Mechanism of Amphotericin B Resistance in Clinical Isolates of Leishmania donovani

Abstract

The clinical value of amphotericin B, the mainstay therapy for visceral leishmaniasis in sodium antimony gluconatenonresponsive zones of Bihar, India, is now threatened by the emergence of acquired drug resistance, and a comprehensive understanding of the underlying mechanisms is the need of the hour. We have selected an amphotericin B-resistant clinical isolate which demonstrated 8-fold-higher 50% lethal doses (LD 50 ) than an amphotericin B-sensitive strain to explore the mechanism of amphotericin B resistance. Fluorimetric analysis demonstrated lower anisotropy in the motion of the diphenylhexatriene fluorescent probe in the resistant strain, which indicated a higher fluidity of the membrane for the resistant strain than for the sensitive strain. The expression patterns of the two transcripts of S-adenosyl-L-methionine:C-24-⌬-sterol methyltransferase and the absence of ergosterol, replaced by cholesta-5,7,24-trien-3␤-ol in the membrane of the resistant parasite, indicate a decreased amphotericin B affinity, which is evidenced by decreased amphotericin B uptake. The expression level of MDR1 is found to be higher in the resistant strain, suggesting a higher rate of efflux of amphotericin B. The resistant parasite also possesses an upregulated tryparedoxin cascade and a more-reduced intracellular thiol level, which helps in better scavenging of reactive oxygen species produced by amphotericin B. The resistance to amphotericin B was partially reverted by the thiol metabolic pathway and ABC transporter inhibitors. Thus, it can be concluded that altered membrane composition, ATP-binding cassette transporters, and an upregulated thiol metabolic pathway have a role in conferring amphotericin B resistance in clinical isolates of Leishmania donovani. L eishmaniasis, or kala azar, is a group of diseases caused by a protozoan parasite of the genus Leishmania. Kala azar is a symptomatic infection of liver, spleen, and bone marrow. The global estimates for the incidence and prevalence of kala azar cases per year are 0.5 and 2.5 million, respectively (57). In India, visceral leishmaniasis (VL) has been reported in parts of West Bengal, Uttar Pradesh, and Bihar. It poses a major health problem in the state of Bihar, which accounts for nearly 90% of the total cases in India (51). Chemotherapy has proven to be the only effective way of controlling infections and is highly dependent upon antimonycontaining drugs, such as sodium stibogluconate (Pentostam). Amphotericin B (AmB) is used as the drug of choice when acquired drug resistance emerges for traditional antimony therapy, and nearly 64% of cases in regions of Bihar where VL is hyperendemic are now resistant to antimonials (28). Hexadecylphosphocholine (miltefosine) is the first orally administered drug for VL and the latest to enter the market. The dosing scheme of hexadecylphosphocholine is 100 mg/kg of body weight/day for 28 days in adults weighing Ն50 kg, 50 mg/kg/day in adults weighing Ͻ50 kg, and 2.5 mg/kg/day in children (maximum dose, 100 mg/day). Major concerns abou...

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The Cytochrome P450 Complement (CYPome) of Leishmania Leads to the Discovery of a Plant Like Cytochrome P450 Sub-Family CYP710C1 Gene

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In vitro antileishmanial activity of trans-stilbene and terphenyl compounds

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