Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Faculty of Science, Khon Kaen University, Khon Kaen, 40002, Thailand.
Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen, 40002, Thailand.
Sci Rep. 2023 Sep 26;13(1):16096. doi: 10.1038/s41598-023-43274-9.
Antimicrobial resistance is a growing health concern. Antimicrobial peptides are a potential solution because they bypass conventional drug resistance mechanisms. Previously, we isolated a peptide from Crocodylus siamensis hemoglobin hydrolysate, which has antimicrobial activity and identified the main peptide from this mixture (QL17). The objective of this work was to evaluate and rationally modify QL17 in order to: (1) control its mechanism of action through bacterial membrane disruption; (2) improve its antimicrobial activity; and (3) ensure it has low cytotoxicity against normal eukaryotic cells. QL17 was rationally designed using physicochemical and template-based methods. These new peptide variants were assessed for: (1) their in vitro inhibition of microbial growth, (2) their cytotoxicity against normal cells, (3) their selectivity for microbes, and (4) the mode of action against bacteria using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and confocal microscopy. The results indicate that all designed peptides have more potent antimicrobial efficacy than QL17 and IL15 peptides. However, only the most rationally modified peptides showed strong antimicrobial activity and minimal toxicity against normal cells. In particular, IL15.3 (hydrophobicity of 47% and net charge of + 6) was a potent antimicrobial agent (MIC = 4-12 μg/mL; MBC = 6-25 μg/mL) and displayed excellent selectivity for microbes (cf. human cells) via FACS assays. Microscopy confirmed that IL15.3 acts against bacteria by disrupting the cell membrane integrity and penetrating into the membrane. This causes the release of intracellular content into the outer environment leading to the death of bacteria. Moreover, IL15.3 can also interact with DNA suggesting it could have dual mode of action. Overall, a novel variant of QL17 is described that increases antimicrobial activity by over 1000-fold (~ 5 μg/mL MIC) and has minimal cytotoxicity. It may have applications in clinical use to treat and safeguard against bacteria.
抗微生物药物耐药性是一个日益严重的健康问题。抗微生物肽是一种潜在的解决方案，因为它们绕过了传统的耐药机制。此前，我们从暹罗鳄血红蛋白水解物中分离出一种具有抗菌活性的肽，并从该混合物中鉴定出主要肽（QL17）。本工作的目的是评估并合理修饰 QL17，以：（1）通过破坏细菌细胞膜来控制其作用机制；（2）提高其抗菌活性；（3）确保其对正常真核细胞的细胞毒性低。QL17 通过物理化学和基于模板的方法进行合理设计。这些新的肽变体的评估包括：（1）其对微生物生长的体外抑制作用，（2）对正常细胞的细胞毒性，（3）对微生物的选择性，以及（4）使用扫描电子显微镜（SEM）、透射电子显微镜（TEM）和共聚焦显微镜对细菌的作用模式。结果表明，所有设计的肽都比 QL17 和 IL15 肽具有更强的抗菌功效。然而，只有经过最合理修饰的肽才显示出对正常细胞的强大抗菌活性和最小毒性。特别是，IL15.3（疏水性为 47%，净电荷为+6）是一种有效的抗菌剂（MIC=4-12μg/mL；MBC=6-25μg/mL），并且通过 FACS 分析显示出对微生物（与人细胞相比）的优异选择性。显微镜证实，IL15.3 通过破坏细胞膜完整性并穿透细胞膜来对抗细菌。这导致细胞内物质释放到外部环境中，导致细菌死亡。此外，IL15.3 还可以与 DNA 相互作用，表明它可能具有双重作用模式。总之，描述了一种新型 QL17 变体，其抗菌活性提高了 1000 多倍（~5μg/mL MIC），且细胞毒性最小。它可能在临床应用中用于治疗和预防细菌感染。
