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

 描述(申请人提供)：弓形虫感染与各种精神障碍和行为改变有关，包括精神分裂症和帕金森氏症。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。因为猫是弓形虫的最终宿主，所以有人提出，进化选择了一种改变啮齿动物行为的寄生虫，以增加其向猫的传播。重要的是，弓形虫在体外增加了神经元中的多巴胺含量和分泌，这表明这种寄生虫可以增加多巴胺的代谢，而不依赖于免疫系统。已有研究表明，弓形虫通过其在慢性感染过程中表达的氨基酸羟化酶2(TgAAH2)改变多巴胺和行为。AAH2催化苯丙氨酸转化为酪氨酸，酪氨酸转化为DOPA，优先于后一种反应。由于酪氨酸转化为DOPA是多巴胺合成的限速步骤，弓形虫可能通过催化多巴胺的直接前体DOPA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。以往的报道表明，弓形虫在慢性感染过程中形成的组织囊壁由双赖氨酸交联物和DOPA组成，提示这种酶具有结构作用。支持这项工作的假设是，AAH2在向包囊形式分化的过程中调节氨基酸代谢，促进囊壁的形成，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对氨基酸代谢和营养是否重要，将通过空斑试验和显微镜观察AAH2突变体和对照菌株在营养丰富和酪氨酸缺乏的培养基中的生长和分化。为了确定AAH2对组织囊壁中合成DOPA和Dityrosine是否重要，将固定AAH2突变株和对照菌株感染的小鼠的大脑，收集并加工成切片，用Dityroine和DOPA抗体进行染色。AAH2突变体和对照包囊在通过胃道的过程中的传染性将通过口服感染幼小鼠和测量血清转换和向大脑传播来测量。囊壁的结构完整性将通过用胃酶消化液处理囊肿并通过菌斑试验测量寄生虫的生存能力来确定。为了确定AAH2在慢性感染期间是否影响神经递质水平，将对感染AAH2突变体的神经元和对照进行高效液相色谱-ED检测。为了确定AAH2是否影响啮齿动物的行为，将测量小鼠在长期感染这些菌株期间围绕山猫或兔子尿液的行为。这项工作的完成将确定AAH2在慢性弓形虫感染中的功能，目前还没有治愈或治疗的方法。此外，它还将测试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.