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 ⌬OMPDC, ⌬UP, and ⌬OMPDC ⌬UP knockout strains were evaluated for pyrimidine auxotrophy, for attenuation of virulence, and for their ability to elicit potent immunity to reinfection. The ⌬UP knockout strain was replication competent and virulent. In contrast, the ⌬OMPDC and ⌬OMPDC ⌬UP strains were uracil auxotrophs that rapidly lost their viability during pyrimidine starvation. Replication of the ⌬OMPDC strain but not the ⌬OMPDC ⌬UP strain was also partially rescued in vitro with uridine or cytidine supplementation. Compared to their hypervirulent parental type I strain, the ⌬OMPDC and ⌬OMPDC ⌬UP knockout strains exhibited extreme attenuation in murine virulence (ϳ8 logs). Genetic complementation of the ⌬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 ⌬OMPDC strain or the ⌬OMPDC ⌬UP knockout strain effectively induced potent protective immunity to lethal challenge infection. The avirulent nonreverting ⌬OMPDC and ⌬OMPDC ⌬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.

Nonreplicating, Cyst-Defective Type II Toxoplasma gondii Vaccine Strains Stimulate Protective Immunity against Acute and Chronic Infection

“…Yeast recombination was used to fuse 3 distinct genetic elements (a 5= target flank, a hypoxanthine-xanthine-guanine phosphoribosyltransferase gene [HXGPRT] selectable marker, and a 3= target flank) in their correct order in the yeast shuttle plasmid vector pRS416. This recombination was performed using 29-to 36-bp crossovers common to pRS416 and the HXGPRT selectable marker or to the gene targeting flanks (see Table S1 in the supplemental material) (18,34). Gene knockout targeting plasmids were engineered to delete a short region of the gene's predicted 5= untranslated region (UTR) and the entire predicted coding region of the targeted genomic locus (www.ToxoDB.org, version 12.0).…”

“…Disruption of the orotidine 5=-monophosphate decarboxylase gene (OMPDC) in the virulent type I RH ⌬ku80 strain (17) also produced a nonreplicating uracil auxotroph mutant that elicited protective immunity to virulent acute infection (18). The extreme attenuation and avirulence of type I T. gondii uracil auxotrophs are exemplified by survival of severely immune-deficient IFN-␥ Ϫ/Ϫ knockout mice (15, 18, 19) and interleukin-2 receptor ␥ knockout (NOD/SCID/IL-2R␥ Ϫ/Ϫ ) mice (20) that were infected with high-dose challenges of CPS.Vaccination with CPS elicits a lifelong CD8 ϩ T cell-dependent immunity against infection with type I strains (15,18,19,21,22 ϩ T cell responses and highly effective protective immunity to Toxoplasma (21).Previously, vaccination with CPS was shown to protect mice against a virulent acute infection after challenge with a type I or type II strain of T. gondii. However, this CPS vaccination did not completely prevent the development of cysts and chronic infection in mice that were challenged with a type II strain (33).…”

“…CPS is a live attenuated mutant that invades host cells normally but fails to proliferate within its vacuole due to nutrient limitation, and this loss of replication ability causes a complete absence of virulence during infection (15). Disruption of the orotidine 5=-monophosphate decarboxylase gene (OMPDC) in the virulent type I RH ⌬ku80 strain (17) also produced a nonreplicating uracil auxotroph mutant that elicited protective immunity to virulent acute infection (18). The extreme attenuation and avirulence of type I T. gondii uracil auxotrophs are exemplified by survival of severely immune-deficient IFN-␥ Ϫ/Ϫ knockout mice (15,18,19) and interleukin-2 receptor ␥ knockout (NOD/SCID/IL-2R␥ Ϫ/Ϫ ) mice (20) that were infected with high-dose challenges of CPS.…”

“…Vaccination with CPS elicits a lifelong CD8 ϩ T cell-dependent immunity against infection with type I strains (15,18,19,21,22 ϩ T cell responses and highly effective protective immunity to Toxoplasma (21).…”

Bioquímica de parásitos protozoarios

“…Targeted gene disruption experiments demonstrate that the pyrimidine biosynthetic pathway of T. gondii is essential for parasite replication and virulence, and its enzymes are potential drug targets (3,4). Bioinformatic analyses suggest that structural differences between T. gondii and human enzymes may be exploited to produce parasite-specific inhibitors.…”