title: Identification of genes expressed during Toxoplasma gondii infection by in vivo-induced antigen technology (IVIAT) with positive porcine sera.
abstract: Infection of pigs with Toxoplasma gondii is a common source of human toxoplasmosis and causes serious economic losses. In vivo-induced antigen technology (IVIAT) is an effective immunological technique to identify the antigens that a pathogen specifically expressed during infection. To discover the genes that are important in T. gondii infection of pigs, we employed IVIAT using sera from infected pigs. Fourteen antigens were identified including microneme protein 11 (MIC11), dense granule protein 5 (GRA5), 18 kDa cyclophilin (C-18), serine proteinase inhibitor (PI), calmodulin (CaM), leucine-rich repeat protein ( LRRP), D-3-phosphoglycerate dehydrogenase (D3PD), elongation factor 1-gamma (EF1), and 6 hypothetical proteins. The increased transcription levels of 5 (MIC11, GRA5, C-18, PI, and CaM) of the 14 molecules identified by IVIAT were confirmed by real-time PCR. The full length or partial proteins encoded by these 5 genes were expressed in Escherichia coli , and their immunogenicity was confirmed by Western blot analysis with positive porcine sera. Further functional studies were conducted with CaM. Suppression of CaM expression by RNA interference decreased T. gondii tachyzoites cell attachment, invasion, and egress but did not influence their replication. The proteins identified in this study are predicted to be involved in cell invasion, ion-protein binding, protein folding, biosynthesis, and metabolism. The results of the functional analysis support the hypothesis that CaM contributes to parasite pathogenesis during infection. These results may have significant implications for the discovery of candidate molecules for the development of potential therapies and preventive measures against toxoplasmosis in pigs.
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, P. R. China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Tao
authors.forename: Jiang
authors.initials: J
authors.lastname: Xiao
authors.forename: Yifan
authors.initials: Y
authors.lastname: Wang
authors.forename: Kun
authors.initials: K
authors.lastname: Fang
authors.forename: Nizhi
authors.initials: N
authors.lastname: Li
authors.forename: Min
authors.initials: M
authors.lastname: Hu
authors.forename: Yanqin
authors.initials: Y
authors.lastname: Zhou
authors.forename: Junlong
authors.initials: J
authors.lastname: Zhao
mesh_terms: D000327:Adsorption; D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood* / Q000378:metabolism; D000953:Antigens, Protozoan / Q000235:genetics* / Q000276:immunology; D002147:Calmodulin / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D016054:DNA, Protozoan / Q000032:analysis; D005786:Gene Expression Regulation; D015723:Gene Library; D017125:Genes, Protozoan; D006801:Humans; D007106:Immune Sera / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D060888:Real-Time Polymerase Chain Reaction; D013552:Swine; D013553:Swine Diseases / Q000235:genetics* / Q000276:immunology / Q000469:parasitology; D014122:Toxoplasma / Q000235:genetics* / Q000276:immunology; D014124:Toxoplasmosis, Animal / Q000235:genetics* / Q000276:immunology
chemical_list: D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D002147:Calmodulin; D016054:DNA, Protozoan; D007106:Immune Sera; D015800:Protozoan Proteins; D012333:RNA, Messenger