Molecular Characterisation of Toxoplasma gondii and Development of Diagnostic Assay for Bradyzoites

Toxoplasma gondii (T. gondii) is a ubiquitous parasite that infects warm-blooded animals and humans. In humans, T. gondii causes encephalitis in AIDS patients, and there is no drug that can eliminate T. gondii infection. T. gondii specifically manipulates the intermediate host’s behaviour favouring its transmission to the definitive feline host. Human T. gondii seropositivity has also been associated with mental disorders. T. gondii has two aromatic amino acid hydroxylases, TgAaaH (1 and 2), that convert phenylalanine to tyrosine, and tyrosine to L- DOPA, the latter being the rate-limiting step of dopamine biosynthesis. Based on this and elevated dopamine levels in brain tissue cysts and infected dopaminergic cells, it has been hypothesised that TgAaaH has a role in altering brain neuromodulation and potentially subsequently in the behavioural changes observed. As TgAaaH genes encode a signal peptide, the location of the enzyme was examined. TgAaaH was localised to outside the parasite both membrane- bound to parasites within the parasitophorous vacuole based on immunofluorescence, fractionation, and trypsin susceptibility of released parasites. Another possible role of TgAaaH in cyst wall generation was examined by testing for dopa-oxidase activity to convert L-DOPA to dopa- quinone. Dopa-oxidase activity was not detectable in infected fibroblasts, yet it remains possible that parasite produced L-DOPA is metabolised to dopaquinone by host cell enzymes such as within feline gut endothelial cells where oocysts are formed. This, and our finding that host cell dopa-decarboxylase is required for dopamine biosynthesis, suggest that the product of parasite-produced L-DOPA may be dependent upon the type of cell infected (ie. dopamine in catecholaminergic cells and dopaquinone in endothelial cells). The limitation of studying the bradyzoite stages of infection due to proliferation of tachyzoite stages was resolved by development of a new culture system with depleted tryptophan. This method was then applied to develop a novel throughput assay to identify bradyzoite inhibitors. The validity of this assay was evaluated using tachyzoite and bradyzoite specific inhibitors. This assay will help in finding an
anti-toxoplasma drug for curing of infection.