Muñoz Alberto, López-García Belén, Marcos Jose F
Instituto de Agroquímica y Tecnología de Alimentos (IATA), Apartado de Correos 73, Burjassot, E-46100 Valencia, Spain.
Antimicrob Agents Chemother. 2006 Nov;50(11):3847-55. doi: 10.1128/AAC.00650-06.
The small antimicrobial peptide PAF26 (Ac-RKKWFW-NH(2)) has been identified by a combinatorial approach and shows preferential activity toward filamentous fungi. In this work, we investigated the mode of action and inhibitory effects of PAF26 on the fungus Penicillium digitatum. The dye Sytox Green was used to demonstrate that PAF26 induced cell permeation. However, microscopic observations showed that sub-MIC concentrations of PAF26 produced both alterations of hyphal morphology (such as altered polar growth and branching) and chitin deposition in areas of no detectable permeation. Analysis of dose-response curves of inhibition and permeation suggested that growth inhibition is not solely a consequence of permeation. In order to shed light on the mode of PAF26 action, its antifungal properties were compared with those of melittin, a well-known pore-forming peptide that kills through cytolysis. While the 50% inhibitory concentrations and MICs of the two peptides against P. digitatum mycelium were comparable, they differed markedly in their fungicidal activities toward conidia and their hemolytic activities toward human red blood cells. Kinetic studies showed that melittin quickly induced Penicillium cell permeation, while PAF26-induced Sytox Green uptake was significantly slower and less efficient. Therefore, the ultimate growth inhibition and morphological alterations induced by PAF26 for P. digitatum are not likely a result of conventional pore formation. Fluorescently labeled PAF26 was used to demonstrate its specific in vivo interaction and translocation inside germ tubes and hyphal cells, at concentrations as low as 0.3 muM (20 times below the MIC), at which no inhibitory, morphological, or permeation effects were observed. Interestingly, internalized PAF26 could bind to cellular RNAs, since in vitro nonspecific RNA binding activity of PAF26 was demonstrated by electrophoretic mobility shift assays. We propose that PAF26 is a short, de novo-designed penetratin-type peptide that has multiple detrimental effects on target fungi, which ultimately result in permeation and killing.
小抗菌肽PAF26（Ac-RKKWFW-NH(2)）已通过组合方法鉴定出来，并对丝状真菌表现出优先活性。在这项工作中，我们研究了PAF26对指状青霉的作用方式和抑制效果。使用染料Sytox Green来证明PAF26诱导细胞渗透。然而，显微镜观察表明，亚抑菌浓度的PAF26既会导致菌丝形态改变（如极性生长和分支改变），也会在未检测到渗透的区域导致几丁质沉积。抑制和渗透的剂量反应曲线分析表明，生长抑制不仅仅是渗透的结果。为了阐明PAF26的作用方式，将其抗真菌特性与蜂毒素进行了比较，蜂毒素是一种通过细胞溶解致死的著名成孔肽。虽然这两种肽对指状青霉菌丝体的50%抑制浓度和最低抑菌浓度相当，但它们对分生孢子的杀真菌活性以及对人红细胞的溶血活性明显不同。动力学研究表明，蜂毒素能迅速诱导青霉细胞渗透，而PAF26诱导的Sytox Green摄取明显较慢且效率较低。因此，PAF26对指状青霉诱导的最终生长抑制和形态改变不太可能是传统成孔的结果。荧光标记的PAF26用于证明其在体内与芽管和菌丝细胞内的特异性相互作用和转运，浓度低至0.3 μM（低于最低抑菌浓度20倍），此时未观察到抑制、形态或渗透作用。有趣的是，内化的PAF26可以与细胞RNA结合，因为通过电泳迁移率变动分析证明了PAF26的体外非特异性RNA结合活性。我们提出，PAF26是一种新设计的短穿膜肽型肽，对靶真菌有多种有害作用，最终导致渗透和杀伤。
