2.2. How Phages Have Adapted to Infect Bacterial Biofilms
2.2.噬菌体如何适应感染细菌生物膜
The long coevolution between phages and bacteria in nature has led them to evolve mechanisms that facilitate their access to the bacterial cell surface, which might be particularly useful in biofilms, where the bacterial cells are encased within the EPS matrix. In fact, it is known that a large number of phage genomes encode enzymes capable of degrading polymeric substances including capsular polysaccharides, exopolysaccharides, or lipopolysaccharides (9, 29). These phage-derived enzymes, called depolymerases, are mostly found as part of the tail fiber or tail spike proteins of phages and are responsible for the depolymerization of bacterial capsules, facilitating phage adsorption (29). Phage depolymerases may also play an important role in phage-host interaction within biofilms by promoting matrix disruption and a consequent easier diffusion of phages through the biofilm structure to the target bacterial cells (6).
噬菌体与细菌在自然界中长期共同进化，使它们进化出了便于进入细菌细胞表面的机制，这在生物膜中可能特别有用，因为在生物膜中，细菌细胞被包裹在 EPS 基质中。事实上，众所周知，大量噬菌体基因组编码的酶能够降解高分子物质，包括胶囊多糖、外多糖或脂多糖 ( 9, 29)。这些噬菌体衍生的酶被称为解聚酶，大多作为噬菌体尾纤或尾穗蛋白的一部分存在，负责细菌胶囊的解聚，从而促进噬菌体的吸附（29）。噬菌体解聚酶还可能在生物膜内的噬菌体-宿主相互作用中发挥重要作用，它能促进基质破坏，从而使噬菌体更容易通过生物膜结构扩散到目标细菌细胞（6）。
In 1998, Hughes et al. (30) reported an Enterobacter agglomerans phage displaying depolymerase activity that was capable of disrupting biofilms, a characteristic that was attributed to the combined effect of EPS degradation caused by the enzyme and the subsequent cell lysis caused by the phage. Similarly, studies by Cornelissen et al. (31) showed that although a Pseudomonas putida phage encoding a polysaccharide depolymerase revealed biofilm-degrading properties, phage amplification had a major role in biofilm degradation, as the experiments using phage depolymerase alone did not cause biofilm disruption. However, some studies have highlighted the role of depolymerases in biofilm degradation and dispersion, even when these enzymes are applied alone. For instance, Gutiérrez et al. (32) reported that an EPS depolymerase derived from a S. epidermidis phage was able to prevent and disperse staphylococcal biofilms when applied alone, although the response was dose dependent. In a similar way, Wu et al. (33) expressed a depolymerase encoded by a Klebsiella pneumoniae phage and applied it in mature biofilms, which revealed the biofilm-dispersion ability of the enzyme. The antibiofilm properties of depolymerases may also be enhanced by other phage-encoded enzymes, such as endolysins [lytic enzymes responsible for peptidoglycan degradation and host cell lysis (34)], as described by Olsen et al. (35) in a study targeting Staphylococcus aureus biofilms.
1998 年，Hughes 等人 ( 30) 报道了一种具有解聚酶活性的凝集肠杆菌噬菌体，它能够破坏生物膜，这一特性是由酶引起的 EPS 降解和噬菌体随后引起的细胞裂解共同作用造成的。同样，Cornelissen 等人的研究（31）表明，虽然一种编码多糖解聚酶的假单胞菌噬菌体具有生物膜降解特性，但噬菌体的扩增在生物膜降解中起着主要作用，因为单独使用噬菌体解聚酶的实验并不能导致生物膜破坏。不过，一些研究强调了解聚酶在生物膜降解和分散中的作用，即使单独使用这些酶也是如此。例如，Gutiérrez 等人（32）报告说，一种从表皮葡萄球菌噬菌体中提取的 EPS 解聚酶在单独使用时能够阻止和驱散葡萄球菌生物膜，尽管这种反应与剂量有关。同样，Wu 等人 ( 33) 表达了一种由肺炎克雷伯菌噬菌体编码的解聚酶，并将其应用于成熟的生物膜，结果显示了该酶的生物膜驱散能力。其他噬菌体编码的酶，如内溶酶（负责肽聚糖降解和宿主细胞裂解的溶解酶( 34)），也可能增强解聚酶的抗生物膜特性，Olsen 等人( 35)在一项针对金黄色葡萄球菌生物膜的研究中描述了这一点。
It is also important to highlight that phages can find other ways to penetrate the biofilm structure and reach the bacterial cells. In a study by Vilas Boas et al. (36), a fluorescence molecular probe designed to target the messenger RNA of a phage major capsid was used to track phage-infected cells within a biofilm population. The authors demonstrated that phage diffusion through the biofilm may be mediated by the channels that can be found in some biofilms, as the phage-infected cells were primarily located close to the edges of these structures (36).
同样重要的是，噬菌体还能找到其他方法穿透生物膜结构，到达细菌细胞。在 Vilas Boas 等人的研究中（36），使用了一种针对噬菌体主要外壳信使 RNA 的荧光分子探针来追踪生物膜群体中被噬菌体感染的细胞。作者证明，噬菌体在生物膜中的扩散可能是由某些生物膜中的通道介导的，因为噬菌体感染的细胞主要位于这些结构的边缘附近（36）。
2.3. How Bacteria Evolved to Escape from Phage Predation
2.3.细菌如何进化以躲避噬菌体的捕食
To date, several studies have reported the fast proliferation of bacteriophage-insensitive mutants (BIMs) after biofilm treatment with phages (37–41). Although the mechanisms underlying phage resistance in these studies are not always clear, the genotypic analysis of BIMs frequently reveals mutations in genes encoding phage receptors (39, 41). Nonetheless, other mechanisms can be used by bacteria to counterattack phage evasion, namely in biofilm mode, which include signaling systems or CRISPR-Cas systems.
迄今为止，已有多项研究报道了噬菌体处理生物膜后，噬菌体不敏感突变体（BIMs）的快速增殖（37-41）。虽然这些研究中噬菌体抗性的机制并不总是很清楚，但对 BIMs 的基因型分析经常发现编码噬菌体受体的基因发生了突变 ( 39, 41)。然而，细菌还可以利用其他机制来对抗噬菌体的逃避，即生物膜模式，其中包括信号系统或 CRISPR-Cas 系统。
It is known that bacterial communication relies on signaling molecules, known as autoinducers, which regulate gene expression in response to variations in population density by a process called quorum sensing (QS) (42). Because QS plays an important role in controlling the gene expression of virulence factors and biofilm development (43), this communication system is also relevant to understand the phage-host dynamics in biofilm populations. In fact, QS can be extremely useful when bacterial cells are under phage predation; consequently, it has been linked to increased phage resistance in several ways (44, 45). One example is that QS signals can regulate the genes involved in the production of biofilm matrix (46, 47), which was described above as one of the major factors impairing phage infection. Additionally, QS can modulate the expression of phage receptors in bacterial cell surface as described by Høyland-Kroghsbo et al. (48). Using a model system of E. coli and phage λ, the authors found that the bacterial host reduced the numbers of cell surface receptors in response to QS signals, which resulted in a reduction in phage adsorption rate (48). Similar observations concerning the QS regulation of antiphage mechanisms were also reported by Tan et al. (49) in Vibrio anguillarum. In addition, QS can also influence phage infection by affecting the physiological state of the host cell population, as observed for Pseudomonas aeruginosa (50). There is also increasing evidence that QS can control the regulation of CRISPR-Cas systems of several bacterial species, such as P. aeruginosa (51) or Serratia spp. (52). CRISPR-Cas systems are widely distributed across bacterial genomes and provide them with adaptive immunity against invasive genetic elements, including phages (53). Many other antiphage systems have been described over the past few years (reviewed in 54, 55). These systems result from the long-term evolutionary adaptation of bacteria to survive the constant offense of phages in natural environments. Overall, QS contributes to maintaining population stability when phage densities are relatively high. Other density-dependent mechanisms, such as superinfection immunity, make important contributions for the equilibrium of biofilm populations. This has been explained by the Piggyback-the-Winner (PtW) theory, which proposes that the phenotypic advantages of lysogeny are favored at high host abundances (56).
众所周知，细菌的通讯依赖于信号分子（即自体诱导剂），它们通过一种称为法定量感应（QS）的过程来调节基因表达，以应对种群密度的变化（42）。由于 QS 在控制毒力因子基因表达和生物膜发展方面发挥着重要作用 ( 43)，因此这一通信系统也与了解生物膜种群中的噬菌体-宿主动态有关。事实上，当细菌细胞受到噬菌体捕食时，QS 非常有用；因此，它与噬菌体抗性的增强有多种联系（44、45）。其中一个例子是，QS 信号可以调节参与生物膜基质生成的基因（46、47），而生物膜基质是上文所述的影响噬菌体感染的主要因素之一。此外，如 Høyland-Kroghsbo 等人所述（48），QS 还能调节细菌细胞表面噬菌体受体的表达。作者利用大肠杆菌和噬菌体 λ 的模型系统发现，细菌宿主在 QS 信号的作用下减少了细胞表面受体的数量，从而降低了噬菌体的吸附率 ( 48)。Tan 等人（49）在鳗弧菌（Vibrio anguillarum）中也发现了类似的 QS 调节抗噬菌体机制的现象。此外，QS 还可以通过影响宿主细胞群的生理状态来影响噬菌体的感染，铜绿假单胞菌（50）就是如此。还有越来越多的证据表明，QS 可以控制一些细菌物种的 CRISPR-Cas 系统，如铜绿假单胞菌（51）或沙雷氏菌属（52）。CRISPR-Cas 系统广泛分布于细菌基因组中，为细菌提供了对抗入侵遗传因子（包括噬菌体）的适应性免疫力（53）。过去几年中还描述了许多其他抗噬菌体系统（综述见 54、55）。这些系统是细菌长期进化适应自然环境中噬菌体不断进攻的结果。总体而言，当噬菌体密度相对较高时，QS 有助于维持种群稳定。其他依赖密度的机制，如超级感染免疫，也对生物膜种群的平衡做出了重要贡献。猪背-赢家（PtW）理论对此做出了解释，该理论认为溶菌作用的表型优势在宿主丰度较高时更受青睐 ( 56)。
Because of all these defense mechanisms, and similar to what happens under lab conditions, the presence of phage-resistant bacteria is also expected in biofilms found in natural contexts. However, it is not clear how these resistant populations will interact with phages in biofilms. To better understand the dynamics of a phage-resistant population within biofilms, Simmons et al. (57) set up an experimental model of mixed E. coli biofilms with resistant and susceptible hosts subjected to T7 phage attack, which was analyzed by confocal microscopy. According to the authors, the biofilm structure promotes the coexistence of both phage-resistant and phage-susceptible bacteria. When phage-resistant cells are initially rare in the biofilm, the susceptible cells are cleared by phage and the number of phage-resistant cells will increase and form clusters in the empty space; however, when phage-resistant cells are initially common (at least 60% of the population), the relative fraction of resistant and susceptible bacteria will not substantially change after phage treatment, as the susceptible cells are protected from phage exposure through immobilization of phages in clusters of resistant cells, resulting in a more structured biofilm composed of both populations (57).
由于存在所有这些防御机制，而且与实验室条件下发生的情况类似，在自然环境下发现的生物膜中也会出现对噬菌体有抗性的细菌。然而，目前还不清楚这些耐药菌群将如何与生物膜中的噬菌体相互作用。为了更好地了解生物膜中噬菌体抗性种群的动态，Simmons 等人 ( 57) 建立了一个大肠杆菌混合生物膜实验模型，模型中的抗性宿主和易感宿主都受到了 T7 噬菌体的攻击，并通过共聚焦显微镜进行了分析。作者认为，生物膜结构促进了抗噬菌体和易感噬菌体的共存。当生物膜中抗噬菌体细胞最初很少时，易感细胞会被噬菌体清除，抗噬菌体细胞的数量会增加，并在空隙中形成细胞簇；然而，当抗噬菌体细胞最初很常见（至少占种群的 60%）时，抗噬菌体和易感细菌的相对比例在噬菌体处理后不会发生实质性变化，因为易感细胞通过固定在抗噬菌体细胞簇中的噬菌体而免受噬菌体暴露，从而形成由两种种群组成的结构更合理的生物膜 ( 57)。