Identification of the nicotinamide mononucleotide adenylyltransferase of Trypanosoma cruzi

Abstract

The intracellular parasite Trypanosoma cruzi is the aetiological agent of Chagas disease, a public health concern with an increasing incidence rate. This increase is due, among other reasons, to the parasite's drug resistance mechanisms, which require nicotinamide adenine dinucleotide (NAD+). Furthermore, this molecule is involved in metabolic and intracellular signalling processes necessary for the survival of T. cruzi throughout its life cycle. NAD+ biosynthesis is performed by de novo and salvage pathways, which converge on the step that is catalysed by the enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT) (enzyme commission number: 2.7.7.1). The identification of the NMNAT of T. cruzi is important for the development of future therapeutic strategies to treat Chagas disease. In this study, a hypothetical open reading frame (ORF) for NMNAT was identified in the genome of T. cruzi. The corresponding putative protein was analysed by simulating structural models. The ORF was amplified from genomic DNA by polymerase chain reaction and was further used for the construction of a corresponding recombinant expression vector. The expressed recombinant protein was partially purified and its activity was evaluated using enzymatic assays. These results comprise the first identification of an NMNAT in T. cruzi using bioinformatics and experimental tools and hence represent the first step to understanding NAD+ metabolism in these parasites.

Structural insights into Plasmodium falciparum nicotinamide mononucleotide adenylyltransferase: oligomeric assembly

“… 14 , 15 In the context of eukaryotic parasites, our research group has identified and characterised NMNATs from Leishmania braziliensis (LbNMNAT) 16 , Giardia lamblia (GlNMNAT) 17 , and Trypanosoma cruzi (TcNMNAT). 18 Concerning the NMNAT from Plasmodium falciparum (PfNMNAT), different functional and structural aspects have been studied. 19 , 20 However, its quaternary structure has not yet been determined.…”

Nicotinamide mononucleotide adenylyltransferase of Trypanosoma cruzi (TcNMNAT): a cytosol protein target for serine kinases

“…The purification was monitored by SDS-PAGE as described previously (Niño et al 2015). On the other hand, the inclusion body was solubilised using reported protocols (Sambrook & Rusell 2001).…”

“…Polymerase chain reaction (PCR) amplification was performed using the plasmid TcNMNAT-pET100, which has been previously described previously (Niño et al 2015), as the template. The primers used contained a restriction site for BamHI in the 5′ end (5′-CGG GAT CCC GAT GAG CGA TGA CAC AT-3′) and a restriction site for EcoRI in the 3′ end (5′-CCG GAA TTC CGG TCA ACA ATT TTG AGT ATT-3′).…”

“…Previously, our group identified the enzyme NMNAT in T. cruzi (Niño et al 2015). This study is presented as an approach to its localisation to establish similarities and differences with its human orthologs, with the aim to gain a better understanding of the host-parasite relation and to enable the development of new tools to fight these parasitic infections.…”

Identification and sub-cellular localization of a NAD transporter in Leishmania braziliensis (LbNDT1)

“…This enzyme has been identified in extracellular organisms as Giardia duodenalis that possesses 2 isozymes ( Gd NMNAT1-2) ( Forero-Baena et al., 2015 ) and Saccharomyces cerevisiae which has 3 isozymes ( Sc NMNAT1-3) ( Emanuelli et al., 1999 , 2002 ; Kato and Lin, 2014 ); in Homo sapiens 3 isozymes ( Hs NMNAT1-3) located in the nucleus, the Golgi apparatus and the mitochondria have been reported ( Berger et al., 2005 ). Interestingly, a unique isoenzyme has been identified in intracellular parasites such as Leishmania braziliensis ( Lb NMNAT) ( Contreras et al., 2015 ), Tripanosoma cruzi ( Tc NMNAT) ( Niño et al., 2015 ) and Plasmodium falciparum ( Pf NMNAT) located in the cytoplasm ( O'Hara et al., 2014 ). In these pathogens, the NAD transport mechanisms remain unanalyzed.…”