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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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References

REFERENCES

Anderson, M. E. ( 1998). Glutathione: an overview of biosynthesis and modulation. Chemistry and Biology Interaction 24, 1–14.CrossRefGoogle Scholar
Bellomo, G., Vairetti, M., Stivala, L., Mirabelli, F., Richelmi, P. and Orrenius, S. ( 1992). Demonstration of nuclear compartmentalisation of glutathione in hepatocytes. Proceedings of the National Academy of Sciences, USA 89, 4412–4416.CrossRefGoogle Scholar
Briviba, K., Fraser, G., Sies, H. and Ketterer, B. ( 1993). Distribution of monochlorobimane-glutathione conjugate between nucleus and cytosol in isolated hepatocytes. The Biochemical Journal 294, 631–633.CrossRefGoogle Scholar
Brown, D., Upcroft, J. and Upcroft, P. ( 1995). Free radical detoxification in Giardia duodenalis. Molecular and Biochemical Parasitology 72, 47–56.CrossRefGoogle Scholar
Campbell, A., Robertson, L. and Smith, H. ( 1992). Viability of Cryptosporidium parvum oocysts: Correlation of in vitro excystation with inclusion or exclusion of fluorogenic vital dyes. Applied and Experimental Microbiology 58, 3488–3493.Google Scholar
Cheng, K. C., Cahill, D. S., Kasai, H., Nishimura, S. and Leob, L. A. ( 1992). 8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G-T and A-T substitution. Journal of Biological Chemistry 267, 166–172.Google Scholar
Entrala, E., Mascaro, C. and Barrett, J. ( 1997). Anti-oxidant enzymes in Cryptosporidium parvum oocysts. Parasitology 114, 13–17.CrossRefGoogle Scholar
Fairlamb, A. and Cerami, A. ( 1992). Metabolism and functions of trypanothione in the Kinetoplastida. Annual Review of Microbiology 46, 695–729.CrossRefGoogle Scholar
Fairlamb, A., Blackburn, P., Ulrich, P., Chait, B. and Cerami, A. ( 1985). Trypanothione: a novel bis (glutathionyl) spermidine cofactor for glutathione reductase in trypanosomatids. Science 227, 1485–1487.CrossRefGoogle Scholar
Fischer-Neilsen, A., Jeding, I. B. and Lofts, S. ( 1994). Radiation- induced formation of 8-hydroxy-2-deoxyguanosine and its prevention by scavengers. Carcinogenesis 15, 1609–1612.CrossRefGoogle Scholar
Haugland, R. P. ( 2005). Probes for cell adhesion, chemotaxis, multidrug resistance and glutathione. In The Handbook: A Guide to Fluorescent Probes and Labelling Technologies ( ed. Spence, M. T. Z), pp. 767–776. Molecular Probes Inc., Oregon, USA.
Huang, K. and Yang, S. ( 2002). Inhibitory effect of selenium on Cryptosporidium parvum infection in vitro and in vivo. Biological Trace Element Research 90, 261–272.CrossRefGoogle Scholar
Mansur, D., Kataoka, Y., Gridna, D. and Diamond, A. ( 2001). Radiosensitivity of mamamlian cell lines engineered to overexpress cytosolic glutathione peroxidase. Radiation Research 155, 536–542.CrossRefGoogle Scholar
Meister, A. and Anderson, M. E. ( 1983). Glutathione. Annual Review of Biochemistry 52, 711–760.CrossRefGoogle Scholar
Ribeiro, F., Coelho, P., Vieira, L., Watson, D. and Kusel, J. ( 1998). The effect of praziquantel treatment on glutathione concentration in Schistosoma mansoni. Parasitology 116, 229–236.CrossRefGoogle Scholar
Robertson, L., Campbell, A. and Smith, H. ( 1993). In vitro excystation of Cryptosporidium parvum. Parasitology 106, 13–19.CrossRefGoogle Scholar
Rochelle, P. A., Fallar, D., Marshall, M. M., Montelone, B. A., Upton, S. J. and Woods, K. ( 2004). Irreversible UV inactivation of Cryptosporidium spp. despite the presence of repair genes. Journal of Eukaryotic Microbiology 51, 553–562.Google Scholar
Rochelle, P., Upton, S., Montelone, B. and Woods, K. ( 2005). The response of Cryptosporidium parvum to UV light. Trends in Parasitology 21, 81–87.CrossRefGoogle Scholar
Stommel, E., Cho, E., Steide, J., Seguin, R., Barchowsky, A., Schwartzman, J. and Kasper, L. ( 2001). Identification and role of thiols in Toxoplasma gondii egress. Experimental Biology and Medicine 226, 229–236.CrossRefGoogle Scholar
Tyagi, R., Kumar, A., Tyagi, M., Jha, P., Kumar, H., Sinha, R. and Hader, D. ( 2003). Protective role of certain chemicals against UV-B-induced damage in the nitrogen-fixing cyanobacterium, Nostoc muscorum. Journal of Basic Microbiology 43, 137–147.CrossRefGoogle Scholar
Wilkinson, S., Taylor, M., Touitha, S., Mauricio, I., Meyer, D. and Kelly, J. ( 2002). TcGPXII, a glutathione-dependent Trypanosoma cruzi peroxidase with substrate specificity restricted to fatty acid and phospholipid hydroperoxide, is localised to the endoplasmic reticulum. The Biochemical Journal 364, 787–794.CrossRefGoogle Scholar
Wilkinson, S., Temperton, N., Mondargon, A. and Kelly, J. ( 2000). Distinct mitochondrial and cytosolic enzymes mediate trypanothione-dependent peroxide metabolism in Trypanosoma cruzi. Journal of Biological Chemistry 275, 8220–8225.CrossRefGoogle Scholar
Will, O., Mahler, H., Arrigo, A. and Epe, B. ( 1999). Influence of glutathione levels and heat-shock on the steady-state levels of oxidative DNA base modifications in mammalian cells. Carcinogenesis 20, 333–337.CrossRefGoogle Scholar