In vitro life cycle of pentamidine-resistant amastigotes: stability of the chemoresistant phenotypes is dependent on the level of resistance induced

Abstract

Using a continuous drug pressure protocol, we induced pentamidine resistance in an active and dividing population of amastigote forms of Leishmania mexicana. We selected in vitro two clones with different levels of resistance to pentamidine, with clone LmPENT5 being resistant to 5 M pentamidine, while clone LmPENT20 was resistant to 20 M pentamidine. Resistance indexes (50% inhibitory concentration [IC 50 ] after drug presure/IC 50 before drug pressure) of 2 (LmPENT5) and 6 (LmPENT20) were determined after drug selection. Both resistant clones expressed significant cross-resistance to diminazene aceturate and primaquine. Pentamidine resistance was not reversed by verapamil, a calcium channel blocker known to reverse multidrug resistance (A. J. Bitonti, et al., Science 242:1301-1303, 1988; A. R. C. Safa et al., J. Biol. Chem. 262:7884-7888, 1987). No difference in the in vitro infectivity for resident mouse macrophages was observed between the wildtype clone (clone LmWT) and pentamidine-resistant clones. During in vitro infectivity experiments, when the life cycle was performed starting from the intramacrophagic amastigote stage, the drug resistance of the resulting LmPENT20 amastigotes was preserved even if the intermediate promastigote stage could not be considered resistant to 20 M pentamidine. In the same way, when a complete developmental sequence of L. mexicana was achieved axenically by manipulation of appropriate culture conditions, the resulting axenically grown LmPENT20 amastigotes remained pentamidine resistant, whereas LmPENT5 amastigotes lost their ability to resist pentamidine, with IC 50 s and index of resistance values close to those for the LmWT clone. These results strongly indicate that the level of pentamidine tolerated by resistant amastigotes after the life cycle was dependent on the induced level of resistance. This fact could be significant in the in vivo transmission of drugresistant parasites by Phlebotominae. Particular attention should be given to the finding that the emergence of parasite resistance is a potential risk of the use of inadequate doses as therapy in humans.

Antimonial Resistance in Leishmania donovani Is Associated with Increased In Vivo Parasite Burden

“…The higher virulence of natural SSG-R L. donovani strains could very well contribute their general fitness. This is in contrast to in vitro induced pentamidine- or glibenclamide-resistant L. mexicana strains which had respectively a similar or decreased infectivity compared to the wild type [18], [19]. We cannot exclude that L. mexicana behaves differently compared to L. donovani under SSG-drug pressure.…”

Leishmania spp.: proficiency of drug-resistant parasites

“…Indeed, this is the case for amphotericin-resistant Leishmania mexicana [54], L. mexicana amazonensis resistant to ABC transporter blockers [34,43], glucantime-resistant Leishmania (Viannia) guyanensis [35] and ricin-resistant Leishmania major [55,56]. In contrast, L. mexicana amazonensis resistant to tunicamycin, a drug not relevant for the treatment of leishmaniasis, shows an increased or unchanged virulence compared with the sensitive strain [57][58][59][60]. Whether decreased infectivity is a consequence of lower cell proficiency with few morphological changes is still unknown.…”

Functional Cloning as a Means to Identify Leishmania Genes Involved in Drug Resistance

“…Although this system has already been employed in drug resistance studies [38,39], there are important caveats to this strategy. Firstly, axenic amastigotes are cultivated in essentially the same growth media as promastigotes, although natural promastigotes and amastigotes are exposed to widely different milieus and nutrient sources and supplies.…”