Analysis of the Coxiella burnetii Type IV Secretion System During Infection

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，通常通过气溶胶暴露获得。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。急性形式通常是一种自限性流感样疾病，范围从亚临床到严重。Q热心内膜炎已被认为是非培养性心内膜炎的主要原因。C.贝氏体对环境稳定，感染剂量低，以前曾被用作武器。这些特征导致它被CDC列为潜在生物恐怖威胁的选择代理。然而，通常情况下，动物种群（特别是反刍动物）是C。贝氏体释放环境稳定的传染性颗粒，可感染人类。因此，这种人畜共患病原体对牛、绵羊和山羊产业的从业人员构成重大风险。初始感染发生在肺泡巨噬细胞中。C.贝氏虫在保留许多成熟吞噬溶酶体特征的液泡中复制。C.贝氏疟原虫寄生于宿主细胞的能力在很大程度上是未知的。C. Burnetii（Nine Mile Phase I strain）的基因序列分析表明，该菌株含有与嗜肺军团菌IV型分泌系统（TFSS）同源的基因。贝氏菌具有与宿主细胞相互作用的特化分泌途径。然而，人们对C的TFSS知之甚少。Burnetii或其在感染周期中的可能作用。我们的中心假设是C.贝氏菌TFSS是细菌存活、复制和/或传播所必需的。本项目的总体目标是确定C. Burnetii产生可以作为TFSS的蛋白质，并检查这些蛋白质的时间表达。具体目标1 -表征由C.贝氏体在感染宿主细胞期间。我们将使用逆转录酶PCR（RT-PCR）、真实的实时RT-PCR和引物延伸作为工具来确定C.贝氏体TFSS同源物在体外感染的早期、中期或晚期。具体目标2 -表征C.贝氏菌TFSS同源物。我们将通过生产重组TFSS同源物的特异性抗血清并使用这些探针来评估感染期间细菌中的蛋白表达来实现这一点。对C.在感染过程中检测贝氏体TFSS是理解这种独特病原体在宿主细胞吞噬体的恶劣环境中生存并引起疾病的机制的关键第一步。贝氏柯克斯体（Coxiellaburnetii）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原体，已被CDC指定为B类选择因子。对C的分子间相互作用知之甚少。Burnetii及其宿主细胞。在目前的建议中，我们将描述C。贝氏IV型分泌系统（TFSS）。考虑到TFSS在其他细菌感染和发展中的既定作用，这些研究可能会导致确定Q热检测和干预的独特诊断，治疗和疫苗靶点。

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.