Flow cytometric determination of intracellular non-protein thiols in Leishmania promastigotes using 5-chloromethyl fluorescein diacetate |
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Authors: | Avijit Sarkar Goutam Mandal Neeloo Singh Mitali Chatterjee |
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Affiliation: | a Department of Pharmacology, Institute of Post Graduate Medical Education and Research, Kolkata, 244B Acharya JC Bose Road, Kolkata-700 020, India b Drug Target Discovery & Development Division, Central Drug Research Institute, Lucknow, India c Dept. of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India |
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Abstract: | Leishmania parasites lack catalase and therefore, their anti-oxidant system hinges primarily upon non-protein thiols; accordingly, depletion of thiols could potentially serve as an effective drug target. We have developed a flow cytometry based assay using 5-chloromethyl fluorescein diacetate based upon its selective staining of non-protein thiols. Its specificity was confirmed using buthionine sulphoximine (a γ-glutamyl cysteine synthetase inhibitor), diamide (an oxidizing agent of intracellular thiols) and N-ethylmaleimide (a covalent modifier of cysteine residues) as evidenced by reduction in fluorescence; furthermore, restoration of fluorescence by N-acetyl cysteine corroborated specificity of 5-chloromethyl fluorescein diacetate to measure non-protein thiols. Differences in basal level of thiols in antimony sensitive and antimony resistant Leishmania field isolates were detected. The depletion of non-protein thiols by conventional anti-leishmanial drugs e.g. antimony and miltefosine was demonstrated. Furthermore, fluorescence was unaffected by depletion of ATP in majority of the strains studied, indicating that 5-chloromethyl fluorescein diacetate is not a substrate for the pump operative in most Leishmania donovani strains. Taken together, measurement of 5-chloromethyl fluorescein diacetate fluorescence is an effective method for monitoring non-protein thiols in Leishmania promastigotes. |
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Keywords: | CMFDA Thiols Leishmaniasis Antileishmanial Promastigotes Antimony resistance |
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