Function of MVFR in Pseudomonas Aeruginosa Virulence

描述（由申请人提供）：迫切需要发现抗菌药物的新方法，以对抗由抗生素耐药性或持久性抗生素耐受性细菌引起的不可治疗的感染。该提案的长期目标是开发新的治疗选择，可以预防或减少人类细菌急性和慢性或复发性感染的并发症，并可以作为抗生素的替代品或替代品。 为了实现抗菌治疗的范式转变，我们建议开发控制细菌毒力和抗生素耐受机制的细菌信号通路抑制剂。群体密度依赖性信号传导，通常被称为群体感应（QS），就是这样一种机制。QS调节毒力的多个方面。它对急性感染的发展很重要，最近发现它对耐药性细胞群的形成很重要，这是慢性感染中病原体持续存在的基础过程。该更新申请将测试我们先前鉴定的QS的新型抑制剂可以导致开发具有体内抗感染活性的高效化合物的假设。 我们将通过使用铜绿假单胞菌来验证这一假设，铜绿假单胞菌是一种革兰氏阴性细菌，无法通过抗生素根除，并在医院和重症监护室中消除了当前存在问题的病原体。在上一个资助周期中，我们证明了铜绿假单胞菌的发病机制可以通过干扰多毒力因子调节因子（MvfR）调节子来体内破坏，该调节子是控制毒力的QS电路的一个组成部分，正如我们最近发现的那样，抗生素耐受细胞的形成。我们阐明了MvfR调节子激活的机制，并确定了几个小的化合物，抑制MvfR转录因子和/或干扰MvfR调节子在体内的活性。 使用这些化合物，我们将通过三个具体目标来测试我们的假设：1）使用生物化学，质谱和分子遗传学分析来研究候选化合物的作用机制。2)通过构效关系（SAR）研究改进最有效的QS抑制剂。3)在合适的动物模型中验证SAR改进的抑制剂在减弱铜绿假单胞菌感染中的体内功效。QS信号通路在进化上是保守的，并且在许多不同的人类病原体中在调节毒力机制中发挥核心作用。因此，通过选择性地干扰QS，我们的数据应该产生典型的见解，这些见解通常与开发新类别的抗感染药物相关，这些抗感染药物可以限制细菌病原体中多药耐药性和抗生素耐受性的发展，同时保护有益的肠道细菌。此外，对抗生素耐受性机制的研究和抑制可能对阐明这一未解决的现象产生重大影响，并能够发现针对抗生素耐受性细胞的第一种探针化合物。

DESCRIPTION (provided by applicant): New approaches to antimicrobial drug discovery are urgently needed to combat untreatable infections caused by antibiotic resistant or persistent, antibiotic tolerant, bacteria. The long-term goal of this proposal is to develop novel therapy options that may prevent or reduce the complications of human bacterial acute and chronic or relapsing infections, and that could serve as alternatives or adjuncts to antibiotics. To achieve a paradigm shift in antimicrobial therapy, we propose to develop inhibitors of bacterial signaling pathways that control bacterial virulence and antibiotic tolerance mechanisms. Population density-dependent signaling, generally referred to as quorum sensing (QS), is one such mechanism. QS regulates multiple aspects of virulence. It is important for the development of acute infections and as recently discovered for the formation of antibiotic-tolerant cell populations, a process underlying pathogen persistence in chronic infections. This renewal application will test the hypothesis that novel inhibitors of QS, previously identified by us, can lead to the development of highly potent compounds with anti-infective activity in vivo. We will test this hypothesis by employing Pseudomonas aeruginosa, a recalcitrant Gram-negative bacterium that defies eradication by antibiotics and exemplifies current problematic pathogens in hospitals and intensive care units. In the last funding cycle, we demonstrated that P. aeruginosa pathogenesis can be disrupted in vivo by pharmacologically interfering with the multiple virulence factor regulator (MvfR) regulon, a component of QS circuitry that controls virulence and, as we recently discovered, the formation of antibiotic tolerant cells. We elucidated the mechanism of MvfR regulon activation and identified several small chemical compounds that inhibit the MvfR transcription factor and/or interfere with MvfR regulon activity in vivo. Using these chemical compounds, we will test our hypothesis through three Specific Aims: 1) To study the mechanisms of action of the candidate compounds using biochemical, mass spectrometric, and molecular genetic analyses. 2) To improve the most potent QS inhibitors through structure-activity relationship (SAR) studies. 3) To validate the in vivo efficacy of SAR-improved inhibitors in attenuating P. aeruginosa infection in suitable animal models. QS signaling circuits are evolutionarily conserved and play central roles in modulating virulence mechanisms in many different human pathogens. Therefore, by selectively interfering with QS, our data should yield paradigmatic insights that will be generally relevant for the development of new classes of anti-infectives that could limit development of multi-drug resistance and antibiotic tolerance in bacterial pathogens, while preserving beneficial commensal bacteria. Moreover, the study and inhibition of mechanisms involved in antibiotic tolerance could have a major impact on elucidating this unresolved phenomenon, as well as enabling the discovery of the first probe compounds targeted against antibiotic tolerant cells.