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Isolation of developmentally regulated genes from Toxoplasma gondii by a gene trap with the positive and negative selectable marker hypoxanthine-xanthine-guanine phosphoribosyltransferase.

ABSTRACT: Within its intermediate host, Toxoplasma gondii switches between two forms: a rapidly replicating tachyzoite and an encysted bradyzoite. Bradyzoites persist within the host throughout its life, hidden from antimicrobial agents and the immune system. The signals that mediate switching are poorly understood. A gene trap was employed to isolate genes whose expression is up-regulated early in the switching of bradyzoites via the negative and positive selectable marker hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT). T. gondii was transfected with promoterless HXGPRT and negatively selected with 6-thioxanthine to inhibit the growth of tachyzoites expressing HXGPRT. The surviving tachyzoites were then induced for in vitro bradyzoite formation and treated with mycophenolic acid and xanthine to positively select for parasites in which the construct had integrated downstream of a bradyzoite-specific gene. Strains were checked for their ability to differentiate by using Dolichos biflorus agglutinin (a bradyzoite-specific lectin) and a monoclonal antibody against P36 (a bradyzoite-specific surface antigen). After differentiation, all gene-trapped clones had Dolichos immunofluorescence and all but one expressed P36. The sequences flanking the insertion site of this P36-negative strain were homologous to the Toxoplasma family of surface antigens, strongly suggesting that P36 is encoded by the disruptive gene. Genetic mapping and complementation of the P36-negative strain further indicated that the disrupted gene is P36. Reverse transcriptase PCR and S1 nuclease digestion were used to compare mRNA levels during the tachyzoite and bradyzoite stages. The presumptive P36 gene does not appear to regulate its mRNA levels between the two stages, indicating a posttranscriptional mechanism of regulation for early bradyzoite-specific genes.

SUBMITTER: Knoll LJ

PROVIDER: S-EPMC108792 | biostudies-literature | 1998 Feb

REPOSITORIES: biostudies-literature

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Isolation of developmentally regulated genes from Toxoplasma gondii by a gene trap with the positive and negative selectable marker hypoxanthine-xanthine-guanine phosphoribosyltransferase.

Knoll L J LJ Boothroyd J C JC

Molecular and cellular biology 19980201 2

Within its intermediate host, Toxoplasma gondii switches between two forms: a rapidly replicating tachyzoite and an encysted bradyzoite. Bradyzoites persist within the host throughout its life, hidden from antimicrobial agents and the immune system. The signals that mediate switching are poorly understood. A gene trap was employed to isolate genes whose expression is up-regulated early in the switching of bradyzoites via the negative and positive selectable marker hypoxanthine-xanthine-guanine p ...[more]

PMID: 9447977

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Project description:To date, Plasmodium falciparum is one of the most lethal strains of the malaria parasite. P. falciparum lacks the required enzymes to create its own purines via the de novo pathway, thereby making Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (PfHGXPT) a crucial enzyme in the malaria life cycle. Recently, studies have described iso-mukaadial acetate and ursolic acid acetate as promising antimalarials. However, the mode of action is still unknown, thus, the current study sought to investigate the selective inhibitory and binding actions of iso-mukaadial acetate and ursolic acid acetate against recombinant PfHGXPT using in-silico and experimental approaches. Recombinant PfHGXPT protein was expressed using E. coli BL21 cells and homogeneously purified by affinity chromatography. Experimentally, iso-mukaadial acetate and ursolic acid acetate, respectively, demonstrated direct inhibitory activity towards PfHGXPT in a dose-dependent manner. The binding affinity of iso-mukaadial acetate and ursolic acid acetate on the PfHGXPT dissociation constant (KD), where it was found that 0.0833 µM and 2.8396 µM, respectively, are indicative of strong binding. The mode of action for the observed antimalarial activity was further established by a molecular docking study. The molecular docking and dynamics simulations show specific interactions and high affinity within the binding pocket of Plasmodium falciparum and human hypoxanthine-guanine phosphoribosyl transferases. The predicted in silico absorption, distribution, metabolism and excretion/toxicity (ADME/T) properties predicted that the iso-mukaadial acetate ligand may follow the criteria for orally active drugs. The theoretical calculation derived from ADME, molecular docking and dynamics provide in-depth information into the structural basis, specific bonding and non-bonding interactions governing the inhibition of malarial. Taken together, these findings provide a basis for the recommendation of iso-mukaadial acetate and ursolic acid acetate as high-affinity ligands and drug candidates against PfHGXPT.

Dataset Information

Isolation of developmentally regulated genes from Toxoplasma gondii by a gene trap with the positive and negative selectable marker hypoxanthine-xanthine-guanine phosphoribosyltransferase.

Publications

Isolation of developmentally regulated genes from Toxoplasma gondii by a gene trap with the positive and negative selectable marker hypoxanthine-xanthine-guanine phosphoribosyltransferase.

Similar Datasets

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