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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 (?Glu-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 ?NcGrx1, 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.

SUBMITTER: Song X

PROVIDER: S-EPMC7487798 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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

Song Xingju X Yang Xu X Xue Yangfei Y Yang Congshan C Wu Kaijian K Liu Jing J Liu Qun Q

Frontiers in microbiology 20200831

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 ( ...[more]

PMID: 32983074

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Project description:The invasive stages of apicomplexan parasites enter their host cells through mechanisms which are largely conserved throughout the phylum. Host cell invasion is divided into two distinct events, namely, adhesion onto the host cell surface and the actual host cell entry process. The former is mediated largely through microneme proteins which are secreted at the onset of establishing contact with the host cell surface. Many of the microneme proteins identified so far contain adhesive domains. We here present the genomic and corresponding cDNA sequences coding for a 460-amino-acid (aa) microneme protein in Neospora caninum tachyzoites which, due to its homology to MIC1 in Toxoplasma gondii (TgMIC1), was named NcMIC1. The deduced NcMIC1 polypeptide sequence contains an N-terminal signal peptide of 20 aa followed by two tandemly internal repeats of 48 and 44 aa, respectively. Integrated into each repeat is a CXXXCG sequence motif reminiscent of the thrombospondin-related family of adhesive proteins. The positioning of this motif is strictly conserved in TgMIC1 and NcMIC1. The C-terminal part, comprised of 278 aa, was expressed in Escherichia coli, and antibodies affinity purified on recombinant NcMIC1 were used to confirm the localization within the micronemes by immunofluorescence and immunogold transmission electron microscopy of tachyzoites. Immunohistochemistry of mouse brains infected with tissue cysts showed that expression of this protein is reduced in the bradyzoite stage. Upon initiation of secretion by elevating the temperature to 37 degrees C, NcMIC1 is released into the medium supernatant. NcMIC1 binds to trypsinized, rounded Vero cells, as well as to Vero cell monolayers. Removal of glycosaminoglycans from the host cell surface and modulation of host cell surface glycosaminoglycan sulfation significantly reduces the binding of NcMIC1 to the host cell surface. Solid-phase binding assays employing defined glycosaminoglycans confirmed that NcMIC1 binds to sulfated glycosaminoglycans.

Project description:Iron-sulfur [Fe-S] clusters are one of the most ancient and functionally versatile natural biosynthetic prosthetic groups required by various proteins involved in important metabolic processes, including the oxidative phosphorylation of proteins, electron transfer, energy metabolism, DNA/RNA metabolism, and protein translation. Apicomplexan parasites harbor two possible [Fe-S] cluster assembly pathways: the iron-sulfur cluster (ISC) pathway in the mitochondria and the sulfur formation (SUF) pathway in the apicoplast. Glutaredoxin 5 (GRX5) is involved in the ISC pathway in many eukaryotes. However, the cellular roles of GRX5 in apicomplexan parasites remain to be explored. Here, we showed that Neospora caninum mitochondrial GRX5 (NcGRX5) deficiency resulted in aberrant mitochondrial ultrastructure and led to a significant reduction in parasite proliferation and virulence in mice, suggesting that NcGRX5 is important for parasite growth in vitro and in vivo. Comparative proteomics and energy metabolomics were used to investigate the effects of NcGRX5 on parasite growth and mitochondrial metabolism. The data showed that disruption of NcGRX5 downregulated the expression of mitochondrial electron transport chain (ETC) and tricarboxylic acid cycle (TCA) cycle proteins and reduced the corresponding metabolic fluxes. Subsequently, we identified 23 proteins that might be adjacent to or interact with NcGRX5 by proximity-based protein labeling techniques and proteomics. The interactions between NcGRX5 and two iron-sulfur cluster synthesis proteins (ISCS and ISCU1) were further confirmed by coimmunoprecipitation assays. In conclusion, NcGRX5 is important for parasite growth and may regulate mitochondrial energy metabolism by mediating the biosynthesis of iron-sulfur clusters. IMPORTANCE Iron-sulfur [Fe-S] clusters are among the oldest and most ubiquitous prosthetic groups, and they are required for a variety of proteins involved in important metabolic processes. The intracellular parasites in the phylum Apicomplexa, including Plasmodium, Toxoplasma gondii, and Neospora caninum, harbor the ISC pathway involved in the biosynthesis of [Fe-S] clusters in mitochondria. These cofactors are required for a variety of important biological processes. However, little is known about the role of oxidoreductase glutaredoxins in these parasites. Our data indicate that NcGRX5 is an essential protein that plays multiple roles in several biological processes of N. caninum. NcGRX5 interacts with the mitochondrial iron-sulfur cluster synthesis proteins ISCS and ISCU1 and also regulates parasite energy metabolism. These data provide an insider's view of the metabolic regulation and iron-sulfur cluster assembly processes in the apicomplexan parasites.

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

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Glutaredoxin 1 Deficiency Leads to Microneme Protein-Mediated Growth Defects in <i>Neospora caninum</i>.

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