CHARACTERIZATION OF COXIELLA BURNETII ADHESINS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 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。贝氏菌具有传染性（气溶胶），并且对环境应激源（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.制备贝氏体SCV和LCV（肌氨酰分级分离）、分离（等电聚焦和SDS-PAGE）、可视化（银染色）和指纹分析（质谱）。 两种优势SCV特异性OMP被鉴定为CBU 0307和CBU 0311。通过NCBI clusters of orthopathic groups（COG）数据库的BLASTO检索，CBU 0307与ompA样外膜蛋白同源，CBU 0311与COG 3637、不透明蛋白及相关表面抗原同源。 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

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.