Genomics of Gene Regulation in P. gingivalis

描述(由申请人提供)：根据R01 DE015931进行的研究的持续长期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周沟的活体条件。假说是牙龈假单胞菌在宿主-病原体相互作用中的作用是通过它对龈下缝隙中普遍存在的环境条件的反应来表现出来的。像其他细菌一样，在牙龈假单胞菌中，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，第一组分，组氨酸激酶(HK)，通过自动磷酸化ITS序列中指定的组氨酸残基来响应特定的环境刺激。信息是通过将激活的磷酸从香港组氨酸转移到同源反应调节器(RR)中指定的天冬氨酸残基来传递的。然后，激活的RR与其目标基因的启动子(调节子)结合并调节它们的表达。在本申请的具体目标1中，将通过芯片上的芯片和互补分析来鉴定牙龈假单胞菌RRS的调控。在特定目标2中，将通过凝胶移位分析证实RRs与目标基因启动子之间的直接相互作用，并将分析目标启动子序列。与现有的关于基因调控的分子机制的信息相比，关于双组分系统的诱导剂的信息相对有限。这项应用的一个目标是填补这一知识空白，并确定牙龈假单胞菌RRS对环境条件的反应。在该项目的这一点上，关于每个RR及其调节子的验证信息将被用来就触发激活的潜在条件做出明智的决定。因此，在特定目标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剂被认为是很好的治疗靶点，因为它们在细菌生理学中具有多重作用。尽管特异性抑制剂的开发一直很缓慢，但最近在化学文库的高通量筛选中发现了细菌转录调节的小分子抑制剂，重新唤起了人们对这些潜在靶点的兴趣。在未来，这些抑制剂可能会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疗法。 项目简介：这项研究的目标是确定牙周袋内促进细菌生长的条件，这些细菌会导致牙周炎。细菌对这些条件的反应由两个组成部分组成的信号中继系统调节：感知环境变化的组氨酸激酶和协调对这些变化的反应的调节器。这种双组分系统是积极影响人类健康的新抑制剂疗法的主要靶点。

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.