Leishmania iron response. Leishmania amazonensis strain:IFLA/BR/67/PH8

Iron is an essential element required for many metabolic pathways, but when it is present in excess, it is toxic. Thus, iron acquisition and storage must be tightly regulated. From previous studies, we know that Leishmania modulate the ferric iron reductase LFR1, the ferrous iron transporter LIT1, and the heme transporter LHR1 in response to iron deprivation to facilitate iron and heme acquisition. The aforementioned proteins are the only currently known components of Leishmania iron acquisition pathways. To identify more genes involved in the iron acquisition/storage pathways, we have used the iron deprivation model coupled with RNA-Seq. It is important to note that the conditions of the experiment must be precisely controlled because iron deprivation can act as a signal to initiate transformation into the intracellular amastigote forms independent of temperature and pH. A previous study using SL RNA-Seq designed to detect novel components of the iron-responsive pathway was set up in such a fashion that genes responsible for parasite differentiation were also part of the list of differentially expressed genes. In order to alleviate this problem and identify genes directly involved in iron acquisition and storage, the following strategy was used. The parasites were in media depleted of both inorganic iron and heme, or in iron- and heme-replete media. This study used parasites harvested 18 hours post iron deprivation, a time point when there is maximal upregulation of the known iron related genes without modulation of genes involved in differentiation. Overall experimental design: mRNA samples taken from three biological replicates representing iron replete or deplete cells were analyzed using RNA-Seq and differential expression analysis methodology.铁是诸多代谢通路不可或缺的元素，但当铁过量蓄积时，会产生毒性作用。因此，铁的摄取与储存必须受到严格调控。既往研究表明，利什曼原虫（Leishmania）会响应铁匮乏条件，调控铁还原酶（ferric iron reductase）LFR1、亚铁转运蛋白（ferrous iron transporter）LIT1以及血红素转运蛋白（heme transporter）LHR1的表达，以促进铁与血红素的摄取。上述蛋白是目前已知的利什曼原虫铁摄取通路仅有的组成元件。为鉴定更多参与铁摄取/储存通路的基因，本研究采用了铁匮乏模型结合RNA测序（RNA-Seq）技术。需要特别说明的是，实验条件必须得到精准控制，因为铁匮乏可作为独立于温度与pH的信号，诱导寄生虫转化为胞内寄生的无鞭毛体（amastigote）形态。此前一项利用剪接前导RNA测序（SL RNA-Seq）检测铁响应通路新组分的研究，其实验设计使得参与寄生虫分化的基因也被纳入差异表达基因列表。为规避该问题并直接鉴定参与铁摄取与储存的基因，本研究采用如下实验策略：将寄生虫分别培养于无机铁与血红素均匮乏的培养基，或是铁与血红素充足的培养基中。本研究选取铁匮乏处理18小时后的寄生虫样本进行实验，该时间点下，已知铁相关基因的表达上调达到峰值，且未发生分化相关基因的表达调控。整体实验设计如下：从铁充足与铁匮乏的细胞样本中各获取三组生物学重复的mRNA样品，采用RNA-Seq技术与差异表达分析方法完成测序与数据分析。