Thiel Meike, Harder Simone, Wiese Martin, Kroemer Manfred, Bruchhaus Iris
Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Str. 74, 20359 Hamburg, Germany.
Mol Biochem Parasitol. 2008 Apr;158(2):152-62. doi: 10.1016/j.molbiopara.2007.12.005. Epub 2007 Dec 15.
Leishmania promastigote cells transmitted by their insect vector get phagocytosed by macrophages and convert into the amastigote form. In a recently performed proteomic study, a thymidine kinase (TK) was found to be preferentially expressed in amastigotes. Western blot analysis showing a marked increase in TK protein synthesis during stage differentiation from promastigotes to amastigotes confirmed this result. After comparison of the amino acid sequence of Leishmania donovani and Leishmania major thymidine kinases with thymidine kinases of other organisms the Leishmania protein has to be classified as a type II TK. Therefore, in accordance with the nomenclature of other thymidine kinases we named the Leishmania enzymes LdTK1 and LmTK1, respectively. The LdTK1 is localised within the cytoplasm of promastigotes. In amastigotes, increased expression and a clustered distribution of the protein can be observed. Lmtk1 single allele gene replacement mutants have significantly elongated flagellum. In contrast, lmtk1 double allele gene replacement mutants show a remarkably reduced flagellar length, diminished overall size and a deformed body shape. In addition, they have a 12-fold reduced growth rate. For both mutant strains, macrophage infectivity is clearly reduced compared to a L. major wildtype infection.
由其昆虫载体传播的利什曼原虫前鞭毛体细胞被巨噬细胞吞噬并转化为无鞭毛体形式。在最近进行的一项蛋白质组学研究中，发现一种胸苷激酶（TK）在无鞭毛体中优先表达。蛋白质印迹分析显示，从前鞭毛体到无鞭毛体的阶段分化过程中，TK蛋白合成显著增加，证实了这一结果。将杜氏利什曼原虫和硕大利什曼原虫胸苷激酶的氨基酸序列与其他生物体的胸苷激酶进行比较后，利什曼原虫蛋白必须归类为II型TK。因此，根据其他胸苷激酶的命名法，我们分别将利什曼原虫的酶命名为LdTK1和LmTK1。LdTK1定位于前鞭毛体的细胞质中。在无鞭毛体中，可以观察到该蛋白表达增加且呈聚集分布。Lmtk1单等位基因基因替代突变体的鞭毛明显延长。相比之下，lmtk1双等位基因基因替代突变体的鞭毛长度显著缩短，整体尺寸减小，体型变形。此外，它们的生长速率降低了12倍。与硕大利什曼原虫野生型感染相比，两种突变株对巨噬细胞的感染性均明显降低。
