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

Abstract

BackgroundLeishmania 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.Methodology/Principal findingsA 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.Conclusions/SignificanceAll 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.

Metabolomic Profile of BALB/c Macrophages Infected with Leishmania amazonensis: Deciphering L-Arginine Metabolism

“…In contrast, BALB/c infection with the La-arg − parasite showed increased levels of L-arginine, supporting the uptake of amino acids by increased levels of Cat2 and Cat1 mRNA expression [23]. Excess L-arginine availability to the macrophages, which was not metabolized via arginase (absent in parasite) or agmatinase that was not modulated in amastigotes axenic forms, could cause the presence of higher levels of L-argininic acid [37]. Additionally, the absence of parasite arginase reduced the levels of ornithine (as compared to La-WT infected macrophages), although the increased levels of Arg1 in infected La-arg − -macrophages when compared to infected with La-WT [23], highlighting the impact of parasite arginase into the total of L-arginine conversion to ornithine.…”

“…The genomic and transcriptomic networks during Leishmania infection have been providing interesting data regarding how Leishmania can to modulate the gene organization and gene expression of its host [37][38][39][40]. Additionally, we have observed differentially expressed genes profile during the different phases of parasite growth [37,38,41]. The metabolomic profile is poorly explored and it could help to understand the metabolite's consummation and network systems pathway of both the host and parasite.…”

“…The metabolomic profile in La-WT infection showed augmented levels of glucose and glutamine, which can be implicated in the activation of macrophages and glycolytic metabolism to generate ATP via aerobic metabolism in pro-inflammatory M1 macrophages, which reduces the oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) pathways that surpass two points of tricarboxylic acid cycle (TCA) by supplying glutamine and acetyl-CoA, respectively; in contrast, alternatively activated M2 macrophages that display a more flexible metabolic activity since they increase OXPHOS, and increase the production of polyamines for cell proliferation or proline to induce collagen production [49]. The levels of the polyamines ornithine and putrescine also increased, which correlated with the increased levels of Arg1 mRNA and parasite ARG [23] directing to the production of ornithine and putrescine via ODC [23,37] (Figures 4 and 5). Additionally, the increased levels of ornithine correlate with increased levels of proline, trans-4-hidroxiproline, glutamic acid, and glutamine.…”

“…In La-arg − -macrophages, the lower levels of ornithine and proline can correlate, once the levels of ornithine cyclodeaminase, which is an enzyme that converts ornithine in proline, do not modify in La-arg − -amastigote axenic forms as compared to La-WT [37]. Besides, lower levels of ornithine also can reduce their conversion to L-glutamate 5-semihaldehyde and subsequently in glutamate or L-1-pyrroline 5-carboxylate, metabolites that were not differentially modulated in comparison to La-arg − -macrophages versus La-WT-infected.…”

A Real Time PCR Assay for Quantification of Parasite Burden in Murine Models of Leishmaniasis

“…The L. major entry for DRBD3 (LmjF.04.1170) on TriTrypDB indicates that the gene is constitutively expressed between parasite life-cycle stages, is not under immune 100 pressure, and has minimal sequence variation between species (17). RNA-seq experiments in both L. major and L. amazonensis have demonstrated little to no change in expression of this gene between the promastigote and amastigote stage consistent with the characteristics of a constitutively expressed housekeeping gene (18)(19)(20). Additionally, there were no identified epitopes in the Immune Epitope Database (IEDB) corresponding to DRBD3 peptides, suggesting 105 that it is not likely to be influenced by host immune pressure (21).…”