MTM 2: Combining structural informatics and crosslinking mass spectrometry to predict the key protein-protein interactions shaping symbiotic microbial communities

MTM 2：结合结构信息学和交联质谱来预测塑造共生微生物群落的关键蛋白质-蛋白质相互作用

• 批准号: 2025426
• 负责人: Lydia Freddolino
• 金额: $ 290万
• 依托单位: Regents of the University of Michigan - Ann Arbor
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2025426&HistoricalAwards=false
• 关键词: MTM; Combining; structural; informatics; crosslinking

Microbial communities on and inside of animals play profound roles in biology, affecting the behavior, nutritional status, and resilience of the host. Much of the behavior of microbial communities is driven by interactions between proteins of microbes and the host. These interactions are used by microbes to adhere to, attack, or communicate with other cells in their surroundings. Because microbial communities are tremendously complex, it has proven difficult to determine the set of protein-protein interactions that define and stabilize a community or affect other community members. Understanding the mechanics of those interactions is essential to engineer or manipulate microbial communities, e.g., by encouraging the presence of beneficial microbes or driving out those that are problematic. This project will unravel the rules governing how protein-protein interactions establish and maintain microbial communities using a combination of bioinformatics and biochemical approaches. The project will target the interaction of two well-known probiotic (‘good’) bacteria with models of the human intestine. The developed methods will also be applicable to a wide range of other microbial communities, both those involving mammals (with applications to agriculture and medicine) and those involving non-living surfaces (with applications to food processing and infrastructure maintenance). Other broader impacts include outreach activities at a museum and training of the next generation scientists in microbiome research.This project will develop a combination of computational and experimental approaches to determine the identities and functional implications of host-microbe protein-protein interactions (PPIs), in the context of synthetic microbial communities grown on epithelial cell cultures and in human organoids. The studies will focus on colonization by the well-known probiotic strains E. coli Nissle and Lactobacillus rhamnosus GG. The use of a highly controllable, reductionist system to study the roles of PPIs in the broader behavior of microbial communities will permit the investigators to develop, test, and benchmark experimental and computational tools to identify important PPIs and examine their functional implications. The project will be anchored by the development of advanced computational pipelines to enable large-scale high-quality prediction of the structure and function of PPI networks. In parallel, for the target organoid-microbe communities, the researchers will make use of recently developed crosslinking mass spectrometry methods to experimentally examine the landscape of PPIs in vivo, and transposon library profiling experiments to identify the microbial genes that contribute substantially to host colonization or microbe-microbe competition. This will be followed by the identification of PPIs in multi-species communities using mouse fecal extracts and/or anaerobes that play an important role in the gut microbiome. The research proposed here will contribute to our knowledge base and technical capabilities both through the enumeration of key trans-kingdom protein-protein interactions driving bacterial host colonization (including identification of the interactions that are functionally important), and through the development and refinement of a computational framework for high throughput prediction of inter-organism protein-protein interactions and their functional importance. The methods developed here will be generalizable to permit rapid enumeration of key host-microbe and microbe-microbe PPIs in complex microbial communities.This project is funded by the Understanding the Rules of Life: Microbiome Theory and Mechanisms Program, administered as part of NSF's Ten Big Ideas and the Division of Emerging Frontiers in the Directorate for Biological Sciences.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.

动物体表和体内的微生物群落在生物学中发挥着深远的作用，影响着宿主的行为、营养状况和恢复力。微生物群落的许多行为是由微生物蛋白质与宿主之间的相互作用驱动的。这些相互作用被微生物用来粘附，攻击或与周围的其他细胞交流。由于微生物群落非常复杂，因此很难确定定义和稳定群落或影响其他群落成员的蛋白质-蛋白质相互作用的集合。了解这些相互作用的机制对于设计或操纵微生物群落至关重要，例如，通过鼓励有益微生物的存在或驱逐那些有问题的微生物。该项目将利用生物信息学和生物化学方法的结合，揭示蛋白质-蛋白质相互作用如何建立和维持微生物群落的规则。该项目将针对两种著名的益生菌（“好”）与人类肠道模型的相互作用。所开发的方法也将适用于广泛的其他微生物群落，包括涉及哺乳动物的微生物群落（应用于农业和医学）和涉及非生物表面的微生物群落（应用于食品加工和基础设施维护）。其他更广泛的影响包括在博物馆开展的外联活动和对下一代科学家进行微生物组研究的培训。该项目将开发计算和实验方法的组合，以确定在上皮细胞培养物和人类类器官中生长的合成微生物群落的背景下，宿主-微生物蛋白质-蛋白质相互作用（PPI）的身份和功能意义。这些研究将集中在由著名的益生菌菌株E。coliNissle和LactobacillusrhamnosusGG. 使用高度可控的还原系统来研究PPI在微生物群落更广泛行为中的作用，将允许研究人员开发，测试和基准实验和计算工具，以确定重要的PPI并检查其功能意义。该项目将以开发先进的计算管道为基础，以实现对PPI网络结构和功能的大规模高质量预测。同时，对于目标类器官-微生物群落，研究人员将利用最近开发的交联质谱法来实验性地研究PPI在体内的分布，以及转座子文库分析实验，以确定对宿主定植或微生物-微生物竞争有重大贡献的微生物基因。随后将使用在肠道微生物组中发挥重要作用的小鼠粪便提取物和/或厌氧菌，在多物种群落中鉴定PPI。这里提出的研究将有助于我们的知识基础和技术能力，通过枚举关键的跨王国蛋白质-蛋白质相互作用驱动细菌宿主定植（包括识别功能上重要的相互作用），并通过开发和完善的计算框架，用于高通量预测生物体间蛋白质-蛋白质相互作用及其功能的重要性。这里开发的方法将是可推广的，以允许在复杂的微生物群落中快速计数关键的宿主微生物和微生物-微生物PPI。微生物组理论和机制计划，作为NSF的十大理念和生物科学理事会新兴前沿部门的一部分进行管理。该奖项反映了NSF的法定使命，并被认为是值得的通过使用基金会的知识价值和更广泛的影响审查标准进行评估，

项目成果

The Human DNA Mismatch Repair Protein MSH3 Contains Nuclear Localization and Export Signals That Enable Nuclear-Cytosolic Shuttling in Response to Inflammation

• DOI: 10.1128/mcb.00029-20
• 发表时间: 2020-07-01
• 期刊: MOLECULAR AND CELLULAR BIOLOGY
• 影响因子: 5.3
• 作者: Tseng-Rogenski, Stephanie S.;Munakata, Koji;Carethers, John M.
• 通讯作者: Carethers, John M.