Date Published: January 15, 2016
Publisher: Public Library of Science
Author(s): Joana Faria, Inês Loureiro, Nuno Santarém, Sandra Macedo-Ribeiro, Joana Tavares, Anabela Cordeiro-da-Silva, Michael Pollastri. http://doi.org/10.1371/journal.pntd.0004365
Abstract: A growing interest in asparagine (Asn) metabolism has currently been observed in cancer and infection fields. Asparagine synthetase (AS) is responsible for the conversion of aspartate into Asn in an ATP-dependent manner, using ammonia or glutamine as a nitrogen source. There are two structurally distinct AS: the strictly ammonia dependent, type A, and the type B, which preferably uses glutamine. Absent in humans and present in trypanosomatids, AS-A was worthy of exploring as a potential drug target candidate. Appealingly, it was reported that AS-A was essential in Leishmania donovani, making it a promising drug target. In the work herein we demonstrate that Leishmania infantum AS-A, similarly to Trypanosoma spp. and L. donovani, is able to use both ammonia and glutamine as nitrogen donors. Moreover, we have successfully generated LiASA null mutants by targeted gene replacement in L. infantum, and these parasites do not display any significant growth or infectivity defect. Indeed, a severe impairment of in vitro growth was only observed when null mutants were cultured in asparagine limiting conditions. Altogether our results demonstrate that despite being important under asparagine limitation, LiAS-A is not essential for parasite survival, growth or infectivity in normal in vitro and in vivo conditions. Therefore we exclude AS-A as a suitable drug target against L. infantum parasites.
Partial Text: Leishmaniasis is a vector borne human disease, caused by several species of digenetic protozoan parasites belonging to genus Leishmania. The clinical presentations of this neglected tropical disease vary from selfhealing cutaneous manifestations to potentially fatal, if untreated, visceral ailment . The most severe form of the disease, designated as visceral leishmaniasis (VL) is mainly associated to Leishmania donovani or Leishmania infantum. Due to the absence of human vaccines, VL control relies mainly on chemotherapy and appropriate vector control . The traditional therapeutic options are associated with significant limitations (cost, toxicity, complex administration regimes, resistance) averting disease control in endemic areas . As consequence, according to World Health Organization between 20,000 and 30,000 people (mostly children) die every year, rendering the search for novel chemotherapeutic options a priority .
Despite being eukaryotes, trypanosomatids, present AS-A enzymes of bacterial origin. Moreover, these enzymes are aminoacyl-tRNA synthetase paralogues, displaying an AsnRS catalytic core with conserved class II motifs, yet lacking the tRNA binding domain . In this work, we have demonstrated that LiAS-A is able to synthesize Asn using either ammonia or glutamine as nitrogen donors, as previously described for TbAS-A, TcAS-A and LdAS-A [28, 29]. Km values for aspartate and ATP are close to the ones determined for TbAS-A and TcAS-A . As for ammonia, the Km value found for LiAS-A is 5 fold lower in comparison to TbAS-A, TcAS-A and LdAS-A [27, 28]. In the case of LdAS-A, the Km values for aspartate were around 10 fold lower  than the ones obtained for LiAS-A. Regarding the high conservation of the active sites among Leishmania AS-A enzymes, we cannot exclude that the observed kinetic differences may be due to the differences in the amount of protein that is properly folded, especially taking into account they are expressed in a heterologous system. Moreover, it is important to emphasize that the kinetic determinations for LdAS-A were performed using a different experimental set up. Importantly, TbAS-A and TcAS-A use preferably ammonia , whereas LiAS-A seems to use both roughly in the same extent (Table 1). AS-A activity in trypanosomatids more resembles AS-B enzymes, concerning both the optimal pH for enzymatic activity (7.6 instead of 8) and also the ability to use both nitrogen donors. AS-B enzymes use preferably glutamine, with exception of the human enzyme that presents approximately the same affinity for both nitrogen sources [18, 19, 25, 52–57]. This biochemical feature, so far only described for trypanosomatids AS-A enzymes [28, 29], becomes particularly interesting in the context of the presence of an ORF encoding a hypothetical, yet non-classical, AS-B, in the genome of these organisms (L. infantum [LinJ.29.1590], L. major [LmjF.29.1490], T. brucei [Tb927.3.4060] and T. cruzi [Tc00.1047053510001.40]) [42–44]. These sequences contain a Pfam AS domain (pfam00733) and glutamine hydrolysing domains in the C and N-terminus, respectively. BLASTp analysis of L. infantum sequence, for instance, revealed several hits that corresponded to hypothetical proteins from a broad range of eukaryotes. However, we have no evidence AS-B is functional at all.