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。伯氏梭菌通过吸入获得，最初与肺泡巨噬细胞接触，并经历两个阶段的生命周期，涉及结构上不同的病原体。感染通常被认为是由代谢不活跃的环境稳定的小细胞变异(SCV)引发的，然后转化为具有复制能力、代谢活跃的大细胞变异(LCV)。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)基因组中发现了与嗜肺军团菌IV型分泌系统(TFSS)同源的基因，提示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。我们的中心假设是在SCV向LCV转化过程中，伯氏梭菌T4SS的表达和结构/功能发生了变化。本申请中提出的正在进行的研究的目标是(I)表征和定义伯氏弧菌T4SS基因在细菌从SCV向LCV转化的过程中的表达，以及(Ii)确定C.burnetii T4SS中的结构和蛋白质相互作用，特别是强调SCV和LCV形式的病原体之间的差异。我们将通过实现以下特定目标来验证我们的中心假设：目的1.确定T4SS基因在伯氏弧菌感染宿主细胞后从SCV向LCV转化过程中的表达。我们将使用RT-qPCR来确定在低速离心法启动感染后的前36个HPI中伯氏梭菌T4SS基因的时间表达，并使用间接荧光抗体(IFA)和免疫电子显微镜(IEM)来确定和区分在这一时间段内伯氏梭菌T4SS在病原体的小细胞变体(SCV)和大细胞变体(LCV)上的表达。目的2.确定伯氏梭菌T4SS在SCV和LCV中的亚细胞定位、结构和蛋白质-蛋白质相互作用。我们将使用针对伯氏柯萨奇杆菌T4SS蛋白的抗体对细菌部分进行免疫印迹分析，以及对在无细胞培养基中生长的伯氏柯萨奇杆菌进行IFA和IEM分析，以确定T4SS的亚细胞定位、蛋白质相互作用和SCV/LCV特异性。了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毒力机制将使我们能够开发针对这种知之甚少的细菌的对策。 公共卫生相关性：伯氏柯克斯体是一种专性细胞内细菌，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(T4SS)的特征。考虑到T4SS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. Acquired by inhalation, C. burnetii initially contacts alveolar macrophages and undergoes a bi-phasic life cycle involving structurally distinct forms of the pathogen. Infection is thought to normally be initiated by the metabolically inactive, environmentally stable small cell variant (SCV) followed by conversion to the replicative, metabolically active, large cell variant (LCV). 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) revealed 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 T4SS expression and structure/function changes during SCV to LCV conversion. The goals of the ongoing research proposed in this application are to (i) characterize and define the expression of C. burnetii T4SS genes during the conversion of the bacteria from SCVs to LCVs and, (ii) determine the structure and protein interactions within the C. burnetii T4SS with particular emphasis in differences between the SCV and LCV forms of the pathogen. We will test our central hypothesis by accomplishing the following Specific Aims: Aim 1. Define the expression of T4SS genes during the conversion of C. burnetii from SCVs to LCVs after infection of host cells. We will use RT-qPCR to define the temporal expression of C. burnetii T4SS genes during the first 36 hpi following a low speed centrifugation initiated infection and Indirect Fluorescent Antibody (IFA) and Immuno-electron (IEM) microscopy to define and differentiate the C. burnetii T4SS expression on small cell variant (SCV) and large cell variant (LCV) forms of the pathogen during this time period. Aim 2. Determine the sub-cellular localization, structure, and protein-protein interactions of the C. burnetii T4SS in SCVs and LCVs. We will use antibodies against C. burnetii T4SS proteins in immunoblot analysis of bacterial fractions, as well as IFA and IEM analysis of C. burnetii grown in cell-free media to define T4SS sub-cellular localization, protein-protein interactions, and SCV/LCV specificity. Understanding the virulence mechanisms employed by this unique pathogen to survive within the harsh environment of the host cell phagosome and cause disease will enable us to develop countermeasures to this poorly understood bacteria. PUBLIC HEALTH RELEVANCE: 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 (T4SS). Considering the established role of the T4SS 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.