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

Abstract

BackgroundLeishmania 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 findingsRNA-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/SignificanceIn 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.

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

“…La-WT infection in BALB/c-macrophages increases the level of L-arginine via the increased expression of Cat2 and Cat1 and the uptake of amino acids as compared to uninfected macrophages [23]. Besides, the levels of La-aap3 expression also appeared to be increased, but did not increase in BALB/c-macrophages that were infected with La-arg − [22,23]. 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].…”

“…Indeed, intracellular amastigotes can scavenge sugar, lipids, and amino acids, such as L-arginine, from the host phagolysosome [24,26]. L-arginine availability and arginase activity play important roles in the gene expression and metabolites profile from L-arginine and polyamines metabolism in Leishmania promastigote and amastigote forms and it also impacts on parasite survival and growth [22,27,28]. In the host context, the macrophage ARG1 activity can be activated to provide substrates for polyamine pathways, and the parasite shares the L-arginine and polyamine intracellular pool with the macrophage [36,44].…”

“…In Leishmania-infected cells, L-arginine uptakes occur via CAT1 and CAT2 to supply the requirement for host-enzymes NOS2 and ARG1 [18][19][20]. In contrast, L-arginine uptake in Leishmania promastigote and amastigote forms occurs via amino acid permease 3 (AAP3) to supply parasite arginase (ARG) or to NOS-like, to produce polyamines or NO, respectively, which leads to parasite growth [18,[21][22][23]. Amastigote that forms inside the host cell can scavenge the amino acid and other nutrients from phagolysosome, thus interfering in the host cell metabolism [24][25][26].…”

“…Amastigote that forms inside the host cell can scavenge the amino acid and other nutrients from phagolysosome, thus interfering in the host cell metabolism [24][25][26]. The availability of L-arginine establishes a competition between the parasite and macrophage enzymes, altering gene expression regulation and, consequently, NO/polyamines production, as well as macrophage susceptibility to infection [18,[21][22][23]. Previous studies demonstrated that the metabolic fingerprint analysis of L. amazonensis wild type (La-WT) promastigote forms under the starvation of L-arginine and revealed decreased levels of ornithine and putrescine, whereas it did not alter the levels of spermidine, spermine, or agmatine [27].…”

Arginase expression modulates nitric oxide production in Leishmania (Leishmania) amazonensis

“…The depletion of L-arginine in macrophages can inhibit both polyamines and NO production. In addition, the lower infectivity of La-arg - could correlate with lower levels of CAT2B [ 21 ] or AAP3 expression and amino acid uptake [ 70 ], reducing L-arginine availability for ARG1 in infected macrophages, reducing parasite survival. In this way, infectivity could be recovered by putrescine supplementation [ 21 ].…”