Characterization and immunolocalization of an oocyst wall antigen ofCryptosporidium parvum(Protozoa: Apicomplexa)

A monoclonal antibody (OW-IGO) raised against purified excysted oocysts ofCryptosporidium parvumreacted in an immunofluorescence assay with the oocyst wall. The corresponding antigen was localized by immunoelectron microscopy in fibrillous material present in the parasitophorous vacuole of developing macrogametes and in the wall of both single and double layered sporulating oocysts. Gold particles were also detected over electron-lucent vesicles of the macrogametes by immunoelectron microscopy. On Western blotting ofC. parvumoocyst extracts, major bands at 250 and 40 kDa and several minor components were recognized by Mab OW-IGO. Almost complete abolition of Western blot reactivity occurred after periodate oxidation of oocyst antigen, suggesting that monoclonal antibody OW-IGO reacts with a carbohydrate epitope. Taken together, our results suggest that a fibrillous glycoproteinic material is released in the parasitophorous vacuole from electron-lucent vesicles during gametogenesis, and later condensed in the oocyst wall.

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Implication of Potential Differential Roles of the Two Phosphoglucomutase Isoforms in the Protozoan Parasite Cryptosporidium parvum

Pathogens ◽

10.3390/pathogens11010021 ◽

2021 ◽

Vol 11 (1) ◽

pp. 21

Author(s):

Jiawen Nie ◽

Jigang Yin ◽

Dongqiang Wang ◽

Chenchen Wang ◽

Guan Zhu

Keyword(s):

Enzyme Activity ◽

Recombinant Proteins ◽

Cryptosporidium Parvum ◽

Plasma Membranes ◽

Parasitophorous Vacuole ◽

Protozoan Parasite ◽

Immunofluorescence Assay ◽

Parasitophorous Vacuole Membrane ◽

Vacuole Membrane ◽

Intracellular Parasites

Phosphoglucomutase 1 (PGM1) catalyzes the conversion between glucose-1-phosphate and glucose-6-phosphate in the glycolysis/glucogenesis pathway. PGM1s are typically cytosolic enzymes in organisms lacking chloroplasts. However, the protozoan Cryptosporidium parasites possess two tandemly duplicated PGM1 genes evolved by a gene duplication after their split from other apicomplexans. Moreover, the downstream PGM1 isoform contains an N-terminal signal peptide, predicting a non-cytosolic location. Here we expressed recombinant proteins of the two PGM1 isoforms from the zoonotic Cryptosporidium parvum, namely CpPGM1A and CpPGM1B, and confirmed their enzyme activity. Both isoforms followed Michaelis–Menten kinetics towards glucose-1-phosphate (Km = 0.17 and 0.13 mM, Vmax = 7.30 and 2.76 μmol/min/mg, respectively). CpPGM1A and CpPGM1B genes were expressed in oocysts, sporozoites and intracellular parasites at a similar pattern of expression, however CpPGM1A was expressed at much higher levels than CpPGM1B. Immunofluorescence assay showed that CpPGM1A was present in the cytosol of sporozoites, however this was enriched towards the plasma membranes in the intracellular parasites; whereas CpPGM1B was mainly present under sporozoite pellicle, although relocated to the parasitophorous vacuole membrane in the intracellular development. These observations indicated that CpPGM1A played a house-keeping function, while CpPGM1B played a different biological role that remains to be defined by future investigations.

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Ultrastructural changes in the oocyst wall during excystation of Cryptosporidium parvum (Apicomplexa; Eucoccidiorida)

Canadian Journal of Zoology ◽

10.1139/z85-281 ◽

1985 ◽

Vol 63 (8) ◽

pp. 1892-1896 ◽

Cited By ~ 28

Author(s):

David W. Reduker ◽

C. A. Speer ◽

John A. Blixt

Keyword(s):

Cryptosporidium Parvum ◽

Outer Layer ◽

Ultrastructural Changes ◽

Oocyst Wall ◽

Thin Electron ◽

Cryptosporidium Parvum Oocysts ◽

Electron Lucent

Cryptosporidium parvum oocysts were obtained from the feces of naturally infected calves and the oocyst wall was examined ultrastructurally. The oocyst wall averaged 49.7 nm thick and was composed of two layers. The outer layer was irregular in thickness, averaging 10 nm. A thin, electron-lucent space (2.5 nm) was interposed between the outer and inner layers. The inner layer had an outer (11.6 nm) and an inner (25.8 nm) zone. A suture that extended partway around the oocyst was observed within the inner layer of the oocyst wall. The suture underwent dissolution during excystation.

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Characterisation of NcGRA7 and NcSAG4 proteins: Immunolocalisation and their role in the host cell invasion by Neospora caninum tachyzoites

Acta Parasitologica ◽

10.2478/s11686-010-0056-9 ◽

2010 ◽

Vol 55 (4) ◽

Cited By ~ 9

Author(s):

Adriana Aguado-Martínez ◽

Gema Álvarez-García ◽

Gereon Schares ◽

Verónica Risco-Castillo ◽

Aurora Fernández-García ◽

...

Keyword(s):

Monoclonal Antibody ◽

Host Cell ◽

Cell Invasion ◽

Neospora Caninum ◽

Parasitophorous Vacuole ◽

Membrane Surface ◽

Chronic Phase ◽

Host Cell Invasion ◽

Invasion Mechanisms ◽

The Matrix

AbstractNeospora caninum negatively impacts bovine reproductive performance around the world. Addressing this problem requires a greater understanding of the parasite’s molecular biology. In this study, monoclonal antibodies against recombinant proteins were successfully developed and employed to characterise two different proteins of N. caninum: the acute phase-associated NcGRA7 and the chronic phase-associated NcSAG4. Immunofluorescence with the anti-rNcGRA7 monoclonal antibody suggested that NcGRA7 trafficks from tachyzoite dense granules to the matrix of the parasitophorous vacuole and parasite’s surroundings. Furthermore, NcGRA7 is also expressed in the bradyzoite stage and localised on the matrix of bradyzoite-positive vacuoles. NcGRA7 appears to be partially involved in the tachyzoite-invasion mechanisms, as an anti-rNcGRA7 monoclonal antibody partially inhibited in vitro tachyzoite-invasion. A monoclonal antibody specific for NcSAG4 confirmed this protein’s bradyzoitespecific expression both by western blot and immunofluorescence. However, some bradyzoite-positive vacuoles only weakly expressed NcSAG4, if it was expressed at all. The specificity of the anti-rNcSAG4 monoclonal antibody was confirmed by the recognition of the NcSAG4 in the membrane surface of Nc-1SAG4c transgenic tachyzoites, which constitutively expresses NcSAG4. Blocking NcSAG4 of Nc-1SAG4c tachyzoites with the monoclonal antibody did not affect host cell invasion. However, its implication on the host cell adhesion or host immune evasion should not be discarded.

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Immunoelectron microscopic localization of an oviductal antigen in hamster zona pellucida by use of a monoclonal antibody.

Journal of Histochemistry & Cytochemistry ◽

10.1177/36.11.3171167 ◽

1988 ◽

Vol 36 (11) ◽

pp. 1441-1447 ◽

Cited By ~ 34

Author(s):

F W Kan ◽

S St-Jacques ◽

G Bleau

Keyword(s):

Monoclonal Antibody ◽

Zona Pellucida ◽

Preimplantation Embryo ◽

Protein A ◽

Cumulus Cells ◽

Gold Particles ◽

Antigenic Sites ◽

Cumulus Oophorus ◽

Entire Thickness ◽

Immunocytochemical Method

The zona pellucida is an extracellular matrix of glycoproteins which surrounds the mammalian oocyte and preimplantation embryo. We have recently developed monoclonal antibodies against oviductal zona pellucida of the golden hamster. We applied the post-embedding immunocytochemical method using a monoclonal antibody (IgGl,k) to determine the precise location of antigenic sites in the cumulus oophorus complex of the superovulated hamster. By applying the high-resolution protein A-gold technique, we demonstrated that the sites of immunoreactivity were exclusively in the zona pellucida encompassing the oocyte. Other structures within the oocyte and neighboring cumulus cells were not labeled by gold particles. Moreover, gold particles were evenly distributed throughout the entire thickness of the zona pellucida, indicating that this extracellular layer is at least in part made up of an antigen recognized by the monoclonal antibody that is uniformly distributed in the zona matrix.

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Preparation, use, and enlargement of ultrasmall gold particles in immunoelectron microscopy

Microscopy Research and Technique ◽

10.1002/(sici)1097-0029(19980701)42:1<66::aid-jemt8>3.0.co;2-p ◽

1998 ◽

Vol 42 (1) ◽

pp. 66-79 ◽

Cited By ~ 35

Author(s):

Werner Baschong ◽

York-Dieter Stierhof

Keyword(s):

Immunoelectron Microscopy ◽

Gold Particles

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Detection of Cryptosporidium parvum in Horses: Thresholds of Acid-Fast Stain, Immunofluorescence Assay, and Flow Cytometry

Journal of Clinical Microbiology ◽

10.1128/jcm.37.2.457-460.1999 ◽

1999 ◽

Vol 37 (2) ◽

pp. 457-460 ◽

Cited By ~ 12

Author(s):

D. J. Cole ◽

K. Snowden ◽

N. D. Cohen ◽

R. Smith

Keyword(s):

Flow Cytometry ◽

Cryptosporidium Parvum ◽

Immunofluorescence Assay ◽

Immunofluorescent Antibody ◽

Acid Fast Staining

Feces collected from three asymptomatic horses and seeded withCryptosporidium parvum oocysts (101 to 106/g of feces) were evaluated by acid-fast staining (AF), an immunofluorescent antibody (IFA) technique, and flow cytometry. The thresholds of detection were 5 × 105 oocysts/g of feces for the IFA and AF techniques and 5 × 104oocysts/g for flow cytometry.

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Detection of UV-Induced Thymine Dimers in Individual Cryptosporidium parvum and Cryptosporidium hominis Oocysts by Immunofluorescence Microscopy

Applied and Environmental Microbiology ◽

10.1128/aem.01251-06 ◽

2006 ◽

Vol 73 (3) ◽

pp. 947-955 ◽

Cited By ~ 23

Author(s):

B. H. Al-Adhami ◽

R. A. B. Nichols ◽

J. R. Kusel ◽

J. O'Grady ◽

H. V. Smith

Keyword(s):

Cryptosporidium Parvum ◽

Immunofluorescence Microscopy ◽

Uv Light ◽

Fluorescein Isothiocyanate ◽

Immunofluorescence Assay ◽

Cryptosporidium Hominis ◽

Specific Fluorescence ◽

Thymine Dimers ◽

Texas Red

ABSTRACT To investigate the effect of UV light on Cryptosporidium parvum and Cryptosporidium hominis oocysts in vitro, we exposed intact oocysts to 4-, 10-, 20-, and 40-mJï¿½cm−2 doses of UV irradiation. Thymine dimers were detected by immunofluorescence microscopy using a monoclonal antibody against cyclobutyl thymine dimers (anti-TDmAb). Dimer-specific fluorescence within sporozoite nuclei was confirmed by colocalization with the nuclear fluorogen 4′,6′-diamidino-2-phenylindole (DAPI). Oocyst walls were visualized using either commercial fluorescein isothiocyanate-labeled anti-Cryptosporidium oocyst antibodies (FITC-CmAb) or Texas Red-labeled anti-Cryptosporidium oocyst antibodies (TR-CmAb). The use of FITC-CmAb interfered with TD detection at doses below 40 mJï¿½cm−2. With the combination of anti-TDmAb, TR-CmAb, and DAPI, dimer-specific fluorescence was detected in sporozoite nuclei within oocysts exposed to 10 to 40 mJï¿½cm−2 of UV light. Similar results were obtained with C. hominis. C. parvum oocysts exposed to 10 to 40 mJï¿½cm−2 of UV light failed to infect neonatal mice, confirming that results of our anti-TD immunofluorescence assay paralleled the outcomes of our neonatal mouse infectivity assay. These results suggest that our immunofluorescence assay is suitable for detecting DNA damage in C. parvum and C. hominis oocysts induced following exposure to UV light.

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