Role of the Coxiella burnetii Cryptic Plasmid in Host Cell Parasitism

描述(申请人提供)：伯氏柯克斯体是一种细胞内细菌病原体，也是人类Q热的病原体，Q热是一种急性衰弱的流感样疾病，可发展为慢性心内膜炎。自从70多年前被发现以来，这种病原体用来寄生宿主细胞的机制仍然知之甚少。在感染过程中，伯氏梭菌主动调节多种寄主过程，包括囊泡运输和细胞存活。介导这些事件的细菌蛋白尚不清楚，但可能通过Dot/ICM IV型分泌系统传递到宿主细胞质。目前的应用旨在从功能上表征伯氏柯萨奇杆菌质粒编码的Dot/ICM底物，并确定它们在毒力中的作用。所有的伯氏梭菌分离株要么含有一个大的隐蔽质粒，要么在它们的染色体上整合了质粒序列，这表明这些分子对病原体生物学至关重要。有趣的是，我们已经鉴定了6种由伯氏柯萨奇杆菌质粒基因编码的Dot/ICM底物，它们被称为柯克斯体质粒效应器A-F(CPEA-F)。其中三个蛋白在所有分离株中都是保守的，三个蛋白是来自人类急性疾病分离株的QpH1质粒特异性的。目的1研究保守的CPEB和CpeD分别与自噬小体和分泌细胞器的相互作用。目标2将定义感染过程中所有六个质粒效应器的要求。此外，这一目标将确定效应器结合宿主蛋白，并确定这些成分对伯氏梭菌感染的需求。目的3在细胞培养和Q热豚鼠感染模型中确定伯氏弧菌对病原菌毒力的要求。总而言之，本申请中的目标将为伯氏梭菌有效寄生宿主细胞所使用的机制提供必要的洞察。这些研究还将提供有关伯氏梭菌隐蔽质粒在病原菌毒力中的作用的新信息。 公共卫生相关性：伯氏柯克斯体是一种细胞内细菌病原体，可引起人畜共患病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。目前，人们对伯氏梭菌毒力决定因素知之甚少。通过Dot/ICM Type IV分泌系统将伯氏梭菌蛋白输送到宿主胞浆的特征将提供对抗Q热的候选治疗靶点，并将为了解伯氏梭菌与其宿主之间的相互作用提供必要的洞察力。本研究的目的是：1)研究伯氏梭菌编码的Dot/ICM底物与宿主蛋白的相互作用；2)确定该质粒在病原菌毒力中的作用。

DESCRIPTION (provided by applicant): Coxiella burnetii is an intracellular bacterial pathogen and the etiologic agent of human Q fever, an acute debilitating flu-like illness that can progress to chronic endocarditis. Since its discovery over 70 years ago, mechanisms used by the pathogen to parasitize host cells remain poorly understood. During infection, C. burnetii actively regulates multiple host processes, including vesicular trafficking and cell survival. The bacterial proteins mediating these events are not known but are likely delivered to the host cytosol by a Dot/Icm type IV secretion system. The current application is designed to functionally characterize C. burnetii plasmid-encoded Dot/Icm substrates and define their role in virulence. All C. burnetii isolates either harbor a large cryptic plasmid or have plasmid sequences integrated into their chromosome, suggesting these molecules are critical for pathogen biology. Interestingly, we have identified six Dot/Icm substrates encoded by C. burnetii plasmid genes that are termed Coxiella plasmid effectors A - F (CpeA - F). Three of these proteins are conserved in all isolates and three are specific to the QpH1 plasmid from a human acute disease isolate. Aim 1 is designed to characterize the interaction of conserved CpeB and CpeD with autophagosomes and secretory organelles, respectively. Aim 2 will define requirements of all six plasmid effectors during infection. Additionally, this aim will identify effector binding host proteins and determine the requirement of these components for C. burnetii infection. Aim 3 will determine the requirement of the C. burnetii plasmid for pathogen virulence in both cell culture and a guinea pig infection model of Q fever. Collectively, the aims in the current application will provide needed insight into the mechanisms used by C. burnetii to efficiently parasitize host cells. These studies will also provide novel information regarding the role of the C. burnetii cryptic plasmid in pathogen virulence. PUBLIC HEALTH RELEVANCE: Coxiella burnetii is an intracellular bacterial pathogen that causes the zoonosis human Q fever, a debilitating acute disease that also presents as chronic endocarditis. Currently, C. burnetii virulence determinants are poorly understood. Characterization of C. burnetii proteins delivered to the host cytosol by the Dot/Icm type IV secretion system will provide candidate therapeutic targets to combat Q fever and will provide needed insight into the interactions between C. burnetii and its host. The goals of the proposed research are to 1) characterize the interaction of C. burnetii plasmid-encoded Dot/Icm substrates with host proteins and 2) determine the role of the plasmid in pathogen virulence.)