Ascorbate peroxidase overexpression protects Leishmania braziliensis against trivalent antimony effects

Abstract

Ascorbate peroxidase (APX) is a redox enzyme of the trypanothione pathway that converts hydrogen peroxide (H2O2) into water molecules. In the present study, the APX gene was overexpressed in Leishmania braziliensis to investigate its contribution to the trivalent antimony (SbIII)-resistance phenotype. Western blot results demonstrated that APX-overexpressing parasites had higher APX protein levels in comparison with the wild-type line (LbWTS). APX-overexpressing clones showed an 8-fold increase in the antimony-resistance index over the parental line. In addition, our results indicated that these clones were approximately 1.8-fold more tolerant to H2O2 than the LbWTS line, suggesting that the APX enzyme plays an important role in the defence against oxidative stress. Susceptibility tests revealed that APX-overexpressing L. braziliensis lines were more resistant to isoniazid, an antibacterial agent that interacts with APX. Interestingly, this compound enhanced the anti-leishmanial SbIII effect, indicating that this combination represents a good strategy for leishmaniasis chemotherapy. Our data demonstrate that APX enzyme is involved in the development of L. braziliensis antimony-resistance phenotype and may be an attractive therapeutic target in the design of new strategies for leishmaniasis treatment.

Genome Analysis of Endotrypanum and Porcisia spp., Closest Phylogenetic Relatives of Leishmania, Highlights the Role of Amastins in Shaping Pathogenicity

“…The most commonly found annotations in these lists were ‘hypothetical protein’ and ‘protein containing domain with unknown function’. However, some well-described proteins were also present, e.g., ornithine decarboxylase implicated in the survival of Leishmania amastigotes [ 112 ] and ascorbate peroxidase, which is essential for the defense against oxidative stress [ 113 ]. No considerable GO enrichment for the positively selected genes was documented in the categories ‘biological process’ or ‘molecular function’ on any of the two branches ( Figure S8 ), which indicated that positive selection pressure affects functionally diverse genes.…”

The ultimate fate determinants of drug induced cell-death mechanisms in Trypanosomatids

“…Hence the emanating question is that if Trypanosomatid peroxidases are tryparedoxin dependent, then how GSH is regulated in these parasites. Ascorbate peroxidase (APx) overexpressing Leishmania promastigotes exhibited enhanced tolerance to oxidative stress-mediated apoptosis in AmB-resistant strains ( Moreira et al., 2018 ). Peroxides and glutathione are known to keep mitochondrial cytochrome c in a reduced state, hence during oxidative stress, the release of cytochrome c into cytoplasm and its concomitant oxidation by cytochrome oxidase makes it bind to ER Ca 2+ channels and disrupt intracellular Ca 2+ homeostasis and thereby trigger cell death in either a caspase-dependent or independent mechanism.…”

“…Ferroptosis, an iron-dependent pathway in mammals, results from inhibition of Glutathione Peroxidases, Gpx4, and is characterized by iron accumulation and cellular lipid peroxidation to lethal levels is suppressed by iron chelators or lipophilic antioxidants ( Mou et al., 2019 ). While mammals harbor catalase, glutathione reductase, thioredoxin reductase, and selenium-dependent peroxidases in their antioxidant repertoire, Leishmania handles it through thiol-dependent peroxidases or heme-dependent ascorbate peroxidases ( Moreira et al., 2018 ). Trypanosomes contain GPx-like glutathione (GSH) peroxidases (Px I–III), but they are type II tryparedoxin peroxidases that reduce tryparedoxin at much higher folds than GSH ( Bogacz and Krauth-Siegel, 2018 ).…”

Application of CRISPR/Cas9-Based Reverse Genetics in Leishmania braziliensis: Conserved Roles for HSP100 and HSP23

“…Studies reporting on the use of homologous recombination in L. braziliensis demonstrate the generation of stable transgenic parasite lines from integration of DNA constructs into the SSU rDNA genomic locus. These include L. braziliensis lines expressing reporter genes, e.g., luciferase or eGFP, which hold potential for parasite tracking and monitoring effects of antileishmanial compounds in vitro and in vivo [ 67 , 68 , 69 ], and over expressing parasite lines for the analysis of gene products, e.g., to assess antimony susceptibility and resistance mechanisms [ 70 , 71 , 72 ]. Moreover, circular extrachromosomal cosmids can be stably introduced into L. braziliensis to over-express stretches of genomic DNA and connect the over expression phenotypes to biological processes such as virulence [ 73 ] and antimony resistance [ 59 ].…”