The role of Toxoplasma gondii amino acid hydroxylase 2 in chronic infection

Stanford University, Stanford CA

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Abstract

? DESCRIPTION (provided by applicant): Toxoplasma infection has been linked to a variety of mental disorders and behavioral alterations, including schizophrenia and Parkinson's disease. Interestingly, chronic Toxoplasma infection has been shown to increase dopamine levels and alter behavior in rodents, causing them to lose their innate fear of cat odors. Because the feline is the definitive host for Toxoplasma, it has been proposed that evolution has selected for a parasite that alters rodent behavior to increase its transmission back to the cat. Importantly, Toxoplasma increases dopamine content and secretion in neurons in vitro, suggesting that the parasite can increase dopamine metabolism independently of the immune system. It has been proposed that Toxoplasma alters dopamine and behavior using amino acid hydroxylase 2 (TgAAH2), which it expresses during chronic infection. AAH2 catalyzes the conversion of phenylalanine to tyrosine and tyrosine to DOPA, with preference for the latter reaction. Because the conversion of tyrosine to DOPA is the rate-limiting step in dopamine synthesis, Toxoplasma may increase dopamine by catalyzing the formation of DOPA, the direct precursor to dopamine. The overarching goal of this work is to determine the role of AAH2 in chronic infection, dopamine metabolism, and behavioral alteration in rodents. Previous reports show that the tissue cyst wall Toxoplasma forms during chronic infection consists of dityrosine crosslinks and DOPA, suggesting a structural role for this enzyme. The hypothesis driving this work is that AAH2 regulates amino acid metabolism during differentiation to the encysted form, contributes to formation of the cyst wall, and/or alters dopamine metabolism and behavior in the rodent host. To determine if AAH2 is important for amino acid metabolism and nutrition, growth and differentiation of the AAH2 mutant and control strains in nutrient-rich and tyrosine-deficient media will be measured by plaque assay and microscopy. To determine if AAH2 is important for synthesizing DOPA and dityrosines in the tissue cyst wall, the brains of mice chronically infected with the AAH2 mutant and control strains will be fixed, harvested, processed into sections, and stained with antibodies for dityrosine and DOPA. The infectivity of the AAH2 mutant and control cysts during passage through the gastric tract will be measured by orally infecting naïve mice and measuring seroconversion and dissemination to the brain. Structural integrity of the cyst wall will be determined by treating cysts with pepsin digestive solution and measuring parasite viability by plaque assay. To determine if AAH2 affects neurotransmitter levels during chronic infection, HPLC-ED will be performed on neurons infected with the AAH2 mutant and controls. To determine if AAH2 affects behavior in rodents, the behavior of mice around bobcat or rabbit urine during chronic infection with these strains will be measured. The completion of this work will identify the function of AAH2 during chronic Toxoplasma infection, for which there is no cure or treatment. Furthermore, it will test whether AAH2 contributes to dopamine dysregulation and behavioral alteration.