Cyclic AMP Signaling in Pseudomonas aeruginosa Virulence

描述（由申请方提供）：铜绿假单胞菌是一种机会性人类病原体，导致近10%的医院获得性感染，是囊性纤维化患者发病和死亡的主要原因。铜绿假单胞菌感染具有高归因死亡率，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引入人类宿主后如何激活协调的毒力程序。这一信息将促进旨在抑制铜绿假单胞菌毒力机制的治疗策略的发展。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许多这些因子并引起疾病的能力取决于细菌产生第二信使腺苷3 '，5'-环一磷酸（cAMP）的能力。我们假设CyaB是一种膜锚定的腺苷酸环化酶，通过响应宿主信号产生cAMP来启动铜绿假单胞菌的毒力程序。CyaB活性由铜绿假单胞菌IV型菌毛趋化系统的组分正向调节，并由藻酸盐胞外多糖调节途径负向调节。此外，环境因素，包括钙浓度，渗透压和CO2实质上影响CyaB依赖的cAMP的生产。一旦产生，cAMP激活控制毒力基因表达的cAMP依赖性转录因子（Vfr）。本提案的目的是确定CyaB的结构和功能之间的关系和感染过程中CyaB依赖性cAMP产生的动力学。具体目的如下：1）对CyaB膜锚定结构域（Membrane-Associated Sensor-2，MASE 2）的结构进行表征，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，MASE 2结构域是酶激活所必需的。2)探讨无机碳（碳酸氢盐和CO2）刺激CyaB催化活性的机制和生物学意义。CyaB是腺苷酸环化酶的无机碳活化家族的成员，并且CyaB催化结构域在体外被HCO 3-刺激，并且当在铜绿假单胞菌中表达时被增加的细胞外CO2刺激。3)确定CyaB活性是否在感染过程中受到宿主线索的调节。缺乏CyaB的突变体在体内被减弱，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，以确定在细胞培养感染期间是否发生细菌细胞内cAMP波动，以及在急性肺炎小鼠模型中cAMP水平是否随时间和疾病进展而变化。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。铜绿假单胞菌感染在免疫功能低下的个体中的流行，加上铜绿假单胞菌菌株中抗生素耐药性的增加，代表了一个紧迫的公共卫生问题。该提案的目标是确定控制环AMP产生的机制，环AMP是铜绿假单胞菌毒力的中心调节因子。这一提议将导致铜绿假单胞菌以及其他具有cAMP依赖性毒力程序的病原菌中新型治疗靶标的鉴定和表征。

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.