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: 6796839
• 负责人: Murray Hackett
• 金额: $ 28.17万
• 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/6796839
• 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.

牙龈卟啉单胞菌是一种革兰氏阴性厌氧菌，是严重成人牙周炎的主要病原体。牙龈卟啉单胞菌拥有许多毒力因子，包括侵入牙龈上皮细胞的能力。在人牙龈上皮细胞（GEC）的原代培养物中，内部细菌迅速定位在细胞质中，主要位于核周区域，在那里它们可以复制并达到高密度。指导这些事件的牙龈卟啉单胞菌分子尚未确定。 GEC 被用作研究人类牙周病中宿主-病原体相互作用的模型系统。我们建议采取一种基于蛋白质组学的综合方法，通过检查病原体入侵期间整体蛋白质表达的变化来研究牙龈卟啉单胞菌的入侵和毒力。对于一组选定的蛋白质，通过质谱法测量蛋白质表达的时间进程，将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。还将使用华盛顿大学开发的非水反相 HPLC 技术和（或）Yates 实验室的 MudPIT 方法（多维蛋白质识别技术）对牙龈卟啉单胞菌疏水性膜结合蛋白的预期变化进行分析，这两种技术都扩大了可分析的蛋白质范围，超出了当前最先进的 2D 凝胶电泳所能完成的范围。 等电点和疏水性。 我们能够直接使用质谱碎片数据以半自动方式将观察到的蛋白质映射到牙龈卟啉单胞菌基因组，从而补充了转录分析。这些实验的数据集将用于深入了解牙龈卟啉单胞菌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，这些治疗剂可抑制牙龈卟啉单胞菌从无害的共生定植菌转变为高度侵袭性的病原体。