Proteomics of P. gingivalis invasion of epithelial cells

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因。牙龈卟啉卟啉具有许多毒力因子，包括侵袭牙龈上皮细胞的能力。在人牙龈上皮细胞（GECs）的原代培养中，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。引导这些事件的牙龈卟啉单胞菌分子尚未确定。gec正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。我们建议采用一种全面的、基于蛋白质组学的方法来研究牙龈假单胞菌的入侵和毒力，通过检测病原体入侵过程中全局蛋白表达的变化。对于选定的一组蛋白质，将用质谱法测量蛋白质表达的时间过程与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。预期p . gingivalis疏水性膜结合蛋白的变化将化验,使用无水的反相高效液相色谱技术发达华盛顿大学和(或)MudPIT方法耶茨(多维蛋白质识别技术)的实验室,这两种蛋白质的范围扩大,可以分析超出了可以用当前最先进的二维凝胶电泳等电点和疏水性。

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.