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

PLOS ONE, Nov 2011

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.

A PDF file should load here. If you do not see its contents the file may be temporarily unavailable at the journal website or you do not have a PDF plug-in installed and enabled in your browser.

Alternatively, you can download the file locally and open with any standalone PDF reader:

http://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0027818&type=printable

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

Floeter-Winter LM (2011) Axenic Leishmania amazonensis Promastigotes Sense both the External and Internal Arginine Pool Distinctly Regulating the Two Transporter-Coding Genes. PLoS ONE 6(11): e27818. doi:10.1371/journal.pone.0027818 Axenic Leishmania amazonensis Promastigotes Sense both the External and Internal Arginine Pool Distinctly Regulating the Two Transporter-Coding Genes Emerson A. Castilho-Martins 0 Maria Fernanda Laranjeira da Silva 0 Marcos G. dos Santos 0 Sandra M. 0 Muxel 0 Lucile M. Floeter-Winter 0 Najib M. El-Sayed, The University of Maryland, United States of America 0 Departamento de Fisiologia, Instituto de Biociencias, Universidade de Sa o Paulo Sa o Paulo , Brazil 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 59 and 39 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 mM) 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-/argDSKL). The arg- and the arg-/argDSKL 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 +/2 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. - Leishmaniasis is a complex parasitic disease that currently affects about 12 million people and an estimated 2 million new cases per year [1]. It is caused by protozoa in the Leishmania genus, which has two distinct phases in its life cycle: the promastigote, an extracellular flagellate present at the gut of sand flies, and the amastigote that lives inside mononuclear phagocytes, mainly macrophages, in a vertebrate host. Arginine is a key amino acid for macrophages because, being the substrate for inducible nitric oxide synthase (iNOS) to produce nitric oxide (NO), it is involved in the macrophage-defense response against pathogen infections. [28]. This amino acid is also a substrate for arginase, which catalyzes the production of urea and ornithine, a product important for polyamine pathway. This pathway is used by Leishmania to replicate and is essential for the parasite to establish infection [912]. It has largely been reported that macrophage or Leishmania modulation of arginine is responsible for parasite survival or its killing in the mammal host [5,1319]. Membrane transporters, present in both Leishmania and macrophages control arginine uptake [2024], to sustain NO production, macrophages increase their expression of the main arginine transporter (CAT2B), which is indicative that the internal pool of arginine is not sufficient to supply arginine to iNOS [25 27]. On the other hand, a high-affinity arginine transporter has been described in L. (L.) donovani. This transporter is LdAAP3, and it has 480 amino acids and 11 predicted trans-membrane domains [22]. With this transporter, Leishmania seems to have mechanisms of sensing arginine decreases and responding with increased arginine uptake [28]. Therefore, the arginine-uptake control appears to be an important limiting factor to parasite survival inside macrophages [17,29]. Leishmania has a polycistronic transcription, and the control of gene expression is mainly performed through protein levels and mRNA stability [30]. In this study, we evaluated the importance of arginine transporter mRNA levels on the physiology of arginine uptake in L. (L.) amazonensis. Our data indicated that these organisms control the arginine transporter expression by regulating the transporter-coding mRNA stability. We also showed that the level of arginine transporter mRNA varies in promastigote development, and, using arginase-deficien (...truncated)


This is a preview of a remote PDF: http://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0027818&type=printable

Emerson A. Castilho-Martins, Maria Fernanda Laranjeira da Silva, Marcos G. dos Santos, Sandra M. Muxel, Lucile M. Floeter-Winter. Axenic Leishmania amazonensis Promastigotes Sense both the External and Internal Arginine Pool Distinctly Regulating the Two Transporter-Coding Genes, PLOS ONE, 2011, 11, DOI: 10.1371/journal.pone.0027818