Mechanobehavior of Pseudomonas aeruginosa in shear flow

摘要 虽然人体内的剪切流限制了细菌病原体的定植，但我们的理解是， 细菌病原体如何感知和响应剪切流的研究是有限的。最近，我结合了 微流体和转录组学来发现人类病原体，铜绿假单胞菌， 通过一个新的过程来感知剪切流，我称之为rheosensing。Rheosensing使用sigma- 因子FroR和抗σ因子FroI调节基因表达以适应剪切流的速度。这里我 提出了一个系统级的方法来表征rheosensing的监管机制。首先我 将使用基于微流体的转录组学来测量基因表达的全基因组变化， 独立修改剪切流的三个参数：流速、通道尺寸和 粘度其次，我将确定电阻传感调节器FroR和FroI的调节目标。 第三，我将荧光激活细胞分选（FACS）与转座子测序（Tn-seq）相结合， 鉴定控制流变感测的其它感觉和调节蛋白。总之，这些目标将 提供靶点、信号传导通路和流量传感器的全基因组表征， 流变传感由于流变传感可能对宿主定殖很重要，因此这项研究将提供新的见解 铜绿假单胞菌在宿主相关剪切流中的毒力。在一起，研究和事业 我在这里概述的发展计划将帮助我获得一个研究型大学的教职， 我的独立研究计划，并允许无缝过渡到我的职业生涯的下一个阶段。

Abstract Although shear flow in the human body restricts colonization by bacterial pathogens, our understanding of how bacterial pathogens sense and respond to shear flow is limited. Recently, I combined microfluidics and transcriptomics to discover that the human pathogen, Pseudomonas aeruginosa, senses shear flow through a novel process, which I named rheosensing. Rheosensing uses the sigma- factor FroR and anti-sigma factor FroI to tune gene expression to the speed of shear flow. Here, I propose a systems-level approach to characterize the regulatory mechanisms of rheosensing. First, I will use microfluidic-based transcriptomics to measure genome-wide changes in gene expression while independently modifying the three parameters of shear flow: flow rate, channel dimensions, and viscosity. Second, I will determine the regulatory targets of the rheosensing regulators FroR and FroI. Third, I will couple fluorescence-activated cell sorting (FACS) with transposon sequencing (Tn-seq) to identify additional sensory and regulatory proteins that control rheosensing. Together, these aims will provide genome-wide characterization of the targets, signaling pathways, and flow sensors that control rheosensing. As rheosensing is likely important for host colonization, this study will provide new insight into the virulence of P. aeruginosa in host-relevant shear flow. Together, the research and career development plans I outline here will help me obtain a faculty position at a research university, launch my independent research program, and allow for a seamless transition to the next stage of my career.