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Analysis of the Coxiella burnetii Type IV Secretion System During Infection

伯氏柯克斯体 IV 型感染过程中分泌系统的分析

基本信息

批准号：
8504113
负责人：
Edward I. Shaw
金额：
$ 2.07万
依托单位：
OKLAHOMA STATE UNIVERSITY STILLWATER
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-04-01 至 2015-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8504113
关键词：
Acute Acute Disease Aerosols Alveolar Macrophages Antibodies Applications Grants Bacteria Binding Biomedical Research Breathing Categories Cell Wall Cells Centers for Disease Control and Prevention (U.S.)Centrifugation Chronic Disease Chronic Hepatitis Clinical Coxiella burnetii Cytoplasm Detection Development Diagnostic Disease Electrons Endocarditis Environment Fatigue Fluorescent Antibody Technique Foundations Future Genes Genome Goals Growth Homologous Gene Hour Human Immunoblot Analysis In Vitro Infection Intervention Lead Legionella pneumophila Life Cycle Stages Membrane Messenger RNA Microscopy Molecular Pathogenesis Pathway interactions Peptides Phagolysosome Phagosomes Phase Play Proteins Q Fever Research Research Training Role Specificity Speed Structure System Testing Time Type IV Secretion System Pathway Vacuole Variant Virulence flu graduate student innovation pathogen periplasm protein protein interaction protein structure therapeutic vaccine trafficking undergraduate student

项目摘要

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.

描述（由申请人提供）：伯纳氏杆菌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因。通过吸入获得，伯纳氏梭菌最初接触肺泡巨噬细胞，并经历两阶段的生命周期，涉及结构上不同形式的病原体。感染通常被认为是由代谢不活跃、环境稳定的小细胞变异（SCV）开始，然后转化为复制、代谢活跃的大细胞变异（LCV）。在感染宿主细胞后，伯氏梭菌在液泡中复制，液泡保留了许多成熟吞噬溶酶体的特征。伯氏疏螺旋体寄生宿主细胞的分子机制在很大程度上是未知的。伯纳氏菌（九哩期I株）的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，提示伯纳氏菌具有与宿主细胞相互作用的特殊分泌途径。然而，对伯纳氏梭菌的TFSS及其在感染周期中的可能作用知之甚少。我们的中心假设是，在SCV到LCV的转化过程中，burnetii 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)表征和定义伯纳蒂C. T4SS基因在细菌从SCV转化为LCV期间的表达，（ii）确定伯纳蒂C. T4SS内部的结构和蛋白质相互作用，特别强调SCV和LCV病原体形式之间的差异。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。确定伯氏梭菌感染宿主细胞后由scv向lcv转化过程中T4SS基因的表达。我们将使用RT-qPCR来确定低速离心启动感染后第一个36 hpi期间伯纳蒂胞杆菌T4SS基因的时间表达，并使用间接荧光抗体（IFA）和免疫电子（IEM）显微镜来确定和区分伯纳蒂胞杆菌T4SS在小细胞变异（SCV）和大细胞变异（LCV）形式的病原体上的表达。目标2。确定伯氏梭菌T4SS在scv和lcv中的亚细胞定位、结构和蛋白-蛋白相互作用。我们将使用针对伯纳蒂胞杆菌T4SS蛋白的抗体进行细菌组分的免疫印迹分析，以及在无细胞培养基中生长的伯纳蒂胞杆菌的IFA和IEM分析，以确定T4SS亚细胞定位、蛋白-蛋白相互作用和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。