Trypsin/α-Amylase Inhibitors from Capsicum chinense Seeds: Characterization and Antifungal Activity against Fungi of Agronomic Importance.

BACKGROUND: Protease inhibitors (PIs) have attracted attention due to their important roles in plant defense. OBJECTIVE: The objective of this work was to characterize and evaluate the antimicrobial activity of the peptides of a family of serine PIs from Capsicum chinense Jacq. seeds. METHODS: Initially, PIs were extracted from the seeds and subjected to purification by chromatography, resulting in three different peptide enriched fractions (PEFs) termed PEF1, PEF2 and PEF3. Subsequently, the PEF3 was subjected to trypsin inhibition assays, α-amylase activity assays, antimicrobial activity assays on phytopathogenic fungi, and assays to determine the likely mechanisms of action. RESULTS: The PEF3 was composed of three protein bands with molecular masses ranging between 6 and 14 kDa. The amino acid residues of the ~6 kDa band showed high similarity with serine PIs. PEF3 inhibited the activity of the enzymes trypsin, human salivary α-amylase, and Tenebrio molitor larval α-amylase and inhibited the growth of phytopathogenic fungi, showing 83.7% loss of viability in Fusarium oxysporum. PEF3 induced reactive oxygen species in Colletotrichum lindemuthianum and F. oxysporum to dissipate their mitochondrial membrane potential and activated caspases in C. lindemuthianum. CONCLUSION: Our results reinforce the importance of PIs in plant defense mechanisms against phytopathogenic fungi as well as in their biotechnological applications for the control of plant pathogens.

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.

Antimicrobial peptides from Capsicum chinense fruits: agronomic alternatives against phytopathogenic fungi.

In recent years, the antimicrobial activity of peptides isolated from a wide variety of organs from plant species has been reported. However, a few studies have investigated the potential of antimicrobial peptides (AMPs) found in fruits, especially Capsicum chinense (pepper). The present study aimed to purify and characterize peptides from Capsicum chinense fruits and evaluate their inhibitory activities against different phytopathogenic fungi and also analyze the possible mechanisms of action involved in microbial inhibition. After fruit protein extraction and high-performance liquid chromatography (HPLC), different fractions were obtained, named F1 to F10. Peptides in the F4 and F5 fractions were sequenced and revealed similarity with the plant antimicrobial peptides like non-specific lipid transfer proteins and defensin-like peptide. The F4 and F5 fractions presented strong antimicrobial activity against the fungus Fusarium solani and Fusarium oxysporum, causing toxic effects on these fungi, leading to membrane permeabilization, endogenous reactive oxygen species increase, activation of metacaspase and loss of mitochondrial function.

Identification and Characterization of Two Defensins from Capsicum annuum Fruits that Exhibit Antimicrobial Activity.

Scientific advances have not been enough to combat the growing resistance to antimicrobial medicines. Antimicrobial peptides (AMPs) are effector molecules of the innate immune defense system in plants and could provide an important source of new antimicrobial drugs. The aim of this work was to extract, purify, characterize, and evaluate the antifungal activities present in fractions obtained from Capsicum annum fruits through reversed-phase chromatography. The fractions named F2 and F3 presented the highest inhibitory activity against Candida and Mycobacterium tuberculosis species. In addition, we identified two sequences of AMPs in the F2 and F3 fractions through mass spectrometry that showed similarity to an already well-characterized family of plant defensins. A plasma membrane permeabilization assay demonstrated that the peptides present in F2, F3, and F4 fractions induced changes in the membrane of some yeast strains, culminating in permeabilization. The production of reactive oxygen species was induced by the fractions in some yeast strains. Fractions F2, F3, and F4 also did not show toxicity in macrophage or monocyte cultures. In conclusion, the obtained data demonstrate that the AMPs, especially those present in the fractions F2 and F3, are promising antimicrobial agents that may be useful to enhance the development of new therapeutic agents for the treatment of diseases.