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Comparison of cultured and sand fly-derived promastigotes by means of RNA-Seq

ABSTRACT: A laboratory colony of Phlebotomus perniciosus sand flies was maintained. Sand flies were infected with cultured Leishmania infantum promastigotes in stationary phase. Ten infected sand flies were dissected after 5 days and promastigotes within the gut pooled. The cells were immediately washed in PBS once and lysed in TRIzol reagent (Life Technologies). RNA isolation was completed according to the manufacturer's instructions, obtaining 63ng. RNA-seq libraries were generated using the spliced leader sequence for second strand synthesis (Cuypers et al., 2017; Haydock et al., 2015), thus allowing for specific amplification of sequences from L. infantum promastigotes, thus avoiding contamination with material from the sand fly gut. Single-end sequencing was performed in an Illumina HiSeq2500 instrument and data analysis was conducted using bowtie2, samtools, featureCounts and Geneious. The main findings are: i) substantial differences in differential gene expression between sand fly-derived (sfPro) and cultured (acPro) promastigotes; and ii) over-expression of genes involved in metacyclogenesis in sfPro vs. acPro, including gp63 genes, autophagy genes, etc.

ORGANISM(S): Leishmania infantum

PROVIDER: GSE102668 | GEO | 2018/08/15

REPOSITORIES: GEO

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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.

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Comparison of cultured and sand fly-derived promastigotes by means of RNA-Seq

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