CHARACTERIZATION OF COXIELLA BURNETII ADHESINS

伯内氏柯克斯体粘附素的表征

• 批准号: 8360164
• 负责人: James Michael Battisti
• 金额: $ 10.47万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8360164
• 关键词: Adherence; Aerosols; Bacteria; Bacterial Adhesins; Binding; Cells; Collagen; Computer Simulation; Coxiella burnetii; Databases; Desiccation; Emerging Communicable Diseases; Enzyme-Linked Immunosorbent Assay; Eukaryotic Cell; Extracellular Matrix; Fingerprint; Funding; Genome; Grant; Human; Infection; Livestock; Mass Spectrum Analysis; Membrane Proteins; National Center for Research Resources; Pathogenesis; Phagolysosome; Principal Investigator; Proteins; Q Fever; Recombinants; Reproduction spores; Research; Research Infrastructure; Resistance; Resources; Reverse Transcriptase Polymerase Chain Reaction; Silver Staining; Source; Surface Antigens; Syndrome; Transcript; Ultraviolet Rays; United States National Institutes of Health; Variant; abortion; cost; environmental stressor; flu; member; pathogen

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Coxiella burnetii is an obligate intracellular pathogen that causes a flu-like syndrome in humans (Q fever) and abortions in livestock. The dormant 'spore-like' small cell variant (SCV) of C. burnetii is infectious (aerosol) and extremely resistant to environmental stressors (e.g., UV light, desiccation). Once inside an acidic phagolysosome-like compartment of a eukaryotic cell, C. burnetii transforms into a vegetative large cell variant (LCV). Identification of dominant outer membrane proteins (OMPs) synthesized by both morphological forms would result in a better understanding of C. burnetii pathogenesis. To this end, OMPs of purified C. burnetii SCVs and LCVs were prepared, separated, visualized by silver staining, and fingerprinted by mass spectrometry. Two dominant SCV-specific OMPs were identified as CBU0307 and CBU0311. Searches of the NCBI clusters of orthologous groups (COG) database showed that CBU0307 is orthologous to ompA-like outer membrane proteins and that CBU0311 is a member of COG3637, opacity protein and related surface antigens. C. burnetii CBU0311 has been the focus of several previous studies and has been termed P1. Further in silico analyses identified 3 additional CBU0307 paralogues (CBU1260, CBU1600, and CBU1814) and 3 additional CBU0311 paralogues (CBU1412, CBU1413 and CBU1414) within the C. burnetii genome. We used quantitative RT-PCR to show that transcript levels of the p1-group [CBU0311 (here termed p1-A), p1-B (CBU1414), p1-C (CBU1413), and p1-D (CBU1412)] were highest at four days post-infection, suggesting that the corresponding proteins are synthesized as the bacterium transitions to the SCV. As orthologues of the p1-group have been shown to serve as adhesins for extracellular matrix (ECM) components, we purified recombinant P1-A and analyzed its ability to bind immobilized ECM components by ELISA. A statistically significant interaction with collagen was observed. Identification of SCV OMP's involved in host cell adherence could provide a frontline defense strategy against C. burnetii infections.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 贝氏柯克斯体是一种专性细胞内病原体，可引起人类流感样综合征（Q热）和牲畜流产。休眠的“孢子样”小细胞变异体（SCV）的C。贝氏菌是传染性的（气溶胶）并且对环境应激源（例如，UV光，干燥）。 一旦进入真核细胞的酸性吞噬溶酶体样区室，C。Burnetii转化为营养大细胞变体（LCV）。 鉴定由两种形态形式合成的优势外膜蛋白（OMPs）将有助于更好地了解C。贝氏菌致病机理 为此，纯化的C.制备、分离贝氏体SCV和LCV，通过银染色可视化，并通过质谱法进行指纹分析。 两种优势SCV特异性OMP被鉴定为CBU 0307和CBU 0311。NCBI邻位组群（COG）数据库的检索显示CBU 0307与ompA样外膜蛋白是邻位的，CBU 0311是COG 3637、不透明蛋白和相关表面抗原的成员。 C. Burnetii CBU 0311是先前几项研究的焦点，并被称为P1。 进一步的计算机模拟分析在C中鉴定了另外3个CBU 0307旁系同源物（CBU 1260、CBU 1600和CBU 1814）和另外3个CBU 0311旁系同源物（CBU 1412、CBU 1413和CBU 1414）。贝氏菌基因组 我们使用定量RT-PCR显示，p1-组[CBU 0311（此处称为p1-A）、p1-B（CBU 1414）、p1-C（CBU 1413）和p1-D（CBU 1412）]的转录水平在感染后4天最高，表明相应的蛋白质在细菌向SCV转变时合成。 由于直向同源的P1-组已被证明作为细胞外基质（ECM）成分的粘附素，我们纯化重组P1-A，并通过ELISA分析其结合固定化ECM成分的能力。 观察到与胶原蛋白的统计学显著相互作用。 鉴定与宿主细胞粘附有关的SCV外膜蛋白可以提供抗C.贝氏体感染