Giner-Llorca Moisés, Locascio Antonella, Del Real Javier Alonso, Marcos Jose F, Manzanares Paloma
Department of Food Biotechnology, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Catedrático Agustín Escardino 7, Paterna, Valencia, 46980, Spain.
Appl Microbiol Biotechnol. 2023 Nov;107(22):6811-6829. doi: 10.1007/s00253-023-12749-0. Epub 2023 Sep 9.
Antifungal proteins (AFPs) from filamentous fungi offer the potential to control fungal infections that threaten human health and food safety. AFPs exhibit broad antifungal spectra against harmful fungi, but limited knowledge of their killing mechanism hinders their potential applicability. PeAfpA from Penicillium expansum shows strong antifungal potency against plant and human fungal pathogens and stands above other AFPs for being active against the yeast Saccharomyces cerevisiae. We took advantage of this and used a model laboratory strain of S. cerevisiae to gain insight into the mode of action of PeAfpA by combining (i) transcriptional profiling, (ii) PeAfpA sensitivity analyses of deletion mutants available in the S. cerevisiae genomic deletion collection and (iii) cell biology studies using confocal microscopy. Results highlighted and confirmed the role of the yeast cell wall (CW) in the interaction with PeAfpA, which can be internalized through both energy-dependent and independent mechanisms. The combined results also suggest an active role of the CW integrity (CWI) pathway and the cAMP-PKA signalling in the PeAfpA killing mechanism. Besides, our studies revealed the involvement of phosphatidylinositol metabolism and the participation of ROX3, which codes for the subunit 19 of the RNA polymerase II mediator complex, in the yeast defence strategy. In conclusion, our study provides clues about both the killing mechanism of PeAfpA and the fungus defence strategies against the protein, suggesting also targets for the development of new antifungals. KEY POINTS: • PeAfpA is a cell-penetrating protein with inhibitory activity against S. cerevisiae. • The CW integrity (CWI) pathway is a key player in the PeAfpA killing mechanism. • Phosphatidylinositol metabolism and ROX3 are involved in the yeast defence strategy.
丝状真菌产生的抗真菌蛋白（AFPs）具有控制威胁人类健康和食品安全的真菌感染的潜力。AFPs对有害真菌具有广泛的抗真菌谱，但对其杀伤机制的了解有限，这阻碍了它们的潜在应用。扩展青霉的PeAfpA对植物和人类真菌病原体显示出强大的抗真菌效力，并且在对酿酒酵母有活性方面优于其他AFPs。我们利用这一点，通过结合（i）转录谱分析、（ii）酿酒酵母基因组缺失文库中可用的缺失突变体的PeAfpA敏感性分析以及（iii）使用共聚焦显微镜的细胞生物学研究，利用酿酒酵母的模型实验室菌株来深入了解PeAfpA的作用模式。结果突出并证实了酵母细胞壁（CW）在与PeAfpA相互作用中的作用，PeAfpA可以通过能量依赖和非依赖机制内化。综合结果还表明CW完整性（CWI）途径和cAMP-PKA信号在PeAfpA杀伤机制中发挥积极作用。此外，我们的研究揭示了磷脂酰肌醇代谢的参与以及编码RNA聚合酶II中介复合物亚基19的ROX3在酵母防御策略中的参与。总之，我们的研究为PeAfpA的杀伤机制和真菌针对该蛋白的防御策略提供了线索，也为新型抗真菌药物的开发提供了靶点。要点：• PeAfpA是一种具有穿透细胞活性且对酿酒酵母有抑制作用的蛋白。• CW完整性（CWI）途径是PeAfpA杀伤机制中的关键因素。• 磷脂酰肌醇代谢和ROX3参与酵母防御策略。
