Characterization of Leishmania donovani Aquaporins Shows Presence of Subcellular Aquaporins Similar to Tonoplast Intrinsic Proteins of Plants

Leishmania donovani, a protozoan parasite, resides in the macrophages of the mammalian host. The aquaporin family of proteins form important components of the parasite-host interface. The parasite-host interface could be a potential target for chemotherapy. Analysis of L. major and L. infantum genomes showed the presence of five aquaporins (AQPs) annotated as AQP9 (230aa), AQP putative (294aa), AQP-like protein (279aa), AQP1 (314aa) and AQP-like protein (596aa). We report here the structural modeling, localization and functional characterization of the AQPs from L. donovani. LdAQP1, LdAQP9, LdAQP2860 and LdAQP2870 have the canonical NPA-NPA motifs, whereas LdAQP putative has a non-canonical NPM-NPA motif. In the carboxyl terminal to the second NPA box of all AQPs except AQP1, a valine/alanine residue was found instead of the arginine. In that respect these four AQPs are similar to tonoplast intrinsic proteins in plants, which are localized to intracellular organelles. Confocal microscopy of L. donovani expressing GFP-tagged AQPs showed an intracellular localization of LdAQP9 and LdAQP2870. Real-time PCR assays showed expression of all aquaporins except LdAQP2860, whose level was undetectable. Three-dimensional homology modeling of the AQPs showed that LdAQP1 structure bears greater topological similarity to the aquaglyceroporin than to aquaporin of E. coli. The pore of LdAQP1 was very different from the rest in shape and size. The cavity of LdAQP2860 was highly irregular and undefined in geometry. For functional characterization, four AQP proteins were heterologously expressed in yeast. In the fps1Δ yeast cells, which lacked the key aquaglyceroporin, LdAQP1 alone displayed an osmosensitive phenotype indicating glycerol transport activity. However, expression of LdAQP1 and LdAQP putative in a yeast gpd1Δ strain, deleted for glycerol production, conferred osmosensitive phenotype indicating water transport activity or aquaporin function. Our analysis for the first time shows the presence of subcellular aquaporins and provides structural and functional characterization of aquaporins in Leishmania donovani.杜氏利什曼原虫（Leishmania donovani）是一种原生动物寄生虫，寄生于哺乳动物宿主的巨噬细胞内。水通道蛋白（aquaporin, AQP）家族是寄生虫-宿主界面的重要组成成分，该界面可作为化疗的潜在靶点。对硕大利什曼原虫（L. major）和婴儿利什曼原虫（L. infantum）基因组的分析显示，其体内存在5种水通道蛋白，分别注释为AQP9（230个氨基酸）、推定AQP（294个氨基酸）、类AQP蛋白（279个氨基酸）、AQP1（314个氨基酸）以及另一种类AQP蛋白（596个氨基酸）。本研究对杜氏利什曼原虫的AQPs开展了结构建模、亚细胞定位及功能表征。其中，LdAQP1、LdAQP9、LdAQP2860与LdAQP2870均携带经典的NPA-NPA基序，而推定的LdAQP仅含有非经典的NPM-NPA基序。除AQP1外，其余所有AQPs的第二个NPA基序的羧基末端区域，均检测到缬氨酸/丙氨酸残基替代精氨酸的特征，这4种AQPs在该特性上与植物的液泡膜内在蛋白（tonoplast intrinsic proteins）高度相似，后者定位于细胞内细胞器。对表达绿色荧光蛋白（GFP）标记AQPs的杜氏利什曼原虫进行共聚焦显微镜观察，结果显示LdAQP9与LdAQP2870呈现细胞内定位模式。实时荧光定量PCR（Real-time PCR）检测表明，除LdAQP2860未检出表达外，其余所有水通道蛋白均有转录表达。对AQPs的三维同源建模结果显示，LdAQP1的拓扑结构与大肠杆菌（E. coli）的水甘油通道蛋白相似性更高，且其孔道的形状与大小均与其余AQPs存在显著差异；LdAQP2860的空腔几何结构高度不规则且边界模糊。为开展功能表征，研究人员将4种AQP蛋白在酵母中进行异源表达。在缺失关键水甘油通道蛋白的fps1Δ酵母菌株中，仅LdAQP1表现出渗透压敏感表型，提示其具备甘油转运活性。而在缺失甘油合成能力的gpd1Δ酵母菌株中，同时表达LdAQP1与推定LdAQP可使其获得渗透压敏感表型，表明这两种蛋白具备水转运活性，即典型水通道蛋白功能。本研究首次证实了亚细胞定位型水通道蛋白的存在，并对杜氏利什曼原虫的水通道蛋白完成了结构与功能层面的系统表征。