title: Analysis of the virulence determination mechanisms in a local Toxoplasma strain (T.gHB1) isolated from central China.
abstract: Several rhoptry proteins (ROPs) have been confirmed to be critical virulence factors of Toxoplasma gondii strains from North America and Europe. The two active kinases ROP17 and ROP18, and pseudokinase ROP5 were thought to be the key determinants of parasites' virulence in laboratory mice. Given the genetic diversity of Toxoplasma strains from different geographical regions, the virulence determinants in other strains, particularly the ones that are phylogenetically distant to the North American and European strains, are yet to be elucidated. In this study, we sought to examine the contribution of three known virulence factors to the virulence of a type I strain (T.gHB1) isolated from Central China. We deleted ROP17 and ROP18 individually, as well as in combination with GRA7 by the CRISPR-Cas9 system in this local isolate. Subsequent virulence tests in mice indicated that deletion of GRA7, ROP17, or ROP18 in T.gHB1showed similar attenuation in mice as the type I RH strain lacking the corresponding proteins. However, in contrast to the reported double knockouts in RH, double deletions of GRA7 plus ROP17 or GRA7 plus ROP18 in T.gHB1 did not show significant further virulence attenuation compared to the ROP17 or ROP18 single knockouts. These results indicated that GRA7, ROP18 and ROP17 may play different roles in virulence determination in genetically diverse strains of Toxoplasma.
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, Hubei, China.|Key Laboratory for development of veterinary diagnostic products, Ministry of Agriculture, Huazhong Agricultural University, Wuhan, 430070, Hubei, China.
authors.forename: Weichao
authors.initials: W
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, Hubei, China.|Key Laboratory for development of veterinary diagnostic products, Ministry of Agriculture, Huazhong Agricultural University, Wuhan, 430070, Hubei, China.
authors.forename: Longjiao
authors.initials: L
authors.lastname: Li
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, Hubei, China.|Key Laboratory for development of veterinary diagnostic products, Ministry of Agriculture, Huazhong Agricultural University, Wuhan, 430070, Hubei, China.
authors.forename: Ningbo
authors.initials: N
authors.lastname: Xia
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, Hubei, China.|Key Laboratory for development of veterinary diagnostic products, Ministry of Agriculture, Huazhong Agricultural University, Wuhan, 430070, Hubei, China.
authors.forename: Yanqin
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, Hubei, China.|Key Laboratory for development of veterinary diagnostic products, Ministry of Agriculture, Huazhong Agricultural University, Wuhan, 430070, Hubei, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Fang
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, Hubei, China.|Key Laboratory for development of veterinary diagnostic products, Ministry of Agriculture, Huazhong Agricultural University, Wuhan, 430070, Hubei, China.
authors.forename: Lan
authors.initials: L
authors.lastname: He
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, Hubei, China.|Key Laboratory for development of veterinary diagnostic products, Ministry of Agriculture, Huazhong Agricultural University, Wuhan, 430070, Hubei, China.
authors.forename: Min
authors.initials: M
authors.lastname: Hu
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, Hubei, China. shenbang@mail.hzau.edu.cn.|Key Laboratory for development of veterinary diagnostic products, Ministry of Agriculture, Huazhong Agricultural University, Wuhan, 430070, Hubei, China. shenbang@mail.hzau.edu.cn.
authors.forename: Bang
authors.initials: B
authors.lastname: Shen
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, Hubei, China. zhaojunlong@mail.hzau.edu.cn.|Key Laboratory for development of veterinary diagnostic products, Ministry of Agriculture, Huazhong Agricultural University, Wuhan, 430070, Hubei, China. zhaojunlong@mail.hzau.edu.cn.|Hubei Cooperative Innovation Center for Sustainable Pig Production, Wuhan, 430070, Hubei, China. zhaojunlong@mail.hzau.edu.cn.
authors.forename: Junlong
authors.initials: J
authors.lastname: Zhao
mesh_terms: D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics / Q000378:metabolism*; D002681:China; D005260:Female; D014644:Genetic Variation; D006801:Humans; D051379:Mice; D008813:Mice, Inbred ICR; D010802:Phylogeny; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D014122:Toxoplasma / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000472:pathogenicity*; D014123:Toxoplasmosis / Q000469:parasitology*; D014774:Virulence; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D000953:Antigens, Protozoan; C112573:GRA7 protein, Toxoplasma gondii; D015800:Protozoan Proteins; D037521:Virulence Factors; D017346:Protein Serine-Threonine Kinases; C556793:ROP18 protein, Toxoplasma gondii; C000611668:rhoptry protein 17, Toxoplasma gondii
keywords: CRISPR-Cas9 system; Gene knockout; Rhoptry proteins (ROPs); Toxoplasma gondii; Virulence factors