Bacteriophage control of host cell DNA transactions by small ORF proteins

这项研究计划旨在帮助我们了解细菌细胞如何防御入侵的病毒，即噬菌体，以及噬菌体是如何进化来克服这些“免疫系统”的。噬菌体是地球上最丰富的生物体。它们的生存依赖于入侵和操纵细菌宿主的能力，以便窃取它们构建和释放更多噬菌体颗粒所需的化学资源和机械。在侵入细菌细胞时，噬菌体部署一系列小蛋白(SmORF)，这些蛋白与宿主细胞中的靶标结合，以控制它们的资源并逃避它们的防御系统。最近的研究发现了这些免疫系统的巨大多样性，它们要么防止噬菌体感染，要么在感染细胞中引发细胞自杀，以保护未感染的种群。RecBCD就是这样的细菌防御系统之一，它是一种大型蛋白质复合体，起到免疫中心的作用。它的功能是识别和降解噬菌体DNA分子，并帮助对入侵的DNA的DNA序列进行编目，以形成对病毒序列的记忆，用于对抗未来的感染。为了逃避RecBCD，噬菌体产生与RecBCD结合并抑制RecBCD的单链ORF蛋白：即所谓的抗RecBCD蛋白。值得注意的是，为了应对这一挑战，细菌细胞随后开发了逆转录病毒；这些基因产生了被称为msDNA的分子，能够感知抗RecBCD蛋白作为病毒感染和引发自杀的指标。这个项目将阐明抗RecBCD蛋白和能够感知它们的msDNA的机制。它是由更广泛的概念推动的，即对smORF蛋白的研究不仅仅是对细菌病毒内部工作原理的纯粹学术兴趣。通过了解噬菌体如何操纵和杀死细菌，以及细菌如何保护自己免受此类攻击，我们可以更好地制定策略，战胜导致人类疾病的病原体。例如，我们已经证明，抗RecBCD蛋白可以增强氟喹诺酮类药物(一种抗生素)的作用，甚至可以恢复临床耐药病原菌的敏感性。因此，科学家们现在正在开发针对RecBCD和相关复合体的药物。众所周知，研究协调噬菌体和细菌之间战斗的分子机制已经发现了一个有用的分子宝库。其中包括蛋白质和酶，它们代表了分子生物学家可用的一些最重要和最有用的工具。例如，所有现代基因编辑方法都是基于通过研究细菌免疫而发现的技术。这一先例，以及大量的smORF蛋白及其细胞靶标仍然被忽视和没有特征的事实，表明还有更多有价值的工具等待发现。

This research proposal is designed to help us understand how bacterial cells defend themselves from invading viruses, called bacteriophage, and how bacteriophage have evolved to overcome these "immunity systems". Bacteriophage are the most abundant biological entity on the planet. Their survival depends on an ability to invade and manipulate the bacterial host in order to steal the chemical resources and machinery they need to build and release more bacteriophage particles. Upon invading the bacterial cell, bacteriophage deploy a range of small proteins (smORFs) which bind to targets in the host cell in order to take control of their resources and evade their defence systems. Recent research has uncovered a huge diversity of these immune systems which either prevent phage infection or trigger cell suicide in the infected cells as a means to preserve the uninfected population.One such bacterial defence system is RecBCD, a large protein complex which acts as an immunity hub. It functions both to recognise and degrade bacteriophage DNA molecules and to help catalogue the DNA sequences of the invading DNA to form a memory of viral sequences that is used to counteract future infection. In order to evade RecBCD, bacteriophage make smORF proteins which bind to and inhibit RecBCD: the so-called anti-RecBCD proteins. Remarkably, in response to this challenge, bacterial cells have subsequently developed retrons; genes that produce molecules called msDNAs which are capable of sensing anti-RecBCD proteins as a indicator of viral infection and triggering suicide.This project will elucidate the mechanisms of anti-RecBCD proteins and the msDNAs which can sense them. It is driven by the broader concept that the study of smORF proteins has value beyond a purely academic interest in the inner workings of bacterial viruses. By understanding how bacteriophage manipulate and kill bacteria, as well as how bacteria defend themselves from such attack, we can better develop strategies to overcome pathogenic bacteria which cause human disease. For example, we have shown that anti-RecBCD proteins potentiate the effect of fluoroquinolones (an antibiotic) and can even restore the sensitivity of clinically-resistant strains of pathogenic bacteria. Consequently, scientists are now developing drugs that target RecBCD and related complexes.It is well-established that studying the molecular machinery which orchestrates the fight between bacteriophage and bacteria has uncovered a treasure-trove of useful molecules. These include proteins and enzymes representing some of the most important and useful tools available to molecular biologists. For example, all modern gene-editing methods are based on technologies that were discovered through the study of bacterial immunity. This precedent, and the fact that vast numbers of smORF proteins and their cellular targets remain overlooked and uncharacterised, suggests that many more valuable tools await discovery.