CHARACTERIZATION OF COXIELLA BURNETII ADHESINS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 伯氏柯克斯体是一种专性细胞内病原体，在人类(Q热)和家畜流产中引起流感样综合征。伯氏梭菌休眠的“孢子状”小细胞变种(SCV)具有传染性(气溶胶)，对环境应激源(紫外线、干燥)具有极强的抵抗力。一旦进入真核细胞的酸性(pH~5)吞噬酶体样隔室，伯氏梭菌就会转变为营养大细胞变体(LCV)。鉴定由这两种形态合成的优势外膜蛋白将有助于更好地理解伯氏卡氏杆菌的致病机理。为此，我们制备了纯化的伯氏梭菌SCV和LCV的OMPS(肌糖分级)、分离(等电聚焦和SDS-PAGE)、可视化(银染)和指纹图谱(质谱学)。鉴定出两个主要的SCV特异性OMP为CBU0307和CBU0311。BLASTO检索COG数据库，发现CBU0307与OmpA样外膜蛋白同源，CBU0311是COG3637、不透明蛋白及相关表面抗原的成员。Burnetii CBU0311一直是以前几个研究的重点，并被命名为P1。进一步的电子分析表明，在伯氏拟青霉基因组中还有3个CBU0307类似物(CBU1260、CBU1600和CBU1814)和另外3个CBU0311类似物(CBU1412、CBU1413和CBU1414)。我们使用定量RT-PCR显示，p1-组[CBU0311(这里称为p1-A)，p1-B(CBU1414)，p1-C(CBU1413)和p1-D(CBU1412)]的转录水平在感染后4天最高，这表明随着细菌向SCV的过渡，相应的蛋白质被合成。由于p1-基团的同源蛋白被证明是细胞外基质(ECM)成分的粘附素，我们纯化了重组的P1-A，并用ELISA法分析了其结合固定化ECM成分的能力。观察到与胶原蛋白的相互作用有统计学意义。识别参与宿主细胞黏附的SCV OMP可提供对抗伯氏梭菌感染的一线防御策略。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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 (UV light, desiccation). Once inside an acidic (pH ~5) 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 (sarkosyl fractionation), separated (isoelectric focusing and SDS-PAGE), visualized (silver staining), and fingerprinted (mass spectrometry). Two dominant SCV-specific OMPs were identified as CBU0307 and CBU0311. BLASTO searches of NCBI clusters of orthologous groups (COG) database show that CBU0307 is orthologous to ompA-like outer membrane proteins and 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)] are 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.