Cyclic AMP Signaling in Pseudomonas aeruginosa Virulence

描述(申请人提供)：铜绿假单胞菌是一种机会人类病原体，导致近10%的医院获得性感染，是囊性纤维化患者发病率和死亡率的主要原因。铜绿假单胞菌感染具有很高的可归因性死亡率，并且由于广泛的抗生素耐药性而极难治疗。我们的长期目标是了解这种无处不在的环境细菌在进入人类宿主后如何激活协调的毒力程序。这些信息将有助于制定旨在抑制铜绿假单胞菌毒力机制的治疗策略。铜绿假单胞菌具有多种毒力因子。在急性感染的动物模型中，表达这些因子并导致疾病的能力取决于细菌产生第二信使3‘，5’-环磷酸腺苷(CAMP)的能力。我们假设CyaB，一种膜锚定的腺苷环化酶，通过产生cAMP来响应宿主提示而启动铜绿假单胞菌的毒力程序。CyaB活性受铜绿假单胞菌IV型菌毛趋化系统各组成部分的正向调节，而受海藻酸胞外多糖调节途径的负调节。此外，包括钙浓度、渗透压和二氧化碳在内的环境信号对依赖CyaB的cAMP的产生有很大影响。一旦产生cAMP，就会激活cAMP依赖的转录因子(Vfr)，控制毒力基因的表达。这项建议的目的是确定感染过程中CyaB的结构和功能之间的关系以及依赖CyaB的cAMP产生的动力学。具体目标如下：1)确定氨基末端CyaB膜锚定结构域的结构，并确定其在CyaB定位、信号检测和催化功能中的作用。初步结果表明，MASE2结构域是酶激活所必需的。2)探讨无机碳(碳酸氢盐和二氧化碳)在体内外刺激CyaB催化活性的机制及生物学意义。CyaB是腺苷环化酶无机碳激活家族中的一员，CyaB催化结构域在体外受到HCO3-的刺激，在铜绿假单胞菌中表达时则受到胞外二氧化碳增加的刺激。3)确定CyaB活性在感染过程中是否受到寄主提示的调节。缺乏CyaB的突变体在体内被减弱，而CyaB在体外模拟细菌-宿主相互作用的条件下被激活。我们将构建报告菌株，以确定细菌细胞内cAMP在细胞培养感染期间是否发生波动，以及cAMP水平是否随时间和急性肺炎小鼠模型的疾病进展而变化。铜绿假单胞菌是引起医院感染的主要原因，也是囊性纤维化患者发病和死亡的主要原因。免疫受损个体中铜绿假单胞菌感染的流行，加上铜绿假单胞菌菌株对抗生素耐药性的增加，是一个紧迫的公共卫生问题。这项提议的目的是定义控制环状AMP产生的机制，环状AMP是铜绿假单胞菌毒力的中央调节因子。这项提议将通过cAMP依赖的毒力计划，在铜绿假单胞菌和其他病原菌中识别和表征新的治疗靶点。

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa is an opportunistic human pathogen that is responsible for nearly 10% of hospital-acquired infections and is the primary cause of morbidity and mortality in cystic fibrosis patients. P. aeruginosa infections have high attributable death rates and are extremely difficult to treat due to widespread antibiotic resistance. Our long-term goal is to understand how this ubiquitous environmental bacterium activates a coordinated virulence program upon introduction into the human host. This information will facilitate the development of therapeutic strategies aimed at inhibiting P. aeruginosa virulence mechanisms. P. aeruginosa possesses a wide variety of virulence factors. The capacity to express many of these factors and to cause disease in an animal model of acute infection depends on the ability of the bacteria to produce the second messenger adenosine 3', 5'-cyclic monophosphate (cAMP). We hypothesize that CyaB, a membrane-anchored adenylate cyclase, initiates a P. aeruginosa virulence program by producing cAMP in response to host cues. CyaB activity is positively regulated by components of the P. aeruginosa Type IV pilus chemotaxis system and negatively regulated by the alginate exopolysaccharide regulatory pathway. In addition, environmental cues including calcium concentration, osmolarity and CO2 substantially affect CyaB-dependent cAMP production. Once produced, cAMP activates a cAMP-dependent transcription factor (Vfr) that controls virulence gene expression. The goal of this proposal is to determine the relationship between structure and function of CyaB and the kinetics of CyaB-dependent cAMP production during infection. The Specific Aims are as follows: 1) Characterize the structure of the amino-terminal CyaB membrane-anchoring domain (termed MASE2 for Membrane-Associated Sensor-2) and determine its role in CyaB localization, signal detection and catalytic function. Preliminary results indicate that the MASE2 domain is required for enzyme activation. 2) Investigate the mechanism and biological significance of inorganic carbon (bicarbonate and CO2)-stimulated CyaB catalytic activity in vitro and in vivo. CyaB is a member of the inorganic carbon-activated family of adenylate cyclases and the CyaB catalytic domain is stimulated by HCO3- in vitro and by increased extracellular CO2 when expressed in P. aeruginosa. 3) Determine whether CyaB activity is modulated by host cues during infection. CyaB-lacking mutants are attenuated in vivo and CyaB is activated under conditions that mimic the bacterial-host interaction in vitro. We will construct reporter strains to determine if bacterial intracellular cAMP fluctuations occur during cell culture infection and whether cAMP levels change with time and disease progression in a mouse model of acute pneumonia. The bacterium Pseudomonas aeruginosa is a major cause nosocomial infection and is the primary cause of morbidity and mortality in individuals with cystic fibrosis. The prevalence of P. aeruginosa infection in immunocompromised individuals coupled with increasing antibiotic resistance among P. aeruginosa strains represents an urgent public health issue. The goal of this proposal is to define the mechanisms controlling the production of cyclic AMP, a central regulator of P. aeruginosa virulence. This proposal will result in the identification and characterization of novel therapeutic targets in P. aeruginosa as well as other pathogenic bacteria with cAMP-dependent virulence programs.