Physics of Bacteria Living in Gels: Host-Bacteria Interactions

这项建议解决了幽门螺杆菌（H。幽门螺杆菌）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殖。这里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，并阐明形状对运动和定植的影响。在微流控装置中移动的细菌的趋化性测量具有跨胃细胞上的粘蛋白/粘液层的多个化学梯度，这将有助于开发有用的离体仿生模型。特别是幽门螺杆菌，导致溃疡，胃炎和胃癌的细菌面临着克服酸性环境的额外挑战。PI小组先前的工作确定了H。幽门螺杆菌使用相同的尿素水解生化策略来中和酸并升高pH，以在胃中存活并通过触发凝胶-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集落。初步的细菌跟踪显微镜和基于阻力理论的建模表明，游泳速度的分布是由于形状和大小的多分散性，沿着的速度的双相时间变化，并且螺旋形状在粘性粘蛋白溶液中提供了额外的推进力，导致更快的游泳速度。创新的成像技术将用于研究细菌的运动性和变化，细菌在宿主凝胶中引起的pH值的变化和改变，在其环境的局部微流变学的定量测量。细菌感染的凝胶的流变学研究将使用振荡剪切流变学来表征。微流体几何形状将被设计为模拟离体条件下的胃粘液环境，用粘液或纯化的粘蛋白或明胶或琼脂覆盖的上皮细胞对基底进行分层，并建立pH或离子梯度以检查细菌运动性以及定殖的程度和模式。细菌对周围粘液/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。野生型H.幽门螺杆菌的突变体具有改变的细胞形状或缺乏特定受体或鞭毛，将使评估在这个复杂的生命系统中起作用的许多变量的影响成为可能。实验研究将通过细菌运动性，身体和鞭毛动力学，趋化性和凝胶中图案形成的计算和理论建模来补充，以提供对基础物理学的定量理解。高中生将通过波士顿大学的夏季研究实习，RISE计划参加。将制作关于细菌运动性和简单分析工具的录像，供本科生课程和演示之用。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，包括当地高中，少数民族和其他社区团体进行演讲，并招募妇女和少数民族研究生，本科生和高中生参加这项研究。

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.