Flow cytometric determination of intracellular non-protein thiols in Leishmania promastigotes using 5-chloromethyl fluorescein diacetate.
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 gamma-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.
Sarkar
Avijit
A
Department of Pharmacology, Institute of Post Graduate Medical Education and Research, Kolkata, 244B Acharya JC Bose Road, Kolkata-700 020, India.
Mandal
Goutam
G
Singh
Neeloo
N
Sundar
Shyam
S
Chatterjee
Mitali
M
D000818:Animals; D000967:Antimony Sodium Gluconate / Q000494:pharmacology; D000981:Antiprotozoal Agents / Q000494:pharmacology; D016895:Culture Media, Serum-Free; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D007893:Leishmania donovani / Q000737:chemistry* / Q000187:drug effects; D010419:Pentamidine / Q000494:pharmacology; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000494:pharmacology; D012680:Sensitivity and Specificity; D013438:Sulfhydryl Compounds / Q000032:analysis*
D000981:Antiprotozoal Agents; D016895:Culture Media, Serum-Free; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013438:Sulfhydryl Compounds; D010767:Phosphorylcholine; C069306:5-chloromethylfluorescein; C039128:miltefosine; D010419:Pentamidine; D000967:Antimony Sodium Gluconate