Abstract
BackgroundIn 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 metabolismMethodology/Principal FindingsWe 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/SignificanceHere 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.
Highlights
Leishmaniasis comprises a complex of vector-borne diseases, caused by more than 20 species of the protozoan genus Leishmania, that range from localized skin ulcers to lethal systemic disease [1]
AQP1 transcript levels exhibited consistent down regulation in the field isolates of Leishmania donovani from India [19] and Nepal [20,21] but RNA levels remain unaltered in resistant isolates of L. braziliensis and L. guyanensis [22,23]
In the present study we have tried to elucidate the mechanism of natural antimony resistance in L. donovani isolates, isolated from VL patients in Bihar/eastern UP, India, by analyzing the expression profiles of known genes involved in transport and thiol based redox metabolism followed by characterization of the functionality of efflux pump(s) and related enzymes
Summary
Leishmaniasis comprises a complex of vector-borne diseases, caused by more than 20 species of the protozoan genus Leishmania, that range from localized skin ulcers to lethal systemic disease [1]. AQP1 transcript levels exhibited consistent down regulation in the field isolates of Leishmania donovani from India [19] and Nepal [20,21] but RNA levels remain unaltered in resistant isolates of L. braziliensis and L. guyanensis [22,23]. The precursor protein of spermidine biosynthesis, ODC was amplified at the genetic and protein levels in Indian L. donovani resistant isolates [25] and in L. braziliensis [23], but the gene was down regulated in isolates from Nepal [20]. In the present study we have tried to elucidate the mechanism of natural antimony resistance in L. donovani isolates, isolated from VL patients in Bihar/eastern UP, India, by analyzing the expression profiles of known genes involved in transport and thiol based redox metabolism followed by characterization of the functionality of efflux pump(s) and related enzymes. A parallel comparison was made with a laboratory raised L. donovani mutant strain resistant to 450 μM SbIII
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