Role of mechanosensation in P. aeruginosa virulence and colonization

描述（由申请方提供）：革兰氏阴性菌铜绿假单胞菌是一种机会致病菌，可感染非常广泛的宿主细胞类型。调节铜绿假单胞菌宿主的广泛宿主特异性的机制在很大程度上仍然未知。以前的研究铜绿假单胞菌如何调节其毒力因子的表达主要集中在化学线索，如群体感应和营养物质的可用性。 我的工作的目标是调查是否机械线索调节铜绿假单胞菌的毒力和定植。在感染过程中，细菌遇到各种机械力，例如细菌附着到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，当细胞从游动转变为粘附在非生物表面时，铜绿假单胞菌会激活毒力基因。此外，我以前的工作表明，铜绿假单胞菌响应流体流动的机械效应，表面运动性和表面粘附性发生显著变化，这是宿主定植的决定因素。基于这些发现，我假设特定的机械信号激活信号转导通路，调节铜绿假单胞菌细胞的定植和毒力。 在这项研究中，我将描述如何机械刺激调节毒力和殖民使用跨学科的方法，结合分子生物学，细胞生物学，机械工程和物理学。我得到了霍华德斯通博士的指导，他在流体动力学方面有专长，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。我将测试的假设，铜绿假单胞菌的毒力是由机械线索激活的过渡期间，从游泳到粘附在哺乳动物宿主细胞表面。使用光学镊子，微流体和原子力显微镜，我将测试直接机械刺激细胞是否足以激活毒力。我还将描述铜绿假单胞菌对流体流动的机械刺激的转录反应以及流体流动对铜绿假单胞菌定殖的影响。 铜绿假单胞菌细胞表面。最后，我将检验PilX蛋白质 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体的能力的抗生素治疗新方法提供基础。此外，该研究将代表将机械感觉表征为细菌中毒力和定殖的调节剂的首次尝试之一。

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.