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，是成人重度牙周炎的主要病原菌。牙龈假单胞菌具有多种毒力因子，包括侵袭牙龈上皮细胞的能力。在原代培养的人牙龈上皮细胞(GECs)中，内部细菌迅速定位于细胞质中，主要分布在核周区域，在那里它们可以复制并达到高密度。指导这些事件的牙龈假单胞菌的分子还没有确定。GECs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侵袭和毒力，方法是检测病原体入侵过程中全球蛋白表达的变化。对于选定的一组蛋白质，通过质谱仪对蛋白质表达的时序测量将与使用半定量实时逆转录聚合酶链式反应(RTPCR)和Northern印迹分析测量的mRNA水平进行比较。此外，还将利用华盛顿大学开发的非水反相高效液相技术和(或)耶茨实验室的MudPIT方法(多维蛋白质鉴定技术)来分析牙龈假单胞菌疏水性膜结合蛋白的预期变化，这两种技术都可以扩展可分析的蛋白质范围，使其在等电点和疏水性方面超出了目前最先进的2D凝胶电泳所能完成的范围。 转录分析将得到我们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假单胞菌基因组的能力的补充。这些实验的数据集将被用来深入了解牙龈假单胞菌侵袭性的精确分子决定因素，这将有助于描述治疗剂的潜在靶标，这些治疗剂可能有助于抑制牙龈假单胞菌从无害的共生定殖者转变为高度侵袭性的病原体。

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.