Mechanisms and evolutionary consequences of host immunosuppression by anti-CRISPR phages

CRISPR-Cas是存在于许多细菌中的一种免疫系统，可以保护细菌免受噬菌体病毒的感染。几年前，研究人员令人兴奋地发现，噬菌体已经进化出了对抗这种免疫反应的方法，通过产生与CRISPR-Cas免疫复合物结合的小蛋白质，从而阻断其活性。这些分子被命名为抗crispr (Acr)，在噬菌体感染后立即产生，但我们早期的工作表明，Acr的产生通常“太少，太迟”，因此细菌宿主细胞中的CRISPR-Cas复合物超过了噬菌体。然而，我们的工作也表明，即使最初的噬菌体感染已被清除，Acr仍在细胞中徘徊并继续阻断CRISPR-Cas免疫复合物。这为第二个噬菌体打开了大门，它现在可以成功地感染这个免疫抑制的宿主。一些Acr比其他的“更强”，也就是说，它们更擅长阻断CRISPR-Cas，但我们不知道是什么因素决定了Acr的强度以及相关的免疫抑制。强烈的免疫抑制并不一定对噬菌体更好；当不同种类的噬菌体相互竞争时（这在许多环境中很常见），不产生Acr的噬菌体可以利用那些产生强Acr分子的噬菌体。我们的数据表明，Acr的生产成本很高（例如，因为生产它们需要能量），因此这种开发风险可能意味着在噬菌体竞争期间，产生Acr的噬菌体的适应性降低。在这个项目中，我们希望了解Acr噬菌体免疫抑制宿主的机制，以及在没有和存在噬菌体竞争的情况下，宿主免疫抑制对Acr基因维持的进化后果。我们将通过整合分子水平（Acr如何与CRISPR-Cas免疫复合物结合？）和单细胞水平（免疫抑制能力如何影响单个细菌中噬菌体的感染成功？）的研究来做到这一点。当感染同一细菌的噬菌体相互竞争时，这种情况如何变化？)和种群水平(Acr分子昂贵吗？噬菌体产生多个不同的Acr总是比产生一个Acr更好吗？以及噬菌体竞争如何影响噬菌体适应性，从而长期影响Acr基因的维持？)。研究结果之所以重要，有几个原因。了解Acrs如何与CRISPR-Cas复合物相互作用以及自然选择如何作用于它们对于我们对CRISPR-Cas生物学的基本理解非常重要。CRISPR-Cas也有非常重要的应用，因为它们是基因编辑技术中非常有用的工具，在这种技术中，特定的突变或基因可以被移除或引入生物体的DNA中。这在医疗保健领域具有巨大的潜力，该技术可用于修复导致人类遗传疾病的突变，如囊性纤维化、杜氏肌营养不良症和亨廷顿氏病。但是CRISPR-Cas也用于生态工程，例如减少昆虫传播传染病，以及用于微生物组工程以消除抗菌素耐药性和/或致病菌。对于这些应用来说，拥有控制CRISPR-Cas活性的方法将非常有用（例如，因为我们只希望它在特定时间段或特定组织或细胞中活跃）。目前，我们还没有办法在蛋白水平上控制CRISPR-Cas的活性，但根据需要，使用Acr分子将是一种非常可靠的使CRISPR-Cas暂时失活的方法。这项研究将为我们提供关于Acr - CRISPR-Cas相互作用的机制以及这些相互作用的进化后果的新的、令人兴奋的和重要的见解，这对于成功地将Acrs作为CRISPR-Cas调节工具应用于医疗保健、生命科学研究和农业至关重要。

CRISPR-Cas is an immune system present in many bacteria that protects them against infections with bacterial viruses, called phages. Several years ago researchers made the exciting discovery that phages have evolved ways to counteract this immune response, by producing small proteins that bind to CRISPR-Cas immunity complexes and thereby block their activity. These molecules, named anti-CRISPRs (Acr), are produced immediately upon phage infection, but our earlier work has shown that Acr production is often 'too little, too late" so that phages are outpaced by the CRISPR-Cas complexes in the bacterial host cell. However, our work also shows that Acr linger on in the cell and keeps blocking CRISPR-Cas immunity complexes, even when the initial phage infection has been cleared. This opens the door to a second phage that can now successfully infect this immunosuppressed host. Some Acrs are "stronger" than others, i.e. they are better at blocking CRISPR-Cas, but we do not understand what factors determine how strong an Acr is and the associated immunosuppression. Being able to strongly immunosuppress is not necessarily better for the phage; when different phage species compete with each other (which is very common in many environments), phages that do not produce Acr can exploit those that produce strong Acr molecules. Our data indicate that Acr are costly to produce (for example, because it takes energy to produce them) and this exploitation risk could therefore mean that during phage-phage competition the fitness of the phage producing Acrs is lowered. In the proposed project we wish to understand the mechanism by which Acr-phages immunosuppress their hosts, and the evolutionary consequences of host immunosuppression on maintenance of Acr genes in the absence and presence of phage-phage competition. We will do this by integrating research on the molecular level (how do Acr bind to CRISPR-Cas immunity complexes?), the single-cell level (how does the ability to immunosuppress influence the infection success of phage in individual bacteria? How does this change when phages that infect the same bacterium compete with each other?) and the population level (are Acr molecules costly? is it always better for a phage to produce multiple different Acrs over one Acr? and how does phage-phage competition influence phage fitness and therefore Acr gene maintenance in the long term?). The research outcomes are important for several reasons. Understanding how Acrs interact with CRISPR-Cas complexes and how natural selection acts on them is important for our fundamental understanding of CRISPR-Cas biology. CRISPR-Cas also has extremely important applications, as they are extremely useful tools in a technique called gene editing, where specific mutations or genes can be removed or introduced in the DNA of an organism. This has major potential in healthcare, where the technique could be used to repair mutations that cause genetic diseases in human, such as Cystic Fibrosis, Duchenne muscular dystrophy and Huntington's disease. But CRISPR-Cas is also used for ecological engineering, for example to reduce the spread of infectious diseases by insects, and for microbiome engineering to remove antimicrobial resistance and/or pathogenic bacteria. For these applications, it would very useful to have ways to control CRISPR-Cas activity (for example, because we only want it to be active during a certain time period or in specific tissues or cells). Currently, we have no ways to control CRISPR-Cas activity on protein level, but using Acr molecules would be an extremely reliable way of temporal inactivation of CRISPR-Cas as required. This research will give us new, exciting and important insights in the mechanisms underlying Acr - CRISPR-Cas interactions and the evolutionary consequences of these interactions, which will be crucial for successfully applying Acrs as a CRISPR-Cas regulating tool in healthcare, life sciences research and agriculture.