L-arginine availability and arginase activity: Characterization of amino acid permease 3 in Leishmania amazonensis
L-arginine availability and arginase activity: Characterization of amino acid permease 3 in Leishmania amazonensis
复制标题
L精氨酸利用率和精氨酸酶活性：亚马逊利什曼原虫氨基酸渗透酶3的特性
DOI：
10.1371/journal.pntd.0006025
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发表时间：
2017-10-01
影响因子：
3.8
通讯作者：
Floeter-Winter, Lucile Maria
中科院分区：
文献类型：
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作者：
Aoki, Juliana Ide;Muxel, Sandra Marcia;Floeter-Winter, Lucile Maria
背景利什曼原虫以L-精氨酸为底物，产生尿素和鸟氨酸，多胺途径的最后前体。该途径被寄生虫用于复制，并且对于在哺乳动物宿主中建立感染至关重要。精氨酸不是由寄生虫合成的，所以它的摄取通过氨基酸通透酶3（AAP 3）发生。AAP 3由两个拷贝基因（5.1和4.7拷贝）编码，在寄生虫基因组中串联组织。一个拷贝呈现由L-精氨酸availability.Methodology/Principal findings RNA-seq数据显示14种氨基酸转运蛋白在La-WT与La-arg（-）前鞭毛体和纯性无鞭毛体的比较中差异表达。5.1和4.7 aap 3转录物在La-WT前鞭毛体相对于纯无鞭毛体以及La-WT相对于La-arg-前鞭毛体中下调。相反，其他转运蛋白的转录本在相同的比较中上调。还在从BALB/c和C57 BL/6小鼠以及用La-WT或La-arg（-）感染的人THP-1谱系获得的巨噬细胞的样品制备物中测定了细胞内无鞭毛体的5.1和4.7 aap 3 mRNA的量，揭示了遗传宿主背景也是重要的。我们还确定了aap 3 mRNA和AAP 3蛋白量的前鞭毛体和无鞭毛体在不同的环境生长条件下，不同的pH值，温度和L-精氨酸的可用性。有趣的是，温度的升高增加了质膜中的AAP 3水平，从而增加了L-精氨酸的摄取，而与pH和L-精氨酸的可用性无关。此外，我们还发现AAP 3除了定位于质膜外，还定位于L.结论/意义在本报告中，我们描述了La-WT和La-arg（-）前鞭毛体和纯无鞭毛体的氨基酸转运蛋白的差异转录谱。我们还显示了氨基酸饥饿或L-精氨酸补充降低AAP 3水平的增加。在前鞭毛体分化为无鞭毛体的条件下确定差异AAP 3表达，以及在质膜中检测AAP 3，反映L-精氨酸摄取。我们的数据表明，根据氨基酸池和酶活性，利什曼原虫的感觉，并可能使用替代途径的氨基酸运输响应应力信号。
Background 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. Larginine 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.