Abstract
Neospora caninum, of the Apicomplexa phylum, is a common cause of abortions in cattle and nervous system dysfunction in dogs. Rhoptry proteins of Apicomplexa play an important role in virulence. The objectives of this study were to study functions of NcROP5 in N. caninum by deleting the NcROP5 gene from the wild Nc-1 strain. We selected NcROP5 in ToxoDB and successfully constructed an NcROP5 gene-deleted vector, pTCR-NcROP5-CD KO. Then we screened the NcROP5 knockout strains (ΔNcROP5) at the gene, protein and transcription levels. Plaque assay, host cell invasion assay and intracellular proliferation test showed that the ΔNcROP5 strain had less plaque space, weakened invasion capacity and slower intracellular growth. Animal testing showed significantly lower cerebral load of ΔNcROP5 than the load of the Nc-1 strain, as well as a loss of virulence for the ΔNcROP5 strains. Phenotypic analyses using the label-free LC-MS/MS assay-based proteomic method and KEGG pathway enrichment analysis showed a reduction of NcGRA7 transcription and altered expression of multiple proteins including the apicomplexan family of binding proteins. The present study indicated that ROP5 is a key virulence factor in N. caninum in mice. The proteomic profiling of Nc-1 and ΔNcROP5 provided some data on differential proteins. These data provide a foundation for future research of protein functions in N. caninum.
Highlights
Neospora caninum is a common cause of abortions in cattle and nervous system dysfunction in dogs (Hall et al, 2005; Lyon, 2010)
We analyzed the genome of N. caninum Liverpool strain in ToxoDB2 and identified the sequences with high identity to TgROP5, which were NcLIV_060730, NcLIV_060740, and NcLIV_060741
Compared to the tandem cluster of polymorphic alleles of TgROP5 (TgROP5A, TgROP5B, and TgROP5C) (Behnke et al, 2011), we believed that NcROP5 would contain NcROP5A, NcROP5B, and NcROP5C, coded by the above three genes, respectively
Summary
Neospora caninum is a common cause of abortions in cattle and nervous system dysfunction in dogs (Hall et al, 2005; Lyon, 2010). The parasite has become an international concern due to the connection of the parasite infection to abortions in dairy and beef cattle worldwide (Dubey, 1999). Antibodies to N. caninum have been detected in humans in Brazil, Korea, Northern Ireland, and the United States, viable parasites have not been isolated (Dubey et al, 2007). Neospora caninum and Toxoplasmosis gondii belong to the same Apicomplexa family and share similar morphology, host range and clinical symptoms, they appear to be divergent in their pathogenicity in mice (Lyon, 2010). Rhoptry proteins (ROPs), including TgROP5, TgROP16, TgROP17, and TgROP18 are impotant virulence factors of ROP5 Affects N. caninum Virulence
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