Role of mechanosensation in P. aeruginosa virulence and colonization

描述（由申请人提供）：革兰氏阴性细菌铜绿假单胞菌是一种机会性病原体，可感染非常广泛的宿主细胞类型。调控铜绿假单胞菌宿主广泛特异性的机制在很大程度上仍然未知。先前关于铜绿假单胞菌如何调节其毒力因子表达的研究主要集中在群体感应和营养可利用性等化学线索上。我的工作目标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，如细菌附着于宿主细胞时的粘附力和液体环境中的定向力。因此，检测宿主生物的机械信号可能是感染策略的一部分。我的初步数据表明，铜绿假单胞菌激活毒力基因，当细胞从游动到粘附在非生物表面。此外，我之前的工作表明，P. aeruginosa对流体流动的机械效应作出反应，其表面运动性和表面粘附性发生了显著变化，这是宿主定植的决定因素。基于这些发现，我假设特定的机械线索激活信号转导途径，调节铜绿假单胞菌细胞的定植和毒力。在这项研究中，我将描述机械刺激如何调节毒力和定植使用跨学科的方法，结合分子生物学，细胞生物学，机械工程和物理学。我得到了流体动力学专家霍华德·斯通博士、细菌生物物理学专家约书亚·谢维茨博士和铜绿假单胞菌毒力和生物膜形成专家乔治·奥图尔博士的指导。我将测试假铜绿假单胞菌在哺乳动物宿主细胞表面从游泳到粘附的转变过程中被机械信号激活的假设。使用光学镊子，微流体和原子力显微镜，我将测试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机械刺激的转录反应以及流体流动对铜绿假单胞菌定植的影响。

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.