Drug resistance is a major obstacle to the conquest of parasitic diseases, A thorough understanding of the molecular mechanisms and alterations associated with drug resistance might suggest rational ways that drug resistance in parasitic diseases could be circumvented or prevented. In this application, we propose a thorough biochemical and molecular genetic dissection of one form of drug resistance in Leishmania donovani, gene amplification. Three separate gene amplification systems have been developed in this laboratory: i. a difluoromethylornithine (DFMO)-resistant cell line that overproduces ornithine decarboxylase (ODC); ii. a mycophenolic acid-resistant strain that overexpresses inosine monophosphate dehydrogenase; and iii. a multidrug resistant L. donovani. In this proposal, we will focus on one of these gene amplification systems, the DFMO-resistant L. donovani strain that has amplified the ODC gene and contains two extrachromosomal DNAs. The overall objectives of this proposal are to examine the biochemical and molecular alterations in the drug-resistant cells and to analyze the ODC gene and protein in more detail. We will definitively establish that the NH2 terminus of the ODC protein predicted from the nucleotide sequence of the cloned ODC gene is accurate by purifying ODC and sequencing its NH2 terminus. Whether the two extrachromosomal elements that are present in the DFMO-resistant cells are circular or linear will be authenticated by physical mapping, CHEF electrophoresis, and electron microscopy. In addition, other DFMO-resistant strains,from several Leishmania species will be generated and their amplicons characterized. Homologies between sequences on the two amplified DNAs of DFMO-resistant cells and amplified DNAs from other drug-resistant Leishmania will be ascertained. Restriction maps of both extrachromosomal DNAs and their chromosomal counterparts will be generated by chromosome walking in order to analyze the mechanism by which the amplified DNAs originated as a consequence of selective pressure. Transcriptional activity and mapping of the extrachromosomes will be performed, and the relationship between the two leishmanial ODC transcripts will be determined. Finally, we will insert the leishmanial ODC gene into a bacterial expression vector and transform it into ODC-deficient E, coli to prepare large quantities of recombinant ODC protein for kinetic, structural, and immunological studies. Expression of site directed mutants will permit a genetic dissection of some of the critical structural determinants that are required for catalytic activity and DFMO inactivation of this chemotherapeutically important enzyme.
