| Background: Toxoplasma gondii(T. gondii) is an obligate intracellular haploid protozoan that infects virtually all warm blooded animals, including humans, livestock, birds, marine mammals and a wide range of wildlife. Nearly one-third of the adult human population in the world has been exposed to this protozoan parasite. Human infections mainly due to ingestion of undercooked meat containing tissue cysts or water having contaminated oocysts that shed in the feces of infected cats. It also can cause severe damage to livestock, resulting in huge losses in economics.The genetic diversity of T. gondii in various geographical regions using restriction fragment length polymorphism(RFLP) genotyping has been investigated in previous studies. In North America and Europe, a highly clonal population structure with three major lineages, designated as typeⅠ, Ⅱ and Ⅲ, was described. However, in South America, the genetic polymorphism was complex and comprises a large number of distinct genotypes. An atypical genotype was identified as the predominant clonal lineage in China(Chinese 1, Toxo DB#9).In RFLP-PCR genotyping methodology, most of the markers shown two or three alleles except for a few markers, such as c B21-4 locus, in which investigators have found seven types of alleles. The reason for that might be the specific locus covers a microsatellite region, where multiple allelic variables are much common. Allelic hypervariability in this type of loci might be explained by an accumulation of length modification by polymerase slippage on mutations. As rapidly evolving neutral markers, microsatellites(MS) are wonderful tools for identifying among strains and analyzing the genetic diversity of population structure.Our previous researches revealed a limited diversity with a dominance of Chinese 1 lineage in T. gondii strains. When mice were inoculated with 1000 tachyzoites, the Chinese 1 type isolates showed different virulence to mice and certain isolates generated an abundance of cysts in the brain of infected animals. However, the underlying reason for this distinct phenomenon remains not fully discussed.In the present study, to deeply understand the genetic background of T. gondii isolates from different districts of China, MS markers were used to precisely identify the haplotypes of T. gondii and make clear of the Chinese population structure. Further dose-dependent virulence in mice and cell invasion assay were also experimented. Additionally, the genotype-based dense granule protein 3(GRA3) was constructed into p EGFP-C2 plasmid and then transfected into 293 T cells to explore the biological function of GRA3 protein.Objective: To identify the microsatellite genotyping, phylogeny and virulence divergency of Toxoplasma gondii isolates from China, to briefly explore the potential biological function of GRA3 protein.Methods: Genotyping of 28 Chinese T. gondii isolates were carried out with 15 MS markers. Results were analyzed using population structure by a Bayesian statistical approach. Phylogenetic analysis was obtained from Split-tree application. The virulence analysesof some representative strains were performed by mouse inoculation and cell invasion. The recombinant eukaryotic expression vector p EGFP-GRA3 was constructed and then transfected into 293 T cells. Apoptosis-associated gene PCR arrays were carried out in the transfected eukaryote. Western blotting, quantitative real-time PCR(q RT- PCR), and flow cytometry were performed to observe the potentially apoptosis resistance in p EGFP-GRA3 transfected cells.Results: Three haplotypes were grouped among the twenty-eight isolates although minor genetic difference was found within haplogroup. The majority of isolates belonged to one haplogroup corresponding to the previously described Chinese 1genotype(Toxo DB #9). Phylogenetic networks revealed a limited diversity in Chinese T. gondii strains. The virulence different isolates Wh3 and Wh6 strains shared the common genotype. Dense granule protein3 was found to have a higher expression in low virulent Wh6 strain than that in high virulent Wh3 strain. The recombinant eukaryotic expression vector p EGFPGRA3 was transfected into 293 T. Apoptosis associated gene PCR array showed that 3 genes(tradd, il-10 and grdd45a) were up-regulated and casp4 was dramatically decreased by GRA3 transfection. Western blotting revealed Caspase-3 and PARP-1 were lessened. The q RT-PCR verified the similar results in apoptosis-associated genes.Conclusions: The results of microsatellite genotyping data from Chinese T. gondii strains revealed a high polymorphism and limited genetic diversity in China strains and the phylogenetic network analyses showed limited divergency although the strain virulence differs in the Chinese 1 type of T. gondii which is predominantly prevalent in China. GRA3 protein could be involved in the virulence difference in Chinese 1T.gondii strains and may take an important role in host cell resistance to apoptosis in cyst-forming T. gondii. |
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