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Easy labeling of proliferative phase and sporogonic phase of microsporidia Nosema bombycis in host cells.

ABSTRACT: Microsporidia are eukaryotic, unicellular parasites that have been studied for more than 150 years. These organisms are extraordinary in their ability to invade a wide range of hosts including vertebrates and invertebrates, such as human and commercially important animals. A lack of appropriate labeling methods has limited the research of the cell cycle and protein locations in intracellular stages. In this report, an easy fluorescent labeling method has been developed to mark the proliferative and sporogonic phases of microsporidia Nosema bombycis in host cells. Based on the presence of chitin, Calcofluor White M2R was used to label the sporogonic phase, while ?-tubulin antibody coupled with fluorescence secondary antibody were used to label the proliferative phase by immunofluorescence. This method is simple, efficient and can be used on both infected cells and tissue slices, providing a great potential application in microsporidia research.

SUBMITTER: Chen J

PROVIDER: S-EPMC5480951 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Easy labeling of proliferative phase and sporogonic phase of microsporidia Nosema bombycis in host cells.

Chen Jie J Guo Wei W Dang Xiaoqun X Huang Yukang Y Liu Fangyan F Meng Xianzhi X An Yaoyao Y Long Mengxian M Bao Jialing J Zhou Zeyang Z Xiang Zhonghuai Z Pan Guoqing G

PloS one 20170622 6

Microsporidia are eukaryotic, unicellular parasites that have been studied for more than 150 years. These organisms are extraordinary in their ability to invade a wide range of hosts including vertebrates and invertebrates, such as human and commercially important animals. A lack of appropriate labeling methods has limited the research of the cell cycle and protein locations in intracellular stages. In this report, an easy fluorescent labeling method has been developed to mark the proliferative ...[more]

PMID: 28640848

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Project description:Microsporidia are obligate intracellular parasites that infect a wide variety of host organisms, including humans. The sporoplasm is the initial stage of microsporidian infection and proliferation, but its morphological and molecular characteristics are poorly understood. In this study, the sporoplasm of Nosema bombycis was successfully isolated and characterized after the induction of spore germination in vitro The sporoplasm was spherical, 3.64 ± 0.41 μm in diameter, had the typical two nuclei, and was nonrefractive. Scanning and transmission electron microscopy analyses revealed that the sporoplasm was surrounded by a single membrane, and the cytoplasm was usually filled with relatively homogeneous granules, possibly ribosomes, and contained a vesicular structure comprising a concentric ring and coiled tubules. Propidium iodide staining revealed that the sporoplasm membrane showed stronger membrane permeability than did the cell plasma membrane. Transmission electron microscopy (TEM) revealed that the sporoplasm can gain entry to the host cell by phagocytosis. Transcriptome analysis of mature spores and sporoplasms showed that 541 significantly differentially expressed genes were screened (adjusted P value [Padj] < 0.05), of which 302 genes were upregulated and 239 genes were downregulated in the sporoplasm. The majority of the genes involved in trehalose synthesis metabolism, glycolysis, and the pentose phosphate pathway were downregulated, whereas 10 transporter genes were upregulated, suggesting that the sporoplasm may inhibit its own carbon metabolic activity and obtain the substances required for proliferation through transporter proteins. This study represents the first comprehensive and in-depth investigation of the sporoplasm at the morphological and molecular levels and provides novel insights into the biology of microsporidia and their infection mechanism.IMPORTANCE Once awoken from dormancy, the cellular matter of microsporidia is delivered directly into the host cell cytoplasm through the polar tube. This means that the microsporidia are difficult to study biologically in their active state without a contaminating signal from the host cell. Sporoplasm is a cell type of microsporidia in vitro, but relatively little attention has been paid to the sporoplasm in the past 150 years due to a lack of an effective separation method. Nosema bombycis, the first reported microsporidium, is a type of obligate intracellular parasite that infects silkworms and can be induced to germinate in alkaline solution in vitro We successfully separated the N. bombycis sporoplasm in vitro, and the morphological and structural characteristics were investigated. These results provide important insight into the biology and pathogenesis of microsporidia and potentially provide a possible strategy for genetic manipulation of microsporidia targeting the sporoplasm.

Dataset Information

Easy labeling of proliferative phase and sporogonic phase of microsporidia Nosema bombycis in host cells.

Publications

Easy labeling of proliferative phase and sporogonic phase of microsporidia Nosema bombycis in host cells.

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