Functional Validation of ABCA3 as a Miltefosine Transporter in Human Macrophages: IMPACT ON INTRACELLULAR SURVIVAL OF LEISHMANIA (VIANNIA) PANAMENSIS

Luuk C.T Dohmen,Adriana Navas,Deninson Alejandro Vargas,David J Gregory,Anke Kip,Thomas P.C Dorlo,Maria Adelaida Gomez

doi:10.1074/jbc.m115.688168

Abstract

Within its mammalian host, Leishmania resides and replicates as an intracellular parasite. The direct activity of antileishmanials must therefore depend on intracellular drug transport, metabolism, and accumulation within the host cell. In this study, we explored the role of human macrophage transporters in the intracellular accumulation and antileishmanial activity of miltefosine (MLF), the only oral drug available for the treatment of visceral and cutaneous leishmaniasis (CL). Membrane transporter gene expression in primary human macrophages infected in vitro with Leishmania Viannia panamensis and exposed to MLF showed modulation of ABC and solute liquid carrier transporters gene transcripts. Among these, ABCA3, a lipid transporter, was significantly induced after exposure to MLF, and this induction was confirmed in primary macrophages from CL patients. Functional validation of MLF as a substrate for ABCA3 was performed by shRNA gene knockdown (KD) in THP-1 monocytes. Intracellular accumulation of radiolabeled MLF was significantly higher in ABCA3(KD) macrophages. ABCA3(KD) resulted in increased cytotoxicity induced by MLF exposure. ABCA3 gene expression inversely correlated with intracellular MLF content in primary macrophages from CL patients. ABCA3(KD) reduced parasite survival during macrophage infection with an L. V. panamensis strain exhibiting low in vitro susceptibility to MLF. Confocal microscopy showed ABCA3 to be located in the cell membrane of resting macrophages and in intracellular compartments in L. V. panamensis-infected cells. These results provide evidence of ABCA3 as an MLF efflux transporter in human macrophages and support its role in the direct antileishmanial effect of this alkylphosphocholine drug.

Highlights

IntroductionLeishmania resides and replicates as an intracellular parasite
Within its mammalian host, Leishmania resides and replicates as an intracellular parasite
Exposure to Miltefosine Modulates Expression of Membrane Transporters in Human Macrophages—To identify candidate proteins with putative MLF transport function in human macrophages, we evaluated the effect of MLF exposure on the modulation of expression of transporter genes in L

Summary

Introduction

Leishmania resides and replicates as an intracellular parasite. We explored the role of human macrophage transporters in the intracellular accumulation and antileishmanial activity of miltefosine (MLF), the only oral drug available for the treatment of visceral and cutaneous leishmaniasis (CL). These results provide evidence of ABCA3 as an MLF efflux transporter in human macrophages and support its role in the direct antileishmanial effect of this alkylphosphocholine drug. It has been suggested that the therapeutic response to MLF is potentially linked to species-specific loss of susceptibility [7, 8], recent studies show a wide range of susceptibilities among clinical isolates of the same Leishmania species [9], and therapeutic failure has been reported in visceral leishmaniasis patients infected with drug-susceptible strains [10]. The purpose of this study was to identify the active carriers involved in transport and accumulation of miltefosine in human macrophages and to characterize their impact on intracellular drug-mediated parasite killing

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