Cyst effector gene families and Toxoplasma pathogenesis

描述：该项目的主要目标是确定基因复制和扩展对人类机会性病原体弓形虫造成艾滋病毒/艾滋病患者严重疾病的能力的影响。艾滋病毒/艾滋病患者面临由弓形虫引起的严重疾病的风险，原因是1)原发感染环境稳定的传染性卵囊，2)原发感染未煮熟的肉中的组织囊，以及3)由于艾滋病毒/艾滋病驱动的免疫抑制而缺乏免疫监测，休眠的组织囊重新激活。这些组织包囊，特别是它们在与宿主相互作用过程中分泌的效应蛋白，是这一提议的主要焦点。我们发现这项工作特别重要，因为与感染的急性期相比，人们对弓形虫在慢性感染阶段致病所需的弓形虫效应器知之甚少。这项工作建立在我们之前的研究基础上，确定了重复和多样化的基因，这些基因在弓形虫在动物感染模型中的急性毒力中发挥了关键作用。在此基础上引申 结果，我们已经鉴定并彻底精选了弓形虫基因组中所有重复扩增的基因簇。这项研究发现了两个重要的数据：弓形虫扩增的基因簇显著丰富：1)预测从寄生虫分泌的基因(因此与宿主细胞相互作用)；2)在潜伏期表达的基因。 这对于导致艾滋病毒/艾滋病患者的疾病是至关重要的。在目标1中，我们根据这些孢子表达的扩展基因座(CEELs)在多个寄生虫菌株中的总基因含量来充分描述它们的特征，并对它们编码的基因进行克隆和测序。我们使用表位标记来确定它们在寄生虫和宿主细胞中的推测位置。从寄生虫中分泌的那些基因座将成为目标2的重点。这一目标得益于我们对弓形虫扩展基因座特征的丰富经验，并将提供有关其基因含量的重要新信息，这些信息将对我们和更广泛的弓形虫研究社区有用。在目标2中，我们将对从目标1中选择的基因座进行基因敲除，并确定它们对与宿主相互作用的影响，重点是孢子生物学。我们将在已建立的小鼠感染模型中，确定CEEL缺失对包囊的感染性、形成和潜伏囊重新激活的能力的影响。这些实验的成功完成将确定新的弓形虫胞囊表达的效应物，这些效应物在艾滋病毒/艾滋病患者的疾病中发挥关键作用。在未来的研究中，我们将描述它们的作用机制。

DESCRIPTION: The major goal of this project is to determine the impact of gene duplication and expansion on the ability of the human opportunistic pathogen, Toxoplasma gondii, to cause severe disease in HIV/AIDS patients. HIV/AIDS patients are at risk from severe disease caused by Toxoplasma due to 1) primary infection with environmentally stable infectious oocysts, 2) primary infection from tissue cysts present in undercooked meat and 3) reactivation of dormant tissue cysts due to a lack of immune surveillance due to HIV/AIDS-driven immunosuppression. These tissue cysts, and particularly the effector proteins that they secrete during interactions with the host, are the primary focus of this proposal. We find this work particularly importance since in contrast to the acute phase of infection, much less is known about the T. gondii effectors required for T. gondii to cause disease in the chronic phase of infection. This work builds off of our previous studies identifying tandemly duplicated and diversified genes that play a key role in the acute virulence of T. gondii in an animal model of infection. Extending upon this result, we have identified and thoroughly curated all tandemly expanded gene clusters in the T. gondii genome. Two important pieces of data emerged from this study: T. gondii expanded gene clusters are significantly enriched 1) for genes predicted to be secreted from the parasite (and therefore interact with the host cell) and 2) genes that are expressed during the latent cyst stage that is so crucial for causing disease in HIV/AIDS patients. In Aim 1 we fully characterize these Cyst-Expressed Expanded Loci (CEELs) in terms of their total gene content across multiple parasite strains and clone and sequence the genes that they encode. We use epitope tagging to determine their putative location within the parasite and host cell. Those loci that are secreted from the parasite will then be the focus of Aim 2. This Aim is facilitated by our extensive experience characterizing expanded loci in T. gondii and will provide important new information about their gene content, information which will be useful to us and to the broader Toxoplasma research community. In Aim 2 we will perform gene knockouts for loci selected from Aim 1 and determine their impact on interaction with the host, with a strong focus on cyst biology. We will determine the effect of CEEL deletion on cyst infectivity, formation, and the ability of latent cyss to reactivate in a well-established mouse model of infection. Successful completion of these experiments will identify new Toxoplasma cyst-expressed effectors that play a key role in disease in HIV/AIDS patients. In future studies we will characterize their mechanism of action.