The novel protein, FimL, regulates virulence in Pseudomonas aeruginosa

描述(申请人提供)：铜绿假单胞菌(PA)是一种条件致病菌，也是人类疾病的重要原因。免疫功能受损的人，如艾滋病毒、烧伤、癌症化疗或需要机械呼吸机的疾病患者，特别容易受到急性感染。即使在接受治疗的情况下，死亡率仍然很高，抗生素耐药性越来越常见。此外，PA长期侵袭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，识别新的细菌治疗靶点变得越来越重要。这个项目的长期目标是了解PA中感染过程是如何在信号水平上启动的，这样我们就可以开发预防疾病发生的治疗方法。PA通过使用IV型菌毛(TFP)等毒力因子附着到宿主细胞上启动感染，并使用III型分泌系统(T3SS)向宿主注射有毒效应蛋白。TFP和T3SS的产生受感染过程中激活的环状AMP合成途径控制。恩格尔实验室发现了一种新的基因fim1，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磷酸腺苷的水平。我们建议确定FIML调节细胞内cAMP水平的机制。我们已经发现FIML和TFP和T3SS在细菌中的定位是两极的。我们提出的假设是，FIML的极性定位通过调节环AMP合成蛋白CyaB的定位而导致环AMP及其下游成分的空间生产受到限制。我们将通过1)通过全面和互补的方法确定FIML的互动伙伴来检验我们的假设。我们将通过免疫共沉淀实验和体外蛋白质分析来测试FIML是否与CyaB和其他候选蛋白相互作用。我们还将进行基因筛查，以寻找未知的互动玩家。2)我们还将确定FIML的亚细胞定位需要哪些因子，以及是否需要FIML来正确定位已知的毒力因子和调节因子，包括TFP和T3SS。FIML和该途径中的其他蛋白质可能成为新的、有希望的治疗靶点，以帮助减轻人类受到细菌感染的痛苦。 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，并导致致命的囊性纤维化。我们建议阐明细菌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.