Role of efflux pumps and intracellular thiols in natural antimony resistant isolates of Leishmania donovani.
BACKGROUND
In view of the recent upsurge in the phenomenon of therapeutic failure, drug resistance in Leishmania, developed under natural field conditions, has become a great concern yet little understood. Accordingly, the study of determinants of antimony resistance is urgently warranted. Efflux transporters have been reported in Leishmania but their role in clinical resistance is still unknown. The present study was designed to elucidate the mechanism of natural antimony resistance in L. donovani field isolates by analyzing the functionality of efflux pump(s) and expression profiles of known genes involved in transport and thiol based redox metabolism.


RESULTS
We selected 7 clinical isolates (2 sensitive and 5 resistant) in addition to laboratory sensitive reference and SbIII resistant mutant strains for the present study. Functional characterization using flow cytometry identified efflux pumps that transported substrates of both P-gp and MRPA and were inhibited by the calmodulin antagonist trifluoperazine. For the first time, verapamil sensitive efflux pumps for rhodamine 123 were observed in L. donovani that were differentially active in resistant isolates. RT-PCR confirmed the over-expression of MRPA in isolates with high resistance index only. Resistant isolates also exhibited consistent down regulation of AQP1 and elevated intracellular thiol levels which were accompanied with increased expression of ODC and TR genes. Interestingly, γ-GCS is not implicated in clinical resistance in L. donovani isolates.


CONCLUSIONS
Here we demonstrate for the first time, the role of P-gp type plasma membrane efflux transporter(s) in antimony resistance in L. donovani field isolates. Further, decreased levels of AQP1 and elevated thiols levels have emerged as biomarkers for clinical resistance.
Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow, India.
Smita
S
Rai
Bhaskar
Sudhir K
SK
Goel
Upendra
U
Nath Dwivedi
Shyam
S
Sundar
Neena
N
Goyal
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism*; D000965:Antimony / Q000494:pharmacology*; D001692:Biological Transport; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004789:Enzyme Activation; D015870:Gene Expression; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D007893:Leishmania donovani / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014158:Transcription, Genetic
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D026901:Membrane Transport Proteins; D013438:Sulfhydryl Compounds; D000965:Antimony; D009247:NADH, NADPH Oxidoreductases; C049608:trypanothione reductase