4.4. Phage Engineering 4.4.噬菌体工程
Naturally occurring phages have been the focus of the majority of in vitro and in vivo studies as well as in clinical cases for over a century (78). However, there are some inherent limitations associated with using wild-type phages that can be overcome using a genetic-engineering approach. In the last decade, the synthetic biology revolution and development of new genetic-engineering technologies have paved the way for precise and rapid engineering of phage genomes, allowing the creation of novel designer phages (79, 80). Phage engineering has already been used to enhance properties of naturally occurring phages, including expanding their host ranges, reducing phage resistance in bacteria, increasing phage safety, and improving stability of phages and phage products (78, 81–83).
一个多世纪以来，天然噬菌体一直是大多数体外和体内研究以及临床病例的重点（78）。然而，使用野生型噬菌体存在一些固有的局限性，而基因工程方法可以克服这些局限性。在过去十年中，合成生物学革命和新基因工程技术的发展为精确、快速的噬菌体基因组工程铺平了道路，从而可以创造出新型的设计噬菌体 ( 79, 80)。噬菌体工程已被用于增强天然噬菌体的特性，包括扩大其宿主范围、减少细菌对噬菌体的抗药性、提高噬菌体的安全性以及改善噬菌体和噬菌体产物的稳定性 ( 78, 81- 83)。
In 2019, Dedrick et al. (49) reported the first successful case of human phage therapy using engineered phages. Following a lung transplant, a 15-year-old cystic fibrosis patient acquired a disseminated Mycobacterium abscessus infection that was resistant to multiple antibiotics. The researchers screened hundreds of naturally isolated mycobacterial phages and identified a single lytic phage that efficiently killed the clinical strain as well as two temperate phages with poor killing activity. They genetically engineered the temperate phages by precisely removing their immunity repressor gene. The resulting lytic derivatives of the temperate phages showed enhanced killing activity against the target host strain. A cocktail composed of these three (one natural and two engineered) lytic phages was administered intravenously to the patient, which resulted in clinical improvement and alleviation of the infection (49). This landmark case study clearly exemplifies how genome engineering may be critical to expand the repertoire of phages suitable for clinical applications.
2019 年，Dedrick 等人 ( 49) 报道了首例利用工程噬菌体进行人类噬菌体治疗的成功案例。一名 15 岁的囊性纤维化患者在接受肺移植手术后，感染了对多种抗生素耐药的播散性脓肿分枝杆菌。研究人员筛选了数百种天然分离的分枝杆菌噬菌体，发现了一种能有效杀死临床菌株的溶菌噬菌体，以及两种杀菌活性较差的温带噬菌体。他们通过精确移除温带噬菌体的免疫抑制基因，对其进行了基因工程改造。由此产生的温带噬菌体裂解衍生物对目标宿主菌株的杀伤活性增强。通过静脉注射由这三种（一种天然噬菌体和两种工程噬菌体）致死噬菌体组成的鸡尾酒，患者的临床症状得到了改善，感染也得到了缓解（49）。这一具有里程碑意义的案例研究清楚地说明了基因组工程对于扩大适合临床应用的噬菌体种类至关重要。
In addition to engineering temperate phages to turn them into their lytic derivatives, genetic engineering may also be used to enhance therapeutic activity of lytic phages. However, engineering of lytic phages is hampered by the fact that their genomes do not integrate into the chromosomes of host bacteria and thus cannot be manipulated with existing methods for bacterial genome engineering. Currently, the most popular phage engineering strategy relies on homologous recombination between the phage genome and a DNA-editing template plasmid transformed into host cells used in phage propagation. The recombination frequencies in this method are generally quite low, making it tedious and time-consuming to identify recombinant phages. To enhance the frequency of homologous recombination, recombineering-based methods exploit a phage-encoded recombination system. By heterologous expression of this system within the cell, the recombination template is protected from intracellular degradation and recombination frequency increases. A complementary downstream strategy to facilitate identification of engineered phages is to add a positive selection (e.g., fluorescence markers) or a negative selection (e.g., CRISPR-based system targeting wild-type phages).
除了对温带噬菌体进行工程改造，将其转化为致死噬菌体的衍生物外，基因工程还可用于提高致死噬菌体的治疗活性。然而，噬菌体基因组不会整合到宿主细菌的染色体中，因此无法用现有的细菌基因组工程方法进行操作，这阻碍了噬菌体工程学的发展。目前，最流行的噬菌体工程策略依赖于噬菌体基因组与转化到宿主细胞中用于噬菌体繁殖的 DNA 编辑模板质粒之间的同源重组。这种方法的重组频率通常很低，因此识别重组噬菌体既繁琐又费时。为了提高同源重组的频率，基于重组工程的方法利用了噬菌体编码的重组系统。通过在细胞内异源表达该系统，可保护重组模板不被细胞内降解，从而提高重组频率。为便于识别工程噬菌体，一种补充的下游策略是添加正选择（如荧光标记）或负选择（如针对野生型噬菌体的基于 CRISPR 的系统）。
Recently, new phage genome engineering methods that do not rely on homologous recombination have been described (79, 80). These approaches generate in vitro phage genomic DNA containing the desired genetic changes and reboot these genomes to produce infectious phage particles. There are two approaches to introduce genetic alterations into phage genomes in vitro: by modifying a genome extracted from a phage lysate or by printing and assembling synthetic DNA fragments into complete phage genomes. The assembly techniques can be accomplished within bacterial cells or in vitro. Then, the fully assembled engineered phage genome is rebooted via transformation of the DNA into bacterial hosts or using cell-free transcription-translation systems.
最近，不依赖同源重组的新噬菌体基因组工程方法得到了描述 ( 79, 80)。这些方法可在体外生成含有所需基因改变的噬菌体基因组 DNA，并重新启动这些基因组以产生具有传染性的噬菌体颗粒。在体外将基因改变引入噬菌体基因组的方法有两种：修改从噬菌体裂解液中提取的基因组，或打印合成 DNA 片段并将其组装成完整的噬菌体基因组。组装技术可在细菌细胞内或体外完成。然后，通过将 DNA 转化为细菌宿主或使用无细胞转录-翻译系统，重新启动完全组装好的工程噬菌体基因组。
Phage engineering has been successfully used to increase therapeutic activity of natural lytic phages. For some viruses, host ranges have been modified through engineering of receptor-binding proteins. Yehl et al. (84) utilized a structure-guided approach to identify a specific host-range determining region in the gene encoding the tail fiber (filamentous protein involved in host recognition and binding) in phage T3. Then, they used targeted mutagenesis to generate phage libraries with vast genetic diversity in this region that translated into altered host ranges. Out of this library, they then chose a defined cocktail of 10 phage mutants. The cocktail was able to suppress emergence of phage resistance in E. coli bacteria for 1 week in vitro, whereas bacteria infected with the wild-type phage evolved resistance after merely 12 h. Finally, the study demonstrated that a single phage mutant was able to suppress bacterial growth in vivo in a mouse wound-infection model, which suggested that this phage engineering could be used to minimize evolution of phage resistance during therapy (84).
噬菌体工程已被成功用于提高天然溶菌噬菌体的治疗活性。对于某些病毒来说，宿主范围是通过受体结合蛋白工程学来改变的。Yehl 等人（84 年）利用结构引导法在噬菌体 T3 的尾纤（参与宿主识别和结合的丝状蛋白）编码基因中确定了一个特定的宿主范围决定区。然后，他们利用定向诱变技术生成了噬菌体文库，该文库中的该区域具有巨大的遗传多样性，可转化为改变的宿主范围。在这个文库中，他们选择了由 10 个噬菌体突变体组成的鸡尾酒。最后，研究表明，在小鼠伤口感染模型中，单个噬菌体突变体能够抑制细菌在体内的生长，这表明这种噬菌体工程可用于最大限度地减少治疗过程中噬菌体抗药性的演变 ( 84)。
Bacterial resistance to phage infection does not exclusively arise from receptor mutations. For example, bacterial encapsulation or biofilm formation can restrict phages from accessing their binding target(s) and therefore limit their therapeutic efficacy. Lu et al. (85) engineered a T7 phage to express a biofilm-degrading enzyme (dispersin B) during host infection. When added to an E. coli biofilm model, the engineered T7 phage reduced biofilm-associated cell counts much more efficiently (∼2 orders of magnitude lower biofilm cell counts) relative to wild-type T7 phage. In a different approach that was not constrained to a single target species (i.e., multi-species biofilms), T7 phage was engineered to deliver an enzyme that inactivates a quorum-sensing molecule produced by P. aeruginosa and is involved in biofilm formation. In a coculture of E. coli and P. aeruginosa, presence of the engineered T7 phage effectively inhibited biofilm formation, compared to the wild-type virus (86). These studies demonstrate that biofilm-forming bacteria can be targeted more efficiently using engineered phages, which is highly promising given the tendency for chronic bacterial infections to be associated with biofilm-forming pathogens.
细菌对噬菌体感染的抗药性并不完全来自受体突变。例如，细菌的包裹或生物膜的形成会限制噬菌体接触其结合靶标，从而限制其疗效。Lu 等人 ( 85) 改造了一种 T7 噬菌体，使其在宿主感染期间表达一种生物膜降解酶（分散素 B）。当加入到大肠杆菌生物膜模型中时，与野生型 T7 噬菌体相比，工程化 T7 噬菌体能更有效地减少生物膜相关细胞数量（生物膜细胞数量减少 2 个数量级）。在一种不局限于单一目标物种（即多物种生物膜）的不同方法中，T7噬菌体被设计成能提供一种酶，这种酶能使铜绿假单胞菌产生的参与生物膜形成的法定量感应分子失活。在大肠杆菌和铜绿假单胞菌的共培养过程中，与野生型病毒相比，工程化 T7 噬菌体能有效抑制生物膜的形成 ( 86)。这些研究表明，使用工程噬菌体可以更有效地针对形成生物膜的细菌，鉴于慢性细菌感染往往与形成生物膜的病原体有关，因此这是非常有前景的。
Despite the advances in phage genome-editing methods described above, phage engineering research is still in its infancy. Most of these methods have been developed only for established model phages, and engineering non-model phages can be a lot more complex. Furthermore, many engineering approaches require domesticated bacterial hosts and/or an in-depth molecular understanding of cellular mechanisms and, thus, are not yet broadly applicable for emerging bacterial diseases. Further technological development is still required to provide broadly applicable, efficient, and inexpensive engineering methods.
尽管噬菌体基因组编辑方法取得了上述进展，但噬菌体工程研究仍处于起步阶段。这些方法大多是针对已建立的模式噬菌体开发的，而非模式噬菌体工程学可能要复杂得多。此外，许多工程方法需要驯化细菌宿主和/或对细胞机制有深入的分子了解，因此还不能广泛应用于新出现的细菌疾病。要提供广泛适用、高效和廉价的工程方法，还需要进一步的技术开发。
To increase the efficacy of phage therapies, naturally occurring phages, trained phages, and engineered phages can be combined into a cocktail preparation as opposed to phage monotherapy and can also be co-administered with other antimicrobials (e.g., small molecule antibiotics) to further overwhelm bacteria and reduce their potential to evolve resistance (Figure 2, top).
为了提高噬菌体疗法的疗效，可以将天然噬菌体、训练有素的噬菌体和工程噬菌体组合成鸡尾酒制剂，而不是噬菌体单一疗法，还可以与其他抗菌剂（如小分子抗生素）联合使用，以进一步压制细菌，降低它们产生抗药性的可能性（图 2，上图）。
Given the powerful ability for bacterial populations to defend against lytic phages and to readily evolve increased phage resistance, it may not be possible to always minimize bacterial evolution. Therefore, an equally powerful alternative is to assume that the bacteria will evolve to resist therapeutic phages and devise phage therapies where this evolution is predicted and steered in a biomedically favorable direction that may foster its success.
鉴于细菌种群具有抵御致死噬菌体的强大能力，而且很容易进化出更强的噬菌体抗性，因此可能无法始终最大限度地减少细菌的进化。因此，一个同样有效的替代方法是假定细菌会进化出抵抗治疗性噬菌体的能力，并设计出噬菌体疗法，预测这种进化并将其引向对生物医学有利的方向，以促进其成功。