Document Detail


Genetic identification of essential indels and domains in carbamoyl phosphate synthetase II of Toxoplasma gondii.
MedLine Citation:
PMID:  18992249     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
Barbara A Fox; Jessica G Ristuccia; David J Bzik
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2008-10-21
Journal Detail:
Title:  International journal for parasitology     Volume:  39     ISSN:  1879-0135     ISO Abbreviation:  Int. J. Parasitol.     Publication Date:  2009 Apr 
Date Detail:
Created Date:  2009-02-16     Completed Date:  2010-01-22     Revised Date:  2014-09-10    
Medline Journal Info:
Nlm Unique ID:  0314024     Medline TA:  Int J Parasitol     Country:  England    
Other Details:
Languages:  eng     Pagination:  533-9     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Allosteric Regulation / genetics
Animals
Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / deficiency,  genetics*
DNA, Complementary / genetics
DNA, Protozoan / genetics
Gene Deletion
Genetic Complementation Test
INDEL Mutation / genetics*
Mice
Mice, Inbred C57BL
Mutagenesis, Site-Directed
Plasmids / genetics
Protein Structure, Tertiary / genetics
Toxoplasma / enzymology,  genetics*,  growth & development,  pathogenicity
Transaminases / genetics,  physiology
Transfection
Uracil / biosynthesis
Virulence / genetics
Grant Support
ID/Acronym/Agency:
AI41930/AI/NIAID NIH HHS; R01 AI041930/AI/NIAID NIH HHS; R01 AI041930-11A1/AI/NIAID NIH HHS; R21 AI073142/AI/NIAID NIH HHS; R21 AI075931/AI/NIAID NIH HHS
Chemical
Reg. No./Substance:
0/DNA, Complementary; 0/DNA, Protozoan; 56HH86ZVCT/Uracil; EC 2.6.1.-/Transaminases; EC 2.6.1.15/glutamine-pyruvate aminotransferase; EC 6.3.5.5/Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)
Comments/Corrections

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