Postgraduate Medical Institute, The University of Hull, Room 512, Wolfson building, Cottingham Road, Hull, UK HU6 7RX.
Photochem Photobiol Sci. 2011 Apr;10(4):515-22. doi: 10.1039/c0pp00267d. Epub 2010 Dec 22.
The increasing prevalence of multi-drug resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), necessitates development of alternative modes of bacterial targeting which are not hindered by antibiotic resistance and minimise collateral damage. To achieve this, the FliTrx™ bacterially-displayed peptide library was panned against MRSA and randomly selected clones (n = 20) were DNA sequenced. One selected peptide was synthesised as both cyclic and linear constructs. Binding of the cyclic construct was observed by flow cytometry against isolates of MRSA whilst the linear construct showed low affinity. Low reactivity was observed with other Staphylococcal sp., gram-negative bacteria and human keratinocytes. The selected peptide was also cloned in-frame, within the thioredoxin gene into the pPROTet.E 6xHN vector for protein expression. A porphyrin photosensitiser (5-(4-isothiocyanatophenyl)-10,15,20-tris(4-N-methylpyridiniumyl)porphyrin trichloride) was conjugated to the recombinant protein and the in vitro cytotoxic effect of the resulting bioconjugate was determined against MRSA and other non-specific bacterial and mammalian cell lines. Photoantimicrobial chemotherapy (PACT) using the bioconjugate showed a 66% reduction in MRSA growth in comparison with non-irradiated cells. This work demonstrates the potential to isolate peptides with binding specificity against MRSA that can be used for targeted PACT, providing an effective alternative to antibody targeting.
越来越多的多药耐药菌，如耐甲氧西林金黄色葡萄球菌（MRSA）的出现，促使人们开发出针对细菌的替代方法，这些方法不应受到抗生素耐药性的影响，并且将最小化附带损害。为了实现这一目标，我们针对 MRSA 筛选了 FliTrx™细菌展示肽文库，并对随机选择的 20 个克隆进行了 DNA 测序。选择的一个肽被合成了环状和线性两种结构。通过流式细胞术观察到环状结构与 MRSA 分离株的结合，而线性结构显示出低亲和力。该线性结构与其他葡萄球菌属、革兰氏阴性菌和人角质形成细胞的反应性较低。该选择的肽还被克隆到了 thioredoxin 基因中，通过 pPROTet.E 6xHN 载体进行蛋白表达。将卟啉类光敏剂（5-(4-异硫氰酸根合苯基）-10,15,20-三(4-N-甲基吡啶基)卟啉三氯化物）偶联到重组蛋白上，并测定了该生物缀合物对 MRSA 和其他非特异性细菌和哺乳动物细胞系的体外细胞毒性作用。与非照射细胞相比，使用该生物缀合物进行光抗菌化疗（PACT）可使 MRSA 生长减少 66%。这项工作证明了从 MRSA 中分离出具有结合特异性的肽的潜力，这些肽可用于靶向 PACT，为抗体靶向提供了有效的替代方法。
