抗菌肽OaBac5mini的特性及其对……的杀菌机制
Characteristics of antimicrobial peptide OaBac5mini and its bactericidal mechanism against .
作者信息
Shen Shanshan, Sun Yawei, Ren Fei, Blair Jessica M A, Siasat Pauline, Fan Shuaiqi, Hu Jianhe, He Junping
机构信息
College of Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi, China.
College of Animal Science and Veterinary Medicine, Henan Institute of Science and Technology, Xinxiang, Henan, China.
出版信息
Front Vet Sci. 2023 Feb 23;10:1123054. doi: 10.3389/fvets.2023.1123054. eCollection 2023.
INTRODUCTION
Antimicrobial peptides (AMPs) play an important role in defending against the attack of pathogenic microorganisms. Among them, the proline-rich antibacterial peptides (PrAMPs) have been attracting close attention due to their simple structure, strong antibacterial activity, and low cell toxicity. OaBac5mini is an active fragment of the sheep-derived OaBac5 belonging to the PrAMPs family.
METHODS
In this study, the antibacterial activity of OaBac5mini was investigated by testing the MICs against different stains of and as well as the time-kill curve. The bactericidal mechanism was explored by determining the effect of OaBac5mini on the cell membrane. The stability and biosafety were also evaluated.
RESULTS
The susceptibility test demonstrated that OaBac5mini showed potent antibacterial activity against the multidrug-resistant (MDR) isolates. It is noticeable that the absence of inner membrane protein SbmA in ATCC 25922 caused the MIC of OaBac5mini to increase 4-fold, implying OaBac5mini can enter into the cytoplasm SbmA and plays its antibacterial activity. Moreover, the antibacterial activity of OaBac5mini against ATCC 25922 was not remarkably affected by the serum salts except for CaCl at a physiological concentration, pH, temperature, repeated freeze-thawing and proteases (trypsin < 20 μg/mL, pepsin or proteinase K). Time-kill curve analysis showed OaBac5mini at the concentration of 200 μg/mL (8 × MICs) could effectively kill ATCC 25922 after co-incubation for 12 h. In addition, OaBac5mini was not hemolytic against rabbit red blood cells and also was not cytotoxic to porcine small intestinal epithelial cells (IPEC-J2). Bioinformatic analysis indicated that OaBac5mini is a linear peptide with 8 net positive charges. Furthermore, OaBac5mini significantly increased the outer membrane permeability and impaired the inner membrane integrity and ultrastructure of ATCC25922.
CONCLUSION
OaBac5mini is a stable and potent PrAMP that kills by two different modes of action - inhibiting intracellular target(s) and damaging cell membrane.
引言
抗菌肽（AMPs）在抵御病原微生物攻击中发挥重要作用。其中，富含脯氨酸的抗菌肽（PrAMPs）因其结构简单、抗菌活性强和细胞毒性低而备受关注。OaBac5mini是源自绵羊的OaBac5的活性片段，属于PrAMPs家族。
方法
在本研究中，通过检测对不同菌株的最低抑菌浓度（MICs）以及时间-杀菌曲线来研究OaBac5mini的抗菌活性。通过确定OaBac5mini对细胞膜的影响来探索杀菌机制。还评估了其稳定性和生物安全性。
结果
药敏试验表明，OaBac5mini对多重耐药（MDR）菌株表现出强大的抗菌活性。值得注意的是，大肠埃希菌ATCC 25922中内膜蛋白SbmA的缺失导致OaBac5mini的MIC增加4倍，这意味着OaBac5mini可通过SbmA进入细胞质并发挥其抗菌活性。此外，除生理浓度的CaCl、pH、温度、反复冻融和蛋白酶（胰蛋白酶<20μg/mL、胃蛋白酶或蛋白酶K）外，血清盐对OaBac5mini对大肠埃希菌ATCC 25922的抗菌活性无显著影响。时间-杀菌曲线分析表明，浓度为200μg/mL（8倍MICs）的OaBac5mini与大肠埃希菌ATCC 25922共孵育12小时后可有效杀灭该菌。此外，OaBac5mini对兔红细胞无溶血作用，对猪小肠上皮细胞（IPEC-J2）也无细胞毒性。生物信息学分析表明，OaBac5mini是一种带8个净正电荷的线性肽。此外，OaBac5mini显著增加了大肠埃希菌ATCC25922的外膜通透性，破坏了其内膜完整性和超微结构。
结论
OaBac5mini是一种稳定且有效的PrAMP，通过抑制细胞内靶点和破坏细胞膜两种不同作用模式杀灭大肠埃希菌。
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