Abstract
Neospora caninum (N. caninum) is a major cause of abortions in cattle. During its invasion of host cells, a parasitophorous vacuole (PV) is formed, accompanied by an intravacuolar network (IVN). The IVN takes part in parasite ingesting of nutrients from hosts. The dense granular proteins of N. caninum (NcGRAs) play a key role in forming the PV and the IVN, which may influence virulence during N. caninum invasion. The present study aimed to explore the biological function of NcGRA6 in N. caninum by disrupting the NcGRA6 gene in the Nc-1 strain. We successfully constructed an NcGRA6-targeting CRISPR plasmid (pNc-SAG1-Cas9:U6-SgGRA6) and amplified the DHFR-TS DNA donor. The NcGRA6 knockout mutation (ΔNcGRA6) was generated by co-electroporation of the pNc-SAG1::CAS9-U6::sgGRA6 plasmid and the DHFR-TS DNA donor into the Nc-1 strain, which was then cultured under pyrimethamine selection pressure. The ΔNcGRA6 mutation was further verified by identification of NcGRA6 gene disruption using PCR, measurement of NcGRA6 gene transcription levels using qPCR, assessment of NcGRA6 protein expression levels using western blotting, and observation of NcGRA6 protein location using immunofluorescence and immunoelectron microscopy. The results of in vitro virulence assays, including plaque, invasion, egress, and replication assays, showed that the ΔNcGRA6 strain had smaller plaques, similar invasion and egress ability, and slower intracellular replication ability than the Nc-1 strain. The results of in vivo virulence assays showed that the ΔNcGRA6 strain exhibited reduced virulence and improved survival ability in mice compared with the Nc-1 strain. The parasite burden in ΔNcGRA6 strain-infected mouse tissues, including the heart, brain, liver, spleen, lung, and kidney, was significantly reduced compared with that in mice infected with the Nc-1 strain. These data suggest that we successfully constructed a ΔNcGRA6 strain and verify that NcGRA6 is a critical virulence factor. NcGRA6 gene disruption can slow down N. caninum proliferation and lower the pathogenicity to hosts. Our findings provide a foundation for future research on other targeted N. caninum protein functions and may help in exploring the interaction mechanisms between parasites and hosts.
Highlights
Neospora caninum (N. caninum) is an obligate intracellular parasite of the phylum Apicomplexa that infects a broad range of hosts and leads to neosporosis worldwide [1,2,3,4]
The constructed pNc-SAG1::CAS9-U6::sgGRA6 plasmid and complete dihydrofolate reductase (DHFR)-TS DNA fragment were co-electroporated into the Nc-1 N. caninum strain
For PCR1 and PCR2 amplification, the results indicated that the Nc-1 strain had no band, whereas the NcGRA6 strain produced the target 500 and 1,100 bp bands
Summary
Neospora caninum (N. caninum) is an obligate intracellular parasite of the phylum Apicomplexa that infects a broad range of hosts and leads to neosporosis worldwide [1,2,3,4]. Typical clinical symptoms are abortion in dairy cattle and fetal abnormalities and nervous system dysfunction in calves [5, 6]. This brings significantly economic losses to the breeding industries [7]. There are no effective drugs or vaccines available against N. caninum [8]. Tachyzoites of N. caninum reside in a specialized membranous organelle known as the parasitophorous vacuole (PV). An intravacuolar network (IVN) enhances parasite ingestion of cytosolic material from the hosts [9]. The IVN is made up of highly curved membrane tubules and links parasites and the PV membrane together [10,11,12,13,14]
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