Kink J A, Chang K P
Infect Immun. 1987 Jul;55(7):1692-700. doi: 10.1128/iai.55.7.1692-1700.1987.
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.
一种寄生原生动物墨西哥利什曼原虫亚马逊亚种先前已对衣霉素产生抗性（J.A. 金克和K.-P. 张，《美国国家科学院院刊》84:1253 - 1257，1987）。在本研究中，对六个不同的衣霉素抗性变体与其亲本野生型进行了生物学和生物化学比较，以进一步阐明衣霉素抗性机制及其所观察到的毒力机制。与它们的亲本野生型不同，所有衣霉素抗性变体在含衣霉素的培养基中都能生长和分化。每毫升含10微克或80微克衣霉素的抗性变体的衣霉素50%致死剂量分别比野生型高20倍和100倍。微粒体N - 乙酰葡糖胺 - 1 - 磷酸转移酶的比活性在衣霉素抗性细胞中比其亲本野生型和衣霉素敏感回复株高4至12倍。酶活性水平与耐药程度成正比。抑制动力学研究表明，所有组的酶对该药物的敏感性相同，50%有效浓度为每毫升1至1.3微克衣霉素。因此，变体的衣霉素抗性主要是由其酶水平升高引起的，而其结构未改变。通过掺入2 - D - [³H]甘露糖测定的蛋白质糖基化在衣霉素抗性变体中比其亲本野生型高约两倍。后者中增加的糖基转移酶活性显然使其蛋白质糖基化对衣霉素的抑制不敏感。通过用针对该抗原的单克隆抗体进行免疫沉淀测定，发现利什曼原虫表面63千道尔顿的主要膜糖蛋白（gp63）在衣霉素抗性变体中比野生型高约三倍。用衣霉素处理野生型和衣霉素抗性变体导致该分子的电泳迁移率发生变化，表明在后者细胞中其糖基化程度更高。衣霉素抗性变体在体外比野生型更有效地寄生巨噬细胞，这解释了它们在小鼠中表现出的毒力。因此，高水平的糖基转移酶使衣霉素抗性细胞不仅能够克服衣霉素对蛋白质糖基化的抑制作用，而且可能通过调节对利什曼原虫建立细胞内寄生至关重要的利什曼原虫蛋白质的N - 糖基化来表达其毒力。
