National Food Institute, Technical University of Denmark, Kongens Lyngby, Denmark.
Department of Biology, University of Copenhagen, Copenhagen, Denmark.
PLoS One. 2018 May 31;13(5):e0197742. doi: 10.1371/journal.pone.0197742. eCollection 2018.
Due to the rapid emergence of resistance to classical antibiotics, novel antimicrobial compounds are needed. It is desirable to selectively kill pathogenic bacteria without targeting other beneficial bacteria in order to prevent the negative clinical consequences caused by many broad-spectrum antibiotics as well as reducing the development of antibiotic resistance. Antimicrobial peptides (AMPs) represent an alternative to classical antibiotics and it has been previously demonstrated that Cap18 has high antimicrobial activity against a broad range of bacterial species. In this study we report the design of a positional scanning library consisting of 696 Cap18 derivatives and the subsequent screening for antimicrobial activity against Y. ruckeri, A. salmonicida, S. Typhimurium and L. lactis as well as for hemolytic activity measuring the hemoglobin release of horse erythrocytes. We show that the hydrophobic face of Cap18, in particular I13, L17 and I24, is essential for its antimicrobial activity against S. Typhimurium, Y. ruckeri, A. salmonicida, E. coli, P. aeruginosa, L. lactis, L. monocytogenes and E. faecalis. In particular, Cap18 derivatives harboring a I13D, L17D, L17P, I24D or I24N substitution lost their antimicrobial activity against any of the tested bacterial strains. In addition, we were able to generate species-specific Cap18 derivatives by particular amino acid substitutions either in the hydrophobic face at positions L6, L17, I20, and I27, or in the hydrophilic face at positions K16 and K18. Finally, our data showed the proline residue at position 29 to be essential for the inherent low hemolytic activity of Cap18 and that substitution of the residues K16, K23, or G21 by any hydrophobic residues enhances the hemolytic activity. This study demonstrates the potential of generating species-specific AMPs for the selective elimination of bacterial pathogens.
由于经典抗生素的耐药性迅速出现，因此需要新型抗菌化合物。理想情况下，应选择性地杀死致病菌，而不针对其他有益细菌，以防止许多广谱抗生素引起的负面临床后果，并减少抗生素耐药性的发展。抗菌肽 (AMP) 是经典抗生素的替代品，先前已经证明 Cap18 对广泛的细菌物种具有高抗菌活性。在这项研究中，我们报告了一种由 696 种 Cap18 衍生物组成的位置扫描文库的设计，并随后筛选了针对 Y. ruckeri、A. salmonicida、S. Typhimurium 和 L. lactis 的抗菌活性，以及针对溶血活性（测量马红细胞血红蛋白释放）的活性。我们表明，Cap18 的疏水面，特别是 I13、L17 和 I24，对于其针对 S. Typhimurium、Y. ruckeri、A. salmonicida、E. coli、P. aeruginosa、L. lactis、L. monocytogenes 和 E. faecalis 的抗菌活性至关重要。特别是，携带 I13D、L17D、L17P、I24D 或 I24N 取代的 Cap18 衍生物失去了针对任何测试细菌菌株的抗菌活性。此外，我们能够通过在疏水面上的特定氨基酸取代（位置 L6、L17、I20 和 I27）或在亲水面上的特定氨基酸取代（位置 K16 和 K18）生成具有物种特异性的 Cap18 衍生物。最后，我们的数据表明，位置 29 的脯氨酸残基对于 Cap18 固有的低溶血活性至关重要，并且用任何疏水性残基取代 K16、K23 或 G21 都会增强溶血活性。这项研究表明，生成针对细菌病原体的特异性 AMP 的潜力。
