Purpose

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).

Method

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.

Results and conclusion

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 log₁₀ 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.

中文翻译：

---

Trx-Ib-AMP4和Trx-E50-52抗菌肽的重组生产及体外和体内治疗耐甲氧西林金黄色葡萄球菌的抗菌协同评价

目的

生产替代的新型抗菌剂被认为是应对病原菌间多药耐药性的有效方法。E50-52 和 Ib-AMP4 抗菌肽 (AMP) 已证明具有很好的抗菌作用。本研究的目的是重组生产这些 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。

方法

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，转化大肠杆菌。经过优化生产和纯化步骤，测定重组蛋白对MRSA的MIC（最小抑制浓度）、时间杀伤和生长动力学测试，最后测试体内伤口愈合效率。

结果与结论

记录的重组 Trx-Ib-AMP4、Trx-E50-52 对 MRSA 细菌的 MIC 分别为 0.375 和 0.0875 mg/mL。通过棋盘法生产的 AMP 的组合应用证实了它们的协同活性。时间杀死的结果显示活细胞数量急剧减少，log ₁₀减少超过五次 CFU/mL 通过 Trx-E50-52 和 Trx-IbAMP4 在 2 × MIC 下在 240 分钟内的组合。生长动力学结果证实 Trx-E50-52 和 Trx-IbAMP4 的组合在第一个小时内将 MRSA 悬浮液的浊度降低 50% 以上方面取得了更大的成功。与用 Trx-Ib-AMP4 和 Trx-E50-52 组合治疗的小鼠相比，用 Trx-Ib-AMP4 或 Trx-E50-52 治疗的感染小鼠的伤口愈合测定和组织学分析显示出显着的协同作用。