Fitness effects of DHFR‐TS mutations associated with pyrimethamine resistance in apicomplexan parasites

Fohl, Leah M.; Roos, David S.

doi:10.1046/j.1365-2958.2003.03756.x

Cited by 41 publications

(30 citation statements)

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“…Our initial characterization of the TgIF2α-S71A parasites indicated that the mutants took longer to lyse monolayers of host cells compared with WT. To directly assess whether the mutant parasites had decreased fitness relative to WT, we performed "headto-head" competition assays (17) in which an equal number of WT and TgIF2α-S71A parasites were inoculated into the same culture flask of host cells, human foreskin fibroblasts (HFF) ( Fig. 2A).…”

Section: Resultsmentioning

confidence: 99%

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

Joyce

Queener²,

Wek

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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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

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Section: Resultsmentioning

confidence: 99%

“…The comparative fitness assay was based on a protocol outlined in ref. 17. After filter purification, 5 × 10 5 parental ΔKu80 (WT) and TgIF2α-S71A mutant parasites were mixed in 10 mL DMEM + 1% FBS and added to a T-25 cm 2 flask containing a monolayer of HFF host cells.…”

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

Proc. Natl. Acad. Sci. U.S.A.

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“…More recently, however, we have found that sensitive competition assays enable the detection of subtle fitness defects in other mutants (22). As shown in Fig.…”

Section: Expression Of L Donovani Aprt In T Gondii-to Furthermentioning

confidence: 96%

“…Fitness Assays-Competition assays between the parental wild-type and ⌬HXGPRT and ⌬AK parasites were performed as described (22). For detection of viable wild-type or knockout parasites at various timepoints, plaque assays were performed in 6-well plates in the presence of suitable selective drugs (15).…”

Section: Expression Of Leishmania Donovani Aprt In T Gondii-mentioning

confidence: 99%

Purine Salvage Pathways in the Apicomplexan Parasite Toxoplasma gondii

Chaudhary

Darling

Fohl

et al. 2004

Journal of Biological Chemistry

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106

104

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We have exploited a variety of molecular genetic, biochemical, and genomic techniques to investigate the roles of purine salvage enzymes in the protozoan parasite Toxoplasma gondii. The ability to generate defined genetic knockouts and target transgenes to specific loci demonstrates that T. gondii uses two (and only two) pathways for purine salvage, defined by the enzymes hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) and adenosine kinase (AK). Both HXGPRT and AK are single-copy genes, and either one can be deleted, indicating that either one of these pathways is sufficient to meet parasite purine requirements. Fitness defects suggest both pathways are important for the parasite, however, and that the salvage of adenosine is more important than salvage of hypoxanthine and other purine nucleobases. HXGPRT and AK cannot be deleted simultaneously unless one of these enzymes is provided in trans, indicating that alternative routes of functionally significant purine salvage are lacking. Despite previous reports to the contrary, we found no evidence of adenine phosphoribosyltransferase (APRT) activity when parasites were propagated in APRT-deficient host cells, and no APRT ortholog is evident in the T. gondii genome. Expression of Leishmania donovani APRT in transgenic T. gondii parasites yielded low levels of activity but did not permit genetic deletion of both HXGPRT and AK. A detailed comparative genomic study of the purine salvage pathway in various apicomplexan species highlights important differences among these parasites.Like all parasitic protozoa, the obligate intracellular parasite Toxoplasma gondii lacks the ability to synthesize the purine ring de novo, and thus relies entirely on the salvage of purines from the host cell to meet its nutritional needs (1-3). This requirement, coupled with the shortcomings of conventional therapies for treating congenital toxoplasmosis and opportunistic infections associated with AIDS and other immunosuppressive conditions (4 -8), makes purine salvage an attractive target for chemotherapy.The purine metabolism of T. gondii has previously been examined biochemically, resulting in the identification of various activities capable of assimilating nucleosides and nucleobases from the host cell into the purine nucleotide pools of the parasite (2, 3). (See "Discussion" for a model of the purine salvage pathway in Toxoplasma and other apicomplexan parasites.) Reported salvage activities include the phosphoribosylation of adenine, guanine, hypoxanthine, and xanthine, and the phosphorylation of adenosine. The latter seems to contribute most significantly to parasite purine economy, as adenosine is incorporated into nucleotide pools at a considerably higher rate than any purine nucleobase (2, 3).Most of the reported salvage activities can be accounted for by two enzymes: hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) 1 and adenosine kinase (AK). The genes for both have been cloned and expressed in bacterial systems, and the purified proteins have be...

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“…It has been shown in a wide variety of organisms that acquisition of drug resistance can incur a biological cost (Bjorkman et al ., 2000;Nagaev et al ., 2001;Fohl and Roos, 2003;Lu et al ., 2004;Mariam et al ., 2004). Genetic mutations that alter the structure or function of key proteins in order to evade the actions of deleterious compounds may also compromise the normal function of those proteins.…”

Section: Introductionmentioning

confidence: 99%

pfmdr1 mutations associated with chloroquine resistance incur a fitness cost in Plasmodium falciparum

Hayward

Saliba

Kirk

2005

Molecular Microbiology

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SummaryEfforts to control malaria worldwide have been hindered by the development and expansion of parasite populations resistant to many first-line antimalarial compounds. Two of the best-characterized determinants of drug resistance in the human malaria parasite Plasmodium falciparum are pfmdr1 and pfcrt , although the mechanisms by which resistance is mediated by these genes is still not clear. In order to determine whether mutations in pfmdr1 associated with chloroquine resistance affect the capacity of the parasite to persist when drug pressure is removed, we conducted competition experiments between P. falciparum strains in which the endogenous pfmdr1 locus was modified by allelic exchange. In the absence of selective pressure, the component of chloroquine resistance attributable to mutations at codons 1034, 1042 and 1246 in the pfmdr1 gene also gave rise to a substantial fitness cost in the intraerythrocytic asexual stage of the parasite. The loss of fitness incurred by these mutations was calculated to be 25% with respect to an otherwise genetically identical strain in which wild-type polymorphisms had been substituted at these three codons. At least part of the fitness loss may be attributed to a diminished merozoite viability. These in vitro results support recent in vivo observations that in several countries where chloroquine use has been suspended because of widespread resistance, sensitive strains are re-emerging.

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Fitness effects of DHFR‐TS mutations associated with pyrimethamine resistance in apicomplexan parasites

Cited by 41 publications

References 40 publications

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

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

Purine Salvage Pathways in the Apicomplexan Parasite Toxoplasma gondii

pfmdr1 mutations associated with chloroquine resistance incur a fitness cost in Plasmodium falciparum

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