Regulation of the Pseudomonas aeruginosa protease PrpL by temperature and iron

项目摘要/摘要 铜绿假单胞菌是医院感染和生命的常见原因。 遗传性疾病囊性纤维化患者的肺部感染威胁。铜绿假单胞菌感染很难 由于固有和获得性抗生素耐药性，治疗费用高昂，这突显了新治疗方法的必要性。 更好地了解铜绿假单胞菌是如何导致感染的将有助于这一点。铜绿假单胞菌 在人类宿主中存活，并通过产生多种毒力因子而导致疾病。其中一个致命因素， 丝氨酸蛋白酶PRPL会造成严重的组织损伤，并降解免疫系统的组成部分。 PrPL的表达受铜绿假单胞菌在感染过程中经历的两个环境因素的调节： 铁的利用率和温度都很低。已知的低铁利用率上调了PRPL的表达 机制，与25C相比，37C通过未知机制下调。戈德堡实验室 已经发现这种PRPL温度调节需要转录因子MvaT/MvaU和LasR，但是 PRPL的体温调节机制尚未完全阐明。铁和温度是如何共同调节的 这对铜绿假单胞菌毒力的重要性也是未知的。鉴于铁的利用率很低 当37C条件下调时，铁和温度可能会平衡催乳素的生产和 对铜绿假单胞菌的毒力很重要。基于这些发现，我假设体温调节 PRPL是通过MvaT/MvaU和LasR与PRPL启动子的温度依赖结合而发生的 铁/温度协同调节对铜绿假单胞菌的毒力具有重要意义。我要测试一下这个 使用遗传和生化方法的假说，以及感染的动物模型。在目标1中，我将定义 通过确定MvaT/MvaU和LasR是正向还是负向调节PRPL的体温调节机制 在25C和37C下转录，并通过表征温度对结合的影响 MvaT/MvaU和LasR结合到PRPL启动子上。在目标2中，我将确定温度如何平衡生产 通过测定PRPL基因的表达、PRPL的分泌量和总酶活来检测PRPL的活性 分泌的催乳素在20C-42C范围内的活性。在目标3中，我将描述PRPL铁/温度的作用 感染小菜蛾幼虫对铜绿假单胞菌毒力的协同调节。幼虫会被感染 对于其中PRPL受低铁可利用性调节的铜绿假单胞菌菌株和其中PRPL不受调节的菌株， 来自两组的幼虫将被安置在25C和37C的环境中，以测量铁和温度如何共同调节 PRPL影响铜绿假单胞菌的毒力。对PRPL体温调节的机理研究将解决一个重大差距 了解铜绿假单胞菌毒力因子的温度调节。了解PRPL是如何由 温度和铁促进铜绿假单胞菌的致病也将提供洞察如何这种机会主义 病原体在人体内存活，并导致有问题的感染。这样的洞察力可能有助于 开发治疗铜绿假单胞菌感染的新方法，并改善对感染患者的护理。

PROJECT SUMMARY/ABSTRACT The bacterial pathogen Pseudomonas aeruginosa is a frequent cause of nosocomial infections and life- threatening lung infections in people with the genetic disorder cystic fibrosis. P. aeruginosa infections are difficult and costly to treat due to inherent and acquired antibiotic resistance, underscoring the need for new treatments. A better understanding of how P. aeruginosa causes infections will be instrumental for this. P. aeruginosa survives in a human host and causes disease by producing multiple virulence factors. One such virulence factor, the serine protease PrpL, causes severe tissue damage and degrades components of the immune system. Expression of prpL is regulated by two environmental factors experienced by P. aeruginosa during an infection: low iron availability and temperature. Expression of prpL is upregulated by low iron availability through a known mechanism and downregulated at 37C compared to 25C through an unknown mechanism. The Goldberg Lab has found that the transcription factors MvaT/MvaU and LasR are required for this prpL thermoregulation, but the mechanism of prpL thermoregulation remains to be fully elucidated. How iron and temperature coregulate prpL and the importance of this for P. aeruginosa virulence is also unknown. Given that low iron availability upregulates prpL while 37C conditions downregulate it, iron and temperature may balance PrpL production and be important for P. aeruginosa virulence. Based on these findings, I hypothesize that thermoregulation of prpL occurs through temperature-dependent binding of MvaT/MvaU and LasR to the prpL promoter and that iron/temperature coregulation of prpL is important for P. aeruginosa virulence. I will test this hypothesis using genetic and biochemical approaches, and an animal model of infection. In Aim 1, I will define the prpL thermoregulatory mechanism by determining if MvaT/MvaU and LasR positively or negatively regulate prpL transcription at 25C and 37C, and by characterizing the impact of temperature on the binding of MvaT/MvaU and LasR to the prpL promoter. In Aim 2, I will determine how temperatures balances production and activity of PrpL by measuring prpL gene expression, the amount of PrpL secreted, and the total enzymatic activity of secreted PrpL across a 20C-42C range. In Aim 3, I will characterize the role of prpL iron/temperature coregulation in P. aeruginosa virulence by infecting larvae of the moth Galleria mellonella. Larvae will be infected with a P. aeruginosa strain in which prpL is regulated by low iron availability and a strain in which prpL is not, and larvae from both groups will be housed at 25C and 37C to measure how iron and temperature coregulation of prpL affects P. aeruginosa virulence. A mechanistic study of prpL thermoregulation will address a major gap in the knowledge of virulence factor thermoregulation in P. aeruginosa. Understanding how PrpL is regulated by temperature and iron to facilitate P. aeruginosa pathogenesis will also provide insights into how this opportunistic pathogen survives inside a human and causes problematic infections. Such insights could contribute to the development of new treatments for P. aeruginosa infections and improved care for infected patients.