EnP1 and EnP2, two proteins associated with the Encephalitozoon cuniculi endospore, the chitin-rich inner layer of the microsporidian spore wall

The microsporidian spore extrusion apparatus activates with a calcium influx from Spraguea lophii spore wall/plasma membrane; this influx requires preconditioning with an extrasporular shift in medium pH to the alkaline in the presence of the polyanions mucin or polyglutamate. Undischarged S. lophii spores display calcium bound to the wall/plasma membrane with a characteristic calcium-chlorotetracycline fluorescence; this fluorescence declines significantly during spore discharge. S. lophii spores do not discharge when spore wall/plasma membrane calcium is removed with EGTA. Extrasporular mucin or polyglutamate and a pH shift to the alkaline appear to be necessary preconditions for the triggering of the influx of spore wall/plasma membrane-bound 45Ca2+. Ionophore A-23187 also effectively activates spore discharge without other extrasporular polyanions. Micromolar concentrations of the calcium antagonists lanthanum or verapamil prevent spore discharge, and micromolar concentrations of calmodulin inhibitors chlorpromazine and trifluroperazine prevent spore discharge. Calmodulin, visualized with a calmodulin antibody and a peroxidase conjugate, is localized particularly on the plasma membrane and the polaroplast membranes of the extrusion apparatus.

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Discrimination between Viable and Dead Encephalitozoon cuniculi (Microsporidian) Spores by Dual Staining with Sytox Green and Calcofluor White M2R

Journal of Clinical Microbiology ◽

10.1128/jcm.38.10.3811-3814.2000 ◽

2000 ◽

Vol 38 (10) ◽

pp. 3811-3814 ◽

Cited By ~ 26

Author(s):

L. C. Green ◽

P. J. LeBlanc ◽

E. S. Didier

Keyword(s):

Systemic Disease ◽

Organ Transplant ◽

Spore Wall ◽

Staining Procedure ◽

Excitation Wavelength ◽

Encephalitozoon Cuniculi ◽

Calcofluor White ◽

Sytox Green ◽

Intracellular Parasites ◽

Dual Staining

Microsporidia are obligate intracellular parasites, recognized as causing chronic diarrhea and systemic disease in AIDS patients, organ transplant recipients, travelers, and malnourished children. Species of microsporidia that infect humans have been detected in drinking-water sources, and methods are needed to ascertain if these microsporidia are viable and capable of causing infections. In this study, Calcofluor White M2R and Sytox Green stains were used in combination to differentiate between live (freshly harvested) and dead (boiled)Encephalitozoon cuniculi spores. Calcofluor White M2R binds to chitin in the microsporidian spore wall. Dual-stained live spores appeared as turquoise-blue ovals, while dead spores appeared as white-yellow ovals at an excitation wavelength of 395 to 415 nm used for viewing the Calcofluor stain. Sytox Green, a nuclear stain, is excluded by live spores but penetrates compromised spore membranes. Dual-stained dead spores fluoresced bright yellow-green when viewed at an excitation wavelength of 470 to 490 nm, whereas live spores failed to stain with Sytox Green. After live and dead spores were mixed at various ratios, the number of viably stained spores detected in the dual-staining procedure correlated (P = 0.0025) with the expected numbers of viable spores. Spore mixtures were also assayed for infectivity in a focus-forming assay, and a correlation (P = 0.0002) was measured between the percentage of focus-forming microsporidia and the percentage of expected infectious spores in each mixture. By analysis of variance, no statistically significant differences were measured between the percentage of viably stained microsporidia and the percentage of infectious microsporidia (P = 0.964) in each mixture. These results suggest that Calcofluor White M2R and Sytox Green stains, when used together, may facilitate studies to identify viable microsporidia.

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Branching Network of Proteinaceous Filaments within the Parasitophorous Vacuole ofEncephalitozoon cuniculiandEncephalitozoon hellem

Infection and Immunity ◽

10.1128/iai.01152-10 ◽

2011 ◽

Vol 79 (3) ◽

pp. 1374-1385 ◽

Cited By ~ 8

Author(s):

Kaya Ghosh ◽

Eddie Nieves ◽

Patrick Keeling ◽

Jean-Francois Pombert ◽

Philipp P. Henrich ◽

...

Keyword(s):

Host Cell ◽

Parasitophorous Vacuole ◽

Spore Wall ◽

Human Pathogens ◽

Polar Tube ◽

Molecular Approaches ◽

Resting Spore ◽

Intracellular Parasites ◽

Significant Pathology ◽

Microsporidian Spore

ABSTRACTThe microsporidia are a diverse phylum of obligate intracellular parasites that infect all major animal groups and have been recognized as emerging human pathogens for which few chemotherapeutic options currently exist. These organisms infect every tissue and organ system, causing significant pathology, especially in immune-compromised populations. The microsporidian spore employs a unique infection strategy in which its contents are delivered into a host cell via the polar tube, an organelle that lies coiled within the resting spore but erupts with a force sufficient to pierce the plasma membrane of its host cell. Using biochemical and molecular approaches, we have previously identified components of the polar tube and spore wall of the Encephalitozoonidae. In this study, we employed a shotgun proteomic strategy to identify novel structural components of these organelles inEncephalitozoon cuniculi. As a result, a new component of theE. cuniculideveloping spore wall was identified. Surprisingly, using the same approach, a heretofore undescribed filamentous network within the lumen of the parasitophorous vacuole was discovered. This network was also present in the parasitophorous vacuole ofEncephalitozoon hellem. Thus, in addition to further elucidating the molecular composition of seminal organelles and revealing novel diagnostic and therapeutic targets, proteomic analysis-driven approaches exploring the spore may also uncover unknown facets of microsporidian biology.

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Evaluation of spore wall protein 1 as an alternative antigen for the diagnosis of Encephalitozoon cuniculi infection of farmed foxes using an enzyme-linked immunosorbent assay

Veterinary Parasitology ◽

10.1016/j.vetpar.2014.03.011 ◽

2014 ◽

Vol 203 (3-4) ◽

pp. 331-334 ◽

Cited By ~ 1

Author(s):

Xiangchun Meng ◽

Jun Zheng ◽

Yang Gao ◽

Yanlong Zhang ◽

Honglin Jia

Keyword(s):

Immunosorbent Assay ◽

Spore Wall ◽

Enzyme Linked Immunosorbent Assay ◽

Encephalitozoon Cuniculi

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Evidence for lack of sclerotization in a microsporidian spore wall

Journal of Invertebrate Pathology ◽

10.1016/0022-2011(84)90149-6 ◽

1984 ◽

Vol 43 (2) ◽

pp. 276-277 ◽

Cited By ~ 2

Author(s):

Jeffrey C. Lord ◽

Donald W. Wall

Keyword(s):

Spore Wall ◽

Microsporidian Spore

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Developmental Expression of a Tandemly Repeated, Glycine- and Serine-Rich Spore Wall Protein in the Microsporidian Pathogen Encephalitozoon cuniculi

Infection and Immunity ◽

10.1128/iai.68.4.2268-2275.2000 ◽

2000 ◽

Vol 68 (4) ◽

pp. 2268-2275 ◽

Cited By ~ 66

Author(s):

Wolfgang Bohne ◽

David J. P. Ferguson ◽

Karoline Kohler ◽

Uwe Gross

Keyword(s):

Parasitophorous Vacuole ◽

Mrna Level ◽

Spore Wall ◽

Host Cells ◽

Developmental Expression ◽

Pcr Analysis ◽

Mechanical Resistance ◽

Cdna Expression Library ◽

Encephalitozoon Cuniculi ◽

Expression Library

ABSTRACT Microsporidia are intracellular organisms of increasing importance as opportunistic pathogens in immunocompromised patients. Host cells are infected by the extrusion and injection of polar tubes located within spores. The spore is surrounded by a rigid spore wall which, in addition to providing mechanical resistance, might be involved in host cell recognition and initiation of the infection process. A 51-kDa outer spore wall protein was identified in Encephalitozoon cuniculi with the aid of a monoclonal antibody, and the corresponding gene, SWP1, was cloned by immunoscreening of a cDNA expression library. The cDNA encodes a protein of 450 amino acids which displays no significant similarities to known proteins in databases. The carboxy-terminal region consists of five tandemly arranged glycine- and serine-rich repetitive elements. SWP1is a single-copy gene that is also present in the genomes ofEncephalitozoon intestinalis and Encephalitozoon hellem as demonstrated by Southern analysis. Indirect immunofluorescence and immunoelectron microscopy revealed that SWP1 is differentially expressed during the infection cycle. The protein is absent in replicative meronts until 24 h postinfection, and its expression is first induced in early sporonts at a time when organisms translocate from the periphery to the center of the parasitophorous vacuole. Expression of SWP1 appears to be regulated at the mRNA level, as was shown by reverse transcriptase PCR analysis. Further identification and characterization of stage-specific genes might help to unravel the complex intracellular differentiation process of microsporidia.

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The putative chitin deacetylase ofEncephalitozoon cuniculi: A surface protein implicated in microsporidian spore-wall formation

FEMS Microbiology Letters ◽

10.1016/j.femsle.2005.04.031 ◽

2005 ◽

Vol 247 (1) ◽

pp. 81-90 ◽

Cited By ~ 43

Author(s):

Damien Brosson ◽

Lauriane Kuhn ◽

Christian P. VivarÃ¨s ◽

Catherine Texier

Keyword(s):

Surface Protein ◽

Spore Wall ◽

Chitin Deacetylase ◽

Wall Formation ◽

Microsporidian Spore

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Identification of a New Spore Wall Protein from Encephalitozoon cuniculi

Infection and Immunity ◽

10.1128/iai.74.1.239-247.2006 ◽

2006 ◽

Vol 74 (1) ◽

pp. 239-247 ◽

Cited By ~ 51

Author(s):

Yanji Xu ◽

Peter Takvorian ◽

Ann Cali ◽

Fang Wang ◽

Hong Zhang ◽

...

Keyword(s):

Spore Wall ◽

Chromosome 1 ◽

Encephalitozoon Cuniculi ◽

Polar Tube ◽

Chitin Deacetylase ◽

Reading Frame ◽

Two Dimensional Electrophoresis ◽

Glycosylation Sites ◽

Spore Walls ◽

Dimensional Electrophoresis

ABSTRACT Microsporidia form environmentally resistant spores that are critical for their host-to-host transmission and persistence in the environment. The spore walls of these organisms are composed of two layers, the exospore and the endospore. Two spore wall proteins (SWP1 and SWP2) have been previously identified in members of the Encephalitozoonidae family. These proteins localize to the exospore. The endospore is known to contain chitin, and a putative glycosylphosphatidylinositol (GPI)-anchored chitin deacetylase has been localized to the plasmalemma-endospore interface. Using proteomic techniques, we have identified a new spore wall protein (SWP3) that is located in the endospore. The gene for this protein is located on chromosome 1 and corresponds to the open reading frame ECU01_1270. SWP3 is predicted to have a signal peptide and to be GPI anchored. Consistent with these modifications, two-dimensional electrophoresis demonstrated that SWP3 has an acidic pI and a molecular mass of <20 kDa. By immunoelectron microscopy, this protein was found on the cell surface during sporogony and in the endospore in mature spores. SWP3 has several potential O-glycosylation sites, and it is possible that it is a mannosylated protein like the major polar tube protein (PTP1).

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