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

• 批准号: 7782444
• 负责人: Edward I. Shaw
• 金额: $ 5.18万
• 依托单位: OKLAHOMA STATE UNIVERSITY STILLWATER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7782444
• 关键词: Acute; Aerosols; Alveolar Macrophages; Animals; Antigens; Applications Grants; Bacteria; Binding; Bioterrorism; Breathing; Categories; Cattle; Cell Communication; Cells; Centers for Disease Control and Prevention (U.S.); Characteristics; Chlamydia; Chronic; Chronic Disease; Chronic Hepatitis; Clinical; Coxiella burnetii; Cultured Cells; Cytoplasm; Cytoplasmic Protein; Data; Detection; Development; Diagnostic; Disease; Dose; Endocarditis; Environment; Europe; Fatigue; Foundations; Future; Gene Structure; Genes; Genetic Transcription; Genome; Goals; Goat; Homologous Gene; Human; Immune; Immune Sera; Immunoblot Analysis; Immunoelectron Microscopy; In Vitro; Industry; Infection; Intervention; Lead; Legionella pneumophila; Length; Life Cycle Stages; Link; Location; Lung; Mammalian Cell; Membrane; Messenger RNA; Methods; Molecular; Molecular Analysis; Nature; Open Reading Frames; Operon; Organism; Pathogenesis; Pathway interactions; Pattern; Persons; Phagolysosome; Phagosomes; Phase; Play; Population; Preparation; Primer Extension; Proteins; Q Fever; Recombinant Proteins; Recombinants; Reporting; Research; Reverse Transcriptase Polymerase Chain Reaction; Rickettsia; Risk; Role; Ruminants; Sequence Homology; Sheep; Site; Staging; Structure; System; Testing; Time; Transcriptional Regulation; Type IV Secretion System Pathway; United States National Institutes of Health; Vacuole; Variant; Virulence; Work; base; flu; genome sequencing; macrophage; molecular array; particle; pathogen; periplasm; protein expression; therapeutic vaccine; tissue culture; tool; trafficking; weapons

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.

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。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热性心内膜炎被认为是不可培养性心内膜炎的主要原因。伯纳蒂菌对环境稳定，感染剂量低，以前曾被开发为一种武器。这些特征使得它被疾控中心列为一种潜在的生物恐怖威胁。然而，通常情况下，动物种群（特别是反刍动物）是伯氏梭菌的宿主，它们释放出环境稳定的传染性颗粒，可以感染人类。因此，这种人畜共患病原体对从事牛、绵羊和山羊产业的人构成重大风险。初始感染发生在肺泡巨噬细胞。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（九英里I期菌株）的基因组测序结果显示，该菌株含有与嗜肺军团菌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。我们的中心假设是伯氏梭菌的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以作为TFSS的蛋白质，并检查这些蛋白质的时间表达。具体目的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。我们将使用逆转录酶PCR （RT-PCR）、实时RT-PCR和引物延伸作为工具来确定伯纳氏梭菌TFSS同源物在体外感染的早期、中期或晚期的表达。特异性目的2 -表征伯纳氏梭菌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同源物的特异性抗血清来实现这一目标，并使用这些探针来评估感染期间细菌中的蛋白质表达。在感染过程中表征伯氏梭菌的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的第一步。伯氏克希菌是一种专性胞内细菌，是急性Q热和慢性疾病的病原体。这是一种人畜共患病原体，被疾控中心指定为B类精选病原体。我们对伯纳氏梭菌与宿主细胞的分子相互作用知之甚少。在目前的提案中，我们将表征伯氏C. 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鉴定出q热检测和干预的独特诊断、治疗和疫苗靶点。