Construction of luciferase-expressing Neospora caninum and drug screening
Abstract Background Neospora caninum is an apicomplexan parasite that is particularly responsible for abortions in cattle and neuromuscular disease in dogs. Due to the limited effectiveness of currently available drugs, there is an urgent need for new therapeutic approaches to control neosporosis. L...
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| Format: | Article |
| Language: | English |
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BMC
2024-03-01
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| Series: | Parasites & Vectors |
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| Online Access: | https://doi.org/10.1186/s13071-024-06195-8 |
| _version_ | 1797266905437831168 |
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| author | Fei Wang Yangfei Xue Yanqun Pei Meng Yin Zhepeng Sun Zihui Zhou Jing Liu Qun Liu |
| author_facet | Fei Wang Yangfei Xue Yanqun Pei Meng Yin Zhepeng Sun Zihui Zhou Jing Liu Qun Liu |
| author_sort | Fei Wang |
| collection | DOAJ |
| description | Abstract Background Neospora caninum is an apicomplexan parasite that is particularly responsible for abortions in cattle and neuromuscular disease in dogs. Due to the limited effectiveness of currently available drugs, there is an urgent need for new therapeutic approaches to control neosporosis. Luciferase-based assays are potentially powerful tools in the search for antiprotozoal compounds, permitting the development of faster and more automated assays. The aim of this study was to construct a luciferase-expressing N. caninum and evaluate anti-N. caninum drugs. Methods Luciferase-expressing N. caninum (Nc1-Luc) was constructed using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9). After testing the luciferase expression and phenotype of the Nc1-Luc strains, the drug sensitivity of Nc1-Luc strains was determined by treating them with known positive or negative drugs and calculating the half-maximal inhibitory concentration (IC50). The selective pan-rapidly accelerated fibrosarcoma (pan-RAF) inhibitor TAK-632 was then evaluated for anti-N. caninum effects using Nc1-Luc by luciferase activity reduction assay and other in vitro and in vivo studies. Results The phenotypes and drug sensitivity of Nc1-Luc strains were consistent with those of the parental strains Nc1, and Nc1-Luc strains can be used to determine the IC50 for anti-N. caninum drugs. Using the Nc1-Luc strains, TAK-632 showed promising activity against N. caninum, with an IC50 of 0.6131 μM and a selectivity index (SI) of 62.53. In vitro studies demonstrated that TAK-632 inhibited the invasion, proliferation, and division of N. caninum tachyzoites. In vivo studies showed that TAK-632 attenuated the virulence of N. caninum in mice and significantly reduced the parasite burden in the brain. Conclusions In conclusion, a luciferase-expressing N. caninum strain was successfully constructed, which provides an effective tool for drug screening and related research on N. caninum. In addition, TAK-632 was found to inhibit the growth of N. caninum, which could be considered as a candidate lead compound for new therapeutics for neosporosis. Graphical Abstract |
| first_indexed | 2024-04-25T01:08:07Z |
| format | Article |
| id | doaj.art-600efb0f6e1947d2b7c54c44d52355a5 |
| institution | Directory Open Access Journal |
| issn | 1756-3305 |
| language | English |
| last_indexed | 2024-04-25T01:08:07Z |
| publishDate | 2024-03-01 |
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| series | Parasites & Vectors |
| spelling | doaj.art-600efb0f6e1947d2b7c54c44d52355a52024-03-10T12:07:00ZengBMCParasites & Vectors1756-33052024-03-0117111610.1186/s13071-024-06195-8Construction of luciferase-expressing Neospora caninum and drug screeningFei Wang0Yangfei Xue1Yanqun Pei2Meng Yin3Zhepeng Sun4Zihui Zhou5Jing Liu6Qun Liu7National Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural UniversityNational Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural UniversityNational Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural UniversityNational Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural UniversityNational Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural UniversityNational Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural UniversityNational Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural UniversityNational Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural UniversityAbstract Background Neospora caninum is an apicomplexan parasite that is particularly responsible for abortions in cattle and neuromuscular disease in dogs. Due to the limited effectiveness of currently available drugs, there is an urgent need for new therapeutic approaches to control neosporosis. Luciferase-based assays are potentially powerful tools in the search for antiprotozoal compounds, permitting the development of faster and more automated assays. The aim of this study was to construct a luciferase-expressing N. caninum and evaluate anti-N. caninum drugs. Methods Luciferase-expressing N. caninum (Nc1-Luc) was constructed using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9). After testing the luciferase expression and phenotype of the Nc1-Luc strains, the drug sensitivity of Nc1-Luc strains was determined by treating them with known positive or negative drugs and calculating the half-maximal inhibitory concentration (IC50). The selective pan-rapidly accelerated fibrosarcoma (pan-RAF) inhibitor TAK-632 was then evaluated for anti-N. caninum effects using Nc1-Luc by luciferase activity reduction assay and other in vitro and in vivo studies. Results The phenotypes and drug sensitivity of Nc1-Luc strains were consistent with those of the parental strains Nc1, and Nc1-Luc strains can be used to determine the IC50 for anti-N. caninum drugs. Using the Nc1-Luc strains, TAK-632 showed promising activity against N. caninum, with an IC50 of 0.6131 μM and a selectivity index (SI) of 62.53. In vitro studies demonstrated that TAK-632 inhibited the invasion, proliferation, and division of N. caninum tachyzoites. In vivo studies showed that TAK-632 attenuated the virulence of N. caninum in mice and significantly reduced the parasite burden in the brain. Conclusions In conclusion, a luciferase-expressing N. caninum strain was successfully constructed, which provides an effective tool for drug screening and related research on N. caninum. In addition, TAK-632 was found to inhibit the growth of N. caninum, which could be considered as a candidate lead compound for new therapeutics for neosporosis. Graphical Abstracthttps://doi.org/10.1186/s13071-024-06195-8Neospora caninumFirefly luciferaseCRISPR/Cas9TAK-632Inhibition |
| spellingShingle | Fei Wang Yangfei Xue Yanqun Pei Meng Yin Zhepeng Sun Zihui Zhou Jing Liu Qun Liu Construction of luciferase-expressing Neospora caninum and drug screening Parasites & Vectors Neospora caninum Firefly luciferase CRISPR/Cas9 TAK-632 Inhibition |
| title | Construction of luciferase-expressing Neospora caninum and drug screening |
| title_full | Construction of luciferase-expressing Neospora caninum and drug screening |
| title_fullStr | Construction of luciferase-expressing Neospora caninum and drug screening |
| title_full_unstemmed | Construction of luciferase-expressing Neospora caninum and drug screening |
| title_short | Construction of luciferase-expressing Neospora caninum and drug screening |
| title_sort | construction of luciferase expressing neospora caninum and drug screening |
| topic | Neospora caninum Firefly luciferase CRISPR/Cas9 TAK-632 Inhibition |
| url | https://doi.org/10.1186/s13071-024-06195-8 |
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