Molecular and Medicine Research Center, Arak University of Medical Sciences, Arak, Iran.
Molecular and Medicine Research Center, Arak University of Medical Sciences, Arak, Iran; Department of Microbiology and Immunology, School of Medicine, Arak University of Medical Sciences, Arak, Iran.
Protein Expr Purif. 2021 Dec;188:105949. doi: 10.1016/j.pep.2021.105949. Epub 2021 Jul 26.
The production of alternative novel antimicrobial agents is considered an efficient way to cope with multidrug resistance among pathogenic bacteria. E50-52 and Ib-AMP4 antimicrobial peptides (AMPs) have illustrated great proven antibacterial effects. The aim of this study was recombinant production of these AMPs and investigation of their synergistic effects on methicillin-resistant Staphylococcus aureus (MRSA).
At first, the codon optimized sequences of the Ib-AMP4 (UniProt: 024006 (PRO_0000020721), and E50-52 (UniProtKB: P85148) were individually ligated into the pET-32α vector and transformed into E. coli. After the optimization of production and purification steps, the MIC (Minimum inhibitory concentration), time kill and growth kinetic tests of recombinant proteins were determined against MRSA. Finally, the in vivo wound healing efficiency was tested.
The recorded MIC of recombinant Trx-Ib-AMP4, Trx-E50-52 against MRSA bacterium were 0.375 and 0.0875 mg/mL respectively. The combination application of the produced AMPs by the checkerboard method confirmed their synergic activity. The results of the time-kill showed sharply decrease of the number of viable cells with over five time reductions in log CFU/mL by the combination of Trx-E50-52 and Trx-IbAMP4 at 2 × MIC within 240 min. The growth kinetic results confirmed the combination of Trx-E50-52 and Trx-IbAMP4 had much greater success in the reduction of over 50 % of MRSA suspensions' turbidity within the first hour. Wound healing assay and histological analysis of infected mice treated with Trx-Ib-AMP4 or Trx-E50-52 compared with those treated with a combination of Trx-Ib-AMP4 and Trx-E50-52 showed significant synergic effects.
生产新型抗菌药物被认为是应对致病菌多药耐药的有效方法。E50-52 和 Ib-AMP4 抗菌肽 (AMP) 已被证明具有很强的抗菌作用。本研究旨在重组生产这些 AMP，并研究它们对耐甲氧西林金黄色葡萄球菌 (MRSA) 的协同作用。
首先，将 Ib-AMP4（UniProt: 024006 (PRO_0000020721) 和 E50-52（UniProtKB: P85148）的密码子优化序列分别连接到 pET-32α 载体中，并转化到大肠杆菌中。优化生产和纯化步骤后，测定重组蛋白对 MRSA 的最小抑菌浓度 (MIC)、时间杀伤和生长动力学试验。最后，测试体内伤口愈合效率。
重组 Trx-Ib-AMP4、Trx-E50-52 对 MRSA 细菌的 MIC 分别为 0.375 和 0.0875 mg/mL。棋盘法联合应用这两种 AMP 证实了它们的协同活性。时间杀伤试验结果表明，在 2×MIC 下，Trx-E50-52 和 Trx-IbAMP4 联合应用 240 min 内活菌数减少了 5 个对数 CFU/mL。生长动力学试验结果证实，Trx-E50-52 和 Trx-IbAMP4 联合应用在最初 1 小时内能更有效地降低超过 50%的 MRSA 悬浮液浊度。与单独使用 Trx-Ib-AMP4 或 Trx-E50-52 相比，用 Trx-Ib-AMP4 和 Trx-E50-52 联合治疗感染小鼠的伤口愈合试验和组织学分析显示出明显的协同作用。
