The novel protein, FimL, regulates virulence in Pseudomonas aeruginosa

描述（由申请方提供）：铜绿假单胞菌（PA）是一种机会致病菌，是人类疾病的重要原因。免疫功能低下的人，如那些患有艾滋病毒，烧伤，癌症化疗，或需要机械消毒器的疾病，特别容易受到急性感染。即使有药物治疗，死亡率仍然很高，抗生素耐药性越来越普遍。此外，PA长期定植于患有囊性纤维化的患者，导致严重的肺损伤和死亡。由于多重耐药菌株的报道越来越多，新的细菌治疗靶点的鉴定越来越重要。 该项目的长期目标是了解感染过程是如何在PA的信号水平启动的，以便我们可以开发预防疾病发作的疗法。PA通过使用毒力因子如IV型菌毛（TFP）附着于宿主细胞来启动感染，并使用III型分泌系统（T3SS）向宿主注射毒性效应蛋白。TFP和T3SS的产生由环AMP合成途径控制，该途径在感染过程中被激活。Engel实验室发现了一种新的基因FimL，它对包括TFP和T3SS在内的许多毒力因子的产生和功能都很重要，因为它调节细胞中环AMP的水平。我们建议确定FimL调节细胞中cAMP水平的机制。 我们已经发现FimL在细菌中极性定位，TFP和T3SS也是如此。我们提出的假设，极性定位的FimL导致空间限制生产的环AMP和下游组件通过调节本地化的环AMP合成蛋白，CyaB。我们将测试我们的假设：1）通过全面和互补的方法识别FimL的相互作用伙伴。我们将使用免疫共沉淀实验和体外蛋白质测定来测试FimL是否与CyaB和其他候选蛋白质相互作用。我们还将进行基因筛选，以找到未知的互动玩家。2)我们还将确定哪些因子是FimL亚细胞定位所需的，以及FimL是否是已知毒力因子和调节因子（包括TFP和T3SS）正确亚细胞定位所需的。FimL和该途径中的其他蛋白质可以作为新的和有前途的治疗靶点，以帮助减轻人类遭受细菌感染。 公共卫生相关性：细菌病原体铜绿假单胞菌是医院获得性感染的主要原因，并导致致命的疾病囊性纤维化。我们建议阐明细菌在与人类接触时如何启动致病元素的产生的机制。

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa (PA) is an opportunistic pathogen and an important cause of disease in humans. Immunocompromised individuals such as those suffering from HIV, burn wounds, cancer chemotherapy, or illnesses that require mechanical ventilators are particularly vulnerable to acute infection. Even with medical treatment, mortality remains high and antibiotic resistance is increasingly common. In addition, PA chronically colonizes patients afflicted with Cystic Fibrosis, leading to severe pulmonary damage and death. Because multi-drug resistant isolates are increasingly reported, identification of novel bacterial therapeutic targets is increasingly important. The long-term goal of this project is to understand how the infection process is initiated at the signaling level in PA so we can develop therapies to prevent the onset of disease. PA initiates infection by using virulence factors such as the type IV pilus (TFP) to attach to host cells and injects the host with toxic effector proteins using the Type III secretion system (T3SS). TFP and T3SS production is controlled by the cyclic AMP synthesis pathway, which is activated during the infection process. The Engel lab found a novel gene, FimL that is important for the production and function of many virulence factors including TFP and the T3SS because it regulates the level of cyclic AMP in the cell. We propose to determine the mechanism by which FimL regulates cyclic AMP levels in the cell. We have discovered that FimL is polarly localized in the bacteria, as are the TFP and T3SS. We propose the hypothesis that polar localization of FimL leads to spatially restricted production of cyclic AMP and downstream components by regulating the localization of the cyclic AMP synthesis protein, CyaB. We will test our hypothesis by 1) identifying interacting partners of FimL by comprehensive and complimentary approaches. We will test if FimL interacts with CyaB and other candidate proteins using co- immunoprecipitation experiments, and in vitro protein assays. We will also perform a genetic screen to find unknown interacting players. 2) We will also determine which factors are required for subcellular localization of FimL and if FimL is required for proper subcellular localization of known virulence factors and regulators, including the TFP and T3SS. FimL and other proteins in this pathway could serve as new and promising therapeutic targets to aid in alleviating human suffering from bacterial infections. PUBLIC HEALTH RELEVANCE: The bacterial pathogen Pseudomonas aeruginosa is the leading cause of hospital-acquired infections and causes the fatal disease Cystic Fibrosis. We propose to elucidate the mechanism of how the bacteria initiates production of disease causing elements when in contact with humans.