Abstract：
The emergence of multidrug-resistant pathogens has outpaced the development of new antibiotics, leading to renewed interest in endolysins. Endolysins have been investigated as novel biocontrol agents for Gram-positive bacteria. However, their efficacy against Gram-negative species is limited by the barrier presented by their outer membrane, which prevents endolysin access to the peptidoglycan substrate. Here, we used the translocation domain of botulinum neurotoxin to deliver endolysin across the outer membrane of Gram-negative bacteria. The translocation domain selectively interacts with and penetrates membranes composed of anionic lipids, which have been used in nature to deliver various proteins into animal cells. In addition to the botulinum neurotoxin translocation domain, we have fused bacteriophage-derived receptor binding protein to endolysins. This allows the attached protein to efficiently bind to a broad spectrum of Gram-negative bacteria. By attaching these target-binding and translocation machineries to endolysins, we aimed to develop an engineered endolysin with broad-spectrum targeting and enhanced antibacterial activity against Gram-negative species. To validate our strategy, we designed engineered endolysins using two well-known endolysins, T5 and LysPA26, and tested them against 23 strains from six species of Gram-negative bacteria, confirming that our machinery can act broadly. In particular, we observed a 2.32 log reduction in 30 min with only 0.5 µM against an Acinetobacter baumannii isolate. We also used the SpyTag/SpyCatcher system to easily attach target-binding proteins, thereby improving its target-binding ability. Overall, our newly developed endolysin engineering strategy may be a promising approach to control multidrug-resistant Gram-negative bacterial strains.
摘要：
多重耐药病原体的出现已经超过了新抗生素的开发,导致对内溶素重新产生兴趣。已经研究了内溶素作为革兰氏阳性细菌的新型生物防治剂。然而,它们对革兰氏阴性物种的功效受到其外膜所提供的屏障的限制，这防止内溶素进入肽聚糖底物。这里,我们使用肉毒杆菌神经毒素(BoNT)的易位结构域通过革兰氏阴性菌的外膜递送内溶素.易位结构域选择性地与由阴离子脂质组成的膜相互作用并穿透，它们在自然界中被用来将各种蛋白质传递到动物细胞中。除了BoNT转位域，我们将噬菌体衍生的受体结合蛋白与细胞内溶素融合.这允许附着的蛋白质有效地结合广谱的革兰氏阴性细菌。通过将这些靶标结合和易位机制连接到内溶素上，我们的目标是开发一种具有广谱靶向和增强抗革兰氏阴性物种抗菌活性的工程内溶素。为了验证我们的策略，我们使用两种众所周知的内溶素设计了工程内溶素，T5和LysPA26，并对来自6种革兰氏阴性菌的23株菌株进行了测试，确认我们的机器可以广泛行动。特别是,我们在30分钟内观察到2.32对数减少，仅0.5µM对鲍曼不动杆菌分离株。我们还使用SpyTag/SpyCatcher系统轻松地连接靶结合蛋白，从而提高其靶标结合能力。总的来说,我们新开发的细胞内溶素工程策略可能是控制多重耐药革兰氏阴性菌菌株的一种有前景的方法.
