Aoki Juliana Ide, Muxel Sandra Marcia, Zampieri Ricardo Andrade, Laranjeira-Silva Maria Fernanda, Müller Karl Erik, Nerland Audun Helge, Floeter-Winter Lucile Maria
Department of Physiology, Institute of Bioscience, University of Sao Paulo, Sao Paulo, Brazil.
Department of Clinical Science, University of Bergen, Bergen, Norway.
PLoS Negl Trop Dis. 2017 Oct 27;11(10):e0006026. doi: 10.1371/journal.pntd.0006026. eCollection 2017 Oct.
Leishmania is a protozoan parasite that alternates its life cycle between the sand-fly vector and the mammalian host. This alternation involves environmental changes and leads the parasite to dynamic modifications in morphology, metabolism, cellular signaling and regulation of gene expression to allow for a rapid adaptation to new conditions. The L-arginine pathway in L. amazonensis is important during the parasite life cycle and interferes in the establishment and maintenance of the infection in mammalian macrophages. Host arginase is an immune-regulatory enzyme that can reduce the production of nitric oxide by activated macrophages, directing the availability of L-arginine to the polyamine pathway, resulting in parasite replication. In this work, we performed transcriptional profiling to identify differentially expressed genes in L. amazonensis wild-type (La-WT) versus L. amazonensis arginase knockout (La-arg-) promastigotes and axenic amastigotes.
METHODOLOGY/PRINCIPAL FINDINGS: A total of 8253 transcripts were identified in La-WT and La-arg- promastigotes and axenic amastigotes, about 60% of them codifying hypothetical proteins and 443 novel transcripts, which did not match any previously annotated genes. Our RNA-seq data revealed that 85% of genes were constitutively expressed. The comparison of transcriptome and metabolome data showed lower levels of arginase and higher levels of glutamate-5-kinase in La-WT axenic amastigotes compared to promastigotes. The absence of arginase activity in promastigotes increased the levels of pyrroline 5-carboxylate reductase, but decreased the levels of arginosuccinate synthase, pyrroline 5-carboxylate dehydrogenase, acetylornithine deacetylase and spermidine synthase transcripts levels. These observations can explain previous metabolomic data pointing to the increase of L-arginine, citrulline and L-glutamate and reduction of aspartate, proline, ornithine and putrescine. Altogether, these results indicate that arginase activity is important in Leishmania gene expression modulation during differentiation and adaptation to environmental changes. Here, we confirmed this hypothesis with the identification of differential gene expression of the enzymes involved in biosynthesis of amino acids, arginine and proline metabolism and arginine biosynthesis.
CONCLUSIONS/SIGNIFICANCE: All data provided information about the transcriptomic profiling and the expression levels of La-WT and La-arg- promastigotes and axenic amastigotes. These findings revealed the importance of arginase in parasite survival and differentiation, and indicated the existence of a coordinated response in the absence of arginase activity related to arginine and polyamine pathways.
利什曼原虫是一种原生动物寄生虫，其生命周期在白蛉媒介和哺乳动物宿主之间交替。这种交替涉及环境变化，导致寄生虫在形态、代谢、细胞信号传导和基因表达调控方面发生动态变化，以便快速适应新环境。亚马逊利什曼原虫中的L-精氨酸途径在寄生虫生命周期中很重要，并会干扰其在哺乳动物巨噬细胞中感染的建立和维持。宿主精氨酸酶是一种免疫调节酶，它可以减少活化巨噬细胞产生一氧化氮，将L-精氨酸的可用性导向多胺途径，从而导致寄生虫复制。在这项研究中，我们进行了转录谱分析，以鉴定亚马逊利什曼原虫野生型（La-WT）与亚马逊利什曼原虫精氨酸酶敲除型（La-arg-）前鞭毛体和无菌无鞭毛体中差异表达的基因。
方法/主要发现：在La-WT和La-arg-前鞭毛体及无菌无鞭毛体中总共鉴定出8253个转录本，其中约60%编码假设蛋白和443个新转录本，这些转录本与任何先前注释的基因均不匹配。我们的RNA测序数据显示，85%的基因是组成性表达的。转录组和代谢组数据的比较表明，与前鞭毛体相比，La-WT无菌无鞭毛体中精氨酸酶水平较低，而谷氨酸-5-激酶水平较高。前鞭毛体中精氨酸酶活性的缺失增加了吡咯啉5-羧酸还原酶的水平，但降低了精氨琥珀酸合酶、吡咯啉5-羧酸脱氢酶、乙酰鸟氨酸脱乙酰酶和亚精胺合酶的转录本水平。这些观察结果可以解释先前的代谢组学数据，即L-精氨酸、瓜氨酸和L-谷氨酸增加，而天冬氨酸、脯氨酸、鸟氨酸和腐胺减少。总之，这些结果表明精氨酸酶活性在利什曼原虫分化和适应环境变化过程中的基因表达调节中很重要。在这里，我们通过鉴定参与氨基酸生物合成、精氨酸和脯氨酸代谢以及精氨酸生物合成的酶的差异基因表达，证实了这一假设。
结论/意义：所有数据提供了关于La-WT和La-arg-前鞭毛体及无菌无鞭毛体的转录组分析和表达水平的信息。这些发现揭示了精氨酸酶在寄生虫存活和分化中的重要性，并表明在缺乏与精氨酸和多胺途径相关的精氨酸酶活性时存在协调反应。
