Proteomics of Porphyromonas gingivalis interactions with Fusobacterium nucleatum

描述（由申请人提供）：本项目将研究口腔微生物群落的整体蛋白表达变化及其对模型宿主细胞的反应。我们将选择牙周病原体牙龈假单胞菌作为模式生物，同时选择具有代表性的牙生物膜中常见的具核假单胞菌和戈登假单胞菌作为模式生物，它们能够与牙龈假单胞菌协同作用。除了这些生物聚集成群落时的蛋白质表达差异外，我们还将确定与牙龈上皮细胞接触引起的蛋白质表达变化。这项研究的长期目标包括提高我们在分子水平上对异型口腔生物膜形成以及这些生物膜与宿主细胞相互作用的基本认识。特异性目标1定义了牙龈卟啉单胞菌、戈登卟啉单胞菌和核仁卟啉单胞菌的参考蛋白质组。这将包括使用数据挖掘技术的全局蛋白质修饰分析，以及具体的全局磷酸化化学方法。我们将定义可归因于群落微生物相互作用的蛋白质相对丰度变化（相对于孤立的单个生物体）。因此，我们将建立P. gingivalis-F的蛋白质组。nucleatum-S。Gordonii群落，以及与个体蛋白质组相比对群落发育的差异反应。特异性目的2将确定牙龈上皮细胞对牙龈卟噬菌的影响。nucleatum-S。gordonii社区。这将涉及与Aim 1中相同的实验，但在上皮细胞存在的情况下，也将在微生物方面进行比较翻译后修饰（PTM）分析。这将决定群落对宿主细胞的反应。与Aim 1一样，这包括使用计算方法在全球范围内挖掘PTMs的所有已知修饰的数据集，此外还使用基于化学的实验方法来测量差异的全球磷酸化。在Aims 3中，我们将优先考虑在Aims 1和2中鉴定的受调节蛋白，作为发现的保证，以进一步证实并在相应基因中构建突变体。突变体在群落发育和对上皮细胞的反应中的表型将进一步阐明其功能意义和生物学相关性。

DESCRIPTION (provided by applicant): This project will examine global protein expression changes in oral microbial communities and the community response to model host cells. We will select as model organisms the periodontal pathogen P. gingivalis, along with F. nucleatum and S. gordonii as representative of organism commonly found in dental biofilms and capable of synergistic interactions with P. gingivalis. In addition to differential protein expression as these organisms assemble into communities, we will also determine changes in protein expression induced by contact with gingival epithelial cells. The long-term goals of the study involve improving our fundamental understanding at the molecular level of events surrounding heterotypic oral biofilm formation and the interaction of these biofilms with host cells. Specific Aim 1 defines reference proteomes of P. gingivalis, S. gordonii, and F. nucleatum individually, pairwise and all together. This will include global protein modification analysis using data mining techniques, and a chemical approach to global phosphorylation specifically. We will define protein relative abundance changes (relative to the single organism in isolation) that can be attributed to community microbial interactions. We will thus establish the proteome of the P. gingivalis-F. nucleatum-S. gordonii community, and the differential responses to community development in comparison to individual proteomes. Specific Aim 2 will define the effects of gingival epithelial cells on the P. gingivalis-F. nucleatum-S. gordonii community. This will involve running the same experiments as in Aim 1, but in the presence of epithelial cells, also with comparative posttranslational modification (PTM) analysis on the microbial side. This will define the response of the community to the presence of host cells. As in Aim 1, this includes mining the datasets globally for PTMs for all known modifications using computational approaches, and in addition using a chemically based experimental approach to measuring differential global phosphorylation. In Aim 3 we will prioritize the regulated proteins identified in Aims 1 and 2, as discovery warrants, for additional corroboration and for construction of mutants in the corresponding genes. The phenotype of the mutants in community development and responses to epithelial cells will further elucidate functional meaning and biological relevance.