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

 描述（由申请人提供）：弓形虫感染与多种精神障碍和行为改变有关，包括精神分裂症和帕金森病。有趣的是，慢性弓形虫感染已被证明会增加多巴胺水平并改变啮齿动物的行为，使它们失去对猫气味的天生恐惧。由于猫是弓形虫的最终宿主，因此有人提出进化选择了一种改变啮齿动物行为的寄生虫，以增加其传递回猫。重要的是，弓形虫增加体外神经元中多巴胺的含量和分泌，表明寄生虫可以独立于免疫系统增加多巴胺代谢。已经提出弓形虫改变多巴胺和行为使用氨基酸羟化酶2（TgAAH2），其在慢性感染期间表达。AAH2催化苯丙氨酸转化为酪氨酸和酪氨酸转化为多巴，优选后者反应。由于酪氨酸转化为多巴是多巴胺合成的限速步骤，弓形虫可能通过催化多巴（多巴胺的直接前体）的形成来增加多巴胺。这项工作的首要目标是确定AAH2在啮齿动物慢性感染，多巴胺代谢和行为改变中的作用。先前的报道表明，弓形虫在慢性感染期间形成的组织囊壁由双酪氨酸交联和多巴组成，表明这种酶的结构作用。驱动这项工作的假设是AAH2在分化为包囊形式期间调节氨基酸代谢，有助于形成包囊壁，和/或改变啮齿动物宿主中的多巴胺代谢和行为。为了确定AAH2是否对氨基酸代谢和营养重要，将通过噬菌斑测定和显微镜检查来测量AAH2突变体和对照菌株在营养丰富和酪氨酸缺乏培养基中的生长和分化。为了确定AAH2对于在组织囊壁中合成DOPA和二酪氨酸是否重要，将用AAH2突变体和对照菌株慢性感染的小鼠的脑固定、收获、加工成切片，并用二酪氨酸和DOPA的抗体染色。将通过经口感染未处理小鼠并测量血清转化和向脑的传播来测量AAH2突变体和对照包囊在通过胃肠道期间的感染性。将通过用胃蛋白酶消化液处理包囊并通过空斑试验测量寄生虫活力来确定包囊壁的结构完整性。为了确定AAH2是否影响慢性感染期间的神经递质水平，将对感染AAH2突变体和对照的神经元进行HPLC-ED。为了确定AAH2是否影响啮齿动物的行为，将测量在用这些菌株慢性感染期间在山猫或兔尿周围的小鼠的行为。这项工作的完成将确定慢性弓形虫感染期间AAH2的功能，对此没有治愈或治疗方法。此外，它将测试AAH2是否有助于多巴胺失调和行为改变。

 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.