中文摘要：金黄色葡萄球菌引起人类和动物的各种感染，皮肤是这种病原体的主要宿主。耐甲氧西林金黄色葡萄球菌（MRSA）的广泛出现妨碍了该病原体的消除和治疗。已经证明噬菌体裂解蛋白是对抗金黄色葡萄球菌的有效抗菌剂。本研究构建了12个基于内溶素的工程蛋白，并通过逐步漏斗法评估参数，包括浊度降低、最小抑制浓度（MIC）、杀菌曲线、抗生物膜测定，以及在广泛的储存条件（pH、温度和离子强度）下的稳定性，进行最优选择。结果表明工程化噬菌体细胞裂解酶LysRODI Delta Ami和ClyRODI-H5的特异性裂解活性最高（比其他蛋白高5-50倍），保质期长达6个月，并且在高达50℃的温度和pH3-9范围内保持稳定；LysRODI Delta Ami对所有受试葡萄球菌的MIC值均较低；这两种蛋白都能在5分钟内杀死6个对数单位的金黄色葡萄球菌Sa9，并能去除预先形成的生物膜（分别为76%和65%）。此外，LysRODI Delta Ami在低浓度（0.15-0.6μM）下可抑制生物膜的形成。由于LysRODI Delta Ami抗生物膜的能力增强，因此可用它有效去除完整和受损的角质形成细胞单层中的金黄色葡萄球菌污染。值得注意的是，即使在高浓度（22.1μM）时，该蛋白对人类角质形成细胞也没有任何毒性。最后，使用猪皮离体模型评估了LysRODI Delta Ami（16.5μg/cm2）处理人工污染猪皮的效果，发现其在早期减少了金黄色葡萄球菌后，第二剂则彻底根除了该菌。可见，可以用LysRODI Delta Ami作为预防和治疗葡萄球菌皮肤感染候选抗菌剂。
外文摘要：Staphylococcus aureus causes various infections in humans and animals, the skin being the principal reservoir of this pathogen. The widespread occurrence of methicillin-resistant S. aureus (MRSA) limits the elimination and treatment of this pathogen. Phage lytic proteins have been proven as efficient antimicrobials against S. aureus. Here, a set of 12 engineered proteins based on endolysins were conceptualized to select the most optimal following a stepwise funnel approach assessing parameters including turbidity reduction, minimum inhibitory concentration (MIC), time-kill curves, and antibiofilm assays, as well as testing their stability in a broad range of storage conditions (pH, temperature, and ionic strength). The engineered phage lysins LysRODI Delta Ami and ClyRODI-H5 showed the highest specific lytic activity (5 to 50 times higher than the rest), exhibited a shelf-life up to 6 months and remained stable at temperatures up to 50 degrees C and in a pH range from 3 to 9. LysRODI Delta Ami showed the lower MIC values against all staphylococcal strains tested. Both proteins were able to kill 6 log units of the strain S. aureus Sa9 within 5 min and could remove preformed biofilms (76 and 65%, respectively). Moreover, LysRODI Delta Ami could prevent biofilm formation at low protein concentrations (0.15-0.6 mu M). Due to its enhanced antibiofilm properties, LysRODI Delta Ami was selected to effectively remove S. aureus contamination in both intact and disrupted keratinocyte monolayers. Notably, this protein did not demonstrate any toxicity toward human keratinocytes, even at high concentrations (22.1 mu M). Finally, a pig skin ex vivo model was used to evaluate treatment of artificially contaminated pig skin using LysRODI Delta Ami (16.5 mu g/cm(2)). Following an early reduction of S. aureus, a second dose of protein completely eradicated S. aureus. Overall, our results suggest that LysRODI Delta Ami is a suitable candidate as antimicrobial agent to prevent and treat staphylococcal skin infections.
外文关键词：endolysin；protein engineering；antimicrobial；Staphylococcus aureus；skin decontamination
作者：Gutierrez, D；Rodriguez-Rubio, L；Ruas-Madiedo, P；Fernandez, L；Campelo, AB；Briers, Y；Nielsen, MW；Pedersen, K；Lavigne, R；Garcia, P；Rodriguez, A
作者单位：Inst Prod Lacteos Asturias IPLA CSIC；Inst Invest Santana Principado Asturias；Univ Ghent；Katholieke Univ Leuven；Tech Univ Denmark
期刊名称：FRONTIERS IN MICROBIOLOGY
期刊影响因子：4.236
出版年份：2021
出版刊次：12
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