Functionality of drug efflux pumps in antimonial resistant Leishmania donovani field isolates.
The recent upsurge of antimony (Sb) resistance is a major impediment to successful chemotherapy of visceral leishmaniasis (VL). Mechanisms involved in antimony resistance have demonstrated an upregulation of drug efflux pumps; however, the biological role drug efflux pumps in clinical isolates remains to be substantiated. Thus, in this study, the functionality of drug efflux pumps was measured in promastigotes and axenic amastigotes isolated from VL patients, who were either Sb-sensitive (AG83, 2001 and MC9) or resistant (NS2, 41 and GE1) using rhodamine123 as a substrate for multidrug resistant (MDR) pumps and calcein as a substrate for multidrug resistance-associated proteins (MRP) respectively; their specificity was confirmed using established blockers. Sb-resistant (Sb-R) isolates accumulated higher amounts of R123, as compared to Sb-sensitive (Sb-S) isolates. Verapamil, a MDR inhibitor failed to alter R123 accumulation, suggesting absence of classical MDR activity. In Sb-R isolates, both promastigotes and axenic amastigotes accumulated significantly lower amounts of calcein than Sb-S isolates and probenecid, an established pan MRP blocker, marginally increased calcein accumulation. Depletion of ATP dramatically increased calcein accumulation primarily in Sb-R isolates, indicating existence of a MRP-like pump, which was more active in Sb-R isolates. In conclusion, our data suggested that overfunctioning of a MRP-like pump contributed towards generation of Sb-R phenotype in L. donovani field isolates.
Department of Pharmacology, Institute of Post Graduate Medical Education and Research, Kolkata, 244B Acharya JC Bose Road, Kolkata 700 020 India.
Goutam
G
Mandal
Avijit
A
Sarkar
Piu
P
Saha
Neeloo
N
Singh
Shyam
S
Sundar
Mitali
M
Chatterjee
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000965:Antimony / Q000494:pharmacology*; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D018432:Drug Resistance, Multiple; D005452:Fluoresceins / Q000378:metabolism; D006801:Humans; D007893:Leishmania donovani / Q000187:drug effects* / Q000378:metabolism*; D007898:Leishmaniasis, Visceral / Q000188:drug therapy / Q000469:parasitology / Q000503:physiopathology; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D015242:Ofloxacin / Q000494:pharmacology; D011339:Probenecid / Q000494:pharmacology; D015800:Protozoan Proteins / Q000378:metabolism*; D020112:Rhodamine 123 / Q000378:metabolism; D014700:Verapamil / Q000494:pharmacology
D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate; D000965:Antimony; D000981:Antiprotozoal Agents; D005452:Fluoresceins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D015242:Ofloxacin; D011339:Probenecid; D015800:Protozoan Proteins; D020112:Rhodamine 123; D014700:Verapamil; C007740:fluorexon