From TgO/GABA-AT, GABA, and T-263 Mutant to Conception of Toxoplasma.

Toxoplasma gondii causes morbidity, mortality, and disseminates widely via cat sexual stages. Here, we find T. gondii ornithine aminotransferase (OAT) is conserved across phyla. We solve TgO/GABA-AT structures with bound inactivators at 1.55 Å and identify an inactivator selective for TgO/GABA-AT over human OAT and GABA-AT. However, abrogating TgO/GABA-AT genetically does not diminish replication, virulence, cyst-formation, or eliminate cat's oocyst shedding. Increased sporozoite/merozoite TgO/GABA-AT expression led to our study of a mutagenized clone with oocyst formation blocked, arresting after forming male and female gametes, with "Rosetta stone"-like mutations in genes expressed in merozoites. Mutations are similar to those in organisms from plants to mammals, causing defects in conception and zygote formation, affecting merozoite capacitation, pH/ionicity/sodium-GABA concentrations, drawing attention to cyclic AMP/PKA, and genes enhancing energy or substrate formation in TgO/GABA-AT-related-pathways. These candidates potentially influence merozoite's capacity to make gametes that fuse to become zygotes, thereby contaminating environments and causing disease.

Identification and development of novel inhibitors of Toxoplasma gondii enoyl reductase.

Toxoplasmosis causes significant morbidity and mortality, and yet available medicines are limited by toxicities and hypersensitivity. Because improved medicines are needed urgently, rational approaches were used to identify novel lead compounds effective against Toxoplasma gondii enoyl reductase (TgENR), a type II fatty acid synthase enzyme essential in parasites but not present in animals. Fifty-three compounds, including three classes that inhibit ENRs, were tested. Six compounds have antiparasite MIC(90)s < or = 6 microM without toxicity to host cells, three compounds have IC(90)s < 45 nM against recombinant TgENR, and two protect mice. To further understand the mode of inhibition, the cocrystal structure of one of the most promising candidate compounds in complex with TgENR has been determined to 2.7 A. The crystal structure reveals that the aliphatic side chain of compound 19 occupies, as predicted, space made available by replacement of a bulky hydrophobic residue in homologous bacterial ENRs by Ala in TgENR. This provides a paradigm, conceptual foundation, reagents, and lead compounds for future rational development and discovery of improved inhibitors of T. gondii.

Maternal inheritance and stage-specific variation of the apicoplast in Toxoplasma gondii during development in the intermediate and definitive host.

The structure and location of Toxoplasma gondii apicoplasts were examined in intermediate and definitive hosts and shown to vary in a stage-specific manner. Immunocytochemistry and electron microscopy studies were used to identify changes in the morphology of apicoplasts and in their enoyl reductase (ENR) content during asexual and sexual development. Apicoplasts in tachyzoites were small, multimembraned organelles anterior to nuclei that divided and segregated with the nuclei during endodyogeny. In nonproliferating bradyzoites within mature tissue cysts (1 to 24 months), apicoplasts had high levels of ENR. During coccidian development, asexual multiplication (endopolygeny), resulting in simultaneous formation of up to 30 daughters (merozoites), involved an initial growth phase associated with repeated nuclear divisions during which apicoplasts appeared as single, elongated, branched structures with increased levels of ENR. At initiation of merozoite formation, enlarged apicoplasts divided simultaneously, with constrictions, into portions that segregated to developing daughters. In sexual stages, apicoplast division did not occur during microgametogony, and apicoplasts were absent from the microgametes that were formed. In contrast, during macrogametogony, the apicoplast appeared as a large, branched, perinuclear structure that had very high levels of ENR in the absence of nuclear division. Marked increases in the size of apicoplasts and levels of ENR may be related to requirements of the macrogametocytes to synthesize and store all components necessary for oocyst formation and subsequent extracellular sporulation. Thus, it is shown that apicoplasts are present and contain ENR in all T. gondii life cycle stages except microgametes, which will result in maternal inheritance of the organelle.