chitin-Binding Domains

Cited by 73 publications

References 52 publications

Identification and Biochemical Characterization of Unique Secretory Nucleases of the Human Enteric Pathogen, Entamoeba histolytica

Identification and Biochemical Characterization of Unique Secretory Nucleases of the Human Enteric Pathogen, Entamoeba histolytica

Strategies To Discover the Structural Components of Cyst and Oocyst Walls

Analysis of Acanthamoeba polyphaga surface carbohydrate exposure by FITC-lectin binding and fluorescence evaluation

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