Robert G. K. Donald

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

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.

Origin, targeting, and function of the apicomplexan plastid

The discovery of a plastid in Plasmodium, Toxoplasma and related protozoan parasites provides a satisfying resolution to several long-standing mysteries: the mechanism of action for various surprisingly effective antibiotics; the subcellular location of an enigmatic 35 kb episomal DNA; and the nature of an unusual intracellular structure containing multiple membranes. The apicomplexan plastid highlights the importance of lateral genetic transfer in evolution and provides an accessible system for the investigation of protein targeting to secondary endosymbiotic organelles. Combining molecular genetic identification of targeting signals with whole genome analysis promises to yield a complete picture of organellar metabolic pathways and new targets for drug design.

Shikimate pathway in apicomplexan parasites

The discovery of plastids in apicomplexan parasites raised the possibility that these organelles might harbour plastid-specific metabolic activities that could be blocked by therapeutic agents. Ideally, these agents would inhibit the parasites without harming their vertebrate hosts. Roberts et al. have made the promising dicovery that Apicomplexa are sensitive to the herbicide glyphosate (better known by its trade names of RoundUp, Zero or Tumbleweed), which is an inhibitor of the enzyme 5-enopyruvyl shikimate 3-phosphate synthase. They suggested that production of aromatic amino acids by the pathway involving this enzyme, the shikimate pathway, might be an essential function of the apicomplexan plastid, but here we present evidence that this pathway actually operates in the cytosol of Apicomplexa.

Identification and characterization of differentiation mutants in the protozoan parasite Toxoplasma gondii

Two forms of the protozoan parasite Toxoplasma gondii are associated with intermediate hosts such as humans: rapidly growing tachyzoites are responsible for acute illness, whereas slowly dividing encysted bradyzoites can remain latent within the tissues for the life of the host. In order to identify genetic factors associated with parasite differentiation, we have used a strong bradyzoite‐specific promoter (identified by promoter trapping) to drive the expression of T. gondii hypoxanthine–xanthine–guanine phosphoribosyltransferase (HXGPRT) in stable transgenic parasites, providing a stage‐specific positive/negative selectable marker. Insertional mutagenesis has been carried out on this parental line, followed by bradyzoite induction in vitro and selection in 6‐thioxanthine to identify misregulation mutants. Two different mutants fail to induce the HXGPRT gene efficiently during bradyzoite differentiation. These mutants are also defective in other aspects of differentiation: they replicate well under bradyzoite growth conditions, lysing the host cell monolayer as effectively as tachyzoites. Expression of the major bradyzoite antigen BAG1 is reduced, and staining with Dolichos biflorus lectin shows reduced cyst wall formation. Microarray hybridizations show that these mutants behave more like tachyzoites at a global level, even under bradyzoite differentiation conditions.

Insertional tagging of at least two loci associated with resistance to adenine arabinoside in Toxoplasma gondii, and cloning of the adenosine kinase locus

A genetic approach has been exploited to investigate adenylate salvage pathways in the protozoan parasite Toxoplasma gondii, a purine auxotroph. Using a new insertional mutagenesis vector designed to facilitate the rescue of tagged loci even when multiple plasmids integrate as a tandem array, 15 independent clonal lines resistant to the toxic nucleoside analog adenine arabinoside (AraA) were generated. Approximately two-thirds of these clones lack adenosine kinase (AK) activity. Parallel studies identified an expressed sequence tag (EST) exhibiting a small region of weak similarity to human AK, and this locus was tagged in several AK-deficient insertional mutants. Library screening yielded full-length cDNA and genomic clones. The T. gondii AK gene contains five exons spanning a approximately 3 kb locus, and the predicted coding sequence was employed to identify additional AK genes and cDNAs in the GenBank and dbEST databases. A genomic construct lacking essential coding sequence was used to create defined genetic knock-outs at the T. gondii AK locus, and AK activity was restored using a cDNA-derived minigene. Hybridization analysis of DNA from 13 AraA-resistant insertional mutants reveals three distinct classes: (i) AK-mutants tagged at the AK locus; (ii) AK- mutants not tagged at the AK locus, suggesting the possibility that another locus may be involved in regulating AK expression; and (iii) mutants with normal AK activity (potential transport mutants).

Broadening the spectrum of β-lactam antibiotics through inhibition of signal peptidase type I

ABSTRACT The resistance of methicillin-resistant Staphylococcus aureus (MRSA) to all β-lactam classes limits treatment options for serious infections involving this organism. Our goal is to discover new agents that restore the activity of β-lactams against MRSA, an approach that has led to the discovery of two classes of natural product antibiotics, a cyclic depsipeptide (krisynomycin) and a lipoglycopeptide (actinocarbasin), which potentiate the activity of imipenem against MRSA strain COL. We report here that these imipenem synergists are inhibitors of the bacterial type I signal peptidase SpsB, a serine protease that is required for the secretion of proteins that are exported through the Sec and Tat systems. A synthetic derivative of actinocarbasin, M131, synergized with imipenem both in vitro and in vivo with potent efficacy. The in vitro activity of M131 extends to clinical isolates of MRSA but not to a methicillin-sensitive strain. Synergy is restricted to β-lactam antibiotics and is not observed with other antibiotic classes. We propose that the SpsB inhibitors synergize with β-lactams by preventing the signal peptidase-mediated secretion of proteins required for β-lactam resistance. Combinations of SpsB inhibitors and β-lactams may expand the utility of these widely prescribed antibiotics to treat MRSA infections, analogous to β-lactamase inhibitors which restored the utility of this antibiotic class for the treatment of resistant Gram-negative infections.

Evaluation of a Cyclic GMP-Dependent Protein Kinase Inhibitor in Treatment of Murine Toxoplasmosis: Gamma Interferon Is Required for Efficacy

ABSTRACT The trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl]pyridine (compound 1) is a potent inhibitor of cyclic GMP-dependent protein kinases from Apicomplexan protozoa and displays cytostatic activity against Toxoplasma gondii in vitro. Compound 1 has now been evaluated against T. gondii infections in the mouse and appeared to protect the animals when given intraperitoneally at 50 mg/kg twice daily for 10 days. However, samples from brain, spleen, and lung taken from infected treated mice revealed the presence of parasites after cessation of administration of compound 1, indicating that a transient asymptomatic parasite recrudescence occurs in all survivors. The ability of mice to control Toxoplasma infection after compound 1 treatment has been terminated suggested that the mouse immune system plays a synergistic role with chemotherapy in controlling the infection. To explore this possibility, gamma interferon (IFN-γ)-knockout mice were infected with parasites and treated with compound 1, and survival was compared to that of normal mice. IFN-γ-knockout mice were protected against T. gondii throughout the treatment phase but died during the posttreatment phase in which peak recrudescence was observed in treated immunocompetent mice. These data suggest that an IFN-γ-dependent immune response was essential for controlling and resolving parasite recrudescence in mice treated with compound 1. In addition, when compound 1-cured immunocompetent mice were rechallenged with a lethal dose of T. gondii, all survived (n = 32). It appears that the cytostatic nature of compound 1 provides an “immunization” phase during chemotherapy which allows the mice to survive the recrudescence and any subsequent challenge with a lethal dose of T. gondii.

Expression, purification, and characterization of uracil phosphoribosyltransferase from Toxoplasma gondii

The coding region derived from a full-length CDNA spanning the uracil phosphoribosyltransferase (UPRT) gene of Toxoplasma gondii has been ligated into a bacterial expression vector and overexpressed in E. coli. Recombinant UPRT protein migrated with a molecular mass of 27 kDa on SDS polyacrylamide gels and was purified to homogeneity by conventional protein purification techniques. In solution, UPRT behaved as a monomer and exhibited K(m)app values of 3.5 microM for uracil and 243 microM for phosphoribosylpyrophosphate, respectively. Other naturally occurring pyrimidine or purine bases were not recognized as substrates. [14C]Uracil phosphoribosylation was inhibited by 5-fluorouracil with a Ki value of 25 microM and was not activated by GTP. Ample quantities of recombinant enzyme are now available for biochemical and structural studies, facilitating evaluation of UPRT as a possible therapeutic target.