Boitz Jan M, Jardim Armando, Ullman Buddy
Department of Biochemistry and Molecular Biology, Oregon Health & Science University, 3181 SW Sam Jackson Park Rd., Mail Code L224, Portland, OR 97239, USA.
Institute of Parasitology and Centre for Host-Parasite Interactions, Macdonald Campus of McGill University, 21 111 Lakeshore Road, Ste-Anne-de-Bellevue, Quebec, H9X3V9, Canada.
Mol Biochem Parasitol. 2016 Aug;208(2):74-83. doi: 10.1016/j.molbiopara.2016.06.008. Epub 2016 Jun 22.
Purine acquisition is an essential nutritional process for Leishmania. Although purine salvage into adenylate nucleotides has been investigated in detail, little attention has been focused on the guanylate branch of the purine pathway. To characterize guanylate nucleotide metabolism in Leishmania and create a cell culture model in which the pathways for adenylate and guanylate nucleotide synthesis can be genetically uncoupled for functional studies in intact cells, we created and characterized null mutants of L. donovani that were deficient in either GMP reductase alone (Δgmpr) or in both GMP reductase and its paralog IMP dehydrogenase (Δgmpr/Δimpdh). Whereas wild type parasites were capable of utilizing virtually any purine nucleobase/nucleoside, the Δgmpr and Δgmpr/Δimpdh null lines exhibited highly restricted growth phenotypes. The Δgmpr single mutant could not grow in xanthine, guanine, or their corresponding nucleosides, while no purine on its own could support the growth of Δgmpr/Δimpdh cells. Permissive growth conditions for the Δgmpr/Δimpdh necessitated both xanthine, guanine, or the corresponding nucleosides, and additionally, a second purine that could serve as a source for adenylate nucleotide synthesis. Interestingly, GMPR, like its paralog IMPDH, is compartmentalized to the leishmanial glycosome, a process mediated by its COOH-terminal peroxisomal targeting signal. The restricted growth phenotypes displayed by the L. donovani Δgmpr and Δgmpr/Δimpdh null mutants confirms the importance of GMPR in the purine interconversion processes of this parasite.
嘌呤摄取是利什曼原虫必不可少的营养过程。尽管已对嘌呤挽救生成腺苷酸核苷酸的过程进行了详细研究，但嘌呤途径的鸟苷酸分支却很少受到关注。为了表征利什曼原虫中的鸟苷酸核苷酸代谢，并创建一个细胞培养模型，在该模型中腺苷酸和鸟苷酸核苷酸合成途径可以通过基因解偶联，以便在完整细胞中进行功能研究，我们构建并表征了杜氏利什曼原虫的缺失突变体，这些突变体单独缺乏GMP还原酶（Δgmpr）或同时缺乏GMP还原酶及其旁系同源物IMP脱氢酶（Δgmpr/Δimpdh）。野生型寄生虫几乎能够利用任何嘌呤碱基/核苷，而Δgmpr和Δgmpr/Δimpdh缺失株表现出高度受限的生长表型。Δgmpr单突变体不能在黄嘌呤、鸟嘌呤或其相应核苷中生长，而单独的任何嘌呤都不能支持Δgmpr/Δimpdh细胞的生长。Δgmpr/Δimpdh的允许生长条件需要黄嘌呤、鸟嘌呤或相应的核苷，此外，还需要第二种嘌呤作为腺苷酸核苷酸合成的来源。有趣的是，GMPR与其旁系同源物IMPDH一样，被分隔到利什曼原虫的糖体中，这一过程由其COOH末端过氧化物酶体靶向信号介导。杜氏利什曼原虫Δgmpr和Δgmpr/Δimpdh缺失突变体所显示的受限生长表型证实了GMPR在该寄生虫嘌呤相互转化过程中的重要性。
