Adapting to a changing environment: How surface contact induces virulence factor production in Pseudomonas aeruginosa

项目总结 铜绿假单胞菌(PA)是一种多功能的机会致病菌，是导致医院感染的主要原因。 获得性感染。PA抗生素耐药性持续爆炸，促使新的治疗方法的开发 满足了一个迫切的需求。一个在很大程度上未被探索的治疗场所是不同寻常的大量 感知系统表明PA已经进化。这些信号转导途径使PA能够迅速适应广泛的 各种环境，例如从游泳状态转换到与水面相关的状态。通过基因 Screen，我们和其他人已经在PA中确定了三个系统--IV型菌毛(TFP)，CHP化学感觉 第二信使3‘，5’-环单磷酸(CAMP) 以及其变构调节的蛋白结合伙伴Vfr--这是毒力上调所必需的 影响表面结合的因素。通过TFP收回激活CHP系统导致了一系列 导致膜结合腺苷酸环化酶(CyaB)激活的磷酸化事件 CAMP的主要酶来源。CAMP与转录激活物(Vfr)结合诱导转录 &gt；200个基因，其中许多与人类的毒力有关，并导致急性肺损伤。我们有 研究发现，在对表面结合和回缩做出反应时，TFP作为机械换能器发挥作用 激活CHP磷酸继电器，进而增加跨膜腺苷环化酶的活性 CyaB。使用多种方法，我们发现了该系统不同组件之间的交互 确定了两个关键的信号集成中枢。我们提出了三个具体目标来测试和进一步完善我们的模型 这将加深我们对TFP-CHP-CyaB机械力化学信号通路的理解。目标1.测试 假设PilJ通过协调调节MCS作为MCS的中心积分器 机械输入信号(改变的毛细蛋白单体)，激活CHP磷继电器和 CyaB的激活。我们将使用遗传筛选，体内生理功能分析，体内生化， 以及包括FRET在内的活细胞荧光成像，以定义(A)PilA/PilJ/CyaB/和(B)PilJ/PilH相互作用 风景画。目的2.检验FimV/Fim1/PilG集线器将TFP功能与CHP/CyaB连接的假设 系统。我们将(A)定义FIML/PilG交互环境，并(B)使用PHOS-TAG技术16 在MCS期间体内检测PilG和PilH的磷酸化。目标3.确定关键的空间和时间 生物膜形成过程中TFP-CHP-CyaB机械力化学信号转导特性 极化的肺上皮单层。我们将(A)确定TFP-CHP-CyaB MCS在 生物膜的形成和(B)决定表面激活基因的时间和空间动力学 在生物膜形成过程中的表达。

Project summary Pseudomonas aeruginosa (PA) is a versatile opportunistic pathogen that is a leading cause of hospital- acquired infections. PA antibiotic resistance continues to explode, making development of new therapeutic approaches a critical need. One largely unexplored therapeutic venue is the uncommonly large number of sensing systems that PA has evolved. These signal transduction pathways allow PA to rapidly adapt to a wide variety of environments, such as transitioning from swimming to surface-associated states. Through genetic screens, we and others have identified three systems in PA—the type IV pilus (TFP), the Chp chemosensory system (a complex chemosensory system), and the second messenger 3', 5'-cyclic monophosphate (cAMP) and its allosterically regulated protein binding partner, Vfr-- that are required for upregulation of virulence factors upon surface binding. Activation of the Chp system by TFP retraction leads to a cascade of phosphorylation events that leads to the activation of a membrane-bound adenylate cyclase (CyaB), the primary enzymatic source of cAMP. cAMP binds to a transcriptional activator (Vfr) to induce transcription of >200 genes, many of which are involved in virulence in humans and in causing acute lung damage. We have discovered that in response to surface binding and retraction, the TFP functions as a mechanotransducer to activate the Chp phosphorelay, which in turn increases the activity of the transmembrane adenylate cyclase CyaB. Using multiple approaches, we have uncovered interactions between various components of this system that identify two key signal integrating hubs. We propose 3 specific aims to test and further refine our model and that will deepen our understanding of TFP-Chp-CyaB mechanochemical signaling pathway. Aim 1. Test the hypothesis that PilJ serves as a central integrator of MCS by coordinately regulating the mechanical input signal (altered pilin monomers) with activation of the Chp phosphorelay and with activation of CyaB. We will use genetic screens, in vivo assays of physiologic function, in vivo biochemistry, and live cell fluorescence imaging including FRET to define the (A) PilA/PilJ/CyaB/ and (B) PilJ/PilH interaction landscapes. Aim 2. Test the hypothesis that FimV/FimL/PilG hub links TFP function to the Chp/CyaB system. We will (A) define the FimL/PilG interaction landscape and (B) use Phos-tag technology 16 to examine PilG and PilH phosphorylation in vivo during MCS. Aim 3. Define key spatial and temporal properties of TFP-Chp-CyaB mechanochemical signal transduction during biotic biofilm formation on polarized lung epithelial monolayers. We will (A) Determine the contribution of TFP-Chp-CyaB MCS during biotic biofilm formation and (B) Determine the temporal and spatial dynamics of the surface-activated gene expression during biotic biofilm formation.