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，称为噬菌体，以及噬菌体如何进化以克服这些“免疫系统”。噬菌体是地球上最丰富的生物实体。它们的生存依赖于入侵和操纵细菌宿主的能力，以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器。在入侵细菌细胞后，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，它们可以防止噬菌体感染，也可以触发受感染细胞的细胞自杀，以保护未感染的群体。其中一种细菌防御系统是RecBCD，一种大型蛋白质复合物，作为免疫枢纽。它的功能是识别和降解噬菌体DNA分子，并帮助分类入侵DNA的DNA序列，以形成用于抵抗未来感染的病毒序列记忆。为了逃避RecBCD，噬菌体产生结合并抑制RecBCD的smORF蛋白：所谓的抗RecBCD蛋白。值得注意的是，为了应对这一挑战，细菌细胞随后开发出了逆转录酶;这种基因产生称为msDNA的分子，这种分子能够感应作为病毒感染指示剂的抗RecBCD蛋白并触发自杀。本项目将阐明抗RecBCD蛋白和可以感应它们的msDNA的机制。这是由更广泛的概念驱动的，即对smORF蛋白的研究具有超越细菌病毒内部工作的纯粹学术兴趣的价值。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这种攻击，我们可以更好地制定策略来克服导致人类疾病的病原菌。例如，我们已经证明，抗RecBCD蛋白增强了氟喹诺酮（一种抗生素）的作用，甚至可以恢复致病菌的临床耐药菌株的敏感性。因此，科学家们现在正在开发针对RecBCD和相关复合物的药物。众所周知，研究噬菌体和细菌之间协调战斗的分子机制已经发现了有用分子的宝库。其中包括代表分子生物学家可用的一些最重要和最有用的工具的蛋白质和酶。例如，所有现代基因编辑方法都是基于通过细菌免疫研究发现的技术。这一先例，以及大量smORF蛋白及其细胞靶点仍然被忽视和未被表征的事实，表明还有许多更有价值的工具有待发现。

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.