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

CRISPR-Cas是一种存在于许多细菌中的免疫系统，可以保护它们免受被称为噬菌体的细菌病毒的感染。几年前，研究人员做出了一个令人兴奋的发现，即噬菌体已经进化出各种方法来对抗这种免疫反应，方法是产生与CRISPR-Cas免疫复合体结合的小蛋白，从而阻止它们的活动。这些分子被称为抗CRISPR(ACR)，它们在噬菌体感染后立即产生，但我们早期的工作表明，ACR的产生往往“太少、太晚”，以至于噬菌体被细菌宿主细胞中的CRISPR-Cas复合体超过。然而，我们的工作也表明，即使在最初的噬菌体感染被清除后，ACR仍在细胞内徘徊，并继续阻止CRISPR-Cas免疫复合体。这打开了第二个噬菌体的大门，现在它可以成功地感染这个免疫抑制的宿主。有些ACR比其他的“更强”，即它们在阻断CRISPR-CA方面做得更好，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。能够强烈的免疫抑制对噬菌体来说并不一定是更好的；当不同的噬菌体物种相互竞争时(这在许多环境中非常常见)，不产生ACR的噬菌体可以利用那些产生强大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ACR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，生产ACR的噬菌体的适合度降低。在拟议的项目中，我们希望了解ACR噬菌体免疫抑制宿主的机制，以及在没有噬菌体竞争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？)、单细胞水平(免疫抑制能力如何影响噬菌体在单个细菌中的感染成功？)的研究来做到这一点。当感染相同细菌的噬菌体相互竞争时，这种情况会发生什么变化？)以及种群水平(ACR分子昂贵吗？噬菌体在一个ACR上产生多个不同的ACR总是更好吗？以及噬菌体竞争如何影响噬菌体的适合性，从而影响ACR基因的长期维持？)这些研究成果之所以重要，有几个原因。了解ACRs如何与CRISPR-Cas复合体相互作用，以及自然选择如何作用于它们，对于我们从根本上理解CRISPR-Cas生物学具有重要意义。CRISPR-CA也有极其重要的应用，因为它们是一种名为基因编辑的技术中极其有用的工具，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重大潜力，该技术可用于修复导致人类遗传病的突变，如囊性纤维化、杜兴氏肌营养不良症和亨廷顿氏病。但CRISPR-Cas也用于生态工程，例如减少昆虫传播传染病，以及用于微生物工程以消除抗菌素耐药性和/或病原菌。对于这些应用，控制CRISPR-CAS活性的方法将是非常有用的(例如，因为我们只希望它在特定的时间段或特定的组织或细胞中活跃)。目前，我们还没有办法在蛋白质水平上控制CRISPR-Cas的活性，但根据需要使用ACR分子将是一种非常可靠的CRISPR-Cas暂时失活的方法。这项研究将为我们提供新的、令人兴奋的和重要的见解，了解ACR-CRISPR-Cas相互作用的机制以及这些相互作用的进化后果，这对于成功地将ACR作为CRISPR-Cas调控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.