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。通过吸入获得C.贝氏体最初接触肺泡巨噬细胞，并经历涉及病原体结构上不同形式的双相生命周期。感染通常被认为是由代谢不活跃、环境稳定的小细胞变异体（SCV）引发，然后转化为复制型、代谢活跃的大细胞变异体（LCV）。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）与嗜肺军团菌的IV型分泌系统（TFSS）基因同源，提示嗜肺军团菌可能是贝氏隐球菌。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. Burnetii T4 SS表达和结构/功能在SCV向LCV转化过程中的变化。本申请中提出的正在进行的研究的目标是（i）表征和定义C.贝氏梭菌T4 SS基因在细菌从SCV到LCV的转化过程中，以及（ii）确定C. Burnettii T4 SS，特别强调病原体的SCV和LCV形式之间的差异。我们将通过实现以下具体目标来检验我们的中心假设：目标1。明确T4 SS基因在C.在感染宿主细胞后，贝氏体从SCV转化为LCV。我们将使用RT-qPCR来确定C的时间表达。用免疫电镜和间接荧光抗体（IFA）鉴定贝氏锥虫T4 SS基因。在这段时间内，病原体的小细胞变体（SCV）和大细胞变体（LCV）形式上的贝氏体T4 SS表达。目标2.确定C.在SCV和LCV中的贝氏T4 SS。我们将使用抗C的抗体。Burnetii T4 SS蛋白的免疫印迹分析，以及C.在无细胞培养基中生长的贝氏体中的T4 SS，以确定T4 SS亚细胞定位、蛋白质-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体的恶劣环境中生存并引起疾病的毒力机制，将使我们能够制定针对这种知之甚少的细菌的对策。

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.