Analysis of the Coxiella burnetii Type IV Secretion System During Infection

描述(由申请人提供)：伯氏柯克斯体是一种专性的细胞内细菌病原体，通常通过气雾剂接触获得。它是急性Q热和慢性疾病如心内膜炎、肝炎和慢性疲劳的病原体。急性肺炎通常是一种自限性的流感样疾病，从亚临床到严重不等。Q热心内膜炎被认为是非可培养心内膜炎的主要原因。伯氏梭菌环境稳定，感染量低，以前曾被开发为武器。这些特点使它被美国疾病控制与预防中心列为潜在的生物恐怖威胁。然而，正常情况下，动物种群(特别是反刍动物)是布氏梭菌释放环境稳定的可感染人类的颗粒的蓄水池。因此，这种人畜共患病原体对牛、绵羊和山羊养殖业的从业人员构成重大风险。最初的感染发生在肺泡巨噬细胞。在感染宿主细胞后，伯氏梭菌在保留许多成熟吞噬溶菌体特征的空泡中复制。伯氏隐孢子虫寄生宿主细胞的分子机制在很大程度上是未知的。伯氏梭菌(九里氏I期株)的基因组序列已被测序，并含有与嗜肺军团菌IV型分泌系统(TFSS)同源的基因，表明伯氏梭菌具有与宿主细胞相互作用的特殊分泌途径。然而，关于伯氏梭菌的TFSS或其在感染周期中的可能作用，人们知之甚少。我们的中心假设是，伯氏梭菌TFSS是细菌生存、复制和/或传播所必需的。该项目的总体目标是确定伯氏梭菌是否产生可作为TFSS的蛋白质，并检测这些蛋白质的时间表达。这些目标将通过实现以下特定目标来实现：特定目标1-表征伯氏梭菌在感染宿主细胞过程中合成的TFSS mRNA的表达。我们将使用逆转录-聚合酶链式反应(RT-PCR)、实时RT-聚合酶链式反应(Real time RT-PCR)和引物延伸作为工具，在体外研究伯氏梭菌TFSS同源物在感染早期、中期和晚期的表达情况。特定目的2-研究伯氏梭菌TFSS同源物在感染过程中的蛋白表达和亚细胞定位。我们将通过生产针对重组TFSS同源物的特定抗血清并使用这些探针来评估感染期间细菌中的蛋白质表达来实现这一点。伯氏梭菌TFSS在感染过程中的特征是理解这种独特的病原体在宿主细胞吞噬小体的恶劣环境中生存并导致疾病的关键的第一步。伯氏柯克斯体是一种专性胞内细菌，是急性Q热和慢性疾病的病原体。它是一种人畜共患病病原体，已被疾控中心指定为B类选择试剂。关于伯氏隐杆线虫与其宿主细胞的分子相互作用，人们知之甚少。在目前的提案中，我们将描述伯氏梭菌IV型分泌系统(TFSS)的特征。考虑到TFSS在感染和其他细菌发展中的既定作用，这些研究可能会导致确定Q热检测和干预的独特诊断、治疗和疫苗靶标。

DESCRIPTION (provided by applicant): Coxiella burnetii is an obligate intracellular bacterial pathogen that is typically acquired through aerosol exposure. It is the etiologic agent of acute Q fever and chronic diseases such as endocarditis, hepatitis, and chronic fatigue. The acute form is typically a self-limiting flu-like illness that ranges from sub-clinical to severe. Q fever endocarditis has been implicated as a major cause of non-culturable endocarditis. C. burnetii is environmentally stable, has a low infectious dose, and has previously been developed as a weapon. These characteristics have led to it being listed as a Select Agent by the CDC as a potential bioterror threat. Normally however, animal populations (particularly ruminants) are the reservoirs for C. burnetii shedding environmentally stable infectious particles that can infect humans. As such, this zoonotic pathogen poses a significant risk to persons involved in the cattle, sheep, and goat industries. Initial infection occurs in lung alveolar macrophages. After infecting the host cell, C. burnetii replicates in vacuoles that retain many of the features of mature phagolysosomes. The molecular mechanisms used by C. burnetii to parasitize their host cells are largely unknown. The genome of C. burnetii (Nine Mile Phase I strain) has been sequenced and contains genes homologous to the type IV secretion system (TFSS) of Legionella pneumophila, suggesting that C. burnetii possesses a specialized secretory pathway for interacting with host cells. However, little is known about the TFSS of C. burnetii or its possible role during the infectious cycle. Our central hypothesis is that the C. burnetii TFSS is necessary for bacterial survival, replication and/or dissemination. The overall goals of this project it to determine whether C. burnetii produces proteins that can act as a TFSS and to examine the temporal expression of these proteins. The goals will be achieved by accomplishing the following specific aims: Specific aim 1 - Characterize the expression of TFSS mRNA synthesized by C. burnetii during infection of host cells. We will use reverse-transcriptase PCR (RT-PCR), real time RT-PCR, and primer extension as tools to define the expression of the C. burnetii TFSS homologs in early, mid, or late stages of infection in vitro. Specific aim 2 - Characterize the protein expression and sub-cellular localization of the C. burnetii TFSS homologs during infection. We will accomplish this by producing specific antisera to recombinant TFSS homologs and using these probes to assess protein expression in the bacteria during infection. Characterization of the C. burnetii TFSS during infection is a critical first step towards understanding the mechanisms used by this unique pathogen to survive within the harsh environment of the host cell phagosome and cause disease. Coxiella burnetii is an obligate intracellular bacterium and the causative agent of acute Q fever and chronic diseases. It is a zoonotic pathogen, which has been designated a Category B level Select Agent by the CDC. Very little is known about the molecular interactions of C. burnetii and its host cell. In the current proposal we will characterize the C. burnetii type IV secretion system (TFSS). Considering the established role of the TFSS in infection and development of other bacteria, these studies will likely lead to the identification of unique diagnostic, therapeutic, and vaccine targets for Q-fever detection and intervention.