Collaborative Research: Single-molecule in vivo analysis of mechanosensitive channels in bacteria using force spectroscopy

合作研究：利用力谱对细菌中的机械敏感通道进行单分子体内分析

• 批准号: 2221771
• 负责人: Christoph Schmidt
• 金额: $ 51.27万
• 依托单位: Duke University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2025-07-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2221771&HistoricalAwards=false
• 关键词: Collaborative; Research; Single; molecule; vivo

The objective of this research project is to discover how the mechanical safety valves, so called “mechanosensitive channels”, embedded in the tough outer shells of bacteria function and how they help to protect bacteria against rupture due to excessive internal pressure during sudden changes in their environment. An understanding of this essential bacterial function could impact human health and the control of bacterial disease. Multiple drug resistance is an immense health threat. A detailed understanding of bacterial protective functions may lead to new pharmacological approaches to overcoming these protections. Furthermore, bacteria play crucial roles in commercial agriculture, environmental remediation, and alternative energy production. In all these situations, understanding of growth regulation and reactions to changing environmental conditions is critical. The work of this collaborative research program lies at the intersection of biology, experimental biophysics, and mechanical engineering, and graduate students and postdoctoral researchers in the team will be trained to work at this intersection. Undergraduate students at Duke and UCLA will also participate in this research project. Undergraduates from underrepresented minority groups will participate in summer research experiences at UCLA that focus on computational modeling. Middle and high school students from diverse backgrounds will participate in afterschool and camp experiences at Duke that introduce participants to state-of-the-art cell imaging technologies. Through the educational outreach, this work will increase and diversify the group of undergraduates interested in STEM-based careers, including those from community colleges and Minority Serving Institutions. Bacteria are enclosed by a complex multi-layered cell envelope that enables them to maintain a high internal turgor pressure of one or more atmospheres. When external osmolarity drops significantly, excessive turgor can cause cells to burst. To prevent this, mechano-sensitive channels (MSCs) embedded in the inner lipid membrane act as safety valves and release solutes to decrease turgor. It remains unknown if MSCs in the living cell open only when the lateral tension in the inner cell membrane increases, or if they also react to other mechanical stimuli transmitted through their complex mechanical microenvironments. It also remains unknown how biochemical regulation affects force transmission leading to channel gating. This project will utilize a new approach to observe the opening of single MSCs in live bacteria in response to mechanical compression, essentially between flat plates in an atomic force microscope (AFM). This method can precisely quantify turgor pressure and, at the same time, resolve cell volume changes as small as 0.01 femtoliters, produced by the gating of individual MSCs. The project will study gram-negative E. coli and gram-positive B. subtilis bacteria and compare the behavior of wildtype strains with strains expressing only specific MSCs. Experiments will be combined with analytical and numerical coarse-grained modeling to understand force transmission to MSCs through the complex cell wall structures, including the lipid membrane(s), the proteoglycan layer, and the periplasmic polyelectrolyte layer, with a focus on the role of cell wall defects. The new approach to in vivo characterization of MSCs will help to solve fundamental puzzles about MSC function in their native physiological environment.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

该研究项目的目的是发现，所谓的“机械敏感通道”的机械安全阀如何嵌入细菌功能的坚韧外壳中，以及它们如何帮助保护细菌在其环境中突然变化时由于内部压力过大。对这种基本细菌功能的理解可能会影响人类健康和细菌疾病的控制。多种耐药性是巨大的健康威胁。对细菌保护功能的详细理解可能会导致新的药物方法克服这些保护。此外，细菌在商业协议，环境修复和替代能源生产中起着至关重要的作用。在所有这些情况下，了解生长调节和对不断变化的环境条件的反应至关重要。这项合作研究计划的工作在于生物学，实验生物物理学和机械工程的交集以及团队中的研究生和博士后研究人员将接受培训，可以在此交叉路口工作。杜克大学和加州大学洛杉矶分校的本科生也将参加该研究项目。来自代表性不足的少数群体的本科生将参加加州大学洛杉矶分校的夏季研究经验，专注于计算建模。来自潜水员背景的中学生和高中生将参加杜克大学的课后和露营经验，向参与者介绍最先进的细胞成像技术。通过教育宣传，这项工作将增加并使对基于STEM的职业（包括社区学院和少数民族服务机构的职业）感兴趣的本科生多样化。细菌被一个复杂的多层细胞包膜包围，使它们能够保持一个或多个大气的高内部爆炸压力。当外部渗透压显着下降时，过量的turgor会导致细胞破裂。为了防止这种情况，嵌入脂质内部膜中的机械敏感通道（MSC）作为安全阀和释放溶剂可减少turgor。在内部细胞膜中的横向张力增加时，或者它们也对通过其复杂的机械微环境传播的其他机械刺激反应时，活细胞中的MSC是否仅开放。生化调节如何影响导致通道门控的力传递。该项目将利用一种新方法来观察活细菌中的单个MSC的开放，以应对机械压缩，这基本上是原子力显微镜（AFM）中的平板之间的开放。该方法可以准确地量化turgor压力，同时解决细胞体积的变化，而单个MSC的门控产生的0.01个女性变化。该项目将研究革兰氏阴性大肠杆菌和革兰氏阳性枯草芽孢杆菌细菌，并将野生型菌株的行为与仅表达特定MSC的菌株进行比较。实验将与分析和数值的粗粒化建模相结合，以通过复杂的细胞壁结构（包括脂质膜，蛋白聚糖层和周质多电解质层）来理解向MSC传播的力，侧重于细胞壁缺陷的作用。 MSC的体内特征的新方法将有助于解决有关MSC功能在其本土生理环境中的基本难题。该奖项反映了NSF的法定任务，并通过使用该基金会的知识分子和更广泛的影响来评估NSF的法定任务，并被认为是宝贵的支持。