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.

Figure 1

Domain structure of T. gondii CPSII is shown relative to GATase, Carboxy phosphate (P), and carbamoyl phosphate (P) domains (not to scale). Plasmids Pc 0, Pc 2, and Pc 4 possess 0 kb, 0.5 kb, and 2 kb 5′UTR, respectively, a coding cassette of CPSII cDNA, and 0.92 kb 3′ UTR element.

Figure 2

Virulence of complemented cps1-1 strains. Virulence of strains was determined by intraperitoneal injection of C57BL/6 mice with 2 × 10⁶ tachyzoites of strain cps1-1 (circles), or 2 × 10³ tachyzoites of a cloned isolate of cps-1 complemented with Pc 4 (tiangles), mutant A348 (squares), or mutant 873-910 (diamonds). Groups of 4 mice were infected intraperitoneally with freshly isolated tachyzoites. Data from two experiments is shown as % survival of animals compared to cps1-1 infection.

Figure 3

The T. gondii indels location in the GATase, the CPS.A3 and CPS.B3 domains is illustrated. The CPS domain consists of two halves, A & B, functional synthetase domains which can be subdivided into sections 1, 2, and 3 (A.1, A.2, & A.3 and B.1, B2, & B.3) corresponding to functional subdomains described by the structure of E. coli CPS (Thoden et al., 1999a; Thoden et al., 1999b). A.2 and B.2 correspond to the catalytic subdomains. The binding for MgATP in both domains is wedged between the A.2 and A.3 and B.2 and B.3 domains.