Bachmaier Sabine, Witztum Ronit, Tsigankov Polina, Koren Roni, Boshart Michael, Zilberstein Dan
Ludwig-Maximilian-University Munich (LMU), Faculty of Biology, Genetics, 82152 Martinsried, Germany.
Technion-Israel Institute of Technology, Faculty of Biology, Haifa 32000, Israel.
Int J Parasitol. 2016 Feb;46(2):75-82. doi: 10.1016/j.ijpara.2015.09.003. Epub 2015 Oct 13.
Parasitic protozoa of the genus Leishmania are obligatory intracellular parasites that cycle between the phagolysosome of mammalian macrophages, where they proliferate as intracellular amastigotes, and the midgut of female sand flies, where they proliferate as extracellular promastigotes. Shifting between the two environments induces signaling pathway-mediated developmental processes that enable adaptation to both host and vector. Developmentally regulated expression and phosphorylation of protein kinase A subunits in Leishmania and in Trypanosoma brucei point to an involvement of protein kinase A in parasite development. To assess this hypothesis in Leishmania donovani, we determined proteome-wide changes in phosphorylation of the conserved protein kinase A phosphorylation motifs RXXS and RXXT, using a phospho-specific antibody. Rapid dephosphorylation of these motifs was observed upon initiation of promastigote to amastigote differentiation in culture. No phosphorylated sites were detected in axenic amastigotes. To analyse the kinetics of (re)phosphorylation during axenic reverse differentiation from L. donovani amastigotes to promastigotes, we first established a map of this process with morphological and molecular markers. Upon initiation, the parasites rested for 6-12 h before proliferation of an asynchronous population resumed. After early changes in cell shape, the major changes in molecular marker expression and flagella biogenesis occurred between 24 and 33 h after initiation. RXXS/T re-phosphorylation and expression of the regulatory subunit PKAR1 correlated with promastigote maturation, indicating a promastigote-specific function of protein kinase A signaling. This is supported by the localization of PKAR1 to the flagellum, an organelle reduced to a remnant in amastigote forms. We conclude that a significant increase in protein kinase A-mediated phosphorylation is part of the ordered changes that characterise the amastigote to promastigote differentiation.
利什曼原虫属的寄生原生动物是专性细胞内寄生虫，在哺乳动物巨噬细胞的吞噬溶酶体（它们在其中作为细胞内无鞭毛体增殖）和雌性白蛉的中肠（它们在其中作为细胞外前鞭毛体增殖）之间循环。在两种环境之间转换会诱导信号通路介导的发育过程，从而实现对宿主和载体的适应。利什曼原虫和布氏锥虫中蛋白激酶A亚基的发育调控表达和磷酸化表明蛋白激酶A参与寄生虫发育。为了在杜氏利什曼原虫中评估这一假设，我们使用磷酸特异性抗体确定了保守的蛋白激酶A磷酸化基序RXXS和RXXT磷酸化的全蛋白质组变化。在培养中前鞭毛体开始分化为无鞭毛体时，观察到这些基序的快速去磷酸化。在无菌无鞭毛体中未检测到磷酸化位点。为了分析杜氏利什曼原虫无鞭毛体向前鞭毛体无菌逆向分化过程中（再）磷酸化的动力学，我们首先用形态学和分子标记建立了这一过程的图谱。开始时，寄生虫在异步群体增殖恢复之前静止6至12小时。在细胞形状早期变化之后，分子标记表达和鞭毛生物发生的主要变化发生在开始后24至33小时之间。RXXS/T再磷酸化和调节亚基PKAR1的表达与前鞭毛体成熟相关，表明蛋白激酶A信号传导具有前鞭毛体特异性功能。PKAR1定位于鞭毛，鞭毛在无鞭毛体形式中减少为残余细胞器，这支持了这一点。我们得出结论，蛋白激酶A介导的磷酸化显著增加是无鞭毛体向前鞭毛体分化特征的有序变化的一部分。
