Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Iron uptake controls the generation of Leishmania infective forms through regulation of ROS levels .

ABSTRACT: During its life cycle Leishmania undergoes extreme environmental changes, alternating between insect vectors and vertebrate hosts. In mammals the parasites replicate within parasitophorous vacuoles of macrophages, compartments that contain low concentrations of iron. Here we show that stimulation of iron transport, which is induced in Leishmania amazonensis by an iron-poor environment, triggers the differentiation of avirulent promastigotes into virulent amastigotes. Iron depletion from the culture medium triggered expression of the LIT1 ferrous iron transporter, growth arrest, and differentiation of wild type promastigotes into infective amastigotes. In contrast, LIT1 null promastigotes showed continued exponential growth in iron-poor media, followed by massive cell death. Iron depletion from the medium and LIT1 upregulation increased iron superoxide dismutase activity (FeSOD) in wild type, but not in LIT1 null parasites. Notably, the superoxide-generating drug menadione or H2O2 were sufficient to trigger differentiation of wild type promastigotes into fully infective amastigotes. On the other hand, LIT1 null promastigotes accumulated superoxide radical and initiated amastigote differentiation after exposure to H2O2, but not to menadione. Our results reveal a novel role for FeSOD activity and reactive oxygen species (ROS) in orchestrating the differentiation of Leishmania infective stages, in a process regulated by iron availability. Four samples were obtained in total from wild-type L. amazonensis promastigotes grown for 24 hours in culture. Two of these samples derived from medium with iron and two from mediam without iron.These two samples derived from each culture served as biological replicates.

ORGANISM(S): Leishmania amazonensis

SUBMITTER: Gowthaman Ramasamy

PROVIDER: E-GEOD-41641 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Protozoa of the genus Leishmania are the causative agents of leishmaniasis in humans. These parasites cycle between promastigotes in the sand fly mid-gut and amastigotes in phagolysosome of mammalian macrophages. During infection, host up-regulate nitric oxide synthase and parasite induce host arginase expression, both of which use arginine as a substrate. These elevated activities deplete macrophage arginine pools, a situation that invading Leishmania must overcome since it is an essential amino acid. Leishmania donovani imports exogenous arginine via a mono-specific amino acid transporter (AAP3) and utilizes it primarily through the polyamine pathway to provide precursors for trypanothione biosynthesis. Here we report the discovery of a pathway whereby promastigote and amastigote forms of the Leishmania sense the lack of environmental arginine and respond with rapid up-regulation in AAP3 expression and activity, as well as several other transporters. Significantly, this arginine deprivation response is also activated in parasites during macrophage infection. Phosphoproteomic analyses of L. donovani promastigotes have implicated a Mitogen-Activated Protein Kinase 2 (MPK2)-mediated signaling cascade in this response and L. mexicana mutants lacking MPK2 are unable to respond to arginine deprivation. In this study, we established that Leishmania cells sense the absence of arginine in their environment; both in culture (axenic promastigotes and amastigotes) and in macrophages during infection (amastigotes). This study describes the first amino acid deprivation sensing mechanism and the pathway that transduce this response, and reveals a novel host-pathogen metabolic interplay. Total RNA from Ten Leishmania donovani samples were analyzed using RNA-Seq. Cells from two life stages (promastigotes and amastigotes) were grown in axenic culture in the presence and absense of arginine. For each condition two biological replicates were grown and analyzed. In addition two macrophage grown amastigotes were analyzed.

Project description:Among the most central questions in Leishmania research is why some species remain in the skin dermis at the site of infection by the sand fly vector whereas other species migrate to visceral organs where they cause fatal visceral leishmaniasis. Although L. donovani is the species typically responsible for visceral leishmaniasis, an atypical L. donovani strain is the etiologic agent for cutaneous leishmaniasis in Sri Lanka. To identify molecular determinants for visceral disease, we have analysed the phenotype and genotype of two L. donovani clinical isolates from Sri Lanka where one isolate was derived from a cutaneous leishmaniasis patient (CL) and the other from a visceral leishmaniasis patient (VL). These isolates cause dramatically different pathology when introduced into mice; notably the CL isolate has lost the ability to survive in visceral organs while the VL isolate was highly virulent in visceral organs of BALB/c mice. Whole genome sequencing of the CL and VL isolates revealed that these genomes were very similar as there were no gene deletions and few individual gene amplifications. Indels resulting in frame shifts and loss/gain of stop codons resulted in 13 distinct pseudogenes present in each of the CL and VL isolates. There were 154 non-synonymous SNPs specific to the CL isolate and 193 non-synonymous SNPs specific to the VL isolate. Genome wide gene expression analysis revealed several transcript level differences, including the A2 virulence gene resulting in higher expression of A2 proteins in the VL isolate than in the CL isolate. Genotypic variations relevant to pathology and tropism in Leishmania can be interrogated by reverse genetics. Experimentally increasing A2 expression in the CL isolate through gene transfer significantly increased itM-bM-^@M-^Ys ability to survive in the spleen of BALB/c mice and conversely, down-regulating A2 expression in the VL isolate abrogated attenuated its survival in BALB/c mice. These observations reveal that there are relatively few genetic differences between the CL and VL isolates apart from the A2 genes, but collectively these have profound effects on human disease and experimentally infected mice. 6 Samples in total, 3 each from VL and CL causing isolates were analyzed by Splice Leader RNASeq. These three samples from each of the isolates were grown to form one of the following three lifestages, Promastigotes, Macrophage derived Amastigotes, Axenic Amastigotes.

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Iron uptake controls the generation of Leishmania infective forms through regulation of ROS levels .

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