Abstract
ABSTRACTTrypanosomatid type I nitroreductases (NTRs), i.e., mitochondrial enzymes that metabolise nitroaromatic pro-drugs, are essential for parasite growth, infection, and survival. Here, a type I NTR of non-virulent protozoan Trypanosoma rangeli is described and compared to those of other trypanosomatids. The NTR gene was isolated from KP1(+) and KP1(-) strains, and its corresponding transcript and 5’ untranslated region (5’UTR) were determined. Bioinformatics analyses and nitro-drug activation assays were also performed. The results indicated that the type I NTR gene is present in both KP1(-) and KP1(+) strains, with 98% identity. However, the predicted subcellular localisation of the protein differed among the strains (predicted as mitochondrial in the KP1(+) strain). Comparisons of the domains and 3D structures of the NTRs with those of orthologs demonstrated that the nitroreductase domain of T. rangeli NTR is conserved across all the strains, including the residues involved in the interaction with the FMN cofactor and in the tertiary structure characteristics of this oxidoreductase protein family. mRNA processing and expression were also observed. In addition, T. rangeli was shown to be sensitive to benznidazole and nifurtimox in a concentration-dependent manner. In summary, T. rangeli appears to have a newly discovered functional type I NTR.
Highlights
Given the importance of type I NTRs for pathogenic trypanosomatids, the presence of a type I NTR in avirulent parasite T. rangeli is described and discussed here, with a comparison to the enzymes from other trypanosomatids
The T. rangeli NTR gene was amplified by polymerase chain reaction (PCR) using genomic DNA from the KP1(-) Tre isolate (Puerta et al 2009) and the NTR-FW (5’-GAG AAA TGG CAT AAA AAG AGG CC-3’) and NTR-RV (5’-AAA ACT TTC CCC ACC GAA CCA-3’) primer pair
Fig. 1: multiple alignment-using Geneious and Cobalt - of the deduced type I nitroreductase (NTR) amino acid sequences from trypanosomatids, other protozoa, and bacteria. (A) Alignment of the protein N-terminal region used for subcellular localisation prediction by 12 Web servers (Supplementary data, Table II)
Summary
Given the importance of type I NTRs for pathogenic trypanosomatids, the presence of a type I NTR in avirulent parasite T. rangeli is described and discussed here, with a comparison to the enzymes from other trypanosomatids. Multiple sequence alignment of the pGEM®-T Easy vector-cloned sequence (GenBank accession number AHI85557) with that of putative type I NTR-coding genes from CL Brener and T. cruzi 058PUJ strains (Supplementary data, Table I) revealed identity percentages of 71.6% and 72.4%, respectively.
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