Proteomics of P. gingivalis invasion of epithelial cells

牙龈卟啉单胞菌侵袭上皮细胞的蛋白质组学

• 批准号: 6910861
• 负责人: Murray Hackett
• 金额: $ 28.15万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6910861
• 关键词: Bacteroides gingivalis; bacteria infection mechanism; bacterial proteins; epithelium; gingiva; high performance liquid chromatography; host organism interaction; mass spectrometry; membrane proteins; messenger RNA; northern blottings; periodontitis; polymerase chain reaction; proteomics; two dimensional gel electrophoresis; virulence

Porphyromonas gingivalis, a Gram-negative anaerobe, is a major etiologic agent of severe adult periodontitis. P. gingivalis possesses a number of virulence factors including the ability to invade the epithelial cells of the gingiva. In primary cultures of human gingival epithelial cells (GECs) the internal bacteria rapidly locate in the cytoplasm, predominantly in the perinuclear area, where they can replicate and reach a high density. The molecules of P. gingivalis that direct these events have yet to be determined. GECs are being used as a model system to study host-pathogen interactions involved in human periodontal disease. We propose to take a comprehensive, proteomics based approach to the study of P. gingivalis invasion and virulence by examining changes in global protein expression during invasion for the pathogen. For a select group of proteins, timecourse measurements of protein expression by mass spectrometry will be compared to mRNA levels measured using semi-quantitative real time reverse transcriptase polymerase chain reaction (RTPCR) and Northern blot analyses. Expected changes in P. gingivalis hydrophobic membrane bound proteins will be assayed as well, using non-aqueous reversed-phase HPLC technology developed at the University of Washington and (or) the MudPIT approach (Multidimensional Protein Identification Technology) of the Yates laboratory, both of which expand the range of proteins that can be analyzed beyond what can be done with the current state-of-the-art in 2D gel electrophoresis in terms of isoelectric point and hydrophobicity. The transcription analysis will be complemented by our ability to map observed proteins to the P. gingivalis genome in a semi-automated fashion directly using mass spectral fragmentation data. The data sets from these experiments will be used to gain insights into the precise molecular determinants of P. gingivalis invasiveness, that will aid in the characterization of potential targets for therapeutic agents that could serve to inhibit the transformation of P. gingivalis from a harmless commensal colonizer into a highly invasive pathogen.

牙孢菌素是一种革兰氏阴性肌，是严重成人牙周炎的主要病因。牙龈疟原虫具有许多毒力因子，包括侵入牙龈上皮细胞的能力。在人牙龈上皮细胞（GEC）的原发性培养物中，内部细菌迅速位于细胞质中，主要位于核周区域，它们可以复制并达到高密度。指导这些事件的牙龈疟原虫的分子尚未确定。 GEC被用作研究与人牙周疾病有关的宿主 - 病原体相互作用的模型系统。我们建议通过检查病原体侵袭期间全球蛋白质表达的变化来采用一种基于蛋白质组学的方法来研究牙龈牙周疟原虫的侵袭和毒力。对于一组蛋白质，将质谱法对蛋白表达的时间测量与使用半定量实时逆转录酶聚合酶链反应（RTPCR）和Northern Blot分析测量的mRNA水平进行比较。还将使用在华盛顿大学开发的非相反相位的HPLC技术和（或（或）Yates实验室的MudPIT方法（多维蛋白识别技术），这两种蛋白质可以扩展到蛋白质的范围，并且可以分析的是，并且可以分析的范围是，并且可以分析的是，那么在蛋白质上可以分析，但在蛋白质上可以分析，也可以分析的是，还可以分析质量超出，并且可以分析的是，也可以分析，牙齿牙周牙周疟原虫疏水膜结合的蛋白质的预期变化也将被测定。等电点和疏水性。 转录分析将通过我们使用质谱片段化数据直接以半自动化方式将观察到的蛋白映射到牙龈斑叶基因组的能力得到补充。这些实验中的数据集将用于洞悉牙龈疟原虫侵袭性的精确分子决定因素，这将有助于表征治疗剂的潜在靶标的治疗剂，这些剂量可以抑制牙龈疟原虫从无害的共生结肠转化为高度侵入性病原体。