科研之友
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摘要
The orotidine-5'-monophosphate decarboxylase (OMPDC) gene, encoding the final enzyme of the de novo pyrimidine biosynthesis pathway, was deleted using Toxoplasma gondii KU80 knockouts to develop an avirulent nonreverting pyrimidine auxotroph strain. Additionally, to functionally address the role of the pyrimidine salvage pathway, the uridine phosphorylase (UP) salvage activity was knocked out and a double knockout of UP and OMPDC was also constructed. The nonreverting Delta OMPDC, Delta UP, and Delta OMPDC Delta UP knockout strains were evaluated for pyrimidine auxotrophy, for attenuation of virulence, and for their ability to elicit potent immunity to reinfection. The Delta UP knockout strain was replication competent and virulent. In contrast, the Delta OMPDC and Delta OMPDC Delta UP strains were uracil auxotrophs that rapidly lost their viability during pyrimidine starvation. Replication of the Delta OMPDC strain but not the Delta OMPDC Delta UP strain was also partially rescued in vitro with uridine or cytidine supplementation. Compared to their hypervirulent parental type I strain, the Delta OMPDC and Delta OMPDC Delta UP knockout strains exhibited extreme attenuation in murine virulence (similar to 8 logs). Genetic complementation of the Delta OMPDC strain using a functional OMPDC allele restored normal replication and type I parental strain virulence phenotypes. A single immunization of mice with either the live critically attenuated Delta OMPDC strain or the Delta OMPDC Delta UP knockout strain effectively induced potent protective immunity to lethal challenge infection. The avirulent nonreverting Delta OMPDC and Delta OMPDC Delta UP strains provide new tools for the dissection of the host response to infection and are promising candidates for safe and effective Th1 vaccine platforms that can be easily genetically engineered.
- 出版日期2010-9
