Biological and biochemical characterization of tunicamycin-resistant Leishmania mexicana: mechanism of drug resistance and virulence.

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A parasitic protozoan, Leishmania mexicana amazonensis, was previously made resistant to tunicamycin (J.A. Kink and K.-P. Chang, Proc. Natl. Acad. Sci. USA 84:1253-1257, 1987). In the present study, six different tunicamycin-resistant variants were biologically and biochemically compared with their parental wild type to further delineate the mechanism of tunicamycin resistance and that of their virulence observed. In contrast to their parental wild type, all tunicamycin-resistant variants were found to grow and differentiate in tunicamycin-containing medium. The 50% lethal doses of tunicamycin for variants resistant to 10 or 80 micrograms of tunicamycin per ml were 20- and 100-fold higher, respectively, than that of the wild type. Specific activity of the microsomal N-acetylglucosamine-1-phosphate transferase was 4- to 12-fold higher in the tunicamycin-resistant cells than in their parental wild type and tunicamycin-sensitive revertants. The level of the enzyme activity is proportional to the degree of drug resistance. Inhibition kinetics studies showed that the enzyme from all groups was equally sensitive to the drug, with a 50% effective concentration of 1 to 1.3 micrograms of tunicamycin per ml. Thus, tunicamycin resistance of the variants is caused primarily by an increased level of their enzyme without alteration of its structure. Protein glycosylation determined by the incorporation of 2-D-[3H]mannose was about twofold higher in the tunicamycin-resistant variants than in their parental wild type. The increased glycosyltransferase activity in the latter apparently renders their protein glycosylation insensitive to the inhibition by tunicamycin. A major membrane glycoprotein of 63 kilodaltons (gp63) on the leishmania surface was found to be about threefold higher in the tunicamycin-resistant variants than in the wild type, as determined by immunoprecipitation with a monoclonal antibody specific for this antigen. Tunicamycin treatment of the wild type and tunicamycin-resistant variants caused changes in the electrophoretic mobility of this molecule, indicating a higher degree of its glycosylation in the latter cells. The tunicamycin-resistant variants parasitized macrophages in vitro more effectively than did the wild type, accounting for their virulence seen in mice. Thus, a high level of the glycosyltransferase enables the tunicamycin-resistant cells not only to overcome the inhibitory effect of tunicamycin on protein glycosylation but also to express their virulence, possibly by regulating N glycosylation of leishmanial proteins critical for leishmanias to establish intracellular parasitism.

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