Engineered endolysin-based "Artilysins" to combat multidrug-resistant gram-negative pathogens.
Engineered endolysin-based "Artilysins" to combat multidrug-resistant gram-negative pathogens.
复制标题
基于Endolysin的工程“ Artilysins”来对抗耐多药的革兰氏阴性病原体。
DOI：
10.1128/mbio.01379-14
复制
发表时间：
2014-07-01
期刊：
影响因子：
6.4
通讯作者：
Lavigne R
中科院分区：
文献类型：
--
作者：
Briers Y;Walmagh M;Van Puyenbroeck V;Cornelissen A;Cenens W;Aertsen A;Oliveira H;Azeredo J;Verween G;Pirnay JP;Miller S;Volckaert G;Lavigne R
在过去几年中，新出现的多重耐药细菌对公共卫生构成了全球性威胁，因此有必要开发新的方法来对抗细菌感染。细菌病毒编码的内溶素是一种很有前途的研究方向。这些基于酶的抗菌剂在接触时通过特异性细胞壁水解有效地杀死革兰氏阳性菌。然而，在其开发作为抗革兰氏阴性病原体的抗菌剂的主要障碍是其细胞壁周围的不可渗透的脂多糖层。因此，我们开发并优化了一种方法来工程化这些酶作为外膜穿透内溶素（Artilysins），使它们对革兰氏阴性病原体（包括铜绿假单胞菌和鲍曼不动杆菌）具有高度杀菌性。组合聚阳离子九肽和模块化内溶素的Artilysins能够在体外杀死这些（多药耐药）菌株，在30分钟内减少4至5个对数。我们表明，Artilysins的活性可以进一步增强的肽和内溶素之间的长度增加的接头的存在下，或由聚阳离子和疏水/两亲性肽的组合。延时显微镜证实了聚阳离子Artilysins的作用模式，表明它们通过外膜降解肽聚糖，随后发生细胞裂解。Artilysins在体外（人角质形成细胞）和体内（秀丽隐杆线虫）有效。细菌对大多数常用抗生素的耐药性是21世纪世纪的主要挑战。一线抗生素无法治疗的感染导致发病率和死亡率增加，而卫生保健系统每年花费数百万美元试图控制耐药性细菌并防止耐药性交叉传播。内溶素--来自细菌病毒的酶--代表了一类基于细胞壁水解的全新的、有前途的抗菌剂。具体而言，它们对缺乏保护性外膜且产生抗性的可能性低的革兰氏阳性菌种具有活性。我们通过蛋白质工程修饰内溶素，以产生能够通过外膜并对铜绿假单胞菌和鲍曼不动杆菌（两种最危险的耐药革兰氏阴性病原体）具有活性的Artilysins。
The global threat to public health posed by emerging multidrug-resistant bacteria in the past few years necessitates the development of novel approaches to combat bacterial infections. Endolysins encoded by bacterial viruses (or phages) represent one promising avenue of investigation. These enzyme-based antibacterials efficiently kill Gram-positive bacteria upon contact by specific cell wall hydrolysis. However, a major hurdle in their exploitation as antibacterials against Gram-negative pathogens is the impermeable lipopolysaccharide layer surrounding their cell wall. Therefore, we developed and optimized an approach to engineer these enzymes as outer membrane-penetrating endolysins (Artilysins), rendering them highly bactericidal against Gram-negative pathogens, including Pseudomonas aeruginosa and Acinetobacter baumannii. Artilysins combining a polycationic nonapeptide and a modular endolysin are able to kill these (multidrug-resistant) strains in vitro with a 4 to 5 log reduction within 30 min. We show that the activity of Artilysins can be further enhanced by the presence of a linker of increasing length between the peptide and endolysin or by a combination of both polycationic and hydrophobic/amphipathic peptides. Time-lapse microscopy confirmed the mode of action of polycationic Artilysins, showing that they pass the outer membrane to degrade the peptidoglycan with subsequent cell lysis. Artilysins are effective in vitro (human keratinocytes) and in vivo (Caenorhabditis elegans). Bacterial resistance to most commonly used antibiotics is a major challenge of the 21st century. Infections that cannot be treated by first-line antibiotics lead to increasing morbidity and mortality, while millions of dollars are spent each year by health care systems in trying to control antibiotic-resistant bacteria and to prevent cross-transmission of resistance. Endolysins—enzymes derived from bacterial viruses—represent a completely novel, promising class of antibacterials based on cell wall hydrolysis. Specifically, they are active against Gram-positive species, which lack a protective outer membrane and which have a low probability of resistance development. We modified endolysins by protein engineering to create Artilysins that are able to pass the outer membrane and become active against Pseudomonas aeruginosa and Acinetobacter baumannii, two of the most hazardous drug-resistant Gram-negative pathogens.