RNA-seq transcriptional profiling of Leishmania amazonensis reveals an arginase-dependent gene expression regulation

Abstract

Leishmania is a protozoan parasite that alternates its life cycle between the sand-fly vector and the mammalian host. This alternation involves environmental changes and leads the parasite to dynamic modifications in morphology, metabolism, cellular signaling and regulation of gene expression to allow for a rapid adaptation to new conditions. The L-arginine pathway in L. amazonensis is important during the parasite life cycle and interferes in the establishment and maintenance of the infection in mammalian macrophages. Host arginase is an immune-regulatory enzyme that can reduce the production of nitric oxide by activated macrophages, directing the availability of L-arginine to the polyamine pathway, resulting in parasite replication. In this work, we performed transcriptional profiling to identify differentially expressed genes in L. amazonensis wild-type (La-WT) versus L. amazonensis arginase knockout (La-arg^-) promastigotes and axenic amastigotes. A total of 8253 transcripts were identified in La-WT and La-arg^- promastigotes and axenic amastigotes, about 60% of them codifying hypothetical proteins and 443 novel transcripts, which did not match any previously annotated genes. Our RNA-seq data revealed that 85% of genes were constitutively expressed. The comparison of transcriptome and metabolome data showed lower levels of arginase and higher levels of glutamate-5-kinase in La-WT axenic amastigotes compared to promastigotes. The absence of arginase activity in promastigotes increased the levels of pyrroline 5-carboxylate reductase, but decreased the levels of arginosuccinate synthase, pyrroline 5-carboxylate dehydrogenase, acetylornithine deacetylase and spermidine synthase transcripts levels. These observations can explain previous metabolomic data pointing to the increase of L-arginine, citrulline and L-glutamate and reduction of aspartate, proline, ornithine and putrescine. Altogether, these results indicate that arginase activity is important in Leishmania gene expression modulation during differentiation and adaptation to environmental changes. Here, we confirmed this hypothesis with the identification of differential gene expression of the enzymes involved in biosynthesis of amino acids, arginine and proline metabolism and arginine biosynthesis. All data provided information about the transcriptomic profiling and the expression levels of La-WT and La-arg^- promastigotes and axenic amastigotes. These findings revealed the importance of arginase in parasite survival and differentiation, and indicated the existence of a coordinated response in the absence of arginase activity related to arginine and polyamine pathways. Leishmania are auxotrophic for many essential nutrients, including amino acids. In this way, the parasite needs to uptake the amino acids from the environment. The uptake of amino acids is mediated by amino acid transporters that are unique for Leishmania. As part of polyamine pathway, the arginase converts L-arginine to ornithine and furthermore to putrescine, products which are essential for parasite growth. On the other hand, the absence of arginase activity could alter the metabolism of the parasite to surpass the external signals during the life cycle and the fate of infection. The transcriptional profiling of La-WT and La-arg^- promastigotes and axenic amastigotes revealed 8253 transcripts, 60% encoding hypothetical proteins and 443 novel transcripts. In addition, our data revealed that 85% of the genes were constitutively expressed. Among the 15% (1268 genes) of the differentially expressed genes, we identified genes up- and down-regulated comparing the transcript abundance from different life cycle stages of the parasite and in the presence or absence of arginase. We also combined the transcriptional with metabolic profile that revealed a proportional correlation between enzyme and metabolites in the polyamine pathway. The differentiation of promastigotes to amastigotes alters the expression of enzymes from polyamines biosynthesis, which modulates ornithine, L-glutamate, proline and putrescine levels. In addition, the absence of arginase activity increased the levels of L-arginine, citrulline and L-glutamate and decreased the levels of aspartate, proline, ornithine and putrescine in promastigotes by differential modulation of genes involved in its metabolism. Altogether these data provided additional insights into how Leishmania is able to modulate its biological functions in the presence or absence of arginase activity to survive during environmental changes.