Defensin-like peptides from Capsicum chinense induce increased ROS, loss of mitochondrial functionality, and reduced growth of the fungus Colletotrichum scovillei.

In the present study, we identified and characterized two defensin-like peptides in an antifungal fraction obtained from Capsicum chinense pepper fruits and inhibited the growth of Colletotrichum scovillei, which causes anthracnose. AMPs were extracted from the pericarp of C. chinense peppers and subjected to ion exchange, molecular exclusion, and reversed-phase in a high-performance liquid chromatography system. We investigated the endogenous increase in reactive oxygen species (ROS), the loss of mitochondrial functioning, and the ultrastructure of hyphae. The peptides obtained from the G3 fraction through molecular exclusion chromatography were subsequently fractionated using reverse-phase chromatography, resulting in the isolation of fractions F1, F2, F3, F4, and F5. The F1-Fraction suppressed C. scovillei growth by 90, 70.4, and 44% at 100, 50, and 25 µg mL-1, respectively. At 24 h, the IC50 and minimum inhibitory concentration were 21.5 µg mL-1 and 200 µg mL-1, respectively. We found an increase in ROS, which may have resulted in an oxidative burst, loss of mitochondrial functioning, and cytoplasm retraction, as well as an increase in autophagic vacuoles. MS/MS analysis of the F1-Fraction indicated the presence of two defensin-like proteins, and we were able to identify the expression of three defensin sequences in our C. chinense fruit extract. The F1-Fraction was also found to inhibit the activity of insect α-amylases. In summary, the F1-Fraction of C. chinense exhibits antifungal activity against a major pepper pathogen that causes anthracnose. These defensin-like compounds are promising prospects for further research into antifungal and insecticide biotechnology applications. © 2024 Society of Chemical Industry.

Inhibition of Serine Protease, α-Amylase and Growth of Phytopathogenic Fungi by Antimicrobial Peptides from Capsicum chinense Fruits.

Plant fungal diseases cause major problems for the global economy. Antimicrobial peptides have aroused great interest in the control of phytopathogens, as they are natural molecules and have a broad spectrum of inhibitory activity. Herein, we have tried to identify and characterize antimicrobial peptides present in fruits of Capsicum chinense and to evaluate their enzymatic and antifungal activities. The retained fraction obtained in the anion exchange chromatography with strong antifungal activity was subjected to molecular exclusion chromatography and obtained four fractions named G1, G2, G3, and G4. The 6.0-kDa protein band of G2 showed similarity with protease inhibitors type II, and it was able to inhibit 100% of trypsin and α-amylase activities. The protein band with approximately 6.5 kDa of G3 showed similarity with sequences of protease inhibitors from genus Capsicum and showed growth inhibition of 48% for Colletotrichum lindemuthianum, 49% for Fusarium lateritium, and 51% for F. solani and F. oxysporum. Additionally, G3 causes morphological changes, membrane permeabilization, and ROS increase in F. oxysporum cells. The 9-kDa protein band of G4 fraction was similar to a nsLTP type 1, and a protein band of 6.5 kDa was similar to a nsLTP type 2. The G4 fraction was able to inhibit 100% of the activities of glycosidases tested and showed growth inhibition of 35 and 50% of F. oxysporum and C. lindemuthianum, respectively. C. chinense fruits have peptides with antifungal activity and enzyme inhibition with biotechnological potential.

A new bioinspired peptide on defensin from C. annuum fruits: Antimicrobial activity, mechanisms of action and therapeutical potential.

BACKGROUND: Antimicrobial peptides, natural or synthetic, appear as promising molecules for antimicrobial therapy because of their both broad antimicrobial activity and mechanism of action. Herein, we determine the anti-Candida and antimycobacterial activities, mechanism of action on yeasts, and cytotoxicity on mammalian cells in the presence of the bioinspired peptide CaDef2.1G27-K44. METHODS: CaDef2.1G27-K44 was designed to attain the following criteria: high positive net charge; low molecular weight (<3000 Da); Boman index ≤2.5; and total hydrophobic ratio ≥ 40%. The mechanism of action was studied by growth inhibition, plasma membrane permeabilization, ROS induction, mitochondrial functionality, and metacaspase activity assays. The cytotoxicity on macrophages, monocytes, and erythrocytes were also determined. RESULTS: CaDef2.1G27-K44 showed inhibitory activity against Candida spp. with MIC100 values ranging from 25 to 50 µM and the standard and clinical isolate of Mycobacterium tuberculosis with MIC50 of 33.2 and 55.4 µM, respectively. We demonstrate that CaDef2.1G27-K44 is active against yeasts at different salt concentrations, induced morphological alterations, caused membrane permeabilization, increased ROS, causes loss of mitochondrial functionality, and activation of metacaspases. CaDef2.1G27-K44 has low cytotoxicity against mammalian cells. CONCLUSIONS: The results obtained showed that CaDef2.1G27-K44 has great antimicrobial activity against Candida spp. and M. tuberculosis with low toxicity to host cells. For Candida spp., the treatment with CaDef2.1G27-K44 induces a process of regulated cell death with apoptosis-like features. GENERAL SIGNIFICANCE: We show a new AMP bioinspired with physicochemical characteristics important for selectivity and antimicrobial activity, which is a promising candidate for drug development, mainly to control Candida infections.

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.

Improved smallest peptides based on positive charge increase of the γ-core motif from PνD1 and their mechanism of action against Candida species.

BACKGROUND: Plant defensins have a hallmark γ-core motif (GXCX3-9C) that is related to their antimicrobial properties. The aim of this work was to design synthetic peptides based on the region corresponding to the PvD1 defensin γ-core that are the smallest amino acid sequences that bear the strongest biological activity. METHODS: We made rational substitutions of negatively charged amino acid residues with positively charged ones, and the reduction in length in the selected PvD1 γ-core sequence to verify whether the increased net positive charges and shortened length are related to the increase in antifungal activity. Herein, we opted to evaluate the action mechanism of γ33-41 PvD1 ++ peptide due to its significant inhibitory effect on tested yeasts. In addition, it is the smallest construct comprising only nine amino acid residues, giving it a better possibility to be a prototype for designing a new antifungal drug, with lower costs to the pharmaceutical industry while still maintaining the strongest antimicrobial properties. RESULTS: The γ33-41 PvD1 ++ peptide caused the most toxic effects in the yeast Candida buinensis, leading to membrane permeabilization, viability loss, endogenous reactive oxygen species increase, the activation of metacaspase, and the loss of mitochondrial functionality, suggesting that this peptide triggers cell death via apoptosis. CONCLUSION: We observed that the antifungal activity of PvD1 is not strictly localized in the structural domain, which comprises the γ-core region and that the increase in the net positive charge is directly related to the increase in antifungal activity.

Biochemical characterization of a Kunitz inhibitor from Inga edulis seeds with antifungal activity against Candida spp.

We describe the characterization of IETI, the first trypsin inhibitor purified from Inga edulis, a tree widely distributed in Brazil. Two-step chromatography was used to purify IETI, a protein composed of a single peptide chain of 19,685.10 Da. Amino-terminal sequencing revealed that IETI shows homology with the Kunitz family, as substantiated by its physical-chemical features, such as its thermal (up to 70 °C) and wide-range pH stability (from 2 to 10), and the value of its dissociation constant (6.2 nM). IETI contains a single reactive site for trypsin, maintained by a disulfide bridge; in the presence of DTT, its inhibitory activity was reduced in a time- and concentration-dependent manner. IETI presented activity against Candida ssp., including C. buinensis and C. tropicalis. IETI inhibitory activity triggered yeast membrane permeability, affecting cell viability, thus providing support for the use of IETI in further studies for the control of fungal infections.

Characterization of a Kunitz trypsin inhibitor from Enterolobium timbouva with activity against Candida species.

Scientific advances have not been sufficient to accompany the growing resistance to antimicrobial medicines. High mortality rates due to opportunistic infections have threated human health. The development of new drugs, such as those obtained from plant sources, is a world priority. Herein, we report the purification of a trypsin inhibitor from Enterolobium timbouva seeds (EtTI) with regard to its homology, physico-chemical and inhibitory properties. Furthermore, we evaluated its activity against Candida strains, opportunistic pathogens regularly found in hospital infections. EtTI belongs to the Kunitz family and inhibits two trypsin molecules simultaneously; a feature shared among double-headed Kunitz inhibitors. A high inhibitory activity against trypsin was determined (Ki = 0.5 nM), and refractory to digestion by pepsin. EtTI was candidicidal against C. albicans, C. buinensis and C. tropicalis, triggering disturbances on integrity of the plasma membrane and morphological alterations, presumably mediated via apoptosis. The presence of two reactive sites is an unusual feature detected in EtTI. Numerous diseases and pathologies involve changes in peptidase activities, encouraging studies with multifunctional inhibitors. Accordingly, the further exploration of EtTI could provide new insights into the Kunitz inhibitors and their applications in disease control.