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

动物身上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、营养状况和恢复能力。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附着、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。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化方法的结合，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。该项目将以两种众所周知的益生菌（“好”）细菌与人体肠道模型的相互作用为目标。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（应用于农业和医药）和涉及非生命表面（应用于食品加工和基础设施维护）的微生物群落。其他更广泛的影响包括博物馆的外展活动和下一代微生物组研究科学家的培训。该项目将开发计算和实验相结合的方法，以确定宿主-微生物蛋白质-蛋白质相互作用（PPIs）的身份和功能含义，在上皮细胞培养和人类类器官中生长的合成微生物群落的背景下。研究将集中在众所周知的益生菌菌株E. coli Nissle和Lactobacillus rhamnosus GG的定殖上。使用高度可控的还原系统来研究PPIs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来识别重要的PPIs并检查其功能影响。该项目将以先进计算管道的发展为基础，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方法来实验检查PPIs在体内的景观，并利用转座子文库分析实验来鉴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。接下来将使用小鼠粪便提取物和/或在肠道微生物组中发挥重要作用的厌氧菌在多物种群落中鉴定PPIs。本文提出的研究将通过列举驱动细菌宿主定植的关键跨界蛋白质-蛋白质相互作用（包括识别功能重要的相互作用），以及通过开发和完善用于高通量预测生物间蛋白质-蛋白质相互作用及其功能重要性的计算框架，为我们的知识基础和技术能力做出贡献。这里开发的方法将是可推广的，以便在复杂的微生物群落中快速枚举关键的宿主微生物和微生物-微生物PPIs。该项目由理解生命规则：微生物组理论和机制项目资助，该项目是美国国家科学基金会十大理念和生物科学理事会新兴前沿部门的一部分。该奖项反映了美国国家科学基金会的法定使命，并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，被认为值得支持。

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.