Upregulation of nicotinamide/nicotinate mononucleotide adenylyl transferase increases resistance to oxidative stress and antimony in promastigotes of Leishmania braziliensis

Abstract

Leishmaniasis is a prevalent parasitic neglected disease caused by protozoans of the genus Leishmania. Currently, no vaccines are available for humans, and existing treatments are ineffective because of their toxicity and the emergence of drug-resistant strains. Consequently, it is crucial to identify new potential therapeutic targets, thereby facilitating the development of effective therapies. This study indicates a correlation between the upregulated biosynthesis of nicotinamide adenine dinucleotide (NAD) and the resistance to hydrogen peroxide (H₂O₂) and trivalent antimony exposure (Sb³⁺) in L. braziliensis promastigotes. CRISPR/Cas9 gene-editing of the nicotinamide/nicotinate mononucleotide adenylyl transferase in L. braziliensis (Lbnmnat), the key gene in the NAD biosynthetic pathway, allowed upregulation at both the mRNA and protein levels, as well as elevated NAD and NADP contents in the Lbnmnat-edited parasites. A single guide RNA template, directed against the 5′ end of the Lbnmnat and a donor DNA consisting of the mCherry reporter gene preceded by the 5’ UTR from the Crithidia fasciculata phosphoglycerate kinase (CfPGKB) were electroporated into L. braziliensis promastigotes that constitutively express the Streptococcus pyogenes Cas9 and T7 RNA polymerase. PCR analysis and DNA sequencing demonstrated the successful editing of the Lbnmnat gene. Importantly, dose-dependent oxidative stress susceptibility assays to H₂O₂ and Sb³⁺ revealed higher IC₅₀ values in the Lbnmnat-edited parasites. These findings denote a correlation between the upregulated biosynthesis of NAD in L. braziliensis and its enhanced resistance to oxidative stress, indicating that the Lbnmnat gene may play a significant role in the survival and resistance mechanisms of the parasite against antimony compounds.