Publisher Summary The genetic structure of Toxoplasma gondii is notable chiefly for being relatively conventional— similar to that of its mammalian host cells with respect to gene organization, codon usage, and nucleotide bias. These observations have led several investigators to examine the feasibility of molecular transformation in this parasite. This chapter outlines the use of several of the molecular genetic tools that have recently been developed for the T. gondii system. An introduction to parasite culture techniques is also provided. Recombinant molecules can be expressed either transiently or as stable transformants, as episomes or integrated into the parasite genome, and as single copy or multicopy transgenes. Stable integration can be produced by random nonhomologous recombination, single-site homologous recombination, or perfect gene replacement. Many of these outcomes can be selected specifically using appropriate vectors and transformation conditions. The extraordinarily high frequencies of stable transformation observed permit cloning by complementation, insertional mutagenesis/marker rescue, gene knock-outs, and allelic replacement. In combination with available classical and “cell-genetic” possibilities and physical and genetic mapping strategies, these tools provide a powerful arsenal for investigations into the biology of intracellular parasitism.

Molecular tools for genetic dissection of the protozoan parasite Toxoplasma gondii.

Insertional tagging, cloning, and expression of the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyltransferase gene. Use as a selectable marker for stable transformation.

Purine and pyrimidine metabolism in the trypanosomatidae

Publisher Summary This chapter discusses the metabolism of purine and pyrimidine nucleotides in parasitic protozoa and helminths. They are the monomeric units of both DNA and RNA; ATP serves as the universal cellular energy source, adenine nucleotides are components of three key coenzymes (NAD+, FAD and CoA), they are used to form activated intermediates, such as uridine-diphosphate glucose, and they serve as metabolic regulators. The results of studies by investigators show that purine metabolism in these pathogens differs from that of their mammalian hosts. Whereas mammalian cells synthesize the purine ring de novo from simple precursors, all the parasitic protozoa and helminths that have been investigated lack de novo purine synthesis and utilize preformed purines obtained from their host environment. These pathogens use various purine salvage pathways to supply their purine nucleotide requirements. The chapter explains that the difference in purine metabolism between host and parasite is an area of potential chemotherapeutic intervention that is actively being explored. Unlike purines, pyrimidine metabolism,with the exception of the amitochondrial protists Giardia lamblia, Trichomonas vaginalis and Tritrichomonas foetus, is similar to mammalian cells, all pathogenic protozoa and helminths are able to synthesize pyrimidines de novo. Like mammalian cells, their ability to salvage pyrimidines is limited when compared to purine salvage. The chapter summarizes that with the exception of G.lamblia and T.vaginallis, all the parasitic organisms discussed in the chapter possess ribonucleotide reductase and can synthesize purine deoxynucleotides based on one or more of the following criteria: organisms multiply in the absence of deoxynucleosides, reductase activity can be detected in cell extracts, bases or ribonucleosides are incorporated into DNA or organisms are sensitive to growth inhibition by hyroxyurea (a ribonuleotide reductase inhibitor).

Purine and Pyrimidine Metabolism

Nonhomologous integration vectors have been used to demonstrate the feasibility of insertional mutagenesis in haploid tachyzoites of the protozoan parasite Toxoplasma gondii. Mutant clones resistant to 5-fluorouracil were identified at a frequency of approximately 10(-6) (approximately 2 x 10(-5) of the stable transformants). Four independent mutants were isolated, all of which were shown to lack uracil phosphoribosyl-transferase (UPRT) activity and harbor transgenes integrated at closely linked loci, suggesting inactivation of the UPRT-encoding gene. Genomic DNA flanking the insertion point (along with the integrated vector) was readily recovered by bacterial transformation with restriction-digested, self-ligated total genomic DNA. Screening of genomic libraries with the recovered fragment identified sequences exhibiting high homology to known UPRT-encoding genes from other species, and cDNA clones were isolated that contain a single open reading frame predicted to encode the 244-amino acid enzyme. Homologous recombination vectors were exploited to create genetic knock-outs at the UPRT locus, which are deficient in enzyme activity but can be complemented by transient transformation with wild-type sequences--formally confirming identification of the functional UPRT gene. Mapping of transgene insertion points indicates that multiple independent mutants arose from integration at distinct sites within the UPRT gene, suggesting that nonhomologous integration is sufficiently random to permit tagging of the entire parasite genome in a single transformation.

Insertional mutagenesis and marker rescue in a protozoan parasite : cloning of the uracil phosphoribosyltransferase locus from toxoplasma gondii

Pyrimidine biosynthesis was studied in actively dividing, intracellular Toxoplasma gondii, in mutant Chinese hamster ovary cells blocked in pyrimidine biosynthesis to eliminate any contribution by the host cell. The parasite grew normally in these cells even though pyrimidines were not supplied in the medium. Uninfected, mutant cultures showed negligible pyrimidine synthesis. However, mutant cultures infected wit T. gondii efficiently incorporated 14C from glucose or aspartic acid into the pyrimidine bases of nucleic acids. Thus T. gondii is capable of de novo pyrimidine biosynthesis. The parasite may also be able to use pyrimidines of the host cell, because of pyrazofurin, an antimetabolite that blocks pyrimidine biosynthesis, markedly inhibited only a mutant parasite defective in the salvage of pyrimidines.

Joseph D. Schwartzman

Papers

Pyrimidine synthesis by intracellular Toxoplasma gondii.