Function of MVFR in Pseudomonas Aeruginosa Virulence

描述(由申请人提供)：迫切需要新的抗微生物药物发现方法来对抗由抗生素耐药或持久的、抗生素耐药的细菌引起的无法治疗的感染。这项提议的长期目标是开发新的治疗方案，可以预防或减少人类细菌性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附件。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。依赖于种群密度的信号传递，通常被称为群体感应(QS)，就是这样一种机制。QS调控毒力的多个方面。它对急性感染的发展和最近发现的对抗生素耐受细胞群体的形成非常重要，这是慢性感染病原体持续存在的一个过程。这一新的应用将检验这样一个假设，即我们之前发现的新型QS抑制剂可以在体内导致开发具有抗感染活性的高效化合物。我们将通过使用铜绿假单胞菌来验证这一假设，这是一种顽固的革兰氏阴性细菌，无法被抗生素根除，并举例说明了目前医院和重症监护病房中存在问题的病原体。在上一个资助周期中，我们证明了在体内可以通过药物干扰多重毒力因子调节因子(MvfR)调节子来扰乱铜绿假单胞菌的致病机制，MvfR调节子是QS电路的一个组成部分，控制毒力和我们最近发现的抗生素耐受细胞的形成。我们阐明了MvfR调节子激活的机制，并在体内鉴定了几个抑制MvfR转录因子和/或干扰MvfR调节子活性的小化合物。使用这些化合物，我们将通过三个具体目标来验证我们的假设：1)使用生化、质谱学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)在合适的动物模型上验证经SAR改良的抑制剂在体内减轻铜绿假单胞菌感染的效果。QS信号通路在进化上是保守的，并在调节许多不同人类病原体的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一般性的见解，这些见解将普遍与新型抗感染药物的开发相关，这些药物可能限制细菌病原体的多重耐药和抗生素耐药性的发展，同时保留有益的共生细菌。此外，对抗生素耐受相关机制的研究和抑制可能会对阐明这一尚未解决的现象以及发现第一个针对抗生素耐受细胞的探针化合物产生重大影响。

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.