title: Genetic identification of essential indels and domains in carbamoyl phosphate synthetase II of Toxoplasma gondii.
abstract: New treatments need to be developed for the significant human diseases of toxoplasmosis and malaria to circumvent problems with current treatments and drug resistance. Apicomplexan parasites causing these lethal diseases are deficient in pyrimidine salvage, suggesting that selective inhibition of de novo pyrimidine biosynthesis can lead to a severe loss of uridine 5'-monophosphate (UMP) and thymidine 5'-monophosphate (dTMP) pools, thereby inhibiting parasite RNA and DNA synthesis. Disruption of Toxoplasma gondii carbamoyl phosphate synthetase II (CPSII) induces a severe uracil auxotrophy with no detectable parasite replication in vitro and complete attenuation of virulence in mice. Here we show that a CPSII cDNA minigene efficiently complements the uracil auxotrophy of CPSII-deficient mutants, restoring parasite growth and virulence. Our complementation assays reveal that engineered mutations within, or proximal to, the catalytic triad of the N-terminal glutamine amidotransferase (GATase) domain inactivate the complementation activity of T. gondii CPSII and demonstrate a critical dependence on the apicomplexan CPSII GATase domain in vivo. Surprisingly, indels present within the T. gondii CPSII GATase domain as well as the C-terminal allosteric regulatory domain are found to be essential. In addition, several mutations directed at residues implicated in allosteric regulation in Escherichia coli CPS either abolish or markedly suppress complementation and further define the functional importance of the allosteric regulatory region. Collectively, these findings identify novel features of T. gondii CPSII as potential parasite-selective targets for drug development.
authors.affiliation: Department of Microbiology and Immunology, Dartmouth Medical School, 1 Medical Center Drive, 652E Borwell Building, Lebanon, NH 03756, USA.
authors.forename: Barbara A
authors.initials: BA
authors.lastname: Fox
authors.forename: Jessica G
authors.initials: JG
authors.lastname: Ristuccia
authors.forename: David J
authors.initials: DJ
authors.lastname: Bzik
mesh_terms: D000494:Allosteric Regulation / Q000235:genetics; D000818:Animals; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000172:deficiency / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D017353:Gene Deletion; D005816:Genetic Complementation Test; D054643:INDEL Mutation / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D014122:Toxoplasma / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity; D000637:Transaminases / Q000235:genetics / Q000502:physiology; D014162:Transfection; D014498:Uracil / Q000096:biosynthesis; D014774:Virulence / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D016054:DNA, Protozoan; D014498:Uracil; D000637:Transaminases; C020656:glutamine-pyruvate aminotransferase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)