Role of Net Charge on Catalytic Domain and Influence of Cell Wall Binding Domain on Bactericidal Activity, Specificity, and Host Range of Phage Lysins
Role of Net Charge on Catalytic Domain and Influence of Cell Wall Binding Domain on Bactericidal Activity, Specificity, and Host Range of Phage Lysins
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DOI：
10.1074/jbc.m111.244160
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发表时间：
2011-09-30
影响因子：
4.8
通讯作者：
Liddington, Robert
中科院分区：
文献类型：
--
作者：
Low, Lieh Yoon;Yang, Chen;Liddington, Robert
当外部应用于革兰氏阳性细菌时，裂解性大肠杆菌的重组溶素可以是有效的杀菌剂，通常保持高特异性。它们作为新型抗菌剂的发展提供了许多优于传统抗生素的潜在优势。蛋白质工程可以通过产生适合不同目标群体和环境的新型溶素来进一步开发这种潜力。然而，进入肽聚糖层受到各种次生细胞壁聚合物、化学修饰和（在某些情况下）S层和胶囊的控制。经典的溶素需要细胞壁结合结构域（CBD），其通过结合至次级细胞壁聚合物组分将催化结构域靶向肽聚糖层。革兰氏阳性细菌的细胞壁通常具有负电荷，并且我们注意到在没有CBD的情况下催化结构域上的（正）电荷与溶菌活性之间的相关性（非经典行为）。我们研究了这种相关性的物理基础，通过比较的结构和活性对溶素的裂解活性的每对之一是CBD的独立。我们发现，通过设计催化结构域的净电荷的符号反转，我们可以消除或产生CBD依赖性。我们还提供了证据表明，炭疽芽孢杆菌的S-层作为一个分子筛，主要是大小依赖性的，有利于催化结构域的全长溶素。我们的工作提出了一些简单的方法微调溶素活性，无论是提高或降低特异性/宿主范围和/或杀菌潜力，根据需要。
The recombinant lysins of lytic phages, when applied externally to Gram-positive bacteria, can be efficient bactericidal agents, typically retaining high specificity. Their development as novel antibacterial agents offers many potential advantages over conventional antibiotics. Protein engineering could exploit this potential further by generating novel lysins fit for distinct target populations and environments. However, access to the peptidoglycan layer is controlled by a variety of secondary cell wall polymers, chemical modifications, and (in some cases) S-layers and capsules. Classical lysins require a cell wall-binding domain (CBD) that targets the catalytic domain to the peptidoglycan layer via binding to a secondary cell wall polymer component. The cell walls of Gram-positive bacteria generally have a negative charge, and we noticed a correlation between (positive) charge on the catalytic domain and bacteriolytic activity in the absence of the CBD (nonclassical behavior). We investigated a physical basis for this correlation by comparing the structures and activities of pairs of lysins where the lytic activity of one of each pair was CBD-independent. We found that by engineering a reversal of sign of the net charge of the catalytic domain, we could either eliminate or create CBD dependence. We also provide evidence that the S-layer of Bacillus anthracis acts as a molecular sieve that is chiefly size-dependent, favoring catalytic domains over full-length lysins. Our work suggests a number of facile approaches for fine-tuning lysin activity, either to enhance or reduce specificity/host range and/or bactericidal potential, as required.