Regulation of the Pseudomonas aeruginosa protease PrpL by temperature and iron

项目总结/摘要 细菌病原体铜绿假单胞菌是医院感染和生命的常见原因。 对患有遗传性疾病囊性纤维化的人造成肺部感染的威胁。铜绿假单胞菌感染很难 并且由于固有的和获得的抗生素耐药性而治疗昂贵，强调了对新治疗的需要。 更好地了解铜绿假单胞菌如何引起感染将有助于这一点。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引起疾病。其中一个毒力因子， 丝氨酸蛋白酶PrpL引起严重的组织损伤并降解免疫系统的组分。 prpL的表达受铜绿假单胞菌在感染期间经历的两种环境因素调节： 铁的有效性和温度低。低铁利用率通过已知的铁转运途径上调prpL的表达。 与25 ℃相比，37 ℃下通过未知机制下调。戈德堡实验室 已经发现转录因子MvaT/MvaU和LasR是这种prpL温度调节所必需的，但是 prpL温度调节的机制仍有待完全阐明。铁和温度如何相互调节 prpL和铜绿假单胞菌毒力的重要性也是未知的。鉴于铁的供应量低 上调prpL，而37 ℃条件下调，铁和温度可能平衡PrpL生产， 对铜绿假单胞菌的毒力很重要。基于这些发现，我推测， prpL通过MvaT/MvaU和LasR与prpL启动子的温度依赖性结合而发生， 铁/温度协同调节prpL对铜绿假单胞菌的毒力有重要作用。我来测试一下 使用遗传和生物化学方法的假设，以及感染的动物模型。在目标1中，我定义 通过确定MvaT/MvaU和LasR是否正或负调节prpL体温调节机制 在25 ℃和37 ℃下的prpL转录，并通过表征温度对 MvaT/MvaU和LasR与prpL启动子连接。在目标2中，我将确定温度如何平衡生产 通过测量PrpL基因表达、分泌的PrpL量和总酶活性， 在20 ℃-42 ℃范围内分泌PrpL的活性。在目标3中，我将描述prpL铁/温度的作用。 通过感染大蜡螟幼虫来共同调节铜绿假单胞菌的毒力。幼虫会被感染 对于prpL受低铁可用性调节的铜绿假单胞菌菌株和prpL不受低铁可用性调节的菌株， 两组的幼虫将被分别置于25摄氏度和37摄氏度的环境中， 影响铜绿假单胞菌的毒力。prpL体温调节的机制研究将解决一个主要的差距 在铜绿假单胞菌的毒力因子温度调节的知识。了解PrpL是如何被监管的 温度和铁促进铜绿假单胞菌发病机制也将提供这种机会主义的见解， 病原体在人体内存活并引起有问题的感染。这些见解可能有助于 开发新的治疗铜绿假单胞菌感染的方法和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.