RESUMO
l-Arginine is an essential amino acid in Leishmania (Leishmania) amazonensis metabolism. A key enzyme for parasite l-arginine metabolism is arginase (ARG) that uses arginine to produce urea and ornithine, a precursor of polyamine pathway guaranteeing parasite replication in both insect and mammal hosts. There is an alternative pathway to produce ornithine via l-proline and glutamate, but this mechanism is not described in Leishmania. In the mammal host, two enzymes can use l-arginine as substrate, the host ARG and the induced nitric oxide synthase that produces nitric oxide. The competition between induced nitric oxide synthase and both parasite and host ARG can favor the success of the infection or its control. Here, we established the metabolomics profile of the polyamine pathway of wild type (WT) L. (L.) amazonensis, submitted or not to l-arginine starvation, and compared to the ARG-knockout mutant (arg- ). Our results indicated that arginine starvation induces a decrease in arginine, ornithine, and putrescine, but we could not detect the significative level changes of spermidine, spermine, or agmatine. However, the absence of ARG on the arg- induced an increase of arginine and citrulline levels, but decreased the levels of ornithine and putrescine. Similarly to the WT arginine-starved parasites, the arg- parasites presented lower levels of proline when compared to the WT ones. This could be indicative of an alternative pathway to surpass the enzyme or its substrate absence.
RESUMO
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.