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增对人类机会性病原体弓形虫（Toxoplasma gondii）导致艾滋病毒/艾滋病患者严重疾病的能力的影响。由于以下原因，HIV/AIDS患者面临由弓形虫引起的严重疾病的风险：1)原发感染环境稳定的感染性卵囊，2)原发感染存在于未煮熟的肉类中的组织囊肿，3)由于HIV/AIDS驱动的免疫抑制导致缺乏免疫监测而使休眠组织囊肿重新激活。这些组织囊肿，特别是它们在与宿主相互作用时分泌的效应蛋白，是本提案的主要焦点。我们发现这项工作特别重要，因为与感染的急性期相比，对弓形虫在感染的慢性期引起疾病所需的弓形虫效应物知之甚少。这项工作建立在我们之前的研究基础上，发现了在弓形虫感染动物模型的急性毒力中起关键作用的串联复制和多样化基因。在此基础上延伸

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.