Chen Huixian, Mao Ruoyu, Teng Da, Wang Xiumin, Hao Ya, Feng Xingjun, Wang Jianhua
Institute of Animal Nutrition, Northeast Agricultural University, Harbin, 150030, China.
Key Laboratory of Feed Biotechnology, Ministry of Agriculture, Beijing, 100081, China.
AMB Express. 2017 Dec;7(1):46. doi: 10.1186/s13568-017-0345-x. Epub 2017 Feb 22.
NZ2114 is a promising candidate for therapeutic application owing to its potent activity to Staphylococcus aureus. Our objective was to identify NZ2114 derivatives with improved activity through substitution of His16 and His18 with residues Arginine and Lysine. Eight mutants were designed and expressed in Pichia pastoris X-33 via pPICZαA. Five of them exhibited strong antimicrobial activity against S. aureus at low minimal inhibitory concentrations (MICs) of 0.057-0.454 μM. Among them, H1, H2, and H3 showed ideal pharmacodynamic effects on methicillin-resistant S. aureus ATCC43300. The total protein level of H1, H2, and H3 reached 1.70, 1.77 and 1.54 g/l at 120 h of induction in the 5-l fermenter, respectively. They killed over 99.9% of pathogens within 1.5 h at 2× and 4× MIC. The post antibiotic effect of H1, H2 and H3 to S. aureus ATCC43300 was 2.94, 1.75 and 1.55 h at 2× MIC, which was similar with their original peptide NZ2114 (1.43 h) and vancomycin (1.72 h). The fractional inhibitory concentration index (FICI) indicated indifferent effects between H1, H2, H3 and vancomycin, ampicillin, rifampicin. Additionally, they had low hemolysis and high stability in different environments (temperature, pH, proteases, and saline ions). All results indicate that H1, H2, and H3 can be produced in large-scale and have potential as therapeutic drugs against MRSA.
由于NZ2114对金黄色葡萄球菌具有强大的活性，它是一种很有前景的治疗应用候选物。我们的目标是通过用精氨酸和赖氨酸残基取代His16和His18来鉴定活性得到改善的NZ2114衍生物。设计了八个突变体，并通过pPICZαA在毕赤酵母X-33中表达。其中五个在0.057-0.454μM的低最小抑菌浓度（MIC）下对金黄色葡萄球菌表现出强大的抗菌活性。其中，H1、H2和H3对耐甲氧西林金黄色葡萄球菌ATCC43300显示出理想的药效学效果。在5升发酵罐中诱导120小时时，H1、H2和H3的总蛋白水平分别达到1.70、1.77和1.54 g/l。它们在2倍和4倍MIC浓度下1.5小时内杀死了超过99.9%的病原体。H1、H2和H3对金黄色葡萄球菌ATCC43300在2倍MIC时的抗生素后效应分别为2.94、1.75和1.55小时，这与它们的原始肽NZ2114（1.43小时）和万古霉素（1.72小时）相似。分数抑菌浓度指数（FICI）表明H_1、H_2、H_3与万古霉素、氨苄青霉素、利福平之间的联合作用为无关作用。此外，它们在不同环境（温度、pH值、蛋白酶和盐离子）中具有低溶血率和高稳定性。所有结果表明，H1、H2和H3可以大规模生产，并且具有作为抗MRSA治疗药物的潜力。
