Glutaredoxin 1 Deficiency Leads to Microneme Protein-Mediated Growth Defects in Neospora caninum

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 (H 2 O 2 ) 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 .

Identification and Function of Apicoplast Glutaredoxins in Neospora caninum

“…We identified five putative GRXs in N. caninum . Our previous study showed that the GRX1 (NcGRX1) and GRX3 (NcGRX3) of N. caninum are located in the cytoplasm [ 16 ] and NcGRX5 in mitochondria. In the present study, we found that GRX S14 and GRX C5 localize to the apicoplasts.…”

“…One study showed that NcGRX1 is important for microneme protein-mediated parasite growth, but that NcGRX3 deficiency does not affect parasite growth [ 16 ]. Our study showed that the deletion of GRX S14 or GRX C5 alone did not affect the growth of N. caninum , which was consistent with previous research on Arabidopsis [ 25 ].…”

“…GRX S14 is used as a scaffold protein for (2Fe–2S) cluster assembly because it transfers the complete cluster to the receptor protein regulated by GSH [ 29 , 30 ]. Our previous research revealed that N. caninum GRX1 deficiency decreases the ratio of reduced GSH/GSSG, causing a significant accumulation of hydroxyl radicals in parasites, and increases the number of apoptotic cells under oxidative stress (H 2 O 2 ) conditions [ 16 ]. In the present study, GRX S14 and GRX C5 double-gene deletion did not affect the GSH/GSSG ratio of parasites, nor did it alter levels of ROS and OH.…”

“…The EuPaGDT Library in ToxoDB was used to design the gRNA targeting sites of the plasmids pCRISPR-CAS9- grx S14 and pCRISPR-CAS9- grx C5. The basic plasmid template was CRISPR-CAS9-GRX1, which was constructed in our previous study [ 16 ]. Cas9 upstream and downstream fragments containing gRNA sequences were amplified and ligated by seamless cloning (Vazyme Biotech, Co., Ltd., Nanjing, China).…”

“…Our previous study found that N. caninum has five GRXs (GRX1, GRX3, GRX S14, GRX C5 and GRX5). GRX1 and GRX3 are located in cytoplasm, and GRX1 deficiency resulted in a marked reduction in the secretion of microneme proteins, significantly affecting the invasion and egress processing of the parasites [ 16 ]. In contrast, GRX5 localized to the mitochondria (unpublished), whereas GRX S14 and GRX C5 have yet to be explored.…”