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，但我们对 细菌病原体对剪切流的感知和反应是有限的。最近，我结合了 微流体和转录组学发现人类病原体铜绿假单胞菌， 通过一种新的过程感知剪切流，我称之为流变学。流变传感使用西格玛- FROR因子和抗SIGMA因子FROI调节基因表达以适应剪切流的速度。在这里，我 提出了一种系统水平的方法来表征流变学的调节机制。首先，我 将使用基于微流控的转录组学来测量全基因组范围的基因表达变化，而 独立修改剪切流的三个参数：流量、渠道尺寸和 粘度。其次，我将确定流变监管者FROR和FROI的监管目标。 第三，我将把荧光激活细胞分选(FACS)和转座子测序(TN-SEQ)结合起来，以 确定控制流变学的其他感觉和调节蛋白。这些目标加在一起，将 提供基因组范围的靶标、信号通路和流量传感器的特征 流变学。由于流变学可能对寄主定植很重要，这项研究将提供新的见解 在宿主相关的切变流中进入铜绿假单胞菌的毒力。一起，研究和职业生涯 我在这里概述的发展计划将帮助我在一所研究型大学获得教职， 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.