Abstract
Neospora caninum is an obligate intracellular protozoan parasite that infects a wide range of mammalian species and causes spontaneous abortion in cattle. N. caninum is exposed to oxidative stress during its life cycle. Oxidoreductase is crucial for parasite response to the environmental stresses. Glutaredoxins (Grxs) are small oxidoreductases of the thioredoxin family proteins that catalyze thiol-disulfide exchange reactions by utilizing electrons from the tripeptide glutathione (γGlu-Cys-Gly; GSH). Grxs are key elements in redox signaling and cell signal transduction. However, Grxs are an unexplored set of oxidoreductases in N. caninum. Here, we identified two cytoplasm located glutaredoxin domain-containing proteins (NcGrx1 and NcGrx3) in N. caninum. To better understand the functions of these Grx proteins, we generated NcGrx1 and NcGrx3 deficiency and overexpression strains. The deletion or overexpression of NcGrx3 had no significant effect on the growth of N. caninum in vitro and in vivo. NcGrx1 knockout parasites displayed a significant growth defect, which was due to the influence on invasion and egress abilities. Moreover, NcGrx1 deficiency decreased the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) (GSH/GSSG ratio), caused a significant accumulation of hydroxyl radical in parasites, and an increase in apoptotic cells under oxidative stress (H2O2) condition. To determine the cause of growth defects in ΔNcGrx1, we examined the transcription levels of various invasion-egress related genes as measured by qPCR. We found a significant decrease in MIC1, MIC4, and MIC6 genes. Further investigation found that the secretion of MIC1, MIC4, and MIC6 proteins was significantly affected. Collectively, Ncgrx1 is important for microneme protein-mediated parasite growth, and maybe a potential intervention target for the N. caninum.
Highlights
Neospora caninum (N. caninum) is an obligate intracellular apicomplexan parasite causing neosporosis, which results in spontaneous abortion in cattle and neural system dysfunction in dogs
The results revealed that the secretion of MIC1, MIC4, and MIC6 were significantly reduced in the NcGrx1 as compared to Nc1 parasites (Figure 5C), and the secretions of these proteins were recovered in i NcGrx1 parasites (Figure 5D)
Our results showed that the location of MIC1, MIC4, and MIC6 were not affected by NcGrx1 deletion (Figure 6)
Summary
Neospora caninum (N. caninum) is an obligate intracellular apicomplexan parasite causing neosporosis, which results in spontaneous abortion in cattle and neural system dysfunction in dogs. N. caninum tachyzoites involves invasion, replication, and egress. Tachyzoites enter the host cell through an active invasion mechanism. Egress is triggered, resulting in host cell destruction (Blader et al, 2015; Frénal et al, 2017). Invasion and egress are important due to tissue destruction in the infected host cell. Identification of the proteins required for the invasion and egress processes is important for the development of novel therapeutics against neosporosis. The secretion of motility-associated motors and adhesins from the micronemes are required for the initial invasion, egress and movement to a new host cell and its subsequent invasion (Frénal et al, 2017)
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