Actin, RhoA, and Rab11 Participation during Encystment in<i>Entamoeba invadens</i>

Herrera-Martínez, Mayra; Hernández‐Ramírez, Verónica Ivonne; Lagunes-Guillén, Anel; Chávez‐Munguía, Bibiana; Talamás-Rohana, Patricia

doi:10.1155/2013/919345

Cited by 21 publications

(11 citation statements)

References 28 publications

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“…The bright puncta present at the cyst periphery 6 h after differentiation may mark the presence of vesicles secreted by bradyzoites that carry cyst wall cargo for wall building to the cyst periphery. The actin cytoskeleton and Rab11 traffic vesicles to the cyst walls of Giardia and Entamoeba, supporting a model whereby cyst wall proteins are transported through the cytoskeleton (54,55). In addition, the Giardia Rho GTPase Rac regulates endomembrane organization and cyst wall protein trafficking in Giardia (56).…”

Section: Discussionmentioning

confidence: 73%

Succinylated Wheat Germ Agglutinin Colocalizes with the Toxoplasma gondii Cyst Wall Glycoprotein CST1

Guevara

Fox

Bzik

2020

mSphere

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The glycosylated mucin domain of the Toxoplasma gondii cyst wall glycoprotein CST1 is heavily stained by Dolichos biflorus agglutinin, a lectin that binds to N-acetylgalactosamine. The cyst wall is also heavily stained by the chitin binding lectin succinylated wheat germ agglutinin (s-WGA), which selectively binds to N-acetylglucosamine-decorated structures. Here, we tracked the localization of N-acetylglucosamine-decorated structures that bind to s-WGA in immature and mature in vitro cysts. s-WGA localization was observed at the cyst periphery 6 h after the differentiation of the tachyzoite-stage parasitophorous vacuole. By day 1 and at all later times after differentiation, s-WGA was localized in a continuous staining pattern at the cyst wall. Coinciding with the maturation of the cyst matrix by day 3 of cyst development, s-WGA also localized in a continuous matrix pattern inside the cyst. s-WGA localized in both the outer and inner layer regions of the cyst wall and in a continuous matrix pattern inside mature 7-and 10-day-old cysts. In addition, s-WGA colocalized in the cyst wall with CST1, suggesting that N-acetylglucosamineand N-acetylgalactosamine-decorated molecules colocalized in the cyst wall. In contrast to CST1, GRA4, and GRA6, the relative accumulation of the molecules that bind s-WGA in the cyst wall was not dependent on the expression of GRA2. Our results suggest that GRA2-dependent and GRA2-independent mechanisms regulate the trafficking and accumulation of glycosylated molecules that colocalize in the cyst wall. IMPORTANCE Chronic Toxoplasma gondii infection is maintained in the central nervous system by thick-walled cysts. If host immunity wanes, cysts recrudesce and cause severe and often lethal toxoplasmic encephalitis. Currently, there are no therapies to eliminate cysts, and little biological information is available regarding cyst structure(s). Here, we investigated cyst wall molecules recognized by succinylated wheat germ agglutinin (s-WGA), a lectin that specifically binds to N-acetylglucosamine-decorated structures. N-Acetylglucosamine regulates cell signaling and plays structural roles at the cell surface in many organisms. The cyst wall and cyst matrix were heavily stained by s-WGA in mature cysts and were differentially stained during cyst development. The relative accumulation of molecules that bind to s-WGA in the cyst wall was not dependent on the expression of GRA2. Our findings suggest that glycosylated cyst wall molecules gain access to the cyst wall via GRA2-dependent and GRA2-independent mechanisms and colocalize in the cyst wall.

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Section: Discussionmentioning

confidence: 73%

Succinylated Wheat Germ Agglutinin Colocalizes with the Toxoplasma gondii Cyst Wall Glycoprotein CST1

Guevara

Fox

Bzik

2020

mSphere

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“…We then focused on CF and EB dyes to differentially stain trophozoites and cysts. CF is a fluorochrome that binds to structures containing chitin, a component in the Entamoeba cyst wall, and has, therefore, been used in standard microscopy methods (Arroyo-Begovich et al, 1980 ; Herrera-Martínez et al, 2013 ). EB was tentatively used for staining live Entamoeba cells because it was shown to stain the plasma membrane of viable human neutrophils (Hed et al, 1983 ).…”

Section: Resultsmentioning

confidence: 99%

A Flow Cytometry Method for Dissecting the Cell Differentiation Process of Entamoeba Encystation

Mi-ichi

Miyake

Tam

et al. 2018

Front. Cell. Infect. Microbiol.

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Amoebiasis is caused by Entamoeba histolytica infection, a protozoan parasite belonging to the phylum Amoebozoa. This parasite undergoes a fundamental cell differentiation process from proliferative trophozoite to dormant cyst, termed “encystation.” The cysts formed by encystation are solely responsible for the transmission of amoebiasis; therefore, Entamoeba encystation is an important subject from both biological and medical perspectives. Here, we have established a flow cytometry strategy for not only determining the percentage of formed cysts but also for monitoring changes in cell populations during encystation. This strategy together with fluorescence microscopy enables visualization of the cell differentiation process of Entamoeba encystation. We also standardized another flow cytometry protocol for counting live trophozoites. These two different flow cytometry techniques could be integrated into 96-well plate-based bioassays for monitoring the processes of cyst formation and trophozoite proliferation, which are crucial to maintain the Entamoeba life cycle. The combined two systems enabled us to screen a chemical library, the Pathogen Box of the Medicine for Malaria Venture, to obtain compounds that inhibit either the formation of cysts or the proliferation of trophozoites, or both. This is a prerequisite for the development of new drugs against amoebiasis, a global public health problem. Collectively, the two different 96-well plate-based Entamoeba bioassay and flow cytometry analysis systems (cyst formation and trophozoite proliferation) provide a methodology that can not only overcome the limitations of standard microscopic counting but also is effective in applied as well as basic Entamoeba biology.

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“…Studies using E. invadens have revealed that cysts are typically smaller and rounder than trophozoites [8], quadrinucleated, and possess walls that are rich in chitin [9], chitosan fibrils [10] and chitin-binding proteins (e.g., [11]). The unique cyst wall confers desiccation-, acid-, heat-, and detergent-tolerance to the structure (reviewed in [2]).…”

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confidence: 99%

Flow cytometric characterization of encystation in Entamoeba invadens

Welter

Sehorn

Temesvari

2017

Molecular and Biochemical Parasitology

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Entamoeba histolytica causes dysentery and liver abscess mostly in countries that lack proper sanitation. Infection is acquired by ingestion of the cyst form in contaminated food or water. E. histolytica does not encyst in vitro; thus, E. invadens, a reptilian parasite that encysts in vitro, has been used as a surrogate. Cysts are small and possess chitin-rich walls. These are characteristics that may be exploited by flow cytometry. We stained encysting E. invadens cells with a fluorescent chitin stain, and analyzed fluorescence and forward scatter by flow cytometry. We demonstrate that flow cytometry can be used to track differentiation, reveal unique cell populations, and evaluate encystation inhibitors.

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Actin, RhoA, and Rab11 Participation during Encystment inEntamoeba invadens

Cited by 21 publications

References 28 publications

Succinylated Wheat Germ Agglutinin Colocalizes with the Toxoplasma gondii Cyst Wall Glycoprotein CST1

Succinylated Wheat Germ Agglutinin Colocalizes with the Toxoplasma gondii Cyst Wall Glycoprotein CST1

A Flow Cytometry Method for Dissecting the Cell Differentiation Process of Entamoeba Encystation

Flow cytometric characterization of encystation in Entamoeba invadens

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