Holzknecht Jeanett, Kühbacher Alexander, Papp Csaba, Farkas Attila, Váradi Györgyi, Marcos Jose F, Manzanares Paloma, Tóth Gábor K, Galgóczy László, Marx Florentine
Biocenter, Institute of Molecular Biology, Medical University of Innsbruck, Innrain 80-82, A-6020 Innsbruck, Austria.
Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Közép fasor 52, H-6726 Szeged, Hungary.
J Fungi (Basel). 2020 Aug 19;6(3):141. doi: 10.3390/jof6030141.
Small, cysteine-rich and cationic antimicrobial proteins (AMPs) from filamentous ascomycetes promise treatment alternatives to licensed antifungal drugs. In this study, we characterized the Q176 antifungal protein C (PAFC), which is phylogenetically distinct to the other two antifungal proteins, PAF and PAFB, that are expressed by this biotechnologically important ascomycete. PAFC is secreted into the culture broth and is co-expressed with PAF and PAFB in the exudates of surface cultures. This observation is in line with the suggested role of AMPs in the adaptive response of the host to endogenous and/or environmental stimuli. The structural model predicted five β-strands stabilized by four intramolecular disulfide bonds in PAFC. The functional characterization of recombinant PAFC provided evidence for a promising new molecule in anti- therapy. The thermotolerant PAFC killed planktonic cells and reduced the metabolic activity of sessile cells in pre-established biofilms of two strains, one of which was a fluconazole-resistant clinical isolate showing higher PAFC sensitivity than the fluconazole-sensitive strain. Candidacidal activity was linked to severe cell morphology changes, PAFC internalization, induction of intracellular reactive oxygen species and plasma membrane disintegration. The lack of hemolytic activity further corroborates the potential applicability of PAFC in clinical therapy.
丝状子囊菌中富含半胱氨酸的小分子阳离子抗菌蛋白（AMPs）有望成为已获许可的抗真菌药物的替代治疗方法。在本研究中，我们对Q176抗真菌蛋白C（PAFC）进行了表征，它在系统发育上与这种具有重要生物技术意义的子囊菌所表达的其他两种抗真菌蛋白PAF和PAFB不同。PAFC分泌到培养液中，并与PAF和PAFB在表面培养物的渗出物中共表达。这一观察结果与AMPs在宿主对内源和/或环境刺激的适应性反应中的作用相一致。结构模型预测PAFC中有五条β链由四个分子内二硫键稳定。重组PAFC的功能表征为抗真菌治疗中的一种有前景的新分子提供了证据。耐热的PAFC杀死了浮游细胞，并降低了两种菌株预先形成的生物膜中固着细胞的代谢活性，其中一种是耐氟康唑的临床分离株，其对PAFC的敏感性高于氟康唑敏感株。杀念珠菌活性与严重的细胞形态变化、PAFC内化、细胞内活性氧的诱导和质膜崩解有关。缺乏溶血活性进一步证实了PAFC在临床治疗中的潜在适用性。
