Miltefosine Transporter Research Articles

Treatment Failure and Miltefosine Susceptibility in Dermal Leishmaniasis Caused by Leishmania Subgenus Viannia Species

Treatment failure and parasite drug susceptibility in dermal leishmaniasis caused by Leishmania (Viannia) species are poorly understood. Prospective evaluation of drug susceptibility of strains isolated from individual patients before drug exposure and at clinical failure allows intrinsic and acquired differences in susceptibility to be discerned and analyzed. To determine whether intrinsic susceptibility or loss of susceptibility to miltefosine contributed to treatment failure, we evaluated the miltefosine susceptibility of intracellular amastigotes and promastigotes of six Leishmania (Viannia) braziliensis and six Leishmania (Viannia) panamensis strains isolated sequentially, at diagnosis and treatment failure, from two children and four adults ≥55 years old with concurrent conditions. Four patients presented only cutaneous lesions, one had mucosal disease, and one had disseminated mucocutaneous disease. Expression of the Leishmania drug transporter genes abca2, abca3, abcc2, abcc3, abcg4, abcg6, and LbMT was evaluated by quantitative reverse transcription-PCR (qRT-PCR). Intracellular amastigotes (median 50% effective concentration [EC50], 10.7 μmol/liter) were more susceptible to miltefosine than promastigotes (median EC50, 55.3 μmol/liter) (P < 0.0001). Loss of susceptibility at failure, demonstrated by a miltefosine EC50 of >32 μmol/liter (the upper limit of intracellular amastigote assay), occurred in L. panamensis infection in a child and in L. braziliensis infection in an adult and was accompanied by decreased expression of the miltefosine transporter LbMT (LbMT/β-tubulin, 0.42- to 0.26-fold [P = 0.039] and 0.70- to 0.57-fold [P = 0.009], respectively). LbMT gene polymorphisms were not associated with susceptibility phenotype. Leishmania ABCA3 transporter expression was inversely correlated with miltefosine susceptibility (r = -0.605; P = 0.037). Loss of susceptibility is one of multiple factors involved in failure of miltefosine treatment in dermal leishmaniasis.

Drug Susceptibility in Leishmania Isolates Following Miltefosine Treatment in Cases of Visceral Leishmaniasis and Post Kala-Azar Dermal Leishmaniasis

BackgroundWith widespread resistance to antimonials in Visceral Leishmaniasis (VL) in the Indian subcontinent, Miltefosine (MIL) has been introduced as the first line therapy. Surveillance of MIL susceptibility in natural populations of Leishmania donovani is vital to preserve it and support the VL elimination program.Methodology and Principal FindingsWe measured in vitro susceptibility towards MIL and paromomycin (PMM) in L. donovani isolated from VL and PKDL, pre- and post-treatment cases, using an amastigote-macrophage model. MIL susceptibility of post-treatment isolates from cured VL cases (n = 13, mean IC50±SD = 2.43±1.44 µM), was comparable (p>0.05) whereas that from relapses (n = 3, mean IC50 = 4.72±1.99 µM) was significantly higher (p = 0.04) to that of the pre-treatment group (n = 6, mean IC50 = 1.86±0.75 µM). In PKDL, post-treatment isolates (n = 3, mean IC50 = 16.13±2.64 µM) exhibited significantly lower susceptibility (p = 0.03) than pre-treatment isolates (n = 5, mean IC50 = 8.63±0.94 µM). Overall, PKDL isolates (n = 8, mean IC50 = 11.45±4.19 µM) exhibited significantly higher tolerance (p<0.0001) to MIL than VL isolates (n = 22, mean IC50 = 2.58±1.58 µM). Point mutations in the miltefosine transporter (LdMT) and its beta subunit (LdRos3) genes previously reported in parasites with experimentally induced MIL resistance were not present in the clinical isolates. Further, the mRNA expression profile of these genes was comparable in the pre- and post-treatment isolates. Parasite isolates from VL and PKDL cases were uniformly susceptible to PMM with respective mean IC50 = 7.05±2.24 µM and 6.18±1.51 µM.ConclusionThe in vitro susceptibility of VL isolates remained unchanged at the end of MIL treatment; however, isolates from relapsed VL and PKDL cases had lower susceptibility than the pre-treatment isolates. PKDL isolates were more tolerant towards MIL in comparison with VL isolates. All parasite isolates were uniformly susceptible to PMM. Mutations in the LdMT and LdRos3 genes as well as changes in the expression of these genes previously correlated with experimental resistance to MIL could not be verified for the field isolates.

LeishmaniaResistance to Miltefosine Associated with Genetic Marker

<i>Leishmania</i>Resistance to Miltefosine Associated with Genetic Marker

Disruption of the Lipid-Transporting LdMT-LdRos3 Complex in Leishmania donovani Affects Membrane Lipid Asymmetry but Not Host Cell Invasion

Maintenance and regulation of the asymmetric lipid distribution across eukaryotic plasma membranes is governed by the concerted action of specific membrane proteins controlling lipid movement across the bilayer. Here, we show that the miltefosine transporter (LdMT), a member of the P4-ATPase subfamily in Leishmania donovani, and the Cdc50-like protein LdRos3 form a stable complex that plays an essential role in maintaining phospholipid asymmetry in the parasite plasma membrane. Loss of either LdMT or LdRos3 abolishes ATP-dependent transport of NBD-labelled phosphatidylethanolamine (PE) and phosphatidylcholine from the outer to the inner plasma membrane leaflet and results in an increased cell surface exposure of endogenous PE. We also find that promastigotes of L. donovani lack any detectable amount of phosphatidylserine (PS) but retain their infectivity in THP-1-derived macrophages. Likewise, infectivity was unchanged for parasites without LdMT-LdRos3 complexes. We conclude that exposure of PS and PE to the exoplasmic leaflet is not crucial for the infectivity of L. donovani promastigotes.

Inactivation of the miltefosine transporter, LdMT, causes miltefosine resistance that is conferred to the amastigote stage of Leishmania donovani and persists in vivo

Miltefosine (hexadecylphosphocholine) is the first oral antileishmanial drug. In this study, we addressed the question whether miltefosine-resistant Leishmania donovani promastigotes transform to miltefosine-resistant amastigotes. A promastigote line, M-mutR, showed defective internalisation of miltefosine owing to mutations in LdMT, similar to previously described resistant lines. M-mutR parasites were infective to macrophages in vitro as well as in BALB/c mice in vivo. There was good correlation of in vitro resistance indices between promastigotes and intracellular amastigotes. Most importantly, M-mutR parasites retained the resistant phenotype in vivo, with no decrease of hepatic burden in BALB/c mice following miltefosine treatment up to 30 mg/kg (ca. 90% inhibition in wild-type infections). No cross-resistance to other antileishmanial drugs was observed in M-mutR amastigotes.

Phospholipid Translocation and Miltefosine Potency Require Both L. donovani Miltefosine Transporter and the New Protein LdRos3 in Leishmania Parasites

The antitumor drug miltefosine has been recently approved as the first oral drug active against visceral leishmaniasis. We have previously identified the L. donovani miltefosine transporter (LdMT) as a P-type ATPase involved in phospholipid translocation at the plasma membrane of Leishmania parasites. Here we show that this protein is essential but not sufficient for the phospholipid translocation activity and, thus, for the potency of the drug. Based on recent findings in yeast, we have identified the putative beta subunit of LdMT, named LdRos3, as another protein factor required for the translocation activity. LdRos3 belongs to the CDC50/Lem3 family, proposed as likely beta subunits for P4-ATPases. The phenotype of LdRos3-defective parasites was identical to that of the LdMT-/-, including a defect in the uptake of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-amino)-phosphatidylserine, generally considered as not affected in Lem3p-deficient yeast. Both LdMT and LdRos3 normally localized to the plasma membrane but were retained inside the endoplasmic reticulum in the absence of the other protein or when inactivating point mutations were introduced in LdMT. Modulating the expression levels of either protein independently, we show that any one of them could behave as the protein limiting the level of flippase activity. Thus, LdMT and LdRos3 seem to form part of the same translocation machinery that determines flippase activity and miltefosine sensitivity in Leishmania, further supporting the consideration of CDC50/Lem3 proteins as beta subunits required for the normal functioning of P4-ATPases.