Bifunctional Inhibitors from Capsicum chinense Seeds with Antimicrobial Activity and Specific Mechanism of Action against Phytopathogenic Fungi

Background: Antimicrobial peptides (AMPs) are found in the defense system in virtually all life forms, being present in many, if not all, plant species.

Objective: The present work evaluated the antimicrobial, enzymatic activity and mechanism of action of the PEF2 fraction from Capsicum chinense Jack. seeds against phytopathogenic fungi.

Methods: Peptides were extracted from C. chinense seeds and subjected to reverse-phase chromatography on an HPLC system using a C18 column coupled to a C8 guard column, then the obtained PEF2 fraction was rechromatographed using a C2/C18 column. Two fractions, named PEF2A and PEF2B, were obtained. The fractions were tested for antimicrobial activity on Colletotrichum gloeosporioides, Colletotrichum lindemuthianum, Fusarium oxysporum and Fusarium solani. Trypsin inhibition assays, reverse zymographic detection of protease inhibition and α-amylase activity assays were also performed. The mechanism of action by which PEF2 acts on filamentous fungi was studied through analysis of membrane permeability and production of reactive oxygen species (ROS). Additionally, we investigated mitochondrial functionality and caspase activation in fungal cells.

Results: It is possible to observe that PEF2 significantly inhibited trypsin activity and T. molitor larval α-amylase activity. The PEF2 fraction was able to inhibit the growth of C. gloeosporioides, C. lindemuthianum and F. oxysporum. PEF2A inhibited the growth of C. lindemuthianum (75%) and F. solani (43%). PEF2B inhibited C. lindemuthianum growth (66%) and F. solani (94%). PEF2 permeabilized F. solani cell membranes and induced ROS in F. oxysporum and F. solani. PEF2 could dissipate mitochondrial membrane potential but did not cause the activation of caspases in all studied fungi.

Conclusion: The results may contribute to the biotechnological application of these AMPs in the control of pathogenic microorganisms in plants of agronomic importance.

Keywords: Fusarium; Plant defense; antimicrobial peptides; pepper; protease inhibitor; α-amylase inhibitor.