Drug Uptake and Modulation of Drug Resistance in Leishmania by an Aquaglyceroporin

Abstract

Leishmaniasis is a protozoan parasitic disease that affects 12 million people worldwide. The first line choice for the treatment of this disease is antimonial drugs. In the endemic regions, resistance to this class of drugs is a major impediment to treatment. Microbes often become resistant to drugs by mutation or down-regulation of uptake systems, but the uptake system for the antimonial drugs in Leishmania is unknown. In other organisms, aquaglyceroporins have been shown to facilitate uptake of trivalent metalloids. In this study, we report the identification and characterization of aquaglyceroporins from Leishmania major (LmAQP1) and Leishmania tarentolae (LtAQP1), respectively. These Leishmania proteins have the conserved signature motifs of aquaglyceroporins. Transfection of LmAQP1 into three species of Leishmania, L. tarentolae, Leishmania infantum, and L. major, produced hypersensitivity to both As(III) and Sb(III) in all three strains. Increased production of LmAQP1 was detected by immunoblotting. Drug-resistant parasites with various mutations leading to resistance mechanisms became hypersensitive to both metalloids after expression of LmAQP1. Increased rates of uptake of As(III) or Sb(III) correlated with metalloid sensitivity of the wild type and drug-resistant transfectants. Transfection of LmAQP1 in a Pentostam-resistant field isolate also sensitized the parasite in the macrophage-associated amastigote form. One allele of LmAQP1 was disrupted in L. major, and the resulting cells became 10-fold more resistant to Sb(III). This is the first report of the uptake of a metalloid drug by an aquaglyceroporin in Leishmania, suggesting a strategy to reverse resistance in the field.

KHARON1 Mediates Flagellar Targeting of a Glucose Transporter in Leishmania mexicana and Is Critical for Viability of Infectious Intracellular Amastigotes

“…The LmxMIT::GFP fusion protein was generated by cloning the LmxMIT ORF into the BamHI and EcoRV sites of pXG-ЈGFP ϩ (26). The LmjAQP1 ORF was amplified using previously described primers (27) with KpnI and EcoRV restriction sites and cloned into the pX72-Hyg vector described above. All primer sequences are available upon request.…”

Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India

“…Trivalent form of the antimonial drug (SbIII) is the prodrug that is formed by conversion of pentavalent antimony (SbV) by a putative metalloid reductase present in the macrophages [28]. Antimonial resistance in both laboratory mutants and clinical isolates has been associated with (a) decreased uptake of the drug through aquaglyceroporin (AQP1) that codes for the protein responsible for SbIII transport Nepjol.info/index.php/njb [11,12,20,29] (b) Over expression of ODC and γ-GCS enzymes of the trypanothione biosynthetic pathway [2,25] and (c) increased expression of the ABC transporter MRPA, which sequesters SbIII-thiol conjugate [16,23]. We had earlier reported decreased uptake of antimony in all nine SAG-R isolates used in the present study.…”

“…A loss of drug activation is reported to lead to resistance [10]. The route of entry of SbV into Leishmania cells is still unknown but SbIII has been reported to be transported in Leishmania through aquaglyceroporin (AQP1) [11,12]. Recent evidence has suggested that part of the mode of ©NJB, Biotechnology Society of Nepal 2 Nepjol.info/index.php/njb action of SbV could be in depleting the cells of its reduced thiols [13].…”

Contribution of proteomics of Leishmania spp. to the understanding of differentiation, drug resistance mechanisms, vaccine and drug development

“…An increased protein turnover rate of KMP-11 was observed in the resistant parasites. It was suggested by the authors that the decreased expression/accelerated turnover rate of KMP-11 could lead to an increased export of Sb(III) due to an alteration of putative efflux systems 57 or transporters, such as the aquaglyceroporin 58 .…”