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New molecular tools in Neospora caninum for studying apicomplexan parasite proteins.

ABSTRACT: The development of molecular genetics has greatly enhanced the study of the biology and pathology associated with parasites of the phylum Apicomplexa. We have established a system specifically designed for Neospora caninum, and used this system as a heterologous platform for the expression of foreign genes. Plasmid constructs containing fluorescent proteins or targeted genes of Toxoplasma gondii, driven by N. caninum promoters, have yielded robust expression and correct trafficking of target gene products as assessed by immunofluorescence assays and Western blot analyses. Using this approach, we here demonstrated that N. caninum expressing T. gondii's GRA15 and ROP16 kinase are biologically active and induced immunological phenotypes consistent with T. gondii strains. N. caninum expressing TgGRA15 differentially disturbed the NF-?B pathway, inducing an increased IL-12 production. On the other hand, N. caninum expressing TgROP16 induced host STAT3 phosphorylation and consequent reduction of IL-12 synthesis. These results indicate that heterologous gene expression in N. caninum is a useful tool for the study of specific gene functions and may allow the identification of antigenic targets responsible for the phenotypic differences observed between these two closely related apicomplexan parasites. Additionally, these observations may prove to be useful for the development of vaccine protocols to control toxoplasmosis and/or neosporosis.

SUBMITTER: Mota CM

PROVIDER: S-EPMC5476669 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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New molecular tools in Neospora caninum for studying apicomplexan parasite proteins.

Mota Caroline M CM Chen Allan L AL Wang Kevin K Nadipuram Santhosh S Vashisht Ajay A AA Wohlschlegel James A JA Mineo Tiago W P TWP Bradley Peter J PJ

Scientific reports 20170619 1

The development of molecular genetics has greatly enhanced the study of the biology and pathology associated with parasites of the phylum Apicomplexa. We have established a system specifically designed for Neospora caninum, and used this system as a heterologous platform for the expression of foreign genes. Plasmid constructs containing fluorescent proteins or targeted genes of Toxoplasma gondii, driven by N. caninum promoters, have yielded robust expression and correct trafficking of target gen ...[more]

PMID: 28630403

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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.

Dataset Information

New molecular tools in Neospora caninum for studying apicomplexan parasite proteins.

Publications

New molecular tools in Neospora caninum for studying apicomplexan parasite proteins.

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