Goyard Sophie, Segawa Hiroaki, Gordon Jennifer, Showalter Melissa, Duncan Robert, Turco Salvatore J, Beverley Stephen M
Department of Molecular Microbiology, Washington University Medical School, Campus Box 8230, 660 S Euclid Ave, St. Louis, MO 63110, USA.
Mol Biochem Parasitol. 2003 Aug 11;130(1):31-42. doi: 10.1016/s0166-6851(03)00142-7.
Glycoconjugates have been shown to play important roles in Leishmania development. However, the ability to study these molecules and other processes would benefit greatly from improved methods for genetic manipulation and analysis of the amastigote stage. This is especially challenging for L. donovani, the agent of the most severe form of leishmaniasis, which can rapidly lose virulence during in vitro culture. Here we report on a clonal subline of an L. donovani 1S2D (LdBob or LdB), which differentiates readily from promastigotes to amastigotes in axenic culture, and maintains this ability during extended parasite cultivation in vitro. This derivative can be plated and transfected efficiently while grown as promastigotes or amastigotes. Importantly, LdB maintains the ability to differentiate while undergoing genetic alterations required for creation of gene knockouts and complemented lines. Like virulent L. donovani, LdB exhibits down-regulation of lipophosphoglycan (LPG) synthesis and up-regulation of A2 protein synthesis in amastigotes. We showed that knockouts of LPG2, encoding a Golgi GDP-mannose transporter, eliminated phosphoglycan synthesis in LdB axenic amastigotes. These and other data suggest that LdB axenic amastigotes will be generally useful as a differentiation model in studies of gene expression, virulence, glycoconjugate function and drug susceptibility in L. donovani.
糖缀合物已被证明在利什曼原虫的发育中发挥重要作用。然而，研究这些分子和其他过程的能力将极大地受益于改进的利什曼原虫无鞭毛体阶段的基因操作和分析方法。对于杜氏利什曼原虫（最严重形式的利什曼病的病原体）来说，这尤其具有挑战性，因为它在体外培养过程中会迅速丧失毒力。在这里，我们报告了杜氏利什曼原虫1S2D（LdBob或LdB）的一个克隆亚系，它在无菌培养中很容易从前鞭毛体分化为无鞭毛体，并在体外长期培养寄生虫的过程中保持这种能力。这种衍生物在作为前鞭毛体或无鞭毛体生长时可以有效地铺板和转染。重要的是，LdB在进行基因敲除和互补系构建所需的基因改变时，仍保持分化能力。与有毒力的杜氏利什曼原虫一样，LdB在无鞭毛体中表现出脂磷壁酸（LPG）合成的下调和A2蛋白合成的上调。我们发现，编码高尔基体GDP-甘露糖转运蛋白的LPG2基因敲除消除了LdB无菌无鞭毛体中的磷酸聚糖合成。这些以及其他数据表明，LdB无菌无鞭毛体在杜氏利什曼原虫的基因表达、毒力、糖缀合物功能和药物敏感性研究中作为分化模型将具有普遍用途。
