Functional genomics of P. gingivalis-host interactions

根据DE-10510进行的这项研究的广泛、长期目标是在分子水平上了解细菌-宿主相互作用引发的细菌反应；这些知识对于我们理解微生物的发病机制和宿主防御是至关重要的。牙龈卟啉单胞菌感染的第一步是在牙龈沟衬里的连接上皮的定植。该提案的目的是确定决定这种最初相互作用的牙龈假单胞菌基因和蛋白质。这项研究的一个直接结果是证明了牙龈痛是细菌与人类上皮细胞黏附的关键因素。这些重要发现为解决粘连功能基因组学的新假说奠定了基础。该应用程序的特定目的1测试特定的牙龈痛结构域决定牙龈假单胞菌与上皮细胞的黏附和脱离的假说。这将在黏附竞争和阻断实验中用纯化的重组牙痛蛋白和多肽进行测试。特定目标2解决了蛋白质复合体参与黏附的假设，并将识别该复合体的成分。特定目标3和4引入了一个新的研究领域，其核心是假设在感染期间，一组确定的牙龈假单胞菌基因被表达，以促进面对宿主防御的疾病和有机体的生存；非必需基因的表达被下调，以确保细胞资源的有效利用。因此，在特定的目标3中，将使用消减杂交技术来检测特定刺激后的基因表达，即与上皮细胞接触。特殊目的4使用最先进的牙龈假单胞菌微阵列对全基因组进行分析，继续这项研究。在细菌对抗生素耐药性增加和新的人类病原体出现的背景下，了解感染细菌与其宿主之间的分子相互作用是新药发现的加速器。我们关于牙龈痛和上皮细胞黏附的假说的验证可能会进一步刺激针对这些蛋白酶的第二代抗生素的研究。与致病过程相关的新活动的发现将增加未来靶点的曲目，并推动寻找新的治疗干预措施。

The broad, long-term goal of the research conducted under DE-10510 is to understand, at the molecular level, bacterial responses triggered by bacteria-host interaction; knowledge that is central to our comprehension of microbial pathogenesis and host defense. The first step in Porphyromonas gingivalis infection is colonization of the junctional epithelium lining the gingival sulcus. The objective of the proposal was to identify P. gingivalis genes and proteins that determined this initial interaction. A direct result of the study was the demonstration that gingipains were critical players in adherence of the bacterium to human epithelial cells. These important discoveries set the stage for new hypotheses that address the functional genomics of adhesion. Specific Aim 1 of this application tests the hypothesis that specific gingipain domains determine P. gingivalis adhesion to, and detachment from, epithelial cells. This will be tested with purified recombinant gingipain proteins and peptides in adhesion competition and blocking experiments. Specific Aim 2 addresses the hypothesis that a complex of proteins is involved in adhesion, and components of the complex will be identified. Specific Aims 3 and 4 introduce a new research area centered on the hypotheses that during infection a defined set of P. gingivalis genes are expressed to promote disease and survival of the organism in the face of host defenses; and the expression of non-essential genes is down-regulated to ensure the efficient use of cell resources. Thus, in Specific Aim 3, subtractive hybridization techniques will be used to examine gene expression after a defined stimulus, contact with epithelial cells. Specific Aim 4 continues this investigation with genome-wide analyses using state of the art P. gingivalis microarrays. Within the context of increased bacterial resistance to antibiotics, and the emergence of new human pathogens, an understanding of the molecular interactions between an infecting bacterium and its host is the accelerant to new drug discovery. Validation of our hypotheses regarding gingipains and epithelial cell adhesion may further stimulate searches for second-generation antibiotics directed to these proteinases. The discovery of new activities related to the pathogenic process will increase the repertoire of future targets, and drive the hunt for novel therapeutic interventions.