Abstract
Neospora caninum causes abortions in cattle and nervous system dysfunction in dogs. Dense granular proteins (GRAs) play important roles in virulence; however, studies on NcGRA functions are limited. In the present study, multiple methods, including site-directed mutagenesis; CRISPR/Cas9 gene editing; Western blotting; quantitative polymerase chain reaction; confocal microscopy; plaque, invasion, egress, and replication assays; animal assays of survival rate and parasite burden; and hematoxylin–eosin staining, were used to characterize the NcGRA2 protein, construct an NcGRA2 gene disruption (ΔNcGRA2) strain, and explore its virulence in vivo and vitro. The results showed that NcGRA2 shared 31.31% homology with TgGRA2 and was colocalized with NcGRA6 at the posterior end of tachyzoites and the intravacuolar network of parasitophorous vacuoles (PVs). Cell fractionation analysis showed that NcGRA2 behaved as a transmembrane and membrane-coupled protein. The ΔNcGRA2 strain was constructed by coelectroporation of the NcGRA2-targeting CRISPR plasmid (pNc-SAG1-Cas9:U6-SgGRA2) and DHFR-TS DNA donor and verified at the protein, genome, and transcriptional levels and by immunofluorescence localization analysis. The in vitro virulence results showed that the ΔNcGRA2 strain displayed smaller plaques, similar invasion and egress abilities, and slower intracellular growth. The in vivo virulence results showed a prolonged survival time, lower parasite burden, and mild histopathological changes. Overall, the present study indicates that NcGRA2, as a dense granular protein, forms the intravacuolar network structure of PVs and weakens N. caninum virulence by slowing proliferation. These data highlight the roles of NcGRA2 and provide a foundation for research on other protein functions in N. caninum.
Highlights
Neospora caninum, an obligate intracellular protozoan pathogen, belongs to the phylum Apicomplexa and leads to neosporosis with clinical manifestations of reproductive failure in bovines and nervous system dysfunction in dogs [1,2,3,4,5,6,7]
The NcGRA2 gene was cloned into the pGEX-4T-1 vector, and the positive recombinant plasmid pGEX-NcGRA2 was transformed into Rosetta (DE3)
Similar to the location of NcGRA6, NcGRA2 was mainly distributed in the intravacuolar network (IVN) structure of parasitophorous vacuoles (PVs) [38]
Summary
An obligate intracellular protozoan pathogen, belongs to the phylum Apicomplexa and leads to neosporosis with clinical manifestations of reproductive failure in bovines and nervous system dysfunction in dogs [1,2,3,4,5,6,7]. It has been reported that the total annual loss in the cattle industry caused by N. caninum infection worldwide is as high as US $43.08 billion. GRA2 Virulence in Neospora caninum to US $320.98 billion [8, 9]. As a country with a large amount of animal husbandry, China has begun to pay increasing attention to N. caninum infections. Pretreatment and control of neosporosis are mainly by sulfonamides or attenuated vaccines; these drugs have relatively large negative effects, and resistance to them forms leading to incomplete elimination of N. caninum [10, 11]. It is urgent to look for new targets against neosporosis
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