MTM2:CollaborativeResearch:Microbially-mediated epigenetic modifications alter host phenotypes

共生微生物可以对宿主的健康和进化产生巨大的影响。事实上，我们自己的细胞中含有一种古老微生物共生的残余——线粒体——它继续为我们提供生理上必不可少的代谢能力。了解宿主-微生物相互作用的原因、机制和后果已经成为进化生物学、微生物学、生态学和发展领域的一个重要研究目标。这个项目是基于一项发现，即细菌共生体以一种特殊的方式改变宿主细胞，以保护它免受病毒的侵害。在这个拟议的项目中，研究人员将研究宿主细胞中的这些变化，并确定它们如何改变宿主、细菌共生体和入侵病毒。研究人员将使用果蝇、细菌、沃尔巴克氏体（果蝇微生物组的重要成员）和感染果蝇的辛德比斯病毒作为研究这一现象的模型系统。研究人员将把重点放在细菌对果蝇造成的RNA修饰上，这种修饰会影响病毒感染。新的计算工具和湿实验室协议将产生传播到更广泛的社区。了解共生的分子基础将有助于(a)利用这一机制操纵农业和医学上重要的微生物组（例如益生菌和副微生物），(b)揭示与地球上生命广泛相关的基本生物学，以及(c)更好地模拟这些相互作用的演变。更广泛的影响包括在印第安纳大学开设一门以蚊子载体病毒为重点的项目课程，并通过印第安纳州环境恢复研究所向更广泛的公众推广。成功的宿主相关微生物以对它们有用的方式塑造宿主细胞生物学。通常，宿主生物学的这些变化会改变其他微生物的定植能力，从而为第一批微生物保护宿主的生态位。例如，携带沃尔巴克氏体内共生菌的昆虫定植可以防止病毒感染。这一事实导致在全球范围内部署了感染沃尔巴克氏体的蚊子，以限制病媒疾病的传播。该项目基于沃尔巴克氏体诱导的RNA修饰，直接改变果蝇细胞生物学并影响病毒定植的能力。沃尔巴克氏体上调宿主甲基转移酶以限制病毒复制，初步数据表明病毒基因组本身在沃尔巴克氏体感染的细胞中被修饰，改变mRNA的稳定性。这些数据引出了以下问题：“微生物共生体如何改变宿主细胞的表转录组景观？”该项目将使用直接测序方法（PacBio和ONT）确定沃尔巴克氏体和病毒诱导的宿主DNA和RNA的修饰，并以更成熟的方法（亚硫酸氢盐测序和质谱法）为基准。在此过程中，将开发用于生成纳米孔测序的适当对照的湿实验室协议和用于分析纳米孔测序的表转录组数据集的生物信息学管道。所有资源都将广泛提供和开放获取。最后，这些变化的重要性将通过分子病毒学和果蝇遗传学来确定。总之，这项工作将确定在这个重要的共生系统中，表观转录组修饰如何将基因型与表型联系起来。该项目由理解生命规则：微生物组理论和机制项目资助，该项目是由生物科学理事会新兴前沿部门管理的美国国家科学基金会十大理念的一部分。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。

Symbiotic microbes can have enormous impacts on their hosts’ health and evolution. Indeed, our own cells contain remnants of an ancient microbial symbiosis – the mitochondrion – which continues to provide us with metabolic capabilities essential to our physiology. Understanding the causes, mechanisms, and consequences of host-microbe interactions has emerged as a critical research objective at the interface of evolutionary biology, microbiology, ecology, and development. This project is based on the discovery that a bacterial symbiont changes the host cell in a particular way to protect it from viruses. In this proposed project, the researchers will investigate these changes in the host cell and identify how they alter host, bacterial symbiont, and invading virus. The investigators will use the fruit fly, Drosophila, the bacterium, Wolbachia that is a prominent member of the Drosophila microbiome, and the Sindbis virus which infects Drosophila, as a model system to study this phenomenon. The investigators will focus on RNA modifications to the fruit fly caused by the bacterium, which affect virus infection. Novel computational tools and wet lab protocols will be generated for dissemination to the broader community. Understanding the molecular underpinnings of symbiosis will help to (a) leverage that mechanism in the manipulation of agricultural and medically important microbiomes (e.g. probiotics and paratransgenesis), (b) reveal basic biology of broad relevance to life on the planet, and (c) better model the evolution of these interactions. Broader impacts include the generation of a project-based course at Indiana University focused on mosquito vectored viruses and outreach to the broader public through Indiana’s Environmental Resilience Institute. Successful host-associated microbes sculpt host cell biology in ways that are useful to them. Often, these changes in host biology alter the ability of other microbes to colonize, protecting the host niche for the first microbe. For example, colonization of insects with Wolbachia endosymbionts can preclude infection by viruses. This fact has led to the deployment of Wolbachia-infected mosquitos across the globe to limit the transmission of vectored diseases. This project is based on RNA modifications induced by Wolbachia that directly alter Drosophila cell biology and affect the ability of viruses to colonize. Wolbachia upregulates a host methyltransferase to limit virus replication and preliminary data suggest that the virus genome itself is modified in a Wolbachia infected cell, altering mRNA stability. These data lead to the following question:“How does a microbial symbiont alter the epitranscriptomic landscape of host cells?” This proposed project will identify modifications in both host DNA and RNA induced by Wolbachia and virus using direct sequencing approaches (PacBio and ONT), benchmarked by more established methods (bisulfite sequencing and mass spectrometry). In the process, wet lab protocols for generation of appropriate controls for Nanopore sequencing and bioinformatic pipelines for the analyses of epitranscriptomic datasets from Nanopore sequencing will be developed. All resources will be made broadly available and open access. Finally, the importance of these changes will be identified using molecular virology and Drosophila genetics. In sum, this work will identify how epitranscriptomic modifications link genotype to phenotype in this important symbiotic system.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 through 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.