Cloning, Expression, and Characterization of Iron-Containing Superoxide Dismutase From Neospora Caninum

Abstract

A gene encoding superoxide dismutase (SOD) from Neospora caninum, a causative agent of neosporosis, has been cloned and its gene product functionally expressed and characterized. The gene had an open reading frame of 606 bp and deduced 201 amino acids. Sequence analysis showed that the gene had conserved metal-binding residues and conserved amino acid residues that were found in Fe-SODs. Comparison of the deduced amino acid sequence of the enzyme with previously reported Fe-SOD amino acid sequences of the other parasitic protozoans revealed significant high homology. The coding region of the N. caninum Fe-SOD was cloned and functionally expressed in Escherichia coli. Enzyme activity of the expressed protein was inhibited by hydrogen peroxide but not by sodium azide and potassium cyanide, and the enzyme showed similar biochemical properties with typical Fe-SODs of other parasitic protozoans. Southern blot analysis showed that the SOD gene appears to be present as a single-copy gene in N. caninum genome. Semiquantitative reverse transcription-polymerase chain reaction and immunoblot using antiserum raised against the purified recombinant protein showed that Fe-SOD is expressed in both developmental stages of N. caninum, i.e., in bradyzoites and tachyzoites. In an immunofluorescence assay, the enzyme was localized on the cell surface of N. caninum tachyzoites. These results suggest that Fe-SOD might be essential for the intracellular survival of N. caninum and may play an important role in the pathogenesis of the parasite by protecting the parasite from oxidative killing.

Identification and characterization of a mitochondrial iron–superoxide dismutase of Cryptosporidium parvum

“…The molecular mass of the active rCp-mtSOD was determined to be about 50 kDa by gel filtration (data not shown), indicating that the enzyme forms a homodimer as do other classes of Fe-SODs. The rCp-mtSOD showed similar biochemical properties to those of Fe-SODs from other protozoa parasites (Bécuwe et al 1996;Choi et al 2000;Odberg-Ferragut et al 2000;Ahmed et al 2003;Cho et al 2004). The enzyme showed optimum activity for a broad pH range of 7.0 to 9.5, and maximum level of activity was at pH 8.0 (Fig.…”

“…Superoxide dismutases (SODs; 1.15.1.1) are ubiquitous metalloenzymes that detoxify the highly reactive superoxide anions to hydrogen peroxide and molecular oxygen (Fridovich 1986). SODs have been identified and characterized in various parasitic protozoa and are considered as essential enzymes that protect parasites from endogenous oxidative stress, as well as oxidative killing by host immune effector cells (Bécuwe et al 1996;OdbergFerragut et al 2000;Cho et al 2004;Dufernez et al 2006). The most important feature of protozoan parasite SODs is that they belong to iron-containing SODs (FeSODs), which are not found in their mammalian hosts.…”

Identification ofBesnoitia besnoitiproteins that showed differences in abundance between tachyzoite and bradyzoite stages by difference gel electrophoresis

“…In the present study, an SOD enzyme showed higher abundance in the bradyzoite stage. This protein has been detected in both T. gondii (Odberg-Ferragut et al 2000) and N. caninum (Cho et al 2004) bradyzoites and tachyzoites, suggesting that SOD might be essential for the intracellular growth of different developmental stages, protecting the parasite from oxidative killing (Michalski and Prowse, 1991). The higher abundance of SOD in bradyzoites might be explained by the origin of parasite stages because the bradyzoites originated from an in vivo infection, whereas tachyzoites were propagated in an epithelial monkey-derived cell line.…”

Controle Redox em Neospora caninum: investigação de enzimas do sistema antioxidante

“…O sistema redox de parasitas Apicomplexas tem sido amplamente estudado em Plasmodium e T. gondii, nos quais já foram descritas diversas moléculas e enzimas antioxidantes, como por exemplo: peroxirredoxinas (AKERMAN; MÜLLER, 2003;BOUCHER et al, 2006), tiorredoxina redutase (MCCARTY et al, 2015;XUE et al, 2017), tioredoxina (JORTZIK;BECKER, 2012;BIDDAU et al, 2018) e glutationa redutase (SARMA et al, 2003;HASSAN et al, 2014). Em Neospora caninum, porém, apenas as enzimas SOD1 e Glutarredoxina 1 (CHO et al, 2004a;SONG et al, 2020) haviam sido descritas antes do presente trabalho, existindo ainda uma ampla gama de moléculas antioxidantes a serem investigadas nesse parasita. Evidências de estudos em Plasmodium e T. gondii e N. caninum mostram que o balanço entre defesas antioxidantes e o estresse oxidativo influenciam diretamente na infecção do organismo hospedeiro.…”

“…Ademais, já foi demonstrado que a replicação de N. caninum pode ser inibida através de mecanismos ativados por EROs (MOTA et al, 2020;PEREIRA et al, 2020). No organismo hospedeiro o estudo do estresse oxidativo é embasado em um corpo de evidências de longa data e grande parte de seus componentes são profundamente conhecidos (NORDBERG; ARNÉR, 2001;SORDILLO;AITKEN, 2009), por outro lado, quando olhamos para N. caninum, apenas poucas características de seu sistema redox são conhecidas tanto das proteínas envolvidas como do comportamento frente ao estresse oxidativo (CHO et al, 2004a;MOTA et al, 2020;PEREIRA et al, 2020;SONG et al, 2020. Assim, para contribuir com o entendimento do controle redox em N. caninum e buscar novos alvos de tratamento e vacinas, buscamos descrever duas enzimas conservadas do sistema redox e avaliar o comportamento do parasita frente ao estresse oxidativo.…”