Genomics of Gene Regulation in P. gingivalis

描述（由申请人提供）：在R01 DE015931下进行的持续的长期研究目标是确定促进牙龈假单胞菌生长和毒力从而引发牙周炎的牙龈沟体内条件。假设牙龈假单胞菌在这种宿主-病原体相互作用中的作用是通过其对龈下缝隙中普遍存在的环境条件的反应来表现的。像其他细菌一样，牙龈假单胞菌的这些反应是由双组分信号转导系统调节的。在最简单的系统中，第一个组分，组氨酸激酶（HK），通过在其序列中自动磷酸化指定的组氨酸残基来响应特定的环境刺激。信息通过活化的磷酸盐从HK组氨酸转移到同源反应调节剂（RR）中指定的天冬氨酸残基来传递。激活的RR随后与目标基因的启动子（调控子）结合并调节其表达。在本申请的特异性目的1中，将通过ChIP-on-chip和互补分析来鉴定牙龈假单胞菌rr的调控。在Specific Aim 2中，将通过凝胶移位法确认rr与靶基因启动子之间的直接相互作用，并分析靶启动子序列。与基因调控的分子机制相比，双组分系统诱导剂的信息相对有限。该应用程序的一个目标是填补这一知识空白，并确定牙龈卟啉卟啉卟啉卟啉反应的环境条件。在项目的这一点上，每个RR及其规则的验证信息将用于对触发激活的潜在条件做出明智的决策。因此，在Specific Aim 3中，激活单个HK-RR系统的环境线索将使用报告基因结构来识别。组氨酸激酶和反应调节因子是很好的治疗靶点，因为它们在细菌生理中具有多种作用。尽管特异性抑制剂的开发一直缓慢，但最近在化学文库的高通量筛选中发现了细菌转录调节因子的小分子抑制剂，重新引起了对这些潜在靶点的兴趣。在未来，这种抑制剂可能对一般人类健康产生积极影响，并影响牙周病的治疗，取代效果较差和风险较高的抗生素治疗。

DESCRIPTION (provided by applicant): The continuing long-term goal of research conducted under R01 DE015931 is to identify the in vivo conditions of the gingival sulcus that promote the growth and virulence of P. gingivalis, so triggering periodontitis. The hypothesis is that the role of P. gingivalis in this host-pathogen interaction is manifested through its responses to environmental conditions that prevail in the subgingival crevice. Like other bacteria, in P. gingivalis these responses are regulated by two-component signal transduction systems. In the simplest system the first component, a histidine kinase (HK), responds to a specific environmental stimulus by auto-phosphorylating a designated histidine residue in its sequence. Information is relayed by transfer of activated phosphate from the HK histidine to a designated aspartate residue in the cognate response regulator (RR). The activated RR then binds to promoters of its target genes (the regulon) and regulates their expression. In Specific Aim 1 of this application the regulons of P. gingivalis RRs will be identified by ChIP-on-chip and complementary assays. In Specific Aim 2, direct interaction between RRs and promoters of target genes will be confirmed by gel shift assays, and target promoter sequences will be analyzed. There is relatively limited information on the inducers of two-component systems compared to that available on the molecular mechanisms of gene regulation. A goal of this application is to fill this knowledge gap and identify environmental conditions to which P. gingivalis RRs respond. At this point in the project validated information on each RR and its regulon will be used to make informed decisions on potential conditions that trigger activation. Therefore, in Specific Aim 3 the environmental cues that activate individual HK-RR systems will be identified using reporter gene constructs. It is claimed that histidine kinases and response regulators are good therapeutic targets because of their multiple roles in bacterial physiology. Though the development of specific inhibitors has been slow, small molecule inhibitors for bacterial transcriptional regulators were recently discovered in high through-put screens of chemical libraries, reviving interest in these potential targets. In the future, such inhibitors may have a positive impact on general human health and influence treatments for periodontal disease replacing less effective and riskier antibiotic therapies. PROJECT NARRATIVE: The goal of this research is to identify conditions within the gingival pocket that promote the growth of bacteria that cause periodontitis. Bacterial responses to these conditions are regulated by signal relay systems comprised of two components: histidine kinases that sense changes in the environment and regulators that co- ordinate the response to these changes. Such two-component systems are prime targets for new inhibitor therapeutics that positively impact human health.