In vitro assays elucidate peculiar kinetics of clindamycin action against Toxoplasma gondii

125

141

Intraerythrocytic malaria parasites can obtain nearly their entire amino acid requirement by degrading host cell hemoglobin. The sole exception is isoleucine, which is not present in adult human hemoglobin and must be obtained exogenously. We evaluated two compounds for their potential to interfere with isoleucine utilization. Mupirocin, a clinically used antibacterial, kills Plasmodium falciparum parasites at nanomolar concentrations. Thiaisoleucine, an isoleucine analog, also has antimalarial activity. To identify targets of the two compounds, we selected parasites resistant to either mupirocin or thiaisoleucine. Mutants were analyzed by genome-wide high-density tiling microarrays, DNA sequencing, and copy number variation analysis. The genomes of three independent mupirocin-resistant parasite clones had all acquired either amplifications encompassing or SNPs within the chromosomally encoded organellar (apicoplast) isoleucyl-tRNA synthetase. Thiaisoleucine-resistant parasites had a mutation in the cytoplasmic isoleucyl-tRNA synthetase. The role of this mutation in thiaisoleucine resistance was confirmed by allelic replacement. This approach is generally useful for elucidation of new targets in P. falciparum . Our study shows that isoleucine utilization is an essential pathway that can be targeted for antimalarial drug development.

Section: Thiaisoleucine and Mupirocin Interact With Isoleucine Utilizsupporting

confidence: 63%

Validation of isoleucine utilization targets in Plasmodium falciparum

Istvan

Dharia

Bopp

et al. 2011

125

141

“…At least some of its functions are essential for the parasite and are responsible for its conservation during evolution. Macrolide (and other) antibiotics targeting apicoplast ribosomes (30)(31)(32) and fluoroquinolone antibiotics eliminating the apicoplast genome (33) kill the parasite. Thiolactomycin, an inhibitor of fatty acid elongation specific for the multisubunit enzyme found in the apicoplast, has been shown to prevent the growth of P. falciparum (5).…”

Section: T Gondii Accs and Theirmentioning

confidence: 99%

Subcellular localization of acetyl-CoA carboxylase in the apicomplexan parasite Toxoplasma gondii

Jeleńska

Crawford

Harb

et al. 2001

108

Apicomplexan parasites such as Toxoplasma gondii contain a primitive plastid, the apicoplast, whose genome consists of a 35-kb circular DNA related to the plastid DNA of plants. Plants synthesize fatty acids in their plastids. The first committed step in fatty acid synthesis is catalyzed by acetyl-CoA carboxylase (ACC). This enzyme is encoded in the nucleus, synthesized in the cytosol, and transported into the plastid. In the present work, two genes encoding ACC from T. gondii were cloned and the gene structure was determined. Both ORFs encode multidomain proteins, each with an N-terminal extension, compared with the cytosolic ACCs from plants. The N-terminal extension of one isozyme, ACC1, was shown to target green fluorescent protein to the apicoplast of T. gondii . In addition, the apicoplast contains a biotinylated protein, consistent with the assertion that ACC1 is localized there. The second ACC in T. gondii appears to be cytosolic. T. gondii mitochondria also contain a biotinylated protein, probably pyruvate carboxylase. These results confirm the essential nature of the apicoplast and explain the inhibition of parasite growth in cultured cells by herbicides targeting ACC.

“…Toxoplasma doubling assays were performed as previously described (24). Intracellular ΔKu80 and TgIF2α-S71A parasites were physically removed from host cells by syringe passage.…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation of eukaryotic initiation factor-2α promotes the extracellular survival of obligate intracellular parasite Toxoplasma gondii

Joyce

Queener²,

Wek

et al. 2010

While seeking a new host cell, obligate intracellular parasites, such as the protozoan Toxoplasma gondii, must be able to endure the stress of an extracellular environment. The mechanisms Toxoplasma use to remain viable while deprived of a host cell are not understood. We have previously shown that phosphorylation of Toxoplasma eukaryotic initiation factor-2α (TgIF2α) is a conserved response to stress. Here we report the characterization of Toxoplasma harboring a point mutation (S71A) in TgIF2α that prevents phosphorylation. Results show that TgIF2α phosphorylation is critical for parasite viability because the TgIF2α-S71A mutants are illequipped to cope with life outside the host cell. The TgIF2α-S71A mutants also showed a significant delay in producing acute toxoplasmosis in vivo. We conclude that the phosphorylation of TgIF2α plays a crucial role during the lytic cycle by ameliorating the stress of the extracellular environment while the parasite searches for a new host cell.Apicomplexa | toxoplasmosis | stress | translation control | eIF2