title: Avirulent uracil auxotrophs based on disruption of orotidine-5'-monophosphate decarboxylase elicit protective immunity to Toxoplasma gondii.
abstract: 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 DeltaOMPDC, DeltaUP, and DeltaOMPDC DeltaUP knockout strains were evaluated for pyrimidine auxotrophy, for attenuation of virulence, and for their ability to elicit potent immunity to reinfection. The DeltaUP knockout strain was replication competent and virulent. In contrast, the DeltaOMPDC and DeltaOMPDC DeltaUP strains were uracil auxotrophs that rapidly lost their viability during pyrimidine starvation. Replication of the DeltaOMPDC strain but not the DeltaOMPDC DeltaUP strain was also partially rescued in vitro with uridine or cytidine supplementation. Compared to their hypervirulent parental type I strain, the DeltaOMPDC and DeltaOMPDC DeltaUP knockout strains exhibited extreme attenuation in murine virulence (approximately 8 logs). Genetic complementation of the DeltaOMPDC 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 DeltaOMPDC strain or the DeltaOMPDC DeltaUP knockout strain effectively induced potent protective immunity to lethal challenge infection. The avirulent nonreverting DeltaOMPDC and DeltaOMPDC DeltaUP 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.
authors.affiliation: Department of Microbiology and Immunology, Dartmouth Medical School, 1 Medical Center Drive, Lebanon, NH 03756, USA. barbara.a.fox@dartmouth.edu
authors.forename: Barbara A
authors.initials: BA
authors.lastname: Fox
authors.forename: David J
authors.initials: DJ
authors.lastname: Bzik
mesh_terms: D000818:Animals; D051379:Mice; D008810:Mice, Inbred C57BL; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000502:physiology*; D011743:Pyrimidines / Q000096:biosynthesis; D014122:Toxoplasma / Q000378:metabolism* / Q000472:pathogenicity; D014123:Toxoplasmosis / Q000276:immunology*; D014498:Uracil / Q000378:metabolism*; D014543:Uridine Phosphorylase / Q000502:physiology; D014774:Virulence
chemical_list: D011743:Pyrimidines; D014498:Uracil; D014543:Uridine Phosphorylase; D009964:Orotidine-5'-Phosphate Decarboxylase; C030986:pyrimidine