Role of mechanosensation in P. aeruginosa virulence and colonization

说明(申请人提供：革兰氏阴性杆菌铜绿假单胞菌是一种机会致病菌，可感染异常广泛的宿主细胞类型。调节铜绿假单胞菌宿主广泛性的机制在很大程度上仍不清楚。以往关于铜绿假单胞菌如何调控其毒力因子表达的研究主要集中在群体感应和营养可获得性等化学信号上。我工作的目的是调查机械线索是否调节铜绿假单胞菌的毒力和定植。在感染过程中，细菌会遇到各种机械力，如细菌附着到宿主细胞时的粘附力和液体环境中的方向力。因此，检测寄主生物体中的机械线索可能是感染策略的一部分。我的初步数据显示，当细胞从游动过渡到在非生物表面黏附时，铜绿假单胞菌激活了毒力基因。此外，我以前的工作表明，铜绿假单胞菌对流体流动的机械效应做出了响应，表面运动性和表面粘附性发生了显著变化，这是决定宿主定植的因素。基于这些发现，我假设特定的机械信号激活了调节铜绿假单胞菌细胞定植和毒力的信号转导通路。在这项研究中，我将用一种结合了分子生物学、细胞生物学、机械工程和物理学的跨学科方法来描述机械刺激如何调节毒力和定植。我征求了霍华德·斯通博士、约书亚·沙维茨博士和乔治·奥图尔博士的指导，霍华德·斯通博士在流体力学方面有专长，约书亚·沙维茨博士在细菌生物物理学方面有专长，乔治·奥图尔博士在铜绿假单胞菌毒力和生物膜形成方面有专长。我将测试这一假设，即铜绿假单胞菌的毒力是在哺乳动物宿主细胞表面从游泳到黏附的转变过程中由机械信号激活的。我将使用光学镊子、微流体和原子力显微镜，测试对细胞的直接机械刺激是否足以激活毒力。我还将描述铜绿假单胞菌对流体力学刺激的转录反应，以及流体对铜绿假单胞菌定植的影响。 宿主细胞表面的铜绿假单胞菌。最后，我将测试PilX蛋白的假设 和PilY1是调节毒力和定殖的机械传感器。总之，这些实验将确定机械力在细菌感染过程中的作用。这些见解将为开发干扰铜绿假单胞菌感染广泛宿主生物能力的抗生素治疗的新方法提供基础。此外，这项研究将是首次尝试将机械感觉描述为细菌中毒力和定植的调节因素。

DESCRIPTION (provided by applicant: The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen that infects an exceptionally broad range of host cell types. The mechanisms that regulate the broad host specificity of P. aeruginosa host remain largely unknown. Previous studies of how P. aeruginosa regulates expression of its virulence factors have largely focused on chemical cues such as quorum sensing and nutrient availability. The goal of my work is to investigate whether mechanical cues regulate P. aeruginosa virulence and colonization. During the infection process, bacteria encounter a variety of mechanical forces such as adhesion forces when bacteria attach to host cells and directional forces in liquid environments. Detecting mechanical cues in host organisms could thus be part of an infection strategy. My preliminary data show that P. aeruginosa activates virulence genes as cells transition from swimming to adhesion on abiotic surfaces. In addition, my previous work shows that P. aeruginosa responds to the mechanical effects of fluid flow with striking changes in surface motility and surface adhesion, which are determining factors in host colonization. Based on these findings, I hypothesize that specific mechanical cues activate signal transduction pathways that regulate colonization and virulence in P. aeruginosa cells. In this study, I will characterize how mechanical stimuli regulate virulence and colonization using an interdisciplinary approach that combines molecular biology, cell biology, mechanical engineering, and physics. I have enlisted guidance from Dr. Howard Stone, who has expertise in fluid dynamics, Dr. Joshua Shaevitz, who has expertise in bacterial biophysics, and Dr. George O'Toole, who has expertise in P. aeruginosa virulence and biofilm formation. I will test the hypothesis that P. aeruginosa virulence is activated by mechanical cues during the transition from swimming to adhesion on mammalian host cell surfaces. Using optical tweezers, microfluidics, and atomic force microscopy, I will test whether direct mechanical stimulation of cells is sufficient to activate virulence. I will also characterize the P. aeruginosa transcriptionl response to mechanical stimulation by fluid flow and the effect of fluid flow on colonization of P. aeruginosa cells on host cells surfaces. Finally, I will test the hypothesis that the proteins PilX and PilY1 are the mechanosensors that regulate virulence and colonization. Altogether, these experiments will determine the role of mechanical forces in the bacterial infection process. These insights will provide a foundation for developing novel approaches to antibiotic therapies that perturb the ability of P. aeruginosa to infect a broad range of host organisms. In addition, this study would represent one of the first attempts to characterize mechanosensation as a regulator of virulence and colonization in bacteria.