Modified Phages and Their Therapeutic Applications
改良噬菌体及其治疗应用
Bioengineered phages highly minimize the drawbacks of conventional phage therapies owing to their ability to reach and kill the targeted pathogens or reverse the drug resistance of bacteria. Phage-derived biochemical endolysin (phage lysin) is effective against Gram-positive bacteria and it can be used for bacterial treatment instead of viable phages.5,14
生物工程噬菌体能杀灭目标病原体或逆转细菌的耐药性，因此能最大限度地减少传统噬菌体疗法的缺点。噬菌体衍生的生化内溶酶（噬菌体溶菌酶）对革兰氏阳性细菌有效，可代替有活力的噬菌体用于细菌治疗。 5 14
Modified phages are phages whose specificity is deliberately altered into non-native forms. Their host recognition specificity is conferred by receptor binding domains found on phages.66 Modified phages are used to target non-native hosts; they are designed to serve as a vehicle into which antimicrobials are incorporated or attached to the surface, to suppress the host SOS DNA repair system.67,68 Modifications enable phages to overcome the narrow host range of phages, and can reduce the potential of bacteria to develop resistance, avoid challenges in phage manufacturing, exclude systemic side effects (especially endotoxin release), and prevent the phage being attacked by the immune system. To avoid the release of endotoxins by Gram-negative bacteria, mainly due to lytic phages or antibiotic treatment, phages can be made lysine deficient. For example, MRSA-infected mice were successfully treated by lysine-deficient phages because the bacterium was killed without lysis. Another means of using modified phages is a targeted gene delivery system to the site of infection using engineered filamentous phages.12 Modified phages can bypass the host immune system, persist within the body, and deliver lethal genes to the bacterial host. Many experiments in animal models revealed that engineered phages are efficient in treating infections. Filamentous phages that do not lyse the host are used as a vehicle to provide lethal genes or substances such as holins, lethal transcription regulators, and addiction toxins to induce apoptosis specifically at the site of infection.69
改造噬菌体是特异性被故意改变为非本地形式的噬菌体。它们识别宿主的特异性是由噬菌体上的受体结合域赋予的。 66 经改造的噬菌体被用来针对非本地宿主；它们被设计成一种载体，将抗菌素融入其中或附着在其表面，以抑制宿主的 SOS DNA 修复系统。 67 68 改造能使噬菌体克服宿主范围狭窄的问题，并能降低细菌产生抗药性的可能性，避免噬菌体制造过程中的挑战，排除全身副作用（尤其是内毒素释放），并防止噬菌体受到免疫系统的攻击。为了避免革兰氏阴性细菌释放内毒素（主要是由于溶菌噬菌体或抗生素治疗引起），可以使噬菌体缺乏赖氨酸。例如，缺失赖氨酸的噬菌体可以成功治疗感染 MRSA 的小鼠，因为细菌在没有裂解的情况下就被杀死了。另一种使用改良噬菌体的方法是利用工程丝状噬菌体将基因定向传递到感染部位。 12 改造噬菌体可以绕过宿主免疫系统，在体内持续存在，并向细菌宿主传递致命基因。许多动物模型实验表明，工程噬菌体能有效治疗感染。不溶解宿主的丝状噬菌体被用作一种载体，提供致命基因或物质，如 holins、致命转录调节因子和成瘾毒素，以诱导感染部位的特定细胞凋亡。 69
Phage-Encoded Products 噬菌体编码产品
Phages encode proteins that recognize and adhere to sites on the bacterial surface, such as peptidoglycans, pili, flagella, or efflux pumps, and to specific sugar moieties in lipopolysaccharides.70 They encode two types of lysosomes, porin endolysins and phage tail-associated murein lytic enzymes. These enzymes degrade the cell wall of the host. Endolysins, with the help of holin, lyse the bacterial cell wall from the inside and allow phage progenies to be released.71 The ability of endolysins to bind firmly to substrates on the host cell wall minimizes the turnover of endolysins and the requirement of many endolysin molecules to degrade bonds on the cell of the host.72 Phage tail-associated murein lytic enzymes hydrolyze the cell wall after adsorption of the phage to the host cell wall from the outside;73 their activity is limited to Gram-positive bacteria because Gram-negative bacteria have an outer membrane that blocks direct enzyme contact with the peptidoglycan of the host cell wall. Structurally engineered phage lysosomal molecules that have specific binding and fusing ability with other modified lysosomes have shown encouraging results against Gram-negative bacteria.74
噬菌体编码的蛋白质可识别和粘附细菌表面的位点，如肽聚糖、纤毛、鞭毛或外排泵，以及脂多糖中的特定糖分子。它们编码两种类型的溶酶体：孔蛋白内溶酶体和噬菌体尾部相关的黏蛋白溶解酶。这些酶能降解宿主的细胞壁。内溶酶素在holin的帮助下从内部裂解细菌细胞壁，使噬菌体后代得以释放。 71 内溶酶素能与宿主细胞壁上的底物牢固结合，这就最大限度地减少了内溶酶素的周转次数，也减少了降解宿主细胞上的键所需的许多内溶酶素分子。 72 噬菌体尾部相关的木脂溶解酶在噬菌体从外部吸附到宿主细胞壁后会水解细胞壁； 73 它们的活性仅限于革兰氏阳性细菌，因为革兰氏阴性细菌的外膜会阻止酶与宿主细胞壁的肽聚糖直接接触。经过结构改造的噬菌体溶酶体分子具有与其他改造溶酶体特异性结合和融合的能力，对革兰氏阴性细菌的作用效果令人鼓舞。 74
Phage-encoded products are used to kill pathogens directly. Their merits over viable phage therapy include their enhanced ability to penetrate and diffuse to the site of action by bypassing sequestration by the spleen, lymph nodes, and other organs. Lysine is an example of a phage product assumed as antibacterial weapons. It is safe and efficient against bacteria and resistance to lysine is less frequent compared to antibiotics. They are successful in animal models against Gram-positive bacteria including S. pneumonia, S. pyogenes, B. anthracis, E. faecium, and S. aureus, but not against Gram-negative bacteria, to date.75
噬菌体编码产品用于直接杀死病原体。与可行的噬菌体疗法相比，噬菌体编码产物的优点包括：绕过脾脏、淋巴结和其他器官的阻隔，具有更强的穿透能力和扩散到作用部位的能力。赖氨酸就是噬菌体产品作为抗菌武器的一个例子。它对细菌安全有效，与抗生素相比，赖氨酸的抗药性较少。在动物模型中，噬菌体对革兰氏阳性菌（包括肺炎双球菌、化脓性链球菌、炭疽杆菌、粪大肠杆菌和金黄色葡萄球菌）的抗菌效果很好，但对革兰氏阴性菌的抗菌效果不佳。 75
It is unlikely for a bacterium to evolve resistance to lysins, since lysins target sites on the peptidoglycan, which is vital for bacterial cell viability.76,77 In addition, mass preparation and administration of engineered recombinant lytic proteins is much easier than mass preparation and administration of actual phages. Modified products of phages have more potential than natural phages because viable phages have limitations due to their short shelf life, sequestration by the reticuloendothelial system, and potential to induce neutralizing antibodies.78 Applying phage lysin therapy in combination with antibiotics is more effective than the single use of either lysins or antibiotics.79,80
细菌不太可能进化出对溶菌素的抗药性，因为溶菌素针对的是肽聚糖上的位点，而肽聚糖对细菌细胞的活力至关重要。 76 77 此外，大规模制备和使用工程重组溶菌蛋白要比大规模制备和使用真正的噬菌体容易得多。噬菌体的改良产品比天然噬菌体更有潜力，因为存活的噬菌体由于保存期短、被网状内皮系统封存以及可能诱发中和抗体而受到限制。 78 将噬菌体溶菌素疗法与抗生素结合使用比单独使用溶菌素或抗生素更有效。 79 80