Proteomics of P. gingivalis invasion of epithelial cells

牙龈卟啉单胞菌（Porphyromonasgingivalis）是一种革兰氏阴性厌氧菌，是严重成人牙周炎的主要病原菌。牙龈卟啉单胞菌具有许多毒力因子，包括侵入牙龈上皮细胞的能力。在人牙龈上皮细胞（GECs）的原代培养物中，内部细菌迅速定位于细胞质中，主要在核周区域，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。我们建议采取一个全面的，基于蛋白质组学的方法来研究牙龈卟啉单胞菌的侵袭和毒力，通过检查全球蛋白质表达的变化，在入侵的病原体。对于选定的一组蛋白质，将通过质谱法进行的蛋白质表达的时程测量与使用半定量真实的时间逆转录酶聚合酶链反应（RTPCR）和北方印迹分析测量的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）MudPIT方法测定牙龈卟啉单胞菌疏水膜结合蛋白的预期变化（多维蛋白质鉴定技术），这两种方法都扩大了可以分析的蛋白质的范围，超出了目前的水平。在等电点和疏水性方面，在2D凝胶电泳中是最好的。 转录分析将通过我们直接使用质谱片段化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的能力来补充。来自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点，这些治疗剂可用于抑制牙龈卟啉单胞菌从无害的牙龈定殖菌转化为高度侵袭性病原体。

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.