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.