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.
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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)