Putrescine supplementation shifts macrophage L-arginine metabolism related-genes reducing Leishmania amazonensis infection.

Leishmania is a protozoan that causes leishmaniasis, a neglected tropical disease with clinical manifestations classified as cutaneous, mucocutaneous, and visceral leishmaniasis. In the infection context, the parasite can modulate macrophage gene expression affecting the microbicidal activity and immune response. The metabolism of L-arginine into polyamines putrescine, spermidine, and spermine reduces nitric oxide (NO) production, favoring Leishmania survival. Here, we investigate the effect of supplementation with L-arginine and polyamines in infection of murine BALB/c macrophages by L. amazonensis and in the transcriptional regulation of genes involved in arginine metabolism and proinflammatory response. We showed a reduction in the percentage of infected macrophages upon putrescine supplementation compared to L-arginine, spermidine, and spermine supplementation. Unexpectedly, deprivation of L-arginine increased nitric oxide synthase (Nos2) gene expression without changes in NO production. Putrescine supplementation increased transcript levels of polyamine metabolism-related genes Arg2, ornithine decarboxylase (Odc1), Spermidine synthase (SpdS), and Spermine synthase (SpmS), but reduced Arg1 in L. amazonensis infected macrophages, while spermidine and spermine promoted opposite effects. Putrescine increased Nos2 expression without leading to NO production, while L-arginine plus spermine led to NO production in uninfected macrophages, suggesting that polyamines can induce NO production. Besides, L-arginine supplementation reduced Il-1b during infection, and L-arginine or L-arginine plus putrescine increased Mcp1 at 24h of infection, suggesting that polyamines availability can interfere with cytokine/chemokine production. Our data showed that putrescine shifts L-arginine-metabolism related-genes on BALB/c macrophages and affects infection by L. amazonensis.

L-arginine availability and arginase activity: Characterization of amino acid permease 3 in Leishmania amazonensis.

BACKGROUND: Leishmania uses the amino acid L-arginine as a substrate for arginase, enzyme that produces urea and ornithine, last precursor of polyamine pathway. This pathway is used by the parasite to replicate and it is essential to establish the infection in the mammalian host. L-arginine is not synthesized by the parasite, so its uptake occurs through the amino acid permease 3 (AAP3). AAP3 is codified by two copies genes (5.1 and 4.7 copies), organized in tandem in the parasite genome. One copy presents the expression regulated by L-arginine availability. METHODOLOGY/PRINCIPAL FINDINGS: RNA-seq data revealed 14 amino acid transporters differentially expressed in the comparison of La-WT vs. La-arg- promastigotes and axenic amastigotes. The 5.1 and 4.7 aap3 transcripts were down-regulated in La-WT promastigotes vs. axenic amastigotes, and in La-WT vs. La-arg- promastigotes. In contrast, transcripts of other transporters were up-regulated in the same comparisons. The amount of 5.1 and 4.7 aap3 mRNA of intracellular amastigotes was also determined in sample preparations from macrophages, obtained from BALB/c and C57BL/6 mice and the human THP-1 lineage infected with La-WT or La-arg-, revealing that the genetic host background is also important. We also determined the aap3 mRNA and AAP3 protein amounts of promastigotes and axenic amastigotes in different environmental growth conditions, varying pH, temperature and L-arginine availability. Interestingly, the increase of temperature increased the AAP3 level in plasma membrane and consequently the L-arginine uptake, independently of pH and L-arginine availability. In addition, we demonstrated that besides the plasma membrane localization, AAP3 was also localized in the glycosome of L. amazonensis promastigotes and axenic amastigotes. CONCLUSIONS/SIGNIFICANCE: In this report, we described the differential transcriptional profiling of amino acids transporters from La-WT and La-arg- promastigotes and axenic amastigotes. We also showed the increased AAP3 levels under amino acid starvation or its decrease in L-arginine supplementation. The differential AAP3 expression was determined in the differentiation of promastigotes to amastigotes conditions, as well as the detection of AAP3 in the plasma membrane reflecting in the L-arginine uptake. Our data suggest that depending on the amino acid pool and arginase activity, Leishmania senses and could use an alternative route for the amino acid transport in response to stress signaling.