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Efficient Gene Knockout and Knockdown Systems in Neospora caninum Enable Rapid Discovery and Functional Assessment of Novel Proteins

The development of molecular genetics has greatly enhanced the study of the biology and pathology associated with parasites of the phylum Apicomplexa. While the molecular tools are highly developed for the apicomplexan Toxoplasma gondii, the closely related parasite Neospora caninum lacks efficient...

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Autores principales: Mineo, Tiago W. P., Chern, Jessica H., Thind, Amara C., Mota, Caroline M., Nadipuram, Santhosh M., Torres, Juan A., Bradley, Peter J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8754167/
https://www.ncbi.nlm.nih.gov/pubmed/35019667
http://dx.doi.org/10.1128/msphere.00896-21
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author Mineo, Tiago W. P.
Chern, Jessica H.
Thind, Amara C.
Mota, Caroline M.
Nadipuram, Santhosh M.
Torres, Juan A.
Bradley, Peter J.
author_facet Mineo, Tiago W. P.
Chern, Jessica H.
Thind, Amara C.
Mota, Caroline M.
Nadipuram, Santhosh M.
Torres, Juan A.
Bradley, Peter J.
author_sort Mineo, Tiago W. P.
collection PubMed
description The development of molecular genetics has greatly enhanced the study of the biology and pathology associated with parasites of the phylum Apicomplexa. While the molecular tools are highly developed for the apicomplexan Toxoplasma gondii, the closely related parasite Neospora caninum lacks efficient tools for genetic manipulation. To enable efficient homologous recombination in N. caninum, we targeted the Ku heterodimer DNA repair mechanism in the genomic reference strain, Nc-Liverpool (NcLiv), and show that deletion of Ku80 results in a destabilization and loss of its partner Ku70. Disruption of Ku80 generated parasites in which genes are efficiently epitope tagged and only short homology regions are required for gene knockouts. We used this improved strain to target novel nonessential genes encoding dense granule proteins that are unique to N. caninum or conserved in T. gondii. To expand the utility of this strain for essential genes, we developed the auxin-inducible degron system for N. caninum using parasite-specific promoters. As a proof of concept, we knocked down a novel nuclear factor in both N. caninum and T. gondii and showed that it is essential for survival of both parasites. Together, these efficient knockout and knockdown technologies will enable the field to unravel specific gene functions in N. caninum, which is likely to aid in the identification of targets responsible for the phenotypic differences observed between these two closely related apicomplexan parasites. IMPORTANCE Neospora caninum is a parasite with veterinary relevance, inducing severe disease in dogs and reproductive disorders in ruminants, especially cattle, leading to major losses. The close phylogenetic relationship to Toxoplasma gondii and the lack of pathogenicity in humans drives an interest of the scientific community toward using N. caninum as a model to study the pathogenicity of T. gondii. To enable this comparison, it is important to develop efficient molecular tools for N. caninum, to gain accuracy and save time in genetic manipulation protocols. Here, we have developed base strains and protocols using the genomic reference strain of N. caninum to enable efficient knockout and knockdown assays in this model. We demonstrate that these tools are effective in targeting known and previously unexplored genes. Thus, these tools will greatly improve the study of this protozoan, as well as enhance its ability to serve as a model to understand other apicomplexan parasites.
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spelling pubmed-87541672022-01-14 Efficient Gene Knockout and Knockdown Systems in Neospora caninum Enable Rapid Discovery and Functional Assessment of Novel Proteins Mineo, Tiago W. P. Chern, Jessica H. Thind, Amara C. Mota, Caroline M. Nadipuram, Santhosh M. Torres, Juan A. Bradley, Peter J. mSphere Research Article The development of molecular genetics has greatly enhanced the study of the biology and pathology associated with parasites of the phylum Apicomplexa. While the molecular tools are highly developed for the apicomplexan Toxoplasma gondii, the closely related parasite Neospora caninum lacks efficient tools for genetic manipulation. To enable efficient homologous recombination in N. caninum, we targeted the Ku heterodimer DNA repair mechanism in the genomic reference strain, Nc-Liverpool (NcLiv), and show that deletion of Ku80 results in a destabilization and loss of its partner Ku70. Disruption of Ku80 generated parasites in which genes are efficiently epitope tagged and only short homology regions are required for gene knockouts. We used this improved strain to target novel nonessential genes encoding dense granule proteins that are unique to N. caninum or conserved in T. gondii. To expand the utility of this strain for essential genes, we developed the auxin-inducible degron system for N. caninum using parasite-specific promoters. As a proof of concept, we knocked down a novel nuclear factor in both N. caninum and T. gondii and showed that it is essential for survival of both parasites. Together, these efficient knockout and knockdown technologies will enable the field to unravel specific gene functions in N. caninum, which is likely to aid in the identification of targets responsible for the phenotypic differences observed between these two closely related apicomplexan parasites. IMPORTANCE Neospora caninum is a parasite with veterinary relevance, inducing severe disease in dogs and reproductive disorders in ruminants, especially cattle, leading to major losses. The close phylogenetic relationship to Toxoplasma gondii and the lack of pathogenicity in humans drives an interest of the scientific community toward using N. caninum as a model to study the pathogenicity of T. gondii. To enable this comparison, it is important to develop efficient molecular tools for N. caninum, to gain accuracy and save time in genetic manipulation protocols. Here, we have developed base strains and protocols using the genomic reference strain of N. caninum to enable efficient knockout and knockdown assays in this model. We demonstrate that these tools are effective in targeting known and previously unexplored genes. Thus, these tools will greatly improve the study of this protozoan, as well as enhance its ability to serve as a model to understand other apicomplexan parasites. American Society for Microbiology 2022-01-12 /pmc/articles/PMC8754167/ /pubmed/35019667 http://dx.doi.org/10.1128/msphere.00896-21 Text en Copyright © 2022 Mineo et al. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Research Article
Mineo, Tiago W. P.
Chern, Jessica H.
Thind, Amara C.
Mota, Caroline M.
Nadipuram, Santhosh M.
Torres, Juan A.
Bradley, Peter J.
Efficient Gene Knockout and Knockdown Systems in Neospora caninum Enable Rapid Discovery and Functional Assessment of Novel Proteins
title Efficient Gene Knockout and Knockdown Systems in Neospora caninum Enable Rapid Discovery and Functional Assessment of Novel Proteins
title_full Efficient Gene Knockout and Knockdown Systems in Neospora caninum Enable Rapid Discovery and Functional Assessment of Novel Proteins
title_fullStr Efficient Gene Knockout and Knockdown Systems in Neospora caninum Enable Rapid Discovery and Functional Assessment of Novel Proteins
title_full_unstemmed Efficient Gene Knockout and Knockdown Systems in Neospora caninum Enable Rapid Discovery and Functional Assessment of Novel Proteins
title_short Efficient Gene Knockout and Knockdown Systems in Neospora caninum Enable Rapid Discovery and Functional Assessment of Novel Proteins
title_sort efficient gene knockout and knockdown systems in neospora caninum enable rapid discovery and functional assessment of novel proteins
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8754167/
https://www.ncbi.nlm.nih.gov/pubmed/35019667
http://dx.doi.org/10.1128/msphere.00896-21
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