Axenic Leishmania amazonensis Promastigotes Sense both the External and Internal Arginine Pool Distinctly Regulating the Two Transporter-Coding Genes

Abstract

Leishmania (L.) amazonensis uses arginine to synthesize polyamines to support its growth and survival. Here we describe the presence of two gene copies, arranged in tandem, that code for the arginine transporter. Both copies show similar Open Reading Frames (ORFs), which are 93% similar to the L. (L.) donovani AAP3 gene, but their 5′ and 3′ UTR's have distinct regions. According to quantitative RT-PCR, the 5.1 AAP3 mRNA amount was increased more than 3 times that of the 4.7 AAP3 mRNA along the promastigote growth curve. Nutrient deprivation for 4 hours and then supplemented or not with arginine (400 µM) resulted in similar 4.7 AAP3 mRNA copy-numbers compared to the starved and control parasites. Conversely, the 5.1 AAP3 mRNA copy-numbers increased in the starved parasites but not in ones supplemented with arginine (p<0.05). These results correlate with increases in amino acid uptake. Both Meta1 and arginase mRNAs remained constant with or without supplementation. The same starvation experiment was performed using a L. (L.) amazonensis null knockout for arginase (arg -) and two other mutants containing the arginase ORF with (arg -/ARG) or without the glycosomal addressing signal (arg -/argΔSKL). The arg - and the arg -/argΔSKL mutants did not show the same behavior as the wild-type (WT) parasite or the arg -/ARG mutant. This can be an indicative that the internal pool of arginine is also important for controlling transporter expression and function. By inhibiting mRNA transcription or/and mRNA maturation, we showed that the 5.1 AAP3 mRNA did not decay after 180 min, but the 4.7 AAP3 mRNA presented a half-life decay of 32.6 +/− 5.0 min. In conclusion, parasites can regulate amino acid uptake by increasing the amount of transporter-coding mRNA, possibly by regulating the mRNA half-life in an environment where the amino acid is not present or is in low amounts.

Multi-analytical platform metabolomic approach to study miltefosine mechanism of action and resistance in Leishmania

“…L-arginine accomplishes two major roles in Leishmania, as a biosynthetic precursor for polyamines and building block for protein biosynthesis [70]; as such, L-arginine uptake is carried out by dedicated transporters that sense its intra-and extracellular concentration [70,71]. Under this role, MT-treated parasites either loose the capacity to sense intracellular pools of Arg, otherwise transport capacity is unable to reach an adequate intracellular level of Arg.…”

“…Leishmania senses intracellular pools at least for Arg [71] and Pro [81] with crossed-influence between these metabolites. In this sense, Inbard et al [81] proposed recently an appealing hypothesis consisting of the cross-talk among the different amino acid transporters through their large hydrophilic N-terminal extension, typical in Leishmania, which will be modulated allosterically by these amino acids.…”

A role for adenine nucleotides in the sensing mechanism to purine starvation in Leishmania donovani

“…The reliance of these parasites on the host environment for the provision of a diverse array of essential nutrients, combined with the absence of much of the canonical cell surface sensing machinery described in yeast and higher eukaryotes, support the possibility that the surveillance of intracellular metabolite pools may be a key feature of their nutrient sensing. This supposition is bolstered by a previous study in L. amazonensis in which AAP3 arginine transporter mRNA levels were linked to changes in intracellular arginine levels (Castilho‐Martins et al ., ).…”

Immunopathogenesis of non-healing American cutaneous leishmaniasis and progressive visceral leishmaniasis

“…Parasite growth within these MΦs can be mediated via host arginase-dependent and -independent mechanisms, as illustrated by mouse models of L. amazonensis and L. mexicana infection and hamster models of L. donovani infection [136]. Similarly, parasite-encoded arginase and arginine transporters can serve as virulence factors, subverting MΦ microbicidal activity [70, 161]. Thirdly, host or parasite factors that repress NO or ROS production by infected neutrophils or MΦs, or repress the activation of DC and T cells, will delay parasite clearance and lesion control [46].…”

“…2a), as well as to neutrophil-mediated parasite killing [44]. L. amazonensis may also be unique in sensing both the external and internal arginine pool by regulating two transporter-coding genes, or in utilizing host innate machineries (e.g., the autophagic and lipid metabolic pathways) for their favored intracellular growth [63, 70]. Therefore, L. amazonensis parasites are extraordinary in their ability to repress host cell activation, resist host anti-microbial machinery, and utilize host resources for their own growth (as illustrated in blue in Fig.…”