Sonderegger Christoph, Váradi Györgyi, Galgóczy László, Kocsubé Sándor, Posch Wilfried, Borics Attila, Dubrac Sandrine, Tóth Gábor K, Wilflingseder Doris, Marx Florentine
Biocenter, Division of Molecular Biology, Innsbruck Medical University, Innsbruck, Austria.
Department of Medical Chemistry, Faculty of Medicine, University of Szeged, Szeged, Hungary.
Front Microbiol. 2018 Jul 20;9:1655. doi: 10.3389/fmicb.2018.01655. eCollection 2018.
Small, cysteine-rich and cationic antimicrobial proteins (AMPs) from filamentous ascomycetes represent ideal bio-molecules for the development of next-generation antifungal therapeutics. They are promising candidates to counteract resistance development and may complement or even replace current small molecule-based antibiotics in the future. In this study, we show that a 14 amino acid (aa) long peptide (Pγ) spanning the highly conserved γ-core motif of the antifungal protein (PAF) has antifungal activity against the opportunistic human pathogenic yeast . By substituting specific aa we elevated the positive net charge and the hydrophilicity of Pγ and created the peptide variants Pγ and Pγ with 10-fold higher antifungal activity than Pγ. Similarly, the antifungal efficacy of the PAF protein could be significantly improved by exchanging the respective aa in the γ-core of the protein by creating the protein variants PAFγ and PAFγ. The designed peptides and proteins were investigated in detail for their physicochemical features and mode of action, and were tested for cytotoxicity on mammalian cells. This study proves for the first time the important role of the γ-core motif in the biological function of an AMP from ascomycetes. Furthermore, we provide a detailed phylogenetic analysis that proves the presence and conservation of the γ-core motif in all AMP classes from Eurotiomycetes. We emphasize the potential of this common protein motif for the design of short antifungal peptides and as a protein motif in which targeted aa substitutions enhance antimicrobial activity.
丝状子囊菌中富含半胱氨酸的小分子阳离子抗菌蛋白（AMPs）是开发下一代抗真菌疗法的理想生物分子。它们有望对抗耐药性的产生，未来可能补充甚至取代目前基于小分子的抗生素。在本研究中，我们发现一种由14个氨基酸（aa）组成的肽（Pγ），其跨越抗真菌蛋白（PAF）高度保守的γ-核心基序，对机会性人类致病酵母具有抗真菌活性。通过替换特定氨基酸，我们提高了Pγ的正净电荷和亲水性，并创造了肽变体Pγ和Pγ，其抗真菌活性比Pγ高10倍。同样，通过创建蛋白变体PAFγ和PAFγ，将PAF蛋白γ-核心中的相应氨基酸进行交换，可显著提高其抗真菌功效。对设计的肽和蛋白的物理化学特征和作用方式进行了详细研究，并测试了它们对哺乳动物细胞的细胞毒性。本研究首次证明了γ-核心基序在子囊菌AMPs生物学功能中的重要作用。此外，我们提供了详细的系统发育分析，证明了γ-核心基序在散囊菌纲所有AMP类别中的存在和保守性。我们强调了这种常见蛋白基序在设计短抗真菌肽方面的潜力，以及作为一种通过靶向氨基酸替换可增强抗菌活性的蛋白基序的潜力。
