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Modification of kinetoplast DNA minicircle composition in pentamidine-resistant Leishmania
M Basselin1, M A Badet-Denisot, M Robert-Gero
1Institut de Chimie des Substances Naturelles, C.N.R.S., Gif-sur-Yvette, France. mab6s@udcf.gla.ac.uk
Abstract:
Pentamidine, an antiprotozoal drug, was shown to have various cellular and molecular targets depending on the organism. In Leishmania, ultrastructural modifications of kinetoplast and mitochondria have been observed but no data is available on cellular and molecular events involved in development of pentamidine-resistance. The absence of modification of minicircle DNA in pentamidine treated L. donovani and L. amazonensis promastigotes suggested that topoisomerase II activity is not a target. This result was confirmed by quantitation of the enzyme by immunodetection. Southern blot experiments indicated that the kDNA network was altered in resistant clones. Molecular cloning and sequence analysis of kDNA minicircles showed transkinetoplastidy hitherto reported only for arsenite- and tunicamycin-resistant Leishmania. Comparison of wild-type and resistant sequences showed only 32-51% homology. The AT-rich regions, known as binding sites, of the drug occurred less frequently in the resistant clones and their locations were different. These minicircle sequence modifications leading to decreased binding sites for the drug might contribute to pentamidine-resistance in Leishmania.
Insights
Pentamidine resistance in Leishmania involves changes in kinetoplast DNA (kDNA) minicircles. These modifications reduce drug binding sites, offering a new understanding of antiprotozoal drug resistance mechanisms.
Area of Science:
- Parasitology
- Molecular Biology
- Drug Resistance Studies
Background:
- Pentamidine is an antiprotozoal drug with diverse cellular targets.
- While ultrastructural changes in Leishmania are known, the molecular basis of pentamidine resistance remains unclear.
- Topoisomerase II activity was not identified as a target for pentamidine resistance.
Purpose of the Study:
- To investigate the cellular and molecular mechanisms underlying pentamidine resistance in Leishmania.
- To analyze alterations in kinetoplast DNA (kDNA) in pentamidine-resistant Leishmania clones.
Main Methods:
- Immunodetection to quantify topoisomerase II.
- Southern blot analysis of kDNA networks.
- Molecular cloning and sequence analysis of kDNA minicircles from wild-type and resistant Leishmania.
Main Results:
- Pentamidine treatment did not alter minicircle DNA in promastigotes, and topoisomerase II levels remained unchanged.
- Southern blot revealed alterations in the kDNA network of resistant Leishmania clones.
- Sequence analysis of kDNA minicircles showed significant homology reduction (32-51%) and altered AT-rich regions, indicative of transkinetoplastidy.
Conclusions:
- Pentamidine resistance in Leishmania is associated with modifications in kDNA minicircle sequences.
- Reduced frequency and altered locations of drug-binding sites within kDNA minicircles likely contribute to resistance.
- Transkinetoplastidy, observed in resistant clones, represents a novel mechanism in pentamidine resistance.