Aoki Juliana Ide, Muxel Sandra Marcia, Zampieri Ricardo Andrade, Acuña Stephanie Maia, Fernandes Juliane Cristina Ribeiro, Vanderlinde Rubia Heloisa, Sales Maria Carmen Oliveira de Pinho, Floeter-Winter Lucile Maria
Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil.
PLoS Negl Trop Dis. 2017 Oct 26;11(10):e0006025. doi: 10.1371/journal.pntd.0006025. eCollection 2017 Oct.
Leishmania uses the amino acid L-arginine as a substrate for arginase, enzyme that produces urea and ornithine, last precursor of polyamine pathway. This pathway is used by the parasite to replicate and it is essential to establish the infection in the mammalian host. L-arginine is not synthesized by the parasite, so its uptake occurs through the amino acid permease 3 (AAP3). AAP3 is codified by two copies genes (5.1 and 4.7 copies), organized in tandem in the parasite genome. One copy presents the expression regulated by L-arginine availability.
METHODOLOGY/PRINCIPAL FINDINGS: RNA-seq data revealed 14 amino acid transporters differentially expressed in the comparison of La-WT vs. La-arg- promastigotes and axenic amastigotes. The 5.1 and 4.7 aap3 transcripts were down-regulated in La-WT promastigotes vs. axenic amastigotes, and in La-WT vs. La-arg- promastigotes. In contrast, transcripts of other transporters were up-regulated in the same comparisons. The amount of 5.1 and 4.7 aap3 mRNA of intracellular amastigotes was also determined in sample preparations from macrophages, obtained from BALB/c and C57BL/6 mice and the human THP-1 lineage infected with La-WT or La-arg-, revealing that the genetic host background is also important. We also determined the aap3 mRNA and AAP3 protein amounts of promastigotes and axenic amastigotes in different environmental growth conditions, varying pH, temperature and L-arginine availability. Interestingly, the increase of temperature increased the AAP3 level in plasma membrane and consequently the L-arginine uptake, independently of pH and L-arginine availability. In addition, we demonstrated that besides the plasma membrane localization, AAP3 was also localized in the glycosome of L. amazonensis promastigotes and axenic amastigotes.
CONCLUSIONS/SIGNIFICANCE: In this report, we described the differential transcriptional profiling of amino acids transporters from La-WT and La-arg- promastigotes and axenic amastigotes. We also showed the increased AAP3 levels under amino acid starvation or its decrease in L-arginine supplementation. The differential AAP3 expression was determined in the differentiation of promastigotes to amastigotes conditions, as well as the detection of AAP3 in the plasma membrane reflecting in the L-arginine uptake. Our data suggest that depending on the amino acid pool and arginase activity, Leishmania senses and could use an alternative route for the amino acid transport in response to stress signaling.
利什曼原虫利用氨基酸L-精氨酸作为精氨酸酶的底物，该酶可产生尿素和鸟氨酸，鸟氨酸是多胺途径的最后前体。寄生虫利用该途径进行复制，对于在哺乳动物宿主中建立感染至关重要。寄生虫自身不能合成L-精氨酸，因此其摄取通过氨基酸通透酶3（AAP3）进行。AAP3由两个拷贝的基因（5.1和4.7拷贝）编码，在寄生虫基因组中串联排列。其中一个拷贝的表达受L-精氨酸可用性的调节。
方法/主要发现：RNA测序数据显示，在野生型利什曼原虫（La-WT）与精氨酸缺陷型前鞭毛体（La-arg-）以及无共生体无鞭毛体的比较中，有14种氨基酸转运蛋白差异表达。与无共生体无鞭毛体相比，以及与La-WT与La-arg-前鞭毛体相比，5.1和4.7 aap3转录本在La-WT前鞭毛体中下调。相反，在相同比较中其他转运蛋白的转录本上调。还从感染了La-WT或La-arg-的BALB/c和C57BL/6小鼠以及人THP-1细胞系的巨噬细胞样本制备物中，测定了细胞内无鞭毛体中5.1和4.7 aap3 mRNA的量，结果表明宿主遗传背景也很重要。我们还测定了不同环境生长条件（不同pH、温度和L-精氨酸可用性）下前鞭毛体和无共生体无鞭毛体中aap3 mRNA和AAP3蛋白的量。有趣的是，温度升高会增加质膜中AAP3的水平，从而增加L-精氨酸的摄取，这与pH和L-精氨酸可用性无关。此外，我们证明除了质膜定位外，AAP3还定位于亚马逊利什曼原虫前鞭毛体和无共生体无鞭毛体的糖体中。
结论/意义：在本报告中，我们描述了野生型利什曼原虫和精氨酸缺陷型前鞭毛体以及无共生体无鞭毛体中氨基酸转运蛋白的差异转录谱。我们还显示了在氨基酸饥饿条件下AAP3水平升高，或在补充L-精氨酸时其水平降低。在从前鞭毛体向无鞭毛体分化的条件下确定了AAP3的差异表达，以及在质膜中检测到AAP3反映在L-精氨酸摄取上。我们的数据表明，根据氨基酸库和精氨酸酶活性，利什曼原虫能够感知并可能利用替代途径进行氨基酸转运以应对应激信号。
