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

Abstract

Background: 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.

Methodology/principal findings: 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.

Conclusions/significance: 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.

Fig 1. Arginase (LmxM.34.1480) transcript assembling and annotation.

Total reads coverage aligned in the region of LmxM.34.1480 gene in the lines La-WT and La-arg^-promastigotes (pro) and axenic amastigotes (ama) of the previously annotated L. mexicana genome. (La-WT) L. amazonensis wild-type, (La-arg^-) L. amazonensis arginase knockout.

Fig 2. Transcriptional profiling of La-WT and La-arg^- promastigotes and axenic amastigotes.

Venn diagram of the 1268 differentially expressed genes, showing the number of genes common and non-common from each sample and the number in the overlap. (pro) promastigote, (ama) axenic amastigote, (La-WT) L. amazonensis wild-type, (La-arg^-) L. amazonensis arginase knockout.

Fig 3. Differential gene expression profiling in La-WT and La-arg^- promastigotes and axenic amastigotes.

Count of transcripts up-regulated (light gray) or down-regulated (dark gray) in the comparison of La-WT promastigotes vs. La-arg^-promastigotes, La-WT axenic amastigotes vs. La-arg^- axenic amastigotes, La-WT promastigotes vs. La-WT axenic amastigotes, and La-arg^- promastigotes vs. La-arg^-axenic amastigotes, considering a fold change ≥ 2 and p value ˂ 0.05. (pro) promastigote, (ama) axenic amastigote, (La-WT) L. amazonensis wild-type, (La-arg^-) L. amazonensis arginase knockout.

Fig 4. KEEG enrichment analysis showing the list of the top 20 pathways.

The heatmap shows the list of the 20 pathways regulated in (1) ama La-arg^- vs ama La-WT, (2) ama La-arg^- vs pro La-arg^-, (3) ama La-WT vs pro La-WT and (4) pro La-arg^- vs pro La-WT. The enrichment map is colored by the gradient level of the p value. (pro) promastigote, (ama) axenic amastigote, (La-WT) L. amazonensis wild-type, (La-arg^-) L. amazonensis arginase knockout.

Fig 5. Crossing of transcriptome and metabolome profiles of the arginine pathway, highlighting the enzymes with differential gene expression based on the RNA-seq data.

Representation of the arginine pathway and FPKM expression levels highlighting in gray boxes the enzymes with differential gene expression: (A) argininosuccinate synthase (EC6.3.4.5), (B) acetylornithine deacetylase (EC3.5.1.16), (C) glutamate 5-kinase (EC2.7.2.11), (D) spermidine synthase (EC2.5.1.16), (E) arginase (EC3.5.3.1), (F) pyrroline 5-carboxylase reductase (EC1.5.1.2) and (G) pyrroline 5-carbolylate dehydrogenase (EC1.2.1.88). The arrows indicate the increase or decrease levels, according to our RNA-seq data analyses and the metabolome fingerprints, previously described [9]. Each bar is represented from three independent biological replicates. Statistical analyses were performed using t-test. (*) p < 0.05.