Functional Characterization of Coxiella burnetii Dot/Icm Substrates

描述（由申请方提供）：贝氏柯克斯体是一种专性细胞内细菌病原体，是人类Q热的病原体。Q热通常表现为急性衰弱性流感样疾病，但可进展为严重的慢性心内膜炎。在宿主细胞中，C. Burnetii在溶酶体样的寄生虫空泡中建立了一个复制生态位。虽然发现于70多年前，但我们对病原体感染宿主细胞并引起疾病的机制的理解仍然是零碎的，特别是关于离散毒力因子的身份和活性。在胞内生长过程中，C.贝氏体主动操纵多个宿主过程，包括囊泡运输和细胞存活，以建立有效的感染。然而，介导这些事件的细菌蛋白是未知的。C. Burnetii将细菌蛋白质递送至宿主细胞的分泌系统似乎是Dot/lcm IV型分泌系统。目前的建议是为了功能上的特点C。在感染期间通过Dot/lcm IV型分泌系统递送至宿主胞质溶胶的贝氏体蛋白。为此，特定目标1被设计为表征我们已经鉴定并研究了与这些效应物相互作用的宿主蛋白的Dot/lcm底物的时间表达。这些研究将涉及使用RT-PCR和免疫共沉淀方法来阐明感染和细胞内生长期间的效应子活性。具体目标2将使用三种方法来表征C过程中的Dot/lcm底物功能。贝氏体感染首先，将使用siRNA方法敲低效应子结合宿主蛋白的表达，并分析这种损失对C.贝氏体感染和复制。其次，将检查单个效应物在宿主信号转导的颠覆中的作用，包括抑制凋亡和激活促存活蛋白Akt、Erk 1/2和NF-κ B。第三，在感染过程中，将用特异性多克隆抗血清靶向单个效应物，以研究它们在含有病原体的空泡的适当运输所需的囊泡融合事件中的作用。总的来说，拟议的研究将确定新的C。Burnetii毒力决定因子，并提供了C. Burnetii有效地寄生宿主细胞。此外，这些研究将进一步加深我们对基本宿主细胞信号传导过程和细胞内病原体与宿主相互作用的理解。 相关性（参见说明）：C。贝氏体是CDC类别B选择因子，并引起人畜共患病人类Q热，这是一种使人衰弱的急性疾病，也表现为慢性心内膜炎。对C. Burnettii Dot/lcm效应子将鉴定用于疫苗组分和治疗剂的候选蛋白质以对抗这种疾病，并将提供对这种病原体和宿主之间复杂相互作用的深入了解。

DESCRIPTION (provided by applicant): Coxiella burnetii is an obligate intracellular bacterial pathogen and the etiologic agent of human Q fever. Q fever typically presents as an acute debilitating flu-like illness but can progress to severe, chronic endocarditis. In host cells, C. burnetii establishes a replicative niche in a lysosome-like parasitophorous vacuole. While discovered over 70 years ago, our understanding of mechanisms used by the pathogen to infect host cells and cause disease is still fragmentary, specifically with respect to the identity and activity of discrete virulence factors. During intracellular growth, C. burnetii actively manipulates multiple host processes, including vesicular trafficking and cell survival, to establish an efficient infection. However, the bacterial proteins mediating these events are not known. The major mode of secretion used by C. burnetii to deliver bacterial proteins to the host cell appears to be the Dot/lcm type IV secretion system. The current proposal is designed to functionally characterize C. burnetii proteins delivered to the host cytosol during infection via the Dot/lcm type IV secretion system. To this end, Specific Aim 1 is designed to characterize the temporal expression of Dot/lcm substrates that we have already identified and investigate host proteins that interact with these effectors. These studies will involve the use of RT-PCR and co- immunoprecipitation approaches to elucidate effector activity during infection and intracellular growth. Specific Aim 2 will use three approaches to characterize Dot/lcm substrate function during C. burnetii infection. First, an siRNA approach will be used to knockdown expression of effector binding host proteins and analyze the effect of this loss on C. burnetii infection and replication. Second, individual effectors will be examined for their role in subversion of host signaling, including inhibition of apoptosis and activation of the pro-survival proteins Akt, Erk1/2, and NF-icB. Third, individual effectors will be targeted with specific polyclonal antiserum during infection to investigate their role in vesicular fusion events required for proper trafficking of the pathogen-containing vacuole. Collectively, the proposed studies will identify novel C. burnetii virulence determinants and provide an enhanced understanding of mechanisms used by C. burnetii to efficiently parasitize host cells. Additionally, these studies will further our understanding of basic host cell signaling processes and the interaction of intracellular pathogens with the host. RELEVANCE (See instructions): C. burnetii is a CDC Category B select agent and causes the zoonosis human Q fever, a debilitating acute disease that also presents as chronic endocarditis. Characterization of C. burnetii Dot/lcm effectors will identify candidate proteins for vaccine components and therapeutics to combat this disease and will provide insight into the complex interactions between this pathogen and the host.