López-García Belén, Harries Eleonora, Carmona Lourdes, Campos-Soriano Lidia, López José Javier, Manzanares Paloma, Gandía Mónica, Coca María, Marcos Jose F
Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB. Edifici CRAG, Campus UAB, 08193, Bellaterra, Barcelona, Spain.
Appl Microbiol Biotechnol. 2015 Oct;99(19):8011-21. doi: 10.1007/s00253-015-6541-1. Epub 2015 Apr 7.
There are short cationic and tryptophan-rich antifungal peptides such as the hexapeptide PAF26 (RKKWFW) that have selective toxicity and cell penetration properties against fungal cells. This study demonstrates that concatemeric peptides with tandem repeats of the heptapeptide PAF54 (which is an elongated PAF26 sequence) show increased fungistatic and bacteriostatic activities while maintaining the absence of hemolytic activity of the monomer. The increase in antimicrobial activity of the double-repeated PAF sequences (diPAFs), compared to the nonrepeated PAF, was higher (4-8-fold) than that seen for the triple-repeated sequences (triPAFs) versus the diPAFs (2-fold). However, concatemerization diminished the fungicidal activity against quiescent spores of the filamentous fungus Penicillium digitatum. Peptide solubility and sensitivity to proteolytic degradation were affected by the design of the concatemers: incorporation of the AGPA sequence hinge to separate PAF54 repeats increased solubility while the C-terminal addition of the KDEL sequence decreased in vitro stability. These results led to the design of the triPAF sequence PAF102 of 30 amino acid residues, with increased antimicrobial activity and minimal inhibitory concentration (MIC) value of 1-5 μM depending on the fungus. Further characterization of the mode-of-action of PAF102 demonstrated that it colocalizes first with the fungal cell wall, it is thereafter internalized in an energy dependent manner into hyphal cells of the filamentous fungus Fusarium proliferatum, and finally kills hyphal cells intracellularly. Therefore, PAF102 showed mechanistic properties against fungi similar to the parental PAF26. These observations are of high interest in the future development of PAF-based antimicrobial molecules optimized for their production in biofactories.
存在一些短的阳离子且富含色氨酸的抗真菌肽，比如六肽PAF26（RKKWFW），其对真菌细胞具有选择性毒性和细胞穿透特性。本研究表明，具有七肽PAF54串联重复序列（PAF54是PAF26序列的延伸形式）的串联肽在保持单体无溶血活性的同时，显示出增强的抑菌和杀菌活性。与非重复的PAF相比，双重复PAF序列（diPAFs）抗菌活性的增加幅度（4至8倍）高于三重复序列（triPAFs）相对于双重复序列（2倍）的增加幅度。然而，串联化降低了对丝状真菌指状青霉静止孢子的杀真菌活性。串联肽的设计会影响肽的溶解度和对蛋白水解降解的敏感性：引入AGPA序列铰链来分隔PAF54重复序列可增加溶解度，而在C末端添加KDEL序列会降低体外稳定性。这些结果促成了30个氨基酸残基的triPAF序列PAF102的设计，其抗菌活性增强，最小抑菌浓度（MIC）值根据真菌种类为1至5 μM。对PAF102作用模式的进一步表征表明，它首先与真菌细胞壁共定位，随后以能量依赖的方式内化到丝状真菌轮枝镰孢菌的菌丝细胞中，最终在细胞内杀死菌丝细胞。因此，PAF102显示出与亲本PAF26相似的抗真菌作用机制特性。这些观察结果对于未来基于PAF的抗菌分子在生物工厂中优化生产的开发具有高度意义。
