Roles for mitochondria in pentamidine susceptibility and resistance in Leishmania donovani

https://doi.org/10.1016/j.molbiopara.2005.08.016Get rights and content

Abstract

Pentamidine resistant Leishmania donovani was raised in the laboratory by stepwise exposure to increasing drug pressure until a line capable of growth in 8 μM pentamidine (R8) had been selected. An IC50 value of 40 μM was determined for this line, some 50-fold higher than that recorded for the parental wild-type line. The pentamidine resistant promastigotes were cross-resistant to other toxic diamidine derivatives but not to antimonials or substrates of multidrug resistance pumps. Decreased mitochondrial transmembrane potential was observed in pentamidine resistant promastigotes. A substantial net decrease in accumulation of [3H]-pentamidine accompanied the resistance phenotype. Inhibitors of P-glycoprotein pumps, including prochlorperazine and trifluoperazine, did not reverse this decreased drug uptake, which distinguishes the L. donovani resistant line studied here from L. mexicana promastigotes previously studied for pentamidine resistance. Kinetic analysis identified a carrier with an apparent Km value of 6 μM for pentamidine. No significant difference between wild-type and resistant parasites could be detected with respect to this transporter in rapid uptake experiments. However, in longer-term uptake experiments and also using concentrations of pentamidine up to 1 mM, it was demonstrated that wild-type cells, but not resistant cells, could continue to accumulate pentamidine after apparent saturation via the measured transporter had been reached. Agents that diminish the mitochondrial membrane potential inhibited this secondary route. A fluorescent analogue of pentamidine, 2,5-bis-(4-amidophenyl)-3,4-dimethylfuran (DB99), accumulated in the kinetoplast of wild-type but not resistant parasites indicating that uptake of this cationic compound into mitochondria of wild-type cells was more pronounced than in the resistant line. These data together indicate that resistance to pentamidine in L. donovani is associated with alterations to the mitochondria of the parasites, which lead to reduced accumulation of drug.

Introduction

Leishmania donovani, a flagellated protozoan parasite, is the causative agent of visceral leishmaniasis. Sandflies transmit promastigote forms of the parasite to the mammalian host, where they invade macrophages and transform into amastigotes. Pentavalent antimonials are the standard first-line treatment for leishmaniasis [1], [2], although resistance is a growing problem [3], [4], [5], [6]. The aromatic diamidine pentamidine represents a second line of treatment [7], [8], [9], [10]. Pentamidine is a dicationic drug that has been used for more than 50 years in the therapy and prophylaxis of African trypanosomiasis and Pneumocystis carinii pneumonia in AIDS patients [9], [11] as well as antimony resistant leishmaniasis. However, the mode of action of this drug against parasites is still not understood.

Resistance to pentamidine has been described for trypanosomes [12], [13], L. donovani [3], [5], [14] and other Leishmania species [15], [16], [17], [18], [19], [20], [21]. Yeast are also susceptible to pentamidine [22], and a gene called PNT1, when overexpressed, confers resistance to the drug in Saccharomyces cerevisiae [23]. The protein encoded by this gene localizes to the inner membrane of the mitochondrion and appears to play a role in secretion of proteins from the mitochondrion [24]. Disruption of mitochondrial function has been proposed as a likely toxic effect of pentamidine in S. cerevisiae [24]. The mitochondrion has also been implicated in the action of pentamidine against trypanosomatids. Electron microscopy revealed that treatment of Leishmania species with pentamidine leads to disintegration of the kinetoplast and mitochondrion [25], [26], and a collapse in mitochondrial potential [27], is one of the first manifestations of treatment of these parasites with the drug.

The mechanism of action of pentamidine remains a matter of debate but selective toxicity may relate to high levels of accumulation of the drug in the case of African trypanosomes [28], [29]. Damper and Patton [28], [30] first reported that high affinity transporters that are energy dependent and competitively inhibited by other diamidines mediate the uptake of pentamidine by Trypanosoma brucei brucei bloodstream forms. The P2 amino-purine transporter of Trypanosoma brucei [31] appears to play a key role in the transport of this drug, although the situation is complex [29], [32], with at least two additional transporters also capable of carrying the drug [33]. Loss of P2 nucleoside transporter can mediate resistance to diamidines including diminazene aceturate (berenil) in African trypanosomes [34], [35], [36]. However, the presence of multiple pentamidine transporters in T. brucei ensures that P2-defective parasites are still sensitive to this drug [33]. In Leishmania sp. uptake of pentamidine has also been shown to be saturable, involving a carrier-mediated, energy dependent process [37], [38]. Pentamidine was found to be a competitive inhibitor of arginine transport [39], [40] and a non-competitive inhibitor of putrescine and spermidine transport in L. infantum [41], L. donovani and L. mexicana [42]. However, experiments using radiolabeled pentamidine in L. mexicana, failed to show reciprocal inhibition with arginine or polyamines. The physiological substrate for the transporter that carries pentamidine has yet to be identified in Leishmania. Exclusion of diamidines from the mitochondrion, in a manner associated with diminished mitochondrial membrane potential, has been proposed to contribute to the resistance phenotype in L. mexicana. A separate study showed that episomal expression of a P-glycoprotein could confer low-level resistance to pentamidine in L. major [43]. The subcellular localization of that transporter was not clear. It has recently been proposed [44] that inhibition of respiratory chain complexes can induce apoptosis in L. donovani. Moreover, inhibitors of complex II of the respiratory chain are synergistic with pentamidine's leishmanicidal activity—again pointing to a possible role of the mitochondrion in the action of this drug. While biological phenomena characterized in one Leishmania species are frequently taken as pointers to similar activity in other species of the genus it is important to actually study each individual species. Crucial differences between species exist, and this is clearly manifest in pharmacological responses to drug. For example, L. donovani is markedly more sensitive to a variety of drugs than is L. mexicana [45]. In this study we set out to determine the biochemical basis of resistance to pentamidine in L. donovani, the most important pathogenic Leishmania species that is responsible for visceral leishmaniasis in India. Loss of mitochondrial membrane potential appears to be key to the development of drug resistance in this species, while drug efflux appears to play some role in L. mexicana but not in L. donovani.

Section snippets

Materials

[Ring-3H] pentamidine (98 Ci/mmol, 5 mCi/ml) was custom synthesized by Amersham Pharmacia Biotech (Buckinghamshire, UK). Pentamidine isethionate, rhodamine 123 (Rh123), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), berenil, 4′,6-diamidino-2-phenylindole (DAPI), verapamil, trifluoperazine (TFP), prochlorperazine (PCP), vinblastine, sodium arsenite were purchased from Sigma (St. Louis, MO). Pentostam was a gift from The Wellcome Foundation Ltd., UK, and glucantime from

Characterization of pentamidine resistant parasites

Four cloned lines of L. donovani promastigotes resistant to pentamidine were selected in vitro by increasing drug pressure. The cloned lines, designated R1, R2, R4 and R8, displayed 4-, 22-, 30- and 50-fold resistance to pentamidine, respectively. Doubling time for the exponentially grown wild-type L. donovani was 14 h and 22 h for the R8 resistant promastigotes grown in the presence of 8 μM pentamidine. The phenotype was stable in drug free medium for up to 2 months, although the IC50 of R8 had

Discussion

Pentamidine, a cationic aromatic diamidine, is a drug, which has been widely used as second line of defense against visceral leishmaniasis. Previous studies have given insight into mechanisms of resistance to pentamidine in L. mexicana [55], but less is known of L. donovani. It is tempting to draw conclusions from one Leishmania species across the range of species. However, it is important to learn about what actually happens in each individual species as clear biological differences can be

Acknowledgements

The work carried out in this paper was supported by a joint grant from the Wellcome Trust to Rentala Madhubala and Michael Barrett. A Senior Research Fellowship from the Council of Scientific and Industrial Research, India, supported AM. KP is supported by a grant from the University Grants Commission, New Delhi, India. Mayur was supported by a Collaborative Research grant from the Wellcome Trust. We are grateful to Harry De Koning for providing tritiated pentamidine.

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