Elucidating Pseudomonas aeruginosa non-canonical two component system signaling

描述（由申请人提供）：临床重要病原体中抗生素耐药性的流行率日益增加，这是一个日益严重的威胁，超过了具有新作用机制的抗生素的开发。一种引起越来越多关注的新方法是靶向细菌毒力。一类有吸引力的靶标是双组分系统（TCS），其感测环境中的变化并协调细胞反应。这些细菌靶标在感染期间是重要的，而在哺乳动物宿主中不存在。此外，TCS通常在物种间是保守的，使得抑制剂具有广谱活性的潜力。 铜绿假单胞菌是一种重要的临床病原体，具有不断增加的抗生素耐药性，编码细菌中已知的最大的TCS组之一，并且被认为使用这些TCS来协调外部环境和人类宿主中生长之间的过渡，包括从急性到慢性感染生活方式的过渡。越来越清楚的是，TCS信号在铜绿假单胞菌中是高度复杂的。虽然经典TCS信号传导（定义为通过其同源组氨酸传感器激酶磷酸化反应调节剂）很重要，但越来越多的证据表明非经典TCS信号传导可能同样重要。非规范信令包括许多不同的信令机制，例如1.传感器之间的物理相互作用，而不是磷酸化事件，以调节反应调节剂的活性，2.通过替代响应调节器向传感器发出信号，或3.通过一个传感器激酶通过多个响应调节器进行信号传导（串扰）。 我们已经确定了几个新的TCS以前不知道发挥作用，在急性感染中筛选一套全面的铜绿假单胞菌突变体的58 TCS传感器激酶在一个新的脊椎动物模型的急性感染。我们确定kinB，phoR，bqsS和copS是所需的完整的毒力在斑马鱼（斑马鱼）胚胎以及gacS和retS，这是以前已被证明是需要在其他模型中的感染。我们发现，虽然KinB是急性感染所必需的，但它的信号是非典型的，独立于其同源反应调节因子AlgB和其激酶活性。我们建议阐明KinB的非经典TCS信号转导的机制，并定义其他三种新的TCS（PhoR，BqsS和CopS）的非经典TCS信号转导的程度。了解急性脊椎动物感染期间非经典TCS信号传导的程度以及TCS传感器和反应调节器之间的串扰程度对于评估靶向TCS作为治疗策略的潜力至关重要，并且将提供对病原体用于适应不同环境（包括人类宿主）以引发疾病的机制的深入了解。 公共卫生相关性：了解铜绿假单胞菌等细菌病原体如何感知和响应其环境，包括人类宿主，对于了解它们如何适应宿主微环境并引起疾病至关重要。我们已经确定了几个铜绿假单胞菌信号系统称为两个组成部分的系统，需要在脊椎动物宿主模型中引起致死性。我们建议阐明它们通过细胞内反应调节剂抑制这些信号的机制;因此，通过了解这些分子过程，我们将评估在当前抗生素耐药性增加的时代，靶向这些细菌因子作为新治疗策略的潜在新范例。

DESCRIPTION (provided by applicant): The increasing prevalence of antibiotic resistance among clinically important pathogens is a growing threat, outpacing the development of antibiotics with new mechanisms of action. One novel approach that has drawn increasing attention is targeting bacterial virulence. One attractive class of targets is two component systems (TCSs), which sense changes in the environment and coordinate a cellular response. These bacterial targets are important during infection while absent in mammalian hosts. Moreover, TCSs are often conserved across species so that inhibitors would have the potential to be broad spectrum in activity. Pseudomonas aeruginosa, an important clinical pathogen with increasing antibiotic resistance, encodes one of the largest sets of TCSs known in bacteria and is thought to use these TCSs to coordinate the transition between growth in the external environment and in a human host, including the transition from acute to chronic infection lifestyles. It is becoming increasingly clear that TCS signaling is highly complex in P. aeruginosa. While canonical TCS signaling, defined as phosphorylation of a response regulator by its cognate histidine sensor kinase, is important, increasing evidence suggests that non-canonical TCS signaling may be equally important. Non-canonical signaling includes a number of different signaling mechanisms such as 1. physical interactions between sensors rather than phosphorylation events to modulate the activity of a response regulator, 2. signaling of a sensor through an alternative response regulator, or 3. signaling through multiple response regulators by one sensor kinase (cross-talk). We have identified several novel TCS not previously known to play a role in acute infection by screening a comprehensive set of P. aeruginosa mutants in 58 TCS sensor kinases in a new vertebrate model of acute infection. We identified kinB, phoR, bqsS, and copS as being required for full virulence in Danio rerio (zebrafish) embryos as well as gacS and retS, which have previously been shown to be required for infection in other models. We found that while KinB is required for acute infection, it signals non-canonically, independent both of its cognate response regulator, AlgB, and of its kinase activity. We propose to elucidate the mechanism of KinB's non-canonical TCS signaling and define the degree of non-canonical TCS signaling for the three other novel TCS (PhoR, BqsS, and CopS). Understanding the extent of non-canonical TCS signaling and the degree of cross-talk among TCS sensors and response regulators during acute vertebrate infection is critical for evaluating the potential of targeting TCS as a therapeutic strategy and will provide insight into the mechanisms that pathogens use to adapt to varying environments, including the human host to elicit disease. PUBLIC HEALTH RELEVANCE: Understanding how bacterial pathogens such as Pseudomonas aeruginosa sense and respond to their environments, including the human host, is critical to understanding how they adapt to the host microenvironment and cause disease. We have identified several Pseudomonas aeruginosa signaling systems called two component systems that are required to cause lethality in a vertebrate host model. We propose to elucidate the mechanisms by which they transduce these signals through intracellular response regulators; therefore, by understanding such molecular processes, we will evaluate the potential new paradigm of targeting such bacterial factors as a new therapeutic strategy in this current era of increasing antibiotic resistance.