Conquering CRISPR 征服 CRISPR
CRISPR-Cas is a genome editing system found in bacteria that helps a bacterium to defend against phages by inhibiting the integration of phage DNA to a bacterium via CRISPR and endonuclease activity (Cas). Phages undergo point mutation or deletions to escape bacterial adaptive immunity.42 Therefore, CRISPR-Cas (the effector molecule) fails to recognize and cut the specific genomic sequences of phages that had point mutations and/or deletions. This implies that in CRISPR-Cas systems, a single mutation in the protospacer-adjacent motif is enough to avoid targeting.43–45 Unfortunately, some phage mutations may promote CRISPR immunity by enhancing the gaining of many new spacers.48,49 Phages can evade detection by deleting part of or the entire protospacer target. Despite conquering CRISPR, this strategy can have a fitness cost to the phage, depending on the region deleted.50
CRISPR-Cas 是细菌中的一种基因组编辑系统，通过 CRISPR 和内切酶活性（Cas）抑制噬菌体 DNA 与细菌的整合，从而帮助细菌抵御噬菌体。噬菌体通过点突变或缺失来逃避细菌的适应性免疫。 42 因此，CRISPR-Cas（效应分子）无法识别和切割发生点突变和/或缺失的噬菌体的特定基因组序列。这意味着，在 CRISPR-Cas 系统中，原位相邻基序的单个突变就足以避免靶向。 43–45 不幸的是，一些噬菌体突变可能会通过增强获得许多新的间隔物来促进 CRISPR 免疫。 48 49 噬菌体可以通过删除部分或整个原间隔物靶标来逃避检测。尽管可以征服 CRISPR，但这种策略会使噬菌体付出健康代价，具体代价取决于所删除的区域。 50
Phages produce different proteins that inhibit CRISPR-Cas defense, of which anti-CRISPR (Acr) proteins are the most prominent.51 Inhibition mechanisms of CRISPR-Cas defense include blocking target binding by DNA mimicry or steric blocking and the prevention of DNA cleavage by nucleases.52 An emerging way to inhibit CRISPR-Cas activity is by utilizing phages’ subversion of cellular regulatory pathways that bypass CRISPR-Cas activity.53 Phages may possess regulatory protein homologs to bacterial proteins that suppress bacterial defenses, known as bacterial CRISPR-Cas repressors.54 Otherwise, phages can use proteins that bind and inhibit bacterial regulators. Multiple bacteria modify their CRISPR-Cas activity via quorum sensing, but this behavior may be manipulated by phage-encoded proteins. Genomic modifications are another way for phages to escape from CRISPR-Cas attack.55,56 For example, five distinct anti-CRISPR genes are present in P. aeruginosa temperate phages. These genes encode a small protein that can immediately neutralize the immune system of the host by interfering with the formation or action of CRISPR-Cas ribonucleic protein.57
噬菌体产生不同的蛋白质来抑制CRISPR-Cas防御，其中以抗CRISPR（Acr）蛋白质最为突出。 51 CRISPR-Cas 防御的抑制机制包括通过 DNA 拟态或立体阻断阻止目标结合，以及防止核酸酶切割 DNA。 52 一种新出现的抑制 CRISPR-Cas 活性的方法是利用噬菌体对绕过 CRISPR-Cas 活性的细胞调控途径的颠覆。 53 噬菌体可能拥有与抑制细菌防御功能的细菌蛋白同源的调节蛋白，即细菌 CRISPR-Cas 抑制因子。 54 否则，噬菌体可以使用结合和抑制细菌调节因子的蛋白质。多种细菌通过法定人数感应来改变它们的 CRISPR-Cas 活性，但这种行为可能会被噬菌体编码的蛋白质所操纵。基因组修饰是噬菌体逃避 CRISPR-Cas 攻击的另一种方式。 55 56 例如，铜绿假单胞菌温带噬菌体中有五个不同的抗 CRISPR 基因。这些基因编码一种小蛋白，可通过干扰 CRISPR-Cas 核糖核酸蛋白的形成或作用，立即中和宿主的免疫系统。 57
Two general models have been implemented to manage the risk of bacterial resistance to phage therapy. These involve applying phage cocktails and adapting a single phage to each patient.58 Combining many phages in cocktails provides them a wide host range and improves their effectiveness. Synergizing different phages and targeting different receptors on the bacterial surface reduces bacterial resistance to phages. Such an approach has a major benefit for empirical treatment.59 The personalized phage therapy approach utilizes single phages or targeted phage cocktails directly based on the etiologic agent isolated.60 This approach is more flexible with respect to the phage spectrum, and minimizes the emergence of bacterial resistance effectively, but carries a higher cost for treatment.61
为控制细菌对噬菌体疗法产生抗药性的风险，我们采用了两种通用模式。这两种模式分别是应用噬菌体鸡尾酒疗法和为每位患者调配一种噬菌体。 58 将多种噬菌体组合在鸡尾酒中，使它们的宿主范围更广，并提高其有效性。将不同的噬菌体协同作用，靶向细菌表面的不同受体，可以减少细菌对噬菌体的抗药性。这种方法对经验性治疗大有裨益。 59 个性化噬菌体疗法直接根据分离出的病原体使用单一噬菌体或有针对性的噬菌体鸡尾酒。 60 这种方法在噬菌体谱方面更加灵活，能有效减少细菌耐药性的产生，但治疗成本较高。 61
In summary, although bacteria have the potential to develop resistance to phage therapy via different mechanisms, phages have several mechanisms by which they can escape bacterial resistance against them. Therefore, upgrading current practices and knowledge on phage interactions with phages, bacteria, and humans, is a promising way to treat bacterial infections in the era of increasing incidence and transmission of MDR bacterial species and strains, where the production of new antibiotics is limited.
总之，虽然细菌有可能通过不同的机制对噬菌体疗法产生抗药性，但噬菌体也有多种机制可以摆脱细菌对它们的抗药性。因此，在多重耐药菌种和菌株的发病率和传播率不断上升，而新抗生素的生产又受到限制的时代，提升当前有关噬菌体与噬菌体、细菌和人类之间相互作用的实践和知识，是治疗细菌感染的一条大有可为的途径。