Tunicamycin-resistant Leishmania mexicana amazonensis: expression of virulence associated with an increased activity of N-acetylglucosaminyltransferase and amplification of its presumptive gene.

Abstract

Tunicamycin at 10 micrograms/ml inhibits the growth and infectivity of the parasitic protozoan Leishmania mexicana amazonensis. Tunicamycin-resistant variants of this parasite were produced by gradual acclimatization of cells to increasing concentrations of the drug up to 80 micrograms/ml and a single-step selection of ethyl methanesulfonate-pretreated or differentiating leishmanias with the drug at 10 micrograms/ml. Prolonged exposure to the drug increases stability of drug resistance of those resistant to 10 micrograms/ml. Tunicamycin-resistant cells contain amplified DNA, which hybridizes in proportion to the cells' degree of drug resistance with Alg 7, a cloned DNA probe apparently encoding yeast N-acetylglucosaminyltransferase. This enzyme from all variants remained sensitive to inhibition by tunicamycin, but its specific activity was up to 15-fold higher than that of the wild type. Thus, amplification of the gene encoding this enzyme appears to result in its overproduction in the variants, accounting for their resistance to tunicamycin. The tunicamycin-resistant cells are more virulent to mice than their parental wild type. Thus, leishmanial virulence may be related to amplification or expression of gene(s) encoding enzymes involved in the regulation of N-glycosylation of parasite proteins.