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Experimental evolution reveals post-transcriptional regulation as an important driver of fitness gain in the human parasite Leishmania donovani

ABSTRACT: Leishmania survival inside insect and mammalian hosts depends on the parasite’s capacity to develop into promastigote and amastigote stages, respectively. Promastigotes can be maintained in vitro in culture medium mimicking the insect milieu in which the parasites are surviving, differentiating and proliferating. We previously showed that the maintenance of the parasites in this in vitro environment lead to a progressive loss of infectivity together with genomic changes, notably chromosome amplifications. In contrast to classical eukaryotes Leishmania do not regulate expression of protein coding genes by transcriptional control, thus ruling out regulated gene expression changes in adaptation. In the absence of classical transcriptional regulation, Leishmania has evolved and emphasized other forms of control that are relevant for evolutionary adaptation, notably regulation of RNA abundance by gene dosage variation. In order to identify the biological signals that correlate with the observed fitness trade off we observed during L. donovani culture adaptation we combined DNAseq and RNAseq with label-free quantitative proteomics. Promastigotes freshly derived from splenic amastigotes purified from the spleen of infected hamsters were maintained in culture for 2 (EP) and 20 (LP) passages. Total protein extracts from four EP and four LP promastigotes were prepared for label free MS analysis.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Leishmania Donovani

TISSUE(S): Spleen, Cell Line Cell

DISEASE(S): Leishmaniasis

SUBMITTER: Thibaut Douché

LAB HEAD: Gérald Spaeth

PROVIDER: PXD020236 | Pride | 2022-03-03

REPOSITORIES: Pride

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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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Experimental evolution reveals post-transcriptional regulation as an important driver of fitness gain in the human parasite Leishmania donovani

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