Genomics of Gene Regulation in P. gingivalis

描述（由申请人提供）：根据R 01 DE 015931进行的研究的持续长期目标是确定促进牙龈卟啉单胞菌生长和毒力的龈沟的体内条件，从而引发牙周炎。假设牙龈卟啉单胞菌在宿主-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。与其他细菌一样，在牙龈卟啉单胞菌中，这些反应受双组分信号转导系统调节。在最简单的系统中，第一个组成部分，组氨酸激酶（HK），响应于特定的环境刺激，通过自动磷酸化其序列中指定的组氨酸残基。通过将活化的磷酸盐从HK组氨酸转移到同源反应调节剂（RR）中的指定天冬氨酸残基来传递信息。然后，激活的RR与其靶基因（调节子）的启动子结合并调节其表达。在本申请的具体目标1中，牙龈卟啉单胞菌RR的调节子将通过ChIP芯片和互补测定法鉴定。在特定目的2中，将通过凝胶迁移试验确认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，关于双组分系统的诱导剂的信息相对有限。本申请的目标是填补这一知识空白，并确定牙龈卟啉单胞菌RR响应的环境条件。在项目的这一点上，每个RR及其调节子的验证信息将用于对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，将使用报告基因构建体来鉴定激活个体HK-RR系统的环境线索。由于组氨酸激酶和反应调节剂在细菌生理学中的多重作用，它们被认为是良好的治疗靶点。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，取代效果较差且风险较高的抗生素治疗。 项目叙述：这项研究的目的是确定龈袋内的条件，促进细菌的生长，导致牙周炎。细菌对这些条件的反应由信号中继系统调节，该系统由两种组分组成：感测环境变化的组氨酸激酶和协调对这些变化的反应的调节剂。这种双组分系统是对人类健康产生积极影响的新抑制剂疗法的主要靶标。

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.