Structural and Functional Characterization of New Lipid Transfer Proteins with Chitin-Binding Properties: Insights from Protein Structure Prediction, Molecular Docking, and Antifungal Activity.

Faced with the emergence of multiresistant microorganisms that affect human health, microbial agents have become a serious global threat, affecting human health and plant crops. Antimicrobial peptides have attracted significant attention in research for the development of new microbial control agents. This work's goal was the structural characterization and analysis of antifungal activity of chitin-binding peptides from Capsicum baccatum and Capsicum frutescens seeds on the growth of Candida and Fusarium species. Proteins were initially submitted to extraction in phosphate buffer pH 5.4 and subjected to chitin column chromatography. Posteriorly, two fractions were obtained for each species, Cb-F1 and Cf-F1 and Cb-F2 and Cf-F2, respectively. The Cb-F1 (C. baccatum) and Cf-F1 (C. frutescens) fractions did not bind to the chitin column. The electrophoresis results obtained after chromatography showed two major protein bands between 3.4 and 14.2 kDa for Cb-F2. For Cf-F2, three major bands were identified between 6.5 and 14.2 kDa. One band from each species was subjected to mass spectrometry, and both bands showed similarity to nonspecific lipid transfer protein. Candida albicans and Candida tropicalis had their growth inhibited by Cb-F2. Cf-F2 inhibited the development of C. albicans but did not inhibit the growth of C. tropicalis. Both fractions were unable to inhibit the growth of Fusarium species. The toxicity of the fractions was tested in vivo on Galleria mellonella larvae, and both showed a low toxicity rate at high concentrations. As a result, the fractions have enormous promise for the creation of novel antifungal compounds.

Understanding the mechanism of action of protease inhibitors in controlling the growth of the Candida Genus: potential candidates for development of new antifungal molecules.

There is a growing imperative for research into alternative compounds for the treatment of the fungal infections. Thus, many studies have focused on the analysis of antifungal proteins and peptides from different plant sources. Among these molecules are protease inhibitors (PIs). Previously, PIs present in the peptide-rich fractions called PEF1, PEF2 and PEF3 were identified from Capsicum chinense seeds, which have strong activity against phytopathogenic fungi. The aim of this study was to evaluate the mechanism of action and antimicrobial activity of PIs from PEF2 and PEF3 on the growth of yeasts of the genus Candida. In this work, analyses of their antimicrobial activity and cell viability were carried out. Subsequently, the mechanism of action by which the PIs cause the death of the yeasts was evaluated. Cytotoxicity was assessed in vitro by erythrocytes lysis and in vivo in Galleria mellonella larvae. PEF2 and PEF3 caused 100% of the growth inhibition of C. tropicalis and C. buinensis. For C. albicans inhibition was approximately 60% for both fractions. The PEF2 and PEF3 caused a reduction in mitochondrial functionality of 54% and 46% for C. albicans, 26% and 30% for C. tropicalis, and 71% and 68% for C. buinensis, respectively. These fractions induced morphological alterations, led to membrane permeabilization, elevated ROS levels, and resulted in necrotic cell death in C. tropicalis, whilst demonstrating low toxicity toward host cells. From the results obtained here, we intend to contribute to the understanding of the action of PIs in the control of fungal diseases of medical importance.

Divergence and conservation of defensins and lipid transfer proteins (LTPs) from sugarcane wild species and modern cultivar genomes.

Plant defensins and lipid transfer proteins (LTPs) constitute a large and evolutionarily diverse family of antimicrobial peptides. Defensins and LTPs are two pathogenesis-related proteins (PR proteins) whose characterization may help to uncover aspects about the sugarcane response to pathogens attack. LTPs have also been investigated for their participation in the response to different types of stress. Despite the important roles of defensins and LTPs in biotic and abiotic stresses, scarce knowledge is found about these proteins in sugarcane. By using bioinformatics approaches, we characterized defensins and LTPs in the sugarcane wild species and modern cultivar genomes. The identification of defensins and LTPs showed that all five defensins groups and eight of the nine LTPs have their respective genes loci, although some was only identified in the cultivar genome. Phylogenetic analysis showed that defensins appear to be more conserved among groups of plants than LTPs. Some defensins and LTPs showed opposite expression during pathogenic and benefic bacterial interactions. Interestingly, the expression of defensins and LTPs in shoots and roots was completely different in plants submitted to benefic bacteria or water depletion. Finally, the modeling and comparison of isoforms of LTPs and defensins in wild species and cultivars revealed a high conservation of tertiary structures, with variation of amino acids in different regions of proteins, which could impact their antimicrobial activity. Our data contributed to the characterization of defensins and LTPs in sugarcane and provided new elements for understanding the involvement of these proteins in sugarcane response to different types of stress.

Design of improved synthetic antifungal peptides with targeted variations in charge, hydrophobicity and chirality based on a correlation study between biological activity and primary structure of plant defensin γ-cores.

Microbial resistance to available drugs is a growing health threat imposing the need for the development of new drugs. The scaffold of plant defensins, including their γ-cores, are particularly good candidates for drug design. This work aimed to improve the antifungal activity of a previous design peptide, named A36,42,44γ32-46VuDef (for short DD) against yeasts by altering its biochemical parameters. We explore the correlation of the biological activity and structure of plant defensins and compared their primary structures by superimposition with VuDef1 and DD which indicated us the favorable position and the amino acid to be changed. Three new peptides with modifications in charge, hydrophobicity (RR and WR) and chirality (D-RR) were designed and tested against pathogenic yeasts. Inhibition was determined by absorbance. Viability of mammalian cells was determined by MTT. The three designed peptides had better inhibitory activity against the yeasts with better potency and spectrum of yeast species inhibition, with low toxicity to mammalian cells. WR, the most hydrophobic and cationic, exhibited better antifungal activity and lower toxicity. Our study provides experimental evidence that targeted changes in the primary structure of peptides based on plant defensins γ-core primary structures prove to be a good tool for the synthesis of new compounds that may be useful as alternative antifungal drugs. The method described did not have the drawback of synthesis of several peptides, because alterations are guided. When compared to other methods, the design process described is efficient and viable to those with scarce resources.

Adepamycin: design, synthesis and biological properties of a new peptide with antimicrobial properties.

Antimicrobial peptides (AMP) are molecules with a broad spectrum of activities that have been identified in most living organisms. In addition, synthetic AMPs designed from natural polypeptides have been largely investigated. Here, we designed a novel AMP using the amino acid sequence of a plant trypsin inhibitor from Adenanthera pavonina seeds (ApTI) as a template. The 176 amino acid residues ApTI sequence was cleaved in silico using the Collection of Antimicrobial Peptides (CAMPR3), through the sliding-window method. Further improvements in AMP structure were carried out, resulting in adepamycin, an AMP designed from ApTI. Adepamycin showed antimicrobial activity from 0.9 to 3.6 µM against Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus strains. Moreover, this peptide also displayed activity against Candida albicans and Candida tropicalis. No toxic effects were observed on healthy human cells. Studies on the mechanism of action of adepamycin were carried out using an E. coli and C. tropicalis. Adepamycin triggers membrane disturbances, leading to intracellular nucleic acids release in E. coli. For C. tropicalis, an initial interference with the plasma membrane integrity is followed by the formation of intracellular reactive oxygen species (ROS), leading to apoptosis. Structurally, adepamycin was submitted to circular dichroism spectroscopy, molecular modeling and molecular dynamics simulations, revealing an environment-dependent α-helical structure in the presence of 2,2,2- trifluoroethanol (TFE) and in contact with mimetic vesicles/membranes. Therefore, adepamycin represents a novel lytic AMP with dual antibacterial and antifungal properties.

A synthetic peptide derived of the ß2-ß3 loop of the plant defensin from Vigna unguiculata seeds induces Leishmania amazonensis apoptosis-like cell death.

For medical use of proteins and peptide-based drugs, it is desirable to have small biologically active sequences because they improve stability, reduce side effects, and production costs. Several plant defensins have their biological activities imparted by a sequence named γ-core. Vu-Def, a Vigna unguiculata defensin, has activity against Leishmania amazonensis, which is one etiological agent of leishmaniasis and for which new drugs are needed. Our intention was to understand if the region comprising the Vu-Def γ-core is responsible for the biological activity against L. amazonensis and to unveil its mechanism of action. Different microbiological assays with L. amazonensis in the presence of the synthetic peptide A36,42,44γ32-46Vu-Def were done, as well as ultrastructural and fluorescent analyses. A36,42,44γ32-46Vu-Def showed biological activity similar to Vu-Def. A36,42,44γ32-46Vu-Def (74 µM) caused 97% inhibition of L. amazonensis culture and parasites were unable to regrow in fresh medium. The cells of the treated parasites showed morphological alterations by ultrastructural analysis and fluorescent labelings that corroborate with the data of the organelles alterations. The general significance of our work is based on the description of a small synthetic peptide, A36,42,44γ32-46Vu-Def, which has activity on L. amazonensis and that the interaction between A36,42,44γ32-46Vu-Def-L. amazonensis results in parasite inhibition by the activation of an apoptotic-like cell death pathway.

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.

Insight into the α-Amylase Inhibitory Activity of Plant Lipid Transfer Proteins.

We previously characterized the inhibitory activity of human salivary α-amylase (HSA) and Callosobruchus maculatus intestinal α-amylases by the plant lipid transfer protein from Vigna unguiculata ( Vu-LTP). Herein, we further study this inhibitory activity. First by an analysis of protein α-amylase inhibitors complexed with α-amylase, we find that positively charged amino acids of inhibitors interact with the active site of α-amylases and we know that Vu-LTP is rich in positively charged amino acid residues. For this reason, we model Vu-LTP, and based on its three-dimensional structure, we choose five peptides to be synthesized. Herein, we report that two peptides of Vu-LTP are responsible for HSA inhibition. A comparison of primary and tertiary structures of LTPs with and without inhibitory activity against α-amylase, superimposed with the sequence of Vu-LTP mapped for HSA inhibition, reinforces our suggestion that positively charged amino acids in loops are responsible for the inhibition. To prove our observation, one modified peptide is synthesized in which Arg39 is replaced by Gln. This modified peptide loses the HSA inhibitory property presented by the unmodified peptide. Therefore, we describe a new biological active for Vu-LTP, i.e. the α-amylase inhibitory activity that is not a fortuitous biological activity and probably has evolved to perform a biological function which is still unknown. A good candidate should be defense against insects. The results of this study also expand the possible biotechnological applications of LTPs.

The toxic effect of Vu-Defr, a defensin from Vigna unguiculata seeds, on Leishmania amazonensis is associated with reactive oxygen species production, mitochondrial dysfunction, and plasma membrane perturbation.

Plant defensins are plant antimicrobial peptides that present diverse biological activities in vitro, including the elimination of Leishmania amazonensis. Plant defensins are considered promising candidates for the development of new drugs. This protozoan genus has great epidemiological importance and the mechanism behind the protozoan death by defensins is unknown, thus, we chose L. amazonensis for this study. The aim of the work was to analyze the possible toxic mechanisms of Vu-Defr against L. amazonensis. For analyses, the antimicrobial assay was repeated as previously described, and after 24 h, an aliquot of the culture was tested for viability, membrane perturbation, mitochondrial membrane potential, reactive oxygen species (ROS) and nitric oxide (NO) inductions. The results of these analyses indicated that after interaction with L. amazonensis, the Vu-Defr causes elimination of promastigotes from culture, membrane perturbation, mitochondrial membrane collapse, and ROS induction. Our analysis demonstrated that NO is not produced after Vu-Defr and L. amazonensis interaction. In conclusion, our work strives to help to fill the gap relating to effects caused by plant defensins on protozoan and thus better understand the mechanism of action of this peptide against L. amazonensis.

Purification and characterization of peptides from Capsicum annuum fruits which are α-amylase inhibitors and exhibit high antimicrobial activity against fungi of agronomic importance.

Proteins extracted from Capsicum annuum L. fruits were initially subjected to reversed-phase chromatography on HPLC, resulting in eight peptide-rich fractions. All the fractions obtained were tested for their ability to inhibit porcine trypsin and amylase from both human saliva and from larval insect in vitro. All fractions were also tested for their ability to inhibit growth of the phytopathogenic fungi. Several fractions inhibited the activity of human salivary amylase and larval insect amylase, especially fraction Fa5. No fraction tested was found to inhibit trypsin activity, being Fa2 fraction an exception. Interestingly fraction Fa5 also displayed high antimicrobial activity against the species of the Fusarium genus. Fraction Fa5 was found to have two major protein bands of 17 and 6.5 kDa, and these were sequenced by mass spectrometry. Two peptides were obtained from the 6.5-kDa band, which showed similarity to antimicrobial peptides. Fraction Fa5 was also tested for its ability to permeabilize membranes and induce ROS. Fraction Fa5 was able to permeabilize the membranes of all the fungi tested. Fungi belonging to the genus Fusarium also showed an increase in the endogenous production of ROS when treated with this fraction. Antimicrobial peptides were also identified in the fruits from other Capsicum species.

Interaction between the plant ApDef1 defensin and Saccharomyces cerevisiae results in yeast death through a cell cycle- and caspase-dependent process occurring via uncontrolled oxidative stress.

BACKGROUND: Plant defensins were discovered at beginning of the 90s'; however, their precise mechanism of action is still unknown. Herein, we studied ApDef1-Saccharomyces cerevisiae interaction. METHODS: ApDef1-S. cerevisiae interaction was studied by determining the MIC, viability and death kinetic assays. Viability assay was repeated with hydroxyurea synchronized-yeast and pretreated with CCCP. Plasma membrane permeabilization, ROS induction, chromatin condensation, and caspase activation analyses were assessed through Sytox green, DAB, DAPI and FITC-VAD-FMK, respectively. Viability assay was done in presence of ascorbic acid and Z-VAD-FMK. Ultrastructural analysis was done by electron microscopy. RESULTS: ApDef1 caused S. cerevisiae cell death and MIC was 7.8µM. Whole cell population died after 18h of ApDef1 interaction. After 3h, 98.76% of synchronized cell population died. Pretreatment with CCCP protected yeast from ApDef1 induced death. ApDef1-S. cerevisiae interaction resulted in membrane permeabilization, H2O2 increased production, chromatin condensation and caspase activation. Ascorbic acid prevented yeast cell death and membrane permeabilization. Z-VAD-FMK prevented yeast cell death. CONCLUSIONS: ApDef1-S. cerevisiae interaction caused cell death through cell cycle dependentprocess which requires preserved membrane potential. After interaction, yeast went through uncontrolled ROS production and accumulation, which led to plasma membrane permeabilization, chromatin condensation and, ultimately, cell death by activation of caspase-dependent apoptosis via. GENERAL SIGNIFICANCE: We show novel requirements for the interaction between plant defensin and fungi cells, i.e. cell cycle phase and membrane potential, and we indicate that membrane permeabilization is probably caused by ROS and therefore, it would be an indirect event of the ApDef1-S. cerevisiae interaction.

Isolation, characterization and mechanism of action of an antimicrobial peptide from Lecythis pisonis seeds with inhibitory activity against Candida albicans.

Antimicrobial peptides (AMPs) are produced by a range of organisms as a first line of defense against invaders or competitors. Owing to their broad antimicrobial activity, AMPs have attracted attention as a potential source of chemotherapeutic drugs. The increasing prevalence of infections caused by Candida species as opportunistic pathogens in immunocompromised patients requires new drugs. Lecythis pisonis is a Lecythydaceae tree that grows in Brazil. The AMPs produced by this tree have not been described previously. We describe the isolation of 12 fractions enriched in peptides from L. pisonis seeds. Of the 12 fractions, at 10 µg/ml, the F4 fraction had the strongest growth inhibitory effect (53.7%) in Candida albicans, in addition to a loss of viability of 94.9%. The F4 fraction was separated into seven sub-fractions by reversed-phase chromatography. The F4.7' fraction had the strongest activity at 10 µg/ml, inhibiting C. albicans growth by 38.5% and a 69.3% loss of viability. The peptide in F4.7' was sequenced and was found to be similar to plant defensins. For this reason, the peptide was named L. pisonis defensin 1 (Lp-Def1). The mechanism of action that is responsible for C. albicans inhibition by Lp-Def1 includes a slight increase of reactive oxygen species induction and a significant loss of mitochondrial function. The results described here support the future development of plant defensins, specifically Lp-Def1, as new therapeutic substances against fungi, especially C. albicans.

Bioinspired peptides induce different cell death mechanisms against opportunistic yeasts.

The management of fungal diseases imposes an urgent need for the development of effective antifungal drugs. Among new drug candidates are the antimicrobial peptides, and especially their derivatives. Here, we investigated the molecular mechanism of action of three bioinspired peptides against the opportunistic yeasts Candida tropicalis and Candida albicans. We assessed morphological changes, mitochondrial functionality, chromatin condensation, ROS production, activation of metacaspases, and the occurrence of cell death. Our results indicated that the peptides induced sharply contrasting death kinetics, of 6 h for RR and 3 h for D-RR to C. tropicalis and 1 h for WR to C. albicans. Both peptide-treated yeasts exhibited increased ROS levels, mitochondrial hyperpolarization, cell size reduction, and chromatin condensation. RR and WR induced necrosis in C. tropicalis and C. albicans, but not D-RR in C. tropicalis. The antioxidant ascorbic acid reverted the toxic effect of RR and D-RR, but not WR, suggesting that instead of ROS there is a second signal triggered that leads to yeast death. Our data suggest that RR induced a regulated accidental cell death in C. tropicalis, D-RR induced a programmed cell death metacaspase-independent in C. tropicalis, while WR induced an accidental cell death in C. albicans. Our results were obtained with the LD100 and within the time that the peptides induce the yeast death. Within this temporal frame, our results allow us to gain clarity on the events triggered by the peptide-cell interaction and their temporal order, providing a better understanding of the death process induced by them.

Unveiling the Anatomical and Functional Attributes of Stipular Colleters in Palicourea tetraphylla Cham. & Schltdl. and Palicourea rudgeoides (Müll. Arg.) Standl. (Rubiaceae).

The characterization of colleters in Rubiaceae is crucial for understanding their role in plant function. Analyzing colleters in Palicourea tetraphylla and Palicourea rudgeoides aims to deepen the understanding of these structures morphoanatomical and functional characteristics. The study reveals colleters with palisade epidermis and a parenchymatic central axis, classified as standard type, featuring vascularization and crystals. Colleter secretion, abundant in acidic mucopolysaccharides, proteins, and phenolic compounds, protects against desiccation. The ontogenesis, development, and senescence of the colleters are quite rapid and fulfill their role well in biotic and abiotic protection because these structures are present at different stages of development in the same stipule. Pronounced protrusions on the colleters surface, coupled with the accumulation of secretion in the intercellular and subcuticular spaces, suggest that the secretory process occurs through the wall, driven by pressure resulting from the accumulation of secretion. The microorganisms in the colleters' secretion, especially in microbiota-rich environments such as the Atlantic Forest, provide valuable information about plant-microorganism interactions, such as resistance to other pathogens and organisms and ecological balance. This enhanced understanding of colleters contributes to the role of these structures in the plant and enriches knowledge about biological interactions within specific ecosystems and the family taxonomy.

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.

Activity of recombinant and natural defensins from Vigna unguiculata seeds against Leishmania amazonensis.

Antimicrobial peptides (AMPs), which are differentiated from other antibiotic peptides, such as gramicidins and polymyxins, because they are synthesized by large enzymatic complex and bear modified amino acids including d-amino acids, are short polymers of l-amino acids synthesized by ribosomes upon which all living organisms rely to defend themselves from invaders or competitor microorganisms. AMPs have received a great deal of attention from the scientific community as potential new drugs for neglected diseases such as Leishmaniasis. In plants, they include several families of compounds, including the plant defensins. The aim of the present study was to improve the expression of recombinant defensin from Vigna unguiculata seeds (Vu-Defr) and to test its activity against Leishmania amazonensis promatigotes. Recombinant expression was performed in LB and TB media and under different conditions. The purification of Vu-Defr was achieved by immobilized metal ion affinity and reversed-phase chromatography. The purified Vu-Defr was analyzed by circular dichroism (CD), and its biological activity was tested against L. amazonenis promastigotes. To demonstrate that the recombinant production of Vu-Defr did not interfere with its fold and biological activity, the results of all experiments were compared with the results from the natural defensin (Vu-Def). The CD spectra of both peptides presented good superimposition indicating that both peptides present very similar secondary structure and that the Vu-Defr was correctly folded. L. amazonensis treated with Vu-Defr led to the elimination of 54.3% and 46.9% of the parasites at 24 and 48h of incubation time, respectively. Vu-Def eliminated 50% and 54.8% of the parasites at 24 and 48 h, respectively. Both were used at a concentration of 100 µg/mL. These results suggested the potential for plant defensins to be used as new antiparasitic substances.

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.

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.

Antifungal, Antimycobacterial, Protease and α‒Amylase Inhibitory Activities of a Novel Serine Bifunctional Protease Inhibitor from Adenanthera pavonina L. Seeds.

Antifungal resistance poses a significant challenge to disease management, necessitating the development of novel drugs. Antimicrobial peptides offer potential solutions. This study focused on extraction and characterization of peptides from Adenanthera pavonina seeds with activity against Candida species, Mycobacterium tuberculosis, proteases, and α-amylases. Peptides were extracted in phosphate buffer and heated at 90°C for 10 min to create a peptide rich heated fraction (PRHF). After confirming antimicrobial activity and the presence of peptides, the PRHF underwent ion exchange chromatography, yielding retained and non-retained fractions. These fractions were evaluated for antimicrobial activity and cytotoxicity against murine macrophages. The least toxic and most active fraction underwent reversed-phase chromatography, resulting in ten fractions. These fractions were tested for peptides and antimicrobial activity. The most active fraction was rechromatographed on a reversed-phase column, resulting in two fractions that were assessed for antimicrobial activity. The most active fraction revealed a single band of approximately 6 kDa and was tested for inhibitory effects on proteases and α-amylases. Thermal stability experiments were conducted on the 6 kDa peptide at different temperatures followed by reassessment of antifungal activity and circular dichroism. The 6 kDa peptide inhibited yeasts, M. tuberculosis, human salivary and Tenebrio molitor larvae intestine α-amylases, and proteolytic activity from fungal extracts, and thus named ApPI. Remarkably, ApPI retained antifungal activity and conformation after heating and is primarily composed of α-helices. ApPI is a thermally stable serine protease/α-amylase inhibitor from A. pavonina seeds, offering promise as a foundational molecule for innovative therapeutic agents against fungal infections and tuberculosis.

Structural and Biochemical Characterization of Three Antimicrobial Peptides from Capsicum annuum L. var. annuum Leaves for Anti-Candida Use.

The emergence of resistant microorganisms has reduced the effectiveness of currently available antimicrobials, necessitating the development of new strategies. Plant antimicrobial peptides (AMPs) are promising candidates for novel drug development. In this study, we aimed to isolate, characterize, and evaluate the antimicrobial activities of AMPs isolated from Capsicum annuum. The antifungal potential was tested against Candida species. Three AMPs from C. annuum leaves were isolated and characterized: a protease inhibitor, a defensin-like protein, and a lipid transporter protein, respectively named CaCPin-II, CaCDef-like, and CaCLTP2. All three peptides had a molecular mass between 3.5 and 6.5 kDa and caused morphological and physiological changes in four different species of the genus Candida, such as pseudohyphae formation, cell swelling and agglutination, growth inhibition, reduced cell viability, oxidative stress, membrane permeabilization, and metacaspase activation. Except for CaCPin-II, the peptides showed low or no hemolytic activity at the concentrations used in the yeast assays. CaCPin-II inhibited α-amylase activity. Together, these results suggest that these peptides have the potential as antimicrobial agents against species of the genus Candida and can serve as scaffolds for the development of synthetic peptides for this purpose.