Functional Study Of NcPrx1 For Resistance To Oxidative Stress In Neospora Caninum

Neospora caninum（N.caninum）is a terminal complex phylum intracellular parasite that causes neosporosis in animals with canines as the final host and cattle and sheep as intermediate hosts.N.caninum infections cause abortions,stillbirths and motor nervous system diseases in cattle and sheep as well as in newborn calves,causing huge economic losses to the farming industry.There is no specific drug or vaccine for neosporosis.An in-depth understanding of the pathogenesis of N.caninum will lay the foundation for the development of new strategies for the control of N.caninum and provide potential targets for the development of drugs to treat N.caninum.After the host is infected by the parasite,it initiates a natural immune response and produces large amounts of reactive oxygen species（ROS）,which cause damage to the worm by inducing oxidative stress.The parasite has developed several antioxidant systems to balance its redox status to adapt to the oxidative stress in the host cell,such as Superoxide dismutase（SOD）,Catalase（CAT）,Glutathione reductase（GR）,Thioredoxin reductase（TR）and Peroxiredoxins（Prxs）play important roles in the resistance to host oxidative stress,while the antioxidant stress response of N.caninum has not been fully elucidated and the genes that play a key role in the resistance to reactive oxygen stress are still poorly understood.In this study,we screened key gene of N.caninum against oxidative stress by fluorescence quantitative PCR,and constructed key knockout strains by CRISPR/Cas9 technology to investigate the biological functions of the screened genes and their roles in regulating the host immune response,providing a new theoretical basis for the elucidation of the mechanism of the antioxidant stress response of N.caninum and the control of the disease.The main research contents and results are as follows:1.Screening of key genes for resistance to oxidative stress in N.caninum and their localization in tachyzoites.（1）Screening of key genes for resistance to oxidative stress in N.caninum.N.caninum tachyzoites were stimulated with different concentrations of H₂O₂ to establish a N.caninum oxidative stress model,and the mRNA expression levels of antioxidant-related genes NcPrx1-3,NcCAT,NcTR and NcGR were detected by fluorescence quantitative PCR.The results showed that H₂O₂ stimulation caused up-regulation of NcPrx1,NcPrx2 and NcGR mRNA expression levels,with NcPrx1 being the most significantly up-regulated.（2）NcPrx1 bioinformatics analysis.The amino acid sequence characteristics of NcPrx1 were analyzed using bioinformatics analysis software.The results showed the presence of a predicted alkyl hydroperoxide reductase（AHP1）structural domain in the amino acid sequence of NcPrx1,indicating that NcPrx1 is a member of the Prxs family and exhibits a high degree of consistency and similarity with some protozoan homologs.（3）NcPrx1 Peroxiredoxins activity assay.The NcPrx1 recombinant protein（rNcPrx1）was expressed and purified by constructing a pET-28a-NcPrx1 prokaryotic expression vector,and the results showed that the NcPrx1 protein has peroxidase activity as measured by in vitro enzyme activity.（4）Expression analysis of NcPrx1 in N.caninum and its exocrine antigens.Polyclonal antibodies were obtained from sera of mice immunized with rNcPrx1,and the expression of NcPrx1 in N.caninum whole worm protein and exocrine antigen（ESA）was detected by Western blot.The results showed that NcPrx1 was expressed in both N.caninum and its exocrine antigen.（5）Localization of NcPrx1 in N.caninum.The expression of NcPrx1 in the worm was localized by indirect immunofluorescence using polyclonal antibodies.The test results showed that NcPrx1 was localized in the tachyzoite cytoplasm of N.caninum.2.Construction of NcPrx1 knockout strain and analysis of phenotype and resistance to oxidative stress.（1）Construction of NcPrx1 knockout insect strains.The NcPrx1 knockout strain（ΔNcPrx1）was constructed using the CRISPR/Cas9 knockout system and identified by PCR and Western blot,and the results showed that the NcPrx1 knockout strain was successfully constructed.（2）Phenotypic analysis ofΔNcPrx1 insect strain.The phenotype ofΔNcPrx1 strain was analyzed by plaque,invasion,proliferation and egress assay.The results showed that the deletion of NcPrx1 gene led to a reduction in the in vitro culture growth ability,invasion ability and egress ability of N.caninum,but had no significant effect on proliferation ability.（3）Analysis of the resistance ofΔNcPrx1 strain to oxidative stress.The oxidative stress assay showed that the deletion of the NcPrx1 gene led to a significant reduction in both the proliferation and invasion ability of N.caninum under H₂O₂ stimulation,and theΔNcPrx1 strain was more sensitive to oxidative stress.Subsequent assays of ROS,total antioxidant capacity（T-AOC）and malondialdehyde（MDA）levels inΔNcPrx1 strains showed that NcPrx1gene deletion resulted in the accumulation of ROS and MDA as well as the reduction of T-AOC in N.caninum.（4）Virulence analysis of theΔNcPrx1 strain.The virulence ofΔNcPrx1 strain was analyzed by mouse survival test and pathogenicity test.The results showed that compared with the wild-type（WT）Nc-1 group,the survival rate of C57BL/6 mice infected with theΔNcPrx1 strain was increased,and the infected mice showed reduced load and pathological changes in heart,liver,spleen,lung,kidney and brain tissues,and decreased secretion of IL-6,IL-12,TNF-αand IFN-γin serum.The results indicated that the deletion of NcPrx1 gene led to the reduction of virulence of N.caninum.3.NcPrx1 protein induces pro-inflammatory cytokine secretion from macrophages and the mechanism.（1）Detection of cell viability after stimulation of macrophages by rNcPrx1protein.The safe concentration of rNcPrx1 was screened by cell proliferation and toxicity assay,and the results showed that the safe concentration of rNcPrx1 stimulated mouse peritoneal macrophages was 20μg/m L.（2）rNcPrx1 protein induced pro-inflammatory cytokine assay in macrophages.The secretion levels of pro-inflammatory cytokines IL-6,IL-12 and TNF-αafter rNcPrx1 stimulation of macrophages were detected by ELISA,and the results showed that rNcPrx1 stimulation of macrophages promoted the secretion of IL-6,IL-12 and TNF-α.（3）Effect of TLR2on rNcPrx1 protein-induced pro-inflammatory cytokine secretion by macrophages.The expression of TLR2 after stimulation of macrophages by rNcPrx1was detected using fluorescence quantitative PCR and Western blot,and the results showed that rNcPrx1 protein increased the transcriptional level and expression level of TLR2.Subsequently,TLR2^-/-mouse peritoneal macrophages were further used to detect cytokine secretion by ELISA,and the results showed that TLR2 deficiency decreased the secretion of IL-6,IL-12 and TNF-αin cells compared with wild-type mouse.（4）Analysis of MAPK and AKT pathway activation by rNcPrx1 protein.Changes in the expression and phosphorylation levels of MAPK and AKT signaling pathway-related proteins in rNcPrx1 protein-induced WT and TLR2^-/-mouse peritoneal macrophages were examined by Western blot.The results showed that rNcPrx1 protein caused increased phosphorylation levels of p38,ERK and AKT proteins in peritoneal macrophages of WT mice and decreased in TLR2^-/-mice.Further assay of cytokine secretion after treatment with related pathway inhibitors showed that p38,ERK and AKT inhibitors suppressed rNcPrx1-induced IL-6,IL-12 and TNF-αexpression.In summary,this study screened N.caninum for genes related to resistance to oxidative stress and identified its key regulatory gene NcPrx1.We clarified that NcPrx1is a peroxiredoxin,and elucidated the role of NcPrx1 in N.caninum’s resistance to oxidative stress and its effects on the growth,development and pathogenicity of N.caninum,indicating that NcPrx1 is a N.caninum NcPrx1 was shown to be a virulence factor of N.caninum.It also illustrated that NcPrx1 can activate macrophage MAPK and AKT signaling pathways through TLR2,promote the release of pro-inflammatory cytokines IL-6,IL-12 and TNF-α,and participate in the pathogenic process of N.caninum.It provides a basis for further research on the mechanism of action of N.caninum against oxidative stress,and provides new targets for the development of anti-N.caninum drugs and vaccines.