Physics of Bacteria Living in Gels: Host-Bacteria Interactions

这一提议解决了幽门螺杆菌（h.p ylori）如何突破保护性粘液屏障到达上皮细胞表面并在胃的极端酸性环境中定植的基本难题。本文提出的研究将推进粘弹性凝胶中细菌运动和聚集的基本物理学，并阐明形状对运动和定植的影响。细菌在胃细胞黏液/黏液层上移动的微流体装置中具有多种化学梯度的趋化测量将有助于开发有用的离体仿生模型。许多细菌，如胃肠道中的细菌，必须通过粘膜屏障才能建立菌落。特别是，导致溃疡、胃炎和胃癌的幽门螺杆菌面临着克服酸性环境的额外挑战。PI小组先前的工作证实，幽门螺杆菌使用与尿素水解相同的生化策略来中和酸并提高pH值，以便在胃中生存，并通过触发凝胶-溶液转变而游过。最近对具有细胞形状改变的突变体的研究表明，螺旋形状有利于在琼脂凝胶中建立菌落。初步的细菌跟踪显微镜和基于阻力理论的建模表明，游动速度的分布是由于形状和大小的多分散性，以及速度的双相时间变化，螺旋形状在粘性黏液溶液中提供了额外的推进力，从而导致更快的游动速度。创新的成像技术将用于研究细菌运动和细菌在宿主凝胶中引起的变化，通过定量测量pH值变化和环境局部微流变学的变化。细菌感染凝胶的流变学研究将用振荡剪切流变学来表征。微流控几何结构将被设计成模拟离体条件下的胃粘液环境，将覆盖有粘液或纯化黏液、明胶或琼脂的上皮细胞的底物分层，并建立pH或离子梯度来检查细菌的运动和定植的程度和模式。细菌对周围黏液/粘蛋白的影响将通过荧光染料结合和原子力显微镜来探测。将野生型幽门螺杆菌与细胞形状改变或缺乏特定受体或鞭毛的突变体进行比较研究，将有可能评估在这个复杂的生命系统中起作用的许多变量的影响。实验研究将辅以细菌运动、身体和鞭毛动力学、趋化性和凝胶模式形成的计算和理论模型，以提供对潜在物理的定量理解。高中学生将参加波士顿大学RISE暑期研究实习项目。将开发有关细菌运动和简单分析工具的视频，用于本科课程和演示目的。PI将利用团队的多样性和研究主题的广泛吸引力，开展外联活动，包括向更广泛的受众进行演讲，包括当地高中，少数民族和其他社区团体，并招募妇女和少数民族研究生，本科生和高中生参与这项研究。

This proposal addresses the fundamental puzzle of how Helicobacter pylori (H. pylori) breaches the protective mucus barrier to reach the epithelial cell surface and colonize in the extreme acidic environment of the stomach. The studies proposed here will advance the fundamental physics underlying bacterial motion and aggregation in a viscoelastic gel, and elucidate the influence of shape on motility and colonization. Chemotactic measurements of bacteria moving in microfluidic devices with multiple chemical gradients across mucin/mucus layers on gastric cells will contribute to the development of a useful ex vivo, biomimetic model.Many bacteria, like those in the gastrointestinal tract, have to negotiate mucosal barriers to establish colonies. In particular, Helicobacter pylori, the bacterium that causes ulcers, gastritis and gastric cancer faces the additional challenge of overcoming the acidic environment. Previous work from the PI's group established that H. pylori uses the same biochemical strategy of urea hydrolysis to neutralize the acid and elevate the pH for both survival in the stomach and to swim across by triggering a gel-solution transition. Recent studies with mutants having cell shape alterations have shown that the helical shape is advantageous in establishing colonies in agar gels. Preliminary bacteria tracking microscopy and resistive force theory based modeling show that the distribution of swimming speeds is due to polydispersity of shape and size, along with a biphasic temporal variation of speed, and that the helical shape provides added propulsion in a viscous mucin solution leading to a faster swimming speed. Innovative imaging techniques will be used to investigate bacterial motility and the changes that the bacterium causes in the host gel by quantitative measurements of pH changes and alterations in local micro-rheology of its environment. The rheological studies of bacteria infected gels will be characterized using oscillatory shear rheology. Microfluidic geometries will be designed to mimic the gastric mucus environment under ex-vivo conditions, layering substrates with epithelial cells covered with mucus or purified mucin or gelatin or agar and establish pH or ionic gradients to examine the bacteria motility and the extent and pattern of colonization. The influence of the bacterium on the surrounding mucus /mucin will be probed by fluorescent dye binding and Atomic Force Microscopy. Comparative studies of the wild-type H. pylori with mutants with altered cell shapes or deficient in specific receptors or flagella, will make it possible to assess the effect of the many variables at play in this complex living system. The experimental studies will be complemented by computational and theoretical modeling of bacterial motility, body and flagella dynamics, chemotaxis, and pattern formation in gels to provide a quantitative understanding of the underlying physics. High school students will participate via the Summer research internship, RISE program at Boston University. Videos on bacterial motility and simple analysis tools will be developed for use in undergraduate courses and for demonstration purposes. The PI will take advantage of the diversity of the team, and the broad appeal of the research topic, to do outreach activities involving presentations to a broader audience, including local high schools, and minority and other community groups and to recruit women and minority graduate, undergraduate and high school students to participate in this research.