Discovery of bacterial defense and phage counter-defense strategies

项目总结 细菌编码一系列不同的分子系统来防御感染噬菌体。作为回应， 噬菌体设计了许多反防御策略来克服这种免疫并重新建立感染。 越来越多的证据表明，自然界中的大多数细菌防御系统和噬菌体反防御系统都具有 没有被确认身份。这是一个重大的知识鸿沟，因为这些系统之间的相互作用通常 确定噬菌体是否成功感染其细菌宿主。这些噬菌体感染，反过来又有主要的 对传染病的演变和治疗的影响。例如，细菌中的致病机制通常 由于表达毒素或其他毒力因子的前噬菌体的整合而进化。同时， 噬菌体越来越被视为治疗细菌感染的潜在疗法，特别是在以下情况下 多重耐药使常规治疗不成功。因此，更好地理解 细菌防御的自然多样性和噬菌体的反防御系统。为了满足这一需求，我们将设计 寻找微生物生态系统防御和反防御系统的新的高通量功能选择 以及在合成的噬菌体开放阅读框库中。这种函数式方法不依赖于顺序 相似性来预测防御和反防御系统，从而克服了传统的， 基于同源的发现方法。因此，这一战略预计将确定许多新的防御和 反防御基因超出了目前已知的水平。它对于检查编码的函数特别有价值 在噬菌体基因组和细菌基因组岛中，因为这些来源的大多数基因具有未知的功能。 由于几乎所有的细菌都应该编码抗噬菌体防御系统，而且几乎所有的噬菌体都会编码反噬菌体防御系统。 防御战略，我们期待有许多新的发现。因为这些发现被预测为 新的，我们将使用遗传和功能分析相结合的方法来描述其作用机制。我们 将使用大肠杆菌作为我们功能选择的宿主，不仅是因为这将允许我们构建 大的功能文库，但也因为这种病原体的毒力是由原噬菌体表达的毒素驱动的 因为它的噬菌体是最常用的开发噬菌体疗法的噬菌体之一。因此，我们的发现 不仅与致病机制和跨细菌的噬菌体疗法广泛相关，而且还将产生这些 特别是在这种重要的人类病原体的背景下的洞察力。

PROJECT SUMMARY Bacteria encode a diverse array of molecular systems to defend against infecting phages. In response, phages have devised many counter-defense strategies to overcome this immunity and re-establish infection. Mounting evidence suggests that most bacterial defense systems and phage counter-defenses in nature have not been identified. This is a major knowledge gap because the interplay between these systems often determines whether a phage successfully infects its bacterial host. These phage infections, in turn, have major impacts on the evolution and treatment of infectious disease. For instance, pathogenesis in bacteria often evolves due to the integration of a prophage that expresses a toxin or other virulence factor. At the same time, phages are increasingly viewed as potential therapeutics to treat bacterial infections, especially in cases where multi-drug resistance renders conventional treatments unsuccessful. Thus, it is important to better understand the natural diversity of bacterial defense and phage counter-defense systems. To meet this need, we will devise new high-throughput functional selections to find defense and counter-defense systems in microbial ecosystems and in libraries of synthesized phage open reading frames. This functional approach does not rely on sequence similarity to predict defense and counter-defense systems, so overcomes the limitations of conventional, homology-based discovery methods. This strategy, therefore, is expected to identify many new defense and counter-defense genes beyond what is known currently. It is especially valuable for examining functions encoded in phage genomes and bacterial genomic islands, as most genes from these sources are of unknown function. Since nearly all bacteria should encode anti-phage defense systems, and almost all phages will encode counter- defense strategies, we expect to make many new discoveries. Because these discoveries are predicted to be novel, we will use a combination of genetic and functional assays to describe their mechanisms of action. We will use Escherichia coli as a host for our functional selections, not only because this will allow us to construct large functional libraries, but also because virulence in this pathogen is driven by prophage-expressed toxins and because its phages are among those used most commonly to develop phage therapies. Thus, our findings will not only be broadly relevant to pathogenesis and phage therapy across bacteria, but also will yield these insights specifically in the context of this important human pathogen.