Construction of luciferase-expressing Neospora caninum and drug screening

Abstract

Abstract Background: Neospora caninum is an apicomplexan parasite which is particularly responsible for abortions in cattle and neuromuscular disease in dogs. New therapeutics are urgently needed to control Neosporosis due to the limited effectiveness of currently available drugs. 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: The CRISPR/Cas9 was used to construct the luciferase-expressing N. caninum (Nc1-Luc). After testing the luciferase expression and phenotype of Nc1-Luc strains, we determined the drug sensitivity of Nc1-Luc strains by treating them with known positive or negative drugs and half-maximal inhibitory concentration (IC50) calculation. Then the selective pan-RAF inhibitor TAK-632 was evaluated for anti-N. caninum effects using Nc1-Luc by luciferase activity reduction assay and other in vitro and in vivo pharmacodynamic studies. Results: The phenotypes and drug sensitivity of Nc1-Luc strains were consistent with those of the parent strains Nc1, and Nc1-Luc strains can be used to determine IC50 for anti-N. caninum drugs. Using Nc1-Luc strains, TAK-632 showed promising activities against N. caninum, with an IC50 of 0.6131 mM and a selectivity index (SI) of 62.53. In vitro studies showed that TAK-632 inhibited invasion, proliferation and divison of N. caninum tachyzoites. In vivo studies showed that TAK-632 attenuated the virulence of N. caninum in mice and significantly reduced the parasite burdens 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.