Cathepsin B-like cysteine proteinase-deficient mutants of Leishmania mexicana
Cathepsin B-like cysteine proteinase-deficient mutants of Leishmania mexicana
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DOI：
10.1016/s0166-6851(97)00072-8
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发表时间：
1997-09-01
影响因子：
1.5
通讯作者：
Mottram, JC
中科院分区：
文献类型：
--
作者：
Bart, G;Frame, MJ;Mottram, JC
墨西哥利什曼原虫组织蛋白酶B样半胱氨酸蛋白酶基因（CPC）的突变体无效已产生有针对性的基因破坏。使用聚合酶链反应（PCR）方法与cpc特异性引物和从突变体中分离的基因组DNA确认基因缺失。CPC在无效突变体中从附加型载体重新表达。用特异性抗肽抗血清通过Western印迹法检测酶（CPC）的再表达。cpc无效突变体在无菌培养物中作为前鞭毛体和无鞭毛体正常生长，但它们在体外对巨噬细胞的感染性大大降低，只有低百分比的细胞被感染。cpc在无效突变体中的重新表达增加了体外寄生虫的感染性。无效突变体寄生虫在小鼠中以与野生型寄生虫相似的速率形成病变，尽管产生的病变较小。结果表明，尽管cpc编码的半胱氨酸蛋白酶在寄生虫与巨噬细胞的相互作用中起作用，但它本身对感染性或毒力并不重要。(C)1997年Elsevier Science B.V.
Mutants null for the cathepsin B-like cysteine proteinase gene (cpc) of Leishmania mexicana have been generated by targeted gene disruption. The gene deletion was confirmed using a polymerase chain reaction (PCR) method with cpc-specific primers and genomic DNA isolated from the mutants. cpc was re-expressed in the null mutants from an episomal vector. Re-expression of the enzyme (CPC) was detected by Western blotting with a specific anti-peptide antiserum. The cpc null mutants grew apparently normally as promastigotes and amastigotes in axenic cultures, but they showed greatly reduced infectivity to macrophages in vitro with only a low percentage of the cells being infected. Re-expression of cpc in the null mutant increased the parasite's infectivity in vitro. The null mutant parasites formed lesions in mice at a similar rate as wild type parasites, although somewhat smaller lesions were produced. The results suggest that although the cysteine proteinase encoded by cpc plays a role in the parasite's interaction with macrophages it alone is not crucial for infectivity or virulence. (C) 1997 Elsevier Science B.V.