5.2. Steering Evolution Toward Other Reduced-Virulence Traits
5.2.引导进化走向其他减毒性状
Beyond antibiotic resistance, other virulence factors aid bacterial infection and pathogenicity in humans, such as adherence to host cells, invasion of host tissues, and evasion of host defenses. The cell structures associated with these factors include membrane-embedded proteins and various appendages, as well as polysaccharides forming the outer cell layers such as membranes, cell walls, and capsules. For example, flagella and pili are critical for the formation of bacterial biofilm (24), which protects bacteria from environmental stressors such as phages and antibiotics, and enable bacterial persistence through dormancy in chronic infections. Phages that use virulence factors as receptors, e.g., phages binding to flagella (101–103) or pili (104–107), can select for evolution of resistance that negatively affects functions of these structures, thus reducing bacterial virulence (87).
除抗生素耐药性外，其他毒力因子也有助于细菌感染人类和致病，如附着于宿主细胞、侵入宿主组织和逃避宿主防御。与这些因子相关的细胞结构包括膜嵌入蛋白和各种附属物，以及构成细胞外层（如膜、细胞壁和囊）的多糖。例如，鞭毛和纤毛对细菌生物膜的形成至关重要（24），生物膜可保护细菌免受噬菌体和抗生素等环境压力的影响，并使细菌在慢性感染中通过休眠持续存在。利用毒力因子作为受体的噬菌体，例如与鞭毛（101-103）或纤毛（104-107）结合的噬菌体，可以选择进化抗药性，从而对这些结构的功能产生负面影响，从而降低细菌的毒力（87）。
Phages that drive trade-offs with reduced virulence have already been successfully used in the clinic. Evidence suggests that phage-directed trade-offs that compromise synthesis of the bacterial capsule, a major virulence factor that provides protection from environmental stressors, likely played a crucial role in a well-known case of intravenous phage therapy in the United States. Here, a life-threatening infection of the opportunistic pathogen A. baumanii was resistant to all antibiotic options for the patient. Through emergency treatment approval, several iterations of cocktails composed of different phages were administered to the patient intravenously and eventually led to their full recovery. Follow-up in vitro work revealed that the phages in the cocktail targeted bacterial capsules, the polysaccharide cell envelopes (108). Characterization of patient isolates before and after phage administration suggested that A. baumanii cells with evolved resistance to the administered phages traded off with capsule biosynthesis (108). Notably, this case and the aforementioned use of OMKO1 against MDR P. aeruginosa (96) helped popularize the power of phage therapy in Western medicine.
噬菌体在降低毒力方面的权衡作用已经成功应用于临床。有证据表明，噬菌体引导的权衡会影响细菌胶囊的合成，而细菌胶囊是一种主要的毒力因子，能提供对环境压力的保护，在美国一个著名的静脉噬菌体疗法案例中，噬菌体可能发挥了至关重要的作用。在该病例中，机会性病原体鲍曼不动杆菌的感染危及生命，患者对所有抗生素都产生了耐药性。通过紧急治疗批准，为患者静脉注射了几种由不同噬菌体组成的迭代鸡尾酒，最终使患者完全康复。后续的体外研究表明，鸡尾酒中的噬菌体针对的是细菌的胶囊–多糖细胞包膜（108）。噬菌体给药前后患者分离物的特征表明，鲍曼不动杆菌细胞对给药的噬菌体产生了抗药性，从而取代了胶囊的生物合成（108）。值得注意的是，这一病例和前述使用 OMKO1 治疗 MDR 铜绿假单胞菌的病例（96）有助于在西方医学中推广噬菌体疗法的威力。
Next, we provide examples of in vitro and in vivo studies of phages targeting very diverse pathogenic bacteria that drive trade-offs toward reduced virulence.
接下来，我们举例说明了针对多种致病菌的噬菌体的体外和体内研究，这些研究推动了对降低毒力的权衡。
5.2.1. Reduced bacterial adherence and colonization.
5.2.1.减少细菌附着和定植。
Phages can direct evolution of resistance toward impaired ability of bacteria to adhere to and colonize mammalian host cells. For example, Listeria monocytogenes, an intracellular pathogen, requires a complex glycosylation pattern acquired through teichoic acids in its cell wall for successful infection. Acquiring resistance to a phage specifically recognizing this pattern resulted in loss of these sugars from the bacterial cell surface. The phage-resistant mutants of L. monocytogenes were unable to target the putative receptors on host mammalian cells and, hence, were unable to adhere and infect the host tissue (109). Notably, the phage used in this study was originally a temperate phage that was engineered to be fully lytic. Through this engineering approach, it is evident that temperate phages that drive bacterial evolution in a favorable direction may also provide promising candidates for therapy. Another example of phage-directed modulation of the host-colonization capability of bacteria was reported for Enterococcus faecalis–targeting phages. E. faecalis mutants resistant to a cohort of 19 phages evolved to acquire mutations in the enterococcal polysaccharide antigen (epa) gene cluster, a known virulence factor in enterococci. These epa mutants exhibited altered cell-surface properties (which also made them more susceptible to daptomycin and vancomycin, antibiotics that target the cell wall). Due to reduced fitness of the altered membranes, the mutants were deficient in intestinal colonization and transmission in mice (110).
噬菌体可以引导抗药性的进化，使细菌粘附和定植哺乳动物宿主细胞的能力受损。例如，单核细胞增生李斯特菌是一种细胞内病原体，它需要通过细胞壁中的茶酸获得复杂的糖基化模式才能成功感染。获得对专门识别这种模式的噬菌体的抗性后，细菌细胞表面就会失去这些糖。单核细胞增多性乳酸杆菌的噬菌体抗性突变体无法靶向宿主哺乳动物细胞上的假定受体，因此无法粘附和感染宿主组织（109）。值得注意的是，这项研究中使用的噬菌体原本是一种温带噬菌体，经过改造后具有完全溶菌能力。通过这种工程方法，可以明显看出，温带噬菌体能推动细菌向有利的方向进化，也可能为治疗提供有前途的候选噬菌体。噬菌体定向调节细菌宿主定殖能力的另一个例子是针对粪肠球菌的噬菌体。对 19 种噬菌体具有抗性的粪肠球菌突变体在进化过程中获得了肠球菌多糖抗原（epa）基因簇的突变，epa 基因簇是已知的肠球菌毒力因子。这些 epa 突变体的细胞表面特性发生了改变（这也使它们更容易受到达托霉素和万古霉素这些针对细胞壁的抗生素的影响）。由于改变的细胞膜降低了适存性，这些突变体在小鼠肠道定植和传播方面存在缺陷（110）。
5.2.2. Decreased host invasion.
5.2.2.减少宿主入侵。
Another way phages can steer bacterial evolution toward reduced virulence is by limiting bacterial invasion and spread to additional cells within the infected host. To spread between cells in the human intestine, pathogen Shigella flexneri requires outer membrane protein A (OmpA) as well as the O-antigen (a component of LPS). Shigella phages A1-1 (38) and Sf6 (111) require OmpA for entry into the cell. In a recent study, five spontaneous S. flexneri mutants resistant against A1-1 were isolated (38). Sequencing revealed that two mutants had a deletion in ompA, while the other three genotypes featured mutations in LPS. The phage-resistant mutants, unlike the ancestral wild-type bacteria, were unable to spread between eukaryotic host cells in a tissue-culture pathogenicity model. It was concluded that the phage-resistant mutants had lost their ability to move between eukaryotic cells, indicating that evolved phage resistance compromised bacterial pathogenicity.
噬菌体引导细菌向降低毒力方向进化的另一种方式是限制细菌入侵和扩散到受感染宿主体内的其他细胞。要在人类肠道细胞间传播，病原体志贺氏菌（Shigella flexneri）需要外膜蛋白 A（OmpA）和 O 抗原（LPS 的一种成分）。志贺氏菌噬菌体 A1-1 ( 38) 和 Sf6 ( 111) 需要 OmpA 才能进入细胞。在最近的一项研究中，分离出了五个对 A1-1 具有抗性的自发 S. flexneri 突变体（38）。测序结果显示，两个突变体的 OmpA 发生了缺失，而另外三个基因型的 LPS 发生了突变。与野生型细菌不同，抗噬菌体突变体在组织培养致病性模型中无法在真核宿主细胞间传播。结论是抗噬菌体突变体失去了在真核细胞间移动的能力，这表明噬菌体抗性的进化损害了细菌的致病性。
5.2.3. Impaired ability to evade host immune system.
5.2.3.躲避宿主免疫系统的能力受损。
Phage resistance also has been shown to trade off with reduced virulence by impairing the bacteria’s ability to evade the host immune system. Cai and coworkers (112) found that K. pneumoniae, when subjected to selection by phage GH-K3, evolves to downregulate three glucosyltransferase-encoding genes involved in capsule synthesis. The phage-resistant mutants, defective in capsule synthesis, were less virulent, as measured by mortality counts in murine infection models. Immunofluorescence assays revealed that while a very small fraction of the wild-type K. pneumoniae cells became internalized within macrophages, the phage-resistant mutants had a much higher probability of being endocytosed by macrophages. The authors suggested that the bacterial capsule likely helps in avoiding phagocytosis by macrophages, whereas phage-resistant mutants were less able to evade phagocytosis. Thus, phage resistance may trade off with evasion of host defenses, a topic that deserves further scrutiny.
噬菌体的抗性也被证明可以通过削弱细菌逃避宿主免疫系统的能力来换取毒力的降低。Cai 和同事 ( 112) 发现，肺炎双球菌在受到噬菌体 GH-K3 的选择时，会下调参与胶囊合成的三个葡萄糖基转移酶编码基因。噬菌体抗性突变体在胶囊合成方面存在缺陷，根据小鼠感染模型中的死亡率计算，其毒性较低。免疫荧光测定显示，野生型肺炎克氏菌细胞只有极少部分被巨噬细胞内吞，而抗噬菌体突变体被巨噬细胞内吞的几率要高得多。作者认为，细菌囊可能有助于避免被巨噬细胞吞噬，而抗噬菌体突变体则较难避免被巨噬细胞吞噬。因此，噬菌体的抗性可能会与逃避宿主防御交换，这是一个值得进一步研究的课题。
It is promising that regardless of many species-characteristic differences in surface morphology, host-invasion mechanisms, or virulence capabilities, phages that steer bacterial evolution toward lower virulence can be found. These phages should help in managing the disease progression by ESKAPE pathogens [emerging MDR pathogens involved in hospital-acquired infections: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species (113)] and other bacteria where antibiotic resistance is on the rise.
令人欣喜的是，无论表面形态、宿主入侵机制或毒力能力存在多少物种特征性差异，都能找到引导细菌向低毒力方向进化的噬菌体。这些噬菌体将有助于控制 ESKAPE 病原体（医院感染中新出现的 MDR 病原体：粪肠球菌、金黄色葡萄球菌、肺炎克雷伯菌、鲍曼不动杆菌、铜绿假单胞菌和肠杆菌 ( 113)] 以及其他抗生素耐药性正在上升的细菌。