Collaborative Research: Prophages and how they manipulate model microbiomes

动物的肠道是由微生物群落定植的，肠道微生物群可以影响健康和福祉。肠道中最丰富的微生物是细菌，它们代谢活跃，大多数对动物宿主是友好或良性的。像大多数生物体一样，细菌也会被病毒感染，这些病毒被称为噬菌体或噬菌体。已知这些噬菌体的一个版本可以感染细菌并作为噬菌体整合到细菌的DNA中，并保持被认为是休眠状态。然而，由于它们携带的基因或它们整合的位置，噬菌体可以影响细菌的行为，并可以增加它们对抗生素的耐药性。尽管人们越来越认识到动物微生物群如何影响健康，但对噬菌体如何影响动物微生物群的结构和功能知之甚少。该项目将利用两种模型系统，一种是无脊椎脊索动物Ciona robusta，另一种是脊椎动物斑马鱼Danio rerio，以确定噬菌体如何影响动物微生物群。该项目将整合遗传、预测建模和实验方法，研究前噬菌体对动物微生物组的生物学和生态学影响。这项研究的数据将有助于理解动物肠道内形成稳态的因素。该项目的其他更广泛影响包括在微生物学、生物信息学和免疫学研究方面对学生进行培训和指导，其中一些学生来自代表性不足的社区。调查人员将让K-12年级的学生参与诸如“Skype-a-Scientist”和女生海洋学夏令营等活动。此外，调查人员还将在科学节、博物馆和农贸市场向公众进行宣传。大多数动物与细菌形成了基本的关系。噬菌体（或“噬菌体”）是感染这些细菌的病毒，并且已经确定，通过裂解，“裂解”噬菌体重组细菌群落，而“温带”噬菌体可以作为“前噬菌体”整合到细菌基因组中，将其宿主转化为“溶原”。整合到细菌基因组中可以影响附近的基因，而其他病毒基因或调控区域可以在称为“溶原转化”的过程中影响细菌的生理和适应性。“然而，与我们对细菌在动物微生物组中的不同作用的理解显著提高相比，噬菌体对这些群落的影响在很大程度上仍然被忽视。”这一提议将验证噬菌体塑造其细菌宿主的生物学并驱动动物微生物组结构和功能变化的假设。利用细菌基因组测序和分子遗传学的结合，同时整合生态建模和实验方法，研究人员将研究两个强大的模型系统，被囊动物（Ciona robusta）和斑马鱼（Danio rerio）的微生物组中溶原的生物学和生态学。具体来说，研究人员将从模型微生物组中确定培养的异养菌基因组中噬菌体的分布和位置，通过靶向噬菌体缺失诱变确定噬菌体对细菌宿主特性（游泳、生物膜形成、生长/生理）的影响，并研究噬菌体在体内和硅体内定植和影响肠道微生物组稳定性中的作用。这项研究可能揭示了噬菌体直接影响微生物功能的一般原理，从而导致动物微生物组发生或大或小的变化。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。

The animal gut is colonized by a community of microbes, the gut microbiome, that can impact health and well-being. The most abundant microbes in the gut are bacteria, and they are metabolically active and most of them are friendly or benign to the animal host. Like most organisms, bacteria also get infected by viruses, and these are known as bacteriophages or phages. One version of these phages is known to infect bacteria and integrate into their DNA as prophages, remaining in what is thought to be a dormant state. However, because of the genes they carry or the location of their integration, prophages can impact the behavior of bacteria and can increase their resistance to antibiotics. Despite the increased recognition of how animal microbiomes impact health, little is known about how prophages influence the structure and function of animal microbiomes. This project will leverage two model systems, an invertebrate chordate, Ciona robusta, and a vertebrate, the zebrafish Danio rerio to identify how prophages affect animal microbiomes. The project will integrate genetic, predictive modeling, and experimental approaches to study the biology and ecology of prophage influence on animal microbiomes. Data from this study will contribute to the understanding of factors shaping homeostasis within the gut of animals. Other broader impacts of this project include training and mentoring of students, some from underrepresented communities, in research on microbiology, bioinformatics, and immunology. The investigators will engage K-12 students with activities such as “Skype-a-Scientist” and an oceanography camp for girls. Additionally, the investigators will perform outreach to the general public at science festivals, museums, and farmer markets. Most animals form essential relationships with bacteria. Bacteriophages (or ‘phages’) are viruses that infect these bacteria, and it is well established that through lysis, ‘lytic’ phages restructure bacterial communities while ‘temperate’ phages can integrate into bacterial genomes as ‘prophages,’ transforming their hosts into ‘lysogens’. Integration into bacterial genomes can influence nearby genes, while other viral genes or regulatory regions can influence bacterial physiology and fitness in a process referred to as ‘lysogenic conversion.’ However, compared to our significantly improved understanding of the diverse roles of bacteria in animal microbiomes, the impact of prophages on these communities remains largely overlooked. This proposal will test the hypothesis that prophages shape the biology of their bacterial hosts and drive structural and functional changes in animal microbiomes. Utilizing a combination of bacterial genome sequencing and molecular genetics while integrating ecological modeling and experimental approaches, the investigators will study the biology and ecology of lysogens in the microbiomes of two powerful model systems, the tunicate Ciona robusta, and the zebrafish Danio rerio. Specifically, the investigators will characterize prophage distribution and location in genomes of cultivated heterotrophs from model microbiomes, determine the influence of prophages on bacterial host properties (swimming, biofilm formation, growth / physiology) via targeted prophage deletion mutagenesis, and examine the role of prophages in shaping colonization and influencing the stability of the gut microbiome in vivo and in silico. This research may reveal general principles by which prophages directly affect microbial function, leading to both major and minor changes in animal microbiomes.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.