Interrogating the contributions of novel immune systems to anti-phage defense in their native bacterial hosts

细菌和噬菌体已经在进化军备竞赛中僵持了数十亿年，噬菌体设计出新的进攻策略，而细菌则建立起反击防御。长期以来，人们一直认为细菌具有三种主要的抗病毒防御系统：限制性修饰系统、CRISPR-Cas系统和流产感染系统。然而，最近的工作已经开始揭示在整个细菌世界中存在的更大、更多样化的防御系统。探究这些系统及其对抗病毒防御的贡献是更好地理解细菌-噬菌体相互作用及其对环境和我们的常驻微生物群的影响的下一个主要挑战。迄今为止，这些新型防御系统的少数典型例子涉及生化研究或标准实验室菌株的异源表达。相比之下，防御系统往往是整合在其宿主的生理，并与其他现有的防御系统共存。目前尚不清楚的是，这些防御系统在自然环境下是如何发挥作用的，以及这些系统与其他居民防御系统相比所赋予的保护水平。在这里，我们建议阐明最近发现的防御系统的抗病毒特性，这些防御系统自然地隐藏在菌株中。我们选择Zorya II作为一个引人注目的案例研究，因为该系统在细菌中广泛存在，并编码多种功能未知的蛋白质。关键的是，这些系统存在于许多可培养的、测序的和商业上可用的大肠杆菌菌株中，这些菌株具有其他防御系统，简化了对Zorya II在自然环境中的审讯。我们的操作假设是，Zorya II通过染色体破坏引发细胞自杀，并提供一个非冗余的防御层，进一步保护细菌群体免受噬菌体感染。为了验证这一假设，我们将追求以下两个研究目标：目标1：阐明Zorya II如何实现免疫防御。目的2：确定Zorya II对抗病毒防御的独特贡献。这些目标得到了广泛的初步数据和PI在细菌防御系统、基因组编辑和无细胞系统方面的独特专业知识的支持。如果成功，这项工作将提供一种新的防御系统如何在其原生宿主中促进抗病毒防御的机制描述。这些见解将通过建立细菌抗病毒防御的新概念和机制，直接支持SPP 2330的使命。

Bacteria and phages have been locked in an evolutionary arms race for billions of years, with phages devising new offensive strategies and bacteria mounting countering defenses. Bacteria were long thought to possess three principal systems that confer antiviral defense: restriction-modification systems, CRISPR-Cas systems, and abortive infection systems. However, recent work has begun to reveal a far larger and more diverse repertoire of defense systems present throughout the bacterial world. Interrogating these systems and their contributions to antiviral defense represents the next major challenge to better understand bacterial-phage interactions and their impact on the environment and our resident microbiomes. To date, the few characterized examples of these novel defense systems involved biochemical studies or heterologous expression in standard laboratory strains. In contrast, defense systems are often integrated within the physiology of their host and co-exist with other present defense systems. What remains unclear is how these defense systems function in their natural context and the level of protection conferred by these systems versus the other resident defenses. Here, we propose to elucidate the antiviral properties of recently discovered defense systems in strains harboring these systems naturally. We have selected Zorya II as a compelling case study, as this system is widely prevalent in bacteria and encodes multiple proteins with unknown functions. Critically, these systems are found in numerous culturable, sequenced, and commercially-available strains of Escherichia coli possessing other defense systems, simplifying the interrogation of Zorya II in its natural context. Our operating hypothesis is that Zorya II elicits cell suicide through chromosomal destruction and provides a non-redundant layer of defense that further protects the bacterial population from phage infection. To test this hypothesis, we will pursue the following two research objectives: Objective 1: Elucidate how Zorya II achieves immune defense. Objective 2: Determine the unique contributions of Zorya II to antiviral defense.These objectives are supported by extensive preliminary data and the PI’s unique expertise in bacterial defense systems, genome editing, and cell-free systems. If successful, the proposed work will provide a mechanistic description of how a novel defense system contributes to antiviral defense in its native host. Such insights would directly support the mission of SPP 2330 by establishing new concepts and mechanisms for antiviral defense in bacteria.