Functional genomics of P. gingivalis-host interactions

根据DE-10510进行的研究的广泛、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；这是我们理解微生物发病机制和宿主防御的核心知识。牙龈卟啉单胞菌感染的第一步是龈沟内的结界上皮的定植。该提案的目的是鉴定牙龈假单胞菌基因和蛋白质，决定这种初始相互作用。这项研究的一个直接结果是，牙龈痛是细菌附着在人类上皮细胞上的关键因素。这些重要的发现为解决粘附功能基因组学的新假设奠定了基础。本应用的特异性目的1测试了特定牙龈蛋白酶结构域决定牙龈假单胞菌粘附和脱离上皮细胞的假设。这将用纯化的重组牙龈蛋白酶蛋白和肽在粘附竞争和阻断实验中进行测试。特异性目标2解决了一个蛋白质复合物参与粘附的假设，该复合物的组分将被识别。具体目标3和4介绍了一个新的研究领域，其中心假设是在感染过程中，一组特定的牙龈假单胞菌基因被表达，以促进疾病和生物体在面对宿主防御时的生存；而非必需基因的表达则被下调，以保证细胞资源的有效利用。因此，在Specific Aim 3中，减法杂交技术将用于检测与上皮细胞接触的特定刺激后的基因表达。特异目标4继续这一调查与全基因组分析使用最先进的状态牙龈假单胞菌微阵列。在细菌对抗生素的耐药性增加和新的人类病原体出现的背景下，对感染细菌与其宿主之间分子相互作用的理解是新药发现的催化剂。验证我们关于牙龈疼痛和上皮细胞粘附的假设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.