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Cryptosporidium parvum sporozoites contain glutathione

Published online by Cambridge University Press:  04 July 2006

B. H. AL-ADHAMI ,
R. A. B. NICHOLS ,
J. R. KUSEL ,
J. O'GRADY  and
H. V. SMITH
B. H. AL-ADHAMI
Affiliation:
Scottish Parasite Diagnostic Laboratory, Stobhill Hospital, Glasgow G21 3UW, UK
R. A. B. NICHOLS
Affiliation:
Scottish Parasite Diagnostic Laboratory, Stobhill Hospital, Glasgow G21 3UW, UK
J. R. KUSEL
Affiliation:
Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK
J. O'GRADY
Affiliation:
Department of Immunology, University of Strathclyde, Glasgow G1, UK
H. V. SMITH
Affiliation:
Scottish Parasite Diagnostic Laboratory, Stobhill Hospital, Glasgow G21 3UW, UK
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Abstract

We used the fluorescent dye monochlorobimane (MCB) which binds glutathione (GSH) to localize between 2 and 6 distinctly labelled nuclear and cytoplasmic GSH foci in recently excreted and aged, intact Cryptosporidium parvum oocysts and sporozoites. Buthionine sulfoximine (BSO), a potent and specific inhibitor of GSH, was used to determine whether GSH is synthesized in BSO-treated C. parvum oocysts, by labelling treated oocysts with MCB. Both visual and electronic quantifications were performed. At 5 mM BSO, a significant inhibition of MCB fluorescence, reflecting reduced MCB uptake, was observed in GSH-depleted oocysts (mean±S.D. 35±3·7) compared with controls (3·3±1·2, P=0). This clear reduction occurred only in viable oocysts. 1 mM BSO-treated oocysts exhibited weak or no MCB fluorescence, although they were viable (excluded propidium iodide, PI)), and intact and contained sporozoites by differential interference contrast microscopy (DIC). MCB was used in conjunction with PI to determine C. parvum oocyst viability. Oocysts labelled with MCB/PI or 4′6-diamidino-2-phenyl indole (DAPI)/PI produced comparable labelling patterns. Viable oocysts were labelled with MCB or DAPI whereas dead oocysts were labelled with PI only. The localization of GSH in viable, intact oocysts and excysted sporozoites and UV light-irradiated oocysts and sporozoites revealed no changes in MCB uptake at levels up to 40 mJ.cm−2 irradiation. Although GSH can be detected following MCB localization in both the nucleus and cytoplasm of sporozoites, and can be specifically depleted by BSO treatment, MCB is unlikely to be useful as a surrogate for detecting UV damage in UV-treated Cryptosporidium oocysts.

Keywords

Cryptosporidium parvumoocystssporozoitesglutathionemonocholorobimanebuthionine sulfoximineUV light
Type
Research Article
Information
Parasitology , Volume 133 , Issue 5 , November 2006 , pp. 555 - 563
Copyright
2006 Cambridge University Press

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