Functional Characterization of Coxiella burnetii Dot/Icm Substrates

描述（由申请人提供）：伯纳蒂克希菌是一种专性细胞内细菌病原体，是人类Q热的病原。Q热通常表现为急性衰弱性流感样疾病，但可发展为严重的慢性心内膜炎。在宿主细胞中，伯氏梭菌在溶酶体样寄生物液泡中建立繁殖生态位。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。在细胞内生长过程中，伯氏梭菌积极操纵多个宿主过程，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尚不清楚。伯纳氏梭菌将细菌蛋白传递到宿主细胞的主要分泌方式似乎是Dot/lcm IV型分泌系统。目前的提案旨在功能表征伯氏梭菌蛋白在感染期间通过Dot/lcm IV型分泌系统传递到宿主细胞质。为此，Specific Aim 1旨在表征Dot/lcm底物的时间表达，并研究与这些效应物相互作用的宿主蛋白。这些研究将包括使用RT-PCR和共免疫沉淀方法来阐明在感染和细胞内生长过程中的效应活性。特异性目标2将使用三种方法来表征伯氏杆菌感染期间Dot/lcm底物的功能。首先，将使用siRNA方法敲低效应结合宿主蛋白的表达，并分析这种缺失对伯纳氏梭菌感染和复制的影响。其次，将研究个体效应物在破坏宿主信号传导中的作用，包括抑制细胞凋亡和激活促生存蛋白Akt、Erk1/2和NF-icB。第三，在感染期间，个体效应物将被特异性多克隆抗血清靶向，以研究它们在含有病原体的液泡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伯纳蒂菌毒力决定因素，并提供更好的理解伯纳蒂菌有效寄生宿主细胞的机制。此外，这些研究将进一步加深我们对宿主细胞基本信号传导过程和细胞内病原体与宿主相互作用的理解。

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.