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

Abstract

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.

Chitin‐binding peptides from Capsicum annuum with antifungal activity and low toxicity to mammalian cells and Galleria mellonella larvae

“…Other works have already demonstrated the presence of different peptides with antimicrobial activity in Capsicum seeds, but these peptides have been isolated through methodologies other than chitin affinity columns. [27,39,45,46] Therefore, this is the first work that uses a chitin column to obtain peptide-rich fractions with antimicrobial activity from the seeds of the Capsicum genus. In other plant species, low molecular weight proteins have been purified using a chitin affinity column.…”

“…[23] In recent years, within our group, several Capsicum peptides have already been isolated and characterized, such as thionine-like, [24] defensin, [25] lipid transport proteins (LTPs) [26] and proteinase inhibitors. [27] In this study, for the first time, we used a chitin-binding column to purify peptiderich fractions from seeds of C.annuum with chitin-binding activity and antimicrobial activity against yeasts.…”

Characterization and Antimicrobial Activity of Protease and α-Amylase Inhibitors from Immature Fruits of Capsicum chinense Jacq.

“…Antimicrobial peptides extracted from different plant organs, such as seeds and fruits of the genus Capsicum, have already been described and characterized previously [13,15,[23][24][25][26][27]. Silva and coauthors (2014) [12], showed the antimicrobial potential of protein fractions obtained from seeds of C. chinense Jacq. accession UENF 1755 on the fungi Colletotrichum gloeosporioides, C. lindemunthianum, Fusarium oxysporum and F. solani, in addition to the inhibition of the activity of trypsin and α-amylase enzymes.…”

“…In plants of the genus Capsicum, peptides characterized as inhibitors of trypsin and chymotrypsin-protease with antifungal activity (MIC 50-250 μg.mL -1 ) were identified, mainly in C. annuum and C. chinense [10,11]. The antifungal activity of these AMPs was characterized by visualization of cell agglomeration and pseudohyphae formation or by hyphal morphological changes as well as by membrane permeabilization due to reactive oxygen species (ROS) induction [11,12].…”

Applications of tandem mass spectrometry (MS/MS) in antimicrobial peptides field: Current state and new applications

“…In recent years, desalination (low-pressure chromatography) has emerged as an alternative to dialysis. This technique involves the use of hydrophobic adsorbents, such as phenyl-agarose, reverse phase C8, and C18 [ [107] , [108] , [109] ]. RP-HPLC has become a primary purification method for peptides and other small compounds due to its good selectivity and high separation performance.…”