Synergistic action of synthetic peptides and amphotericin B causes disruption of the plasma membrane and cell wall in Candida albicans.

The objective of this work was to evaluate the combination of synthetic peptides based on the γ-core motif of defensin PvD1 with amphotericin B (AmB) at different concentrations against Candida albicans. We applied the checkerboard assay using different concentrations of the commercial drug AmB and the synthetic peptides γ31-45PvD1++ and γ33-41PvD1++ against C. albicans, aiming to find combinations with synergistic interactions. Between these two interactions involving γ31-45PvD1++ and AmB, an additive effect was observed. One such interaction occurred at concentrations of 0.009 µM of peptide γ31-45PvD1++ and 13.23 µM of AmB and another condition of 0.019 µM of peptide γ31-45PvD1++ and 6.61 µM of AmB. The other two concentrations of the interaction showed a synergistic effect in the combination of synthetic peptide γ31-45PvD1++ and AmB, where the concentrations were 1.40 µM peptide γ31-45PvD1++ and 0.004 µM AmB and 0.70 µM γ31-45PvD1++ peptide and 0.002 µM AmB. We proceeded with analysis of the mechanism of action involving synergistic effects. This examination unveiled a range of impactful outcomes, including the impairment of mitochondrial functionality, compromise of cell wall integrity, DNA degradation, and a consequential decline in cell viability. We also observed that both synergistic combinations were capable of causing damage to the plasma membrane and cell wall, causing leakage of intracellular components. This discovery demonstrates for the first time that the synergistic combinations found between the synthetic peptide γ31-45PvD1++ and AmB have an antifungal effect against C. albicans, acting on the integrity of the plasma membrane and cell wall.

Anti-Candida Potential of Peptides from Immature and Ripe Fruits of Capsicum chinense Jacq.

The objective of this work was to purify and evaluate the antifungal potential of peptides present in immature and ripe fruits of Capsicum chinense Jacq. (accession UENF 1706) on the medical importance yeasts. Initially the proteins of these seedless fruits were extracted, precipitated with ammonium sulfate at 70% saturation, followed by heating at 80 °C. Subsequently, the peptide-rich extract was fractionated by DEAE-Sepharose anion exchange. The whole process was monitored by tricine-SDS-PAGE. The results revealed that the fraction retained in anion exchange column, called D2, of immature and ripe fruits significantly inhibit the growth of Candida albicans and C. tropicalis yeasts. Due to the higher yield, the D2 fraction of immature fruits was selected for further purification by reverse phase chromatography on HPLC, where sixteen different fractions (H1-H16) were obtained and these were subjected to antifungal assay at 50 µg mL-1. Although almost all fractions tested had significant growth inhibition, the HI9 fraction inhibit 99% of the two yeasts tested. The effect of treatment with HI3, HI8, HI9, and HI14 fractions on the viability of yeast cells was analyzed due to their strong growth inhibition. We observed that only 50 µg mL-1 of the HI9 fraction is the lethal dose for 100% of the cells of C. albicans and C. tropicalis in the original assay. Although the HI9 fraction had a fungicidal effect on both tested yeasts, we only observed membrane permeabilization for C. tropicalis cells treated with 50 µg mL-1 of this fraction. Through mass spectrometry, we identified that the 6 kDa peptide band of HI9 fraction showed similarity with antimicrobial peptides belonging to the plant defensin family.

A plant mannose-binding lectin and fluconazole: Key targets combination against Candida albicans.

AIMS: This study aimed to evaluate the combined effect of a mannose-binding lectin Helja with fluconazole (FLC) on Candida albicans and to get insights about the joint action mechanism. METHODS AND RESULTS: The fungal growth was assessed following the optical density at 630 nm. Fungal cell morphology and nucleus integrity were analysed by flow cytometry and confocal laser scanning microscopy using Calcofluor White (CFW) and 4',6-diamidino-2-phenylindole (DAPI) staining respectively. The basis of Helja + FLC action on cell wall and plasma membrane was analysed using perturbing agents. The Helja + FLC combination exhibited an inhibitory effect of fungal growth about three times greater than the sum of both compounds separately and inhibited fungal morphological plasticity, an important virulence attribute associated with drug resistance. Cells treated with Helja + FLC showed morphological changes, nucleus disintegration and formation of multimera structures, leading to cell collapse. CONCLUSIONS: Our findings indicate that the Helja + FLC combination exhibited a potent antifungal activity based on their simultaneous action on different microbial cell targets. SIGNIFICANCE AND IMPACT OF STUDY: The combination of a natural protein with conventional drugs might be helpful for the design of effective therapeutic strategies against Candida, contributing to minimize the development of drug resistance and host cell toxicity.

Differential interactions of the antimicrobial peptide, RQ18, with phospholipids and cholesterol modulate its selectivity for microorganism membranes.

BACKGROUND: Antimicrobial peptides (AMPs) are molecules with potential application for the treatment of microorganism infections. We, herein, describe the structure, activity, and mechanism of action of RQ18, an α-helical AMP that displays antimicrobial activity against Gram-positive and Gram-negative bacteria, and yeasts from the Candida genus. METHODS: A physicochemical-guided design assisted by computer tools was used to obtain our lead peptide candidate, named RQ18. This peptide was assayed against Gram-positive and Gram-negative bacteria, yeasts, and mammalian cells to determine its selectivity index. The secondary structure and the mechanism of action of RQ18 were investigated using circular dichroism, large unilamellar vesicles, and molecular dynamic simulations. RESULTS: RQ18 was not cytotoxic to human lung fibroblasts, peripheral blood mononuclear cells, red blood cells, or Vero cells at MIC values, exhibiting a high selectivity index. Circular dichroism analysis and molecular dynamic simulations revealed that RQ18 presents varying structural profiles in aqueous solution, TFE/water mixtures, SDS micelles, and lipid bilayers. The peptide was virtually unable to release carboxyfluorescein from large unilamellar vesicles composed of POPC/cholesterol, model that mimics the eukaryotic membrane, indicating that vesicles' net charges and the presence of cholesterol may be related with RQ18 selectivity for bacterial and fungal cell surfaces. CONCLUSIONS: RQ18 was characterized as a membrane-active peptide with dual antibacterial and antifungal activities, without compromising mammalian cells viability, thus reinforcing its therapeutic application. GENERAL SIGNIFICANCE: These results provide further insight into the complex process of AMPs interaction with biological membranes, in special with systems that mimic prokaryotic and eukaryotic cell surfaces.

Potent Anti-Candida Fraction Isolated from Capsicum chinense Fruits Contains an Antimicrobial Peptide That is Similar to Plant Defensin and is Able to Inhibit the Activity of Different α-Amylase Enzymes.

Antimicrobial peptides (AMPs) are molecules present in several life forms, possess broad-spectrum of inhibitory activity against pathogenic microorganisms, and are a promising alternative to combat the multidrug resistant pathogens. The aim of this work was to identify and characterize AMPs from Capsicum chinense fruits and to evaluate their inhibitory activities against yeasts of the genus Candida and α-amylases. Initially, after protein extraction from fruits, the extract was submitted to anion exchange chromatography resulting two fractions. Fraction D1 was further fractionated by molecular exclusion chromatography, and three fractions were obtained. These fractions showed low molecular mass peptides, and in fraction F3, only two protein bands of approximately 6.5 kDa were observed. Through mass spectrometry, we identified that the lowest molecular mass protein band of fraction F3 showed similarity with AMPs from plant defensin family. We named this peptide CcDef3 (Capsicum chinense defensin 3). The antifungal activity of these fractions was analyzed against yeasts of the genus Candida. At 200 µg/mL, fraction F1 inhibited the growth of C. tropicalis by 26%, fraction F2 inhibited 35% of the growth of C. buinensis, and fraction F3 inhibited all tested yeasts, exhibiting greater inhibition activity on the growth of the yeast C. albicans (86%) followed by C. buinensis (69%) and C. tropicalis (21%). Fractions F1 and F2 promoted membrane permeabilization of all tested yeasts and increased the endogenous induction of reactive oxygen species (ROS) in C. buinensis and C. tropicalis, respectively. We also observed that fraction F3 at a concentration of 50 µg/mL inhibited the α-amylase activities of Tenebrio molitor larvae by 96% and human salivary by 100%. Thus, our results show that fraction F3, which contains CcDef3, is a very promising protein fraction because it has antifungal potential and is able to inhibit the activity of different α-amylase enzymes.

Biochemical analysis of antimicrobial peptides in two different Capsicum genotypes after fruit infection by Colletotrichum gloeosporioides.

There are several phytosanitary problems that have been causing serious damage to the Capsicum crops, including anthracnose. Upon attack by certain pathogens, various protein molecules are produced, which are known as proteins related to pathogenesis (PR proteins), including antimicrobial peptides such as protease inhibitors, defensins and lipid transfer proteins (LTPs). The objective of this work is to identify antimicrobial proteins and/or peptides of two genotypes from Capsicum annuum fruits infected with Colletotrichum gloeosporioides The fungus was inoculated into Capsicum fruits by the deposition of a spore suspension (106 conidia ml-1), and after 24 and 48 h intervals, the fruits were removed from the humid chamber and subjected to a protein extraction process. Protein analysis of the extracts was performed by tricine gel electrophoresis and Western blotting. The distinctive bands between genotypes in the electrophoresis profiles were subjected to mass spectrometry sequencing. Trypsin inhibition assays, reverse zymographic detection of protease inhibition and ß-1,3-glucanase activity assays were also performed and extracts were also tested for their ability to inhibit the growth of C. gloeosporioides fungi 'in vitro' There were several low molecular weight proteins in all treated samples, and some treatments in which antimicrobial peptides such as defensin, lipid transfer protein (LTP) and protease inhibitor have been identified. It was shown that the green fruits are more responsive to infection, showing the production of antimicrobial peptides in response to injury and inoculation of the fungus, what did not occur in ripe fruits under any treatment.

Coffea canephora Peptides in Combinatorial Treatment with Fluconazole: Antimicrobial Activity against Phytopathogenic Fungus.

The objective of the present study was to evaluate the antimicrobial activity of the Cc-LTP2 and Cc-GRP peptides isolated from Coffea canephora seeds and their possible synergistic activity with the azole drug fluconazole and characterize their mechanisms of action on cells of pathogenic fungi. Cc-LTP2 and Cc-GRP alone or in combination with 20 µg/mL of fluconazole were evaluated for their antimicrobial activity on the fungus Fusarium solani, and the effects of these peptides on the permeability of membranes and the induction of oxidative stress were determined. Our results show that these peptides at a concentration of 400 µg/mL combined with 20 µg/mL of fluconazole were able to inhibit the growth of the tested fungi, promote changes in their growth pattern, permeabilize the membrane, and induce reactive oxygen species (ROS). Some of these results were also observed with the peptides alone or with fluconazole alone, suggesting that the peptides act synergistically, promoting the potentiation of antimicrobial action. In this study, it was shown that Cc-LTP2 and Cc-GRP in combination with fluconazole were able to inhibit the growth of the fungus F. solani, to promote permeabilization of its membrane, and to induce the production of ROS, suggesting a combinatorial activity between the peptides and fluconazole.

Programmed cell death in yeast by thionin-like peptide from Capsicum annuum fruits involving activation of caspases and extracellular H+ flux.

CaThi is a thionin-like peptide isolated from fruits of Capsicum annuum, which has strong antimicrobial activity against bacteria, yeasts and filamentous fungi, and induced reactive oxygen species (ROS) in fungi. ROS are molecules that appear in the early stages of programmed cell death or apoptosis in fungi. Due to this fact, in this work we analyzed some events that may be related to process of apoptosis on yeast induced by CaThi. To investigate this possibility, we evaluated phosphatidylserine (PS) externalization, presence of active caspases and the ability of CaThi to bind to DNA in Candida tropicalis cells. Additionally, we investigated mitochondrial membrane potential, cell surface pH, and extracellular H+ fluxes in C. tropicalis cells after treatment with CaThi. Our results showed that CaThi induced PS externalization in the outer leaflet of the cell membrane, activation of caspases, and it had the ability for DNA binding and to dissipate mitochondrial membrane potential. In addition, the cell surface pH increased significantly when the C. tropicalis cells were exposed to CaThi which corroborates with ~96% inhibition on extracellular H+ efflux. Taking together, these data suggest that this peptide is capable of promoting an imbalance in pH homeostasis during yeast cell death playing a modulatory role in the H+ transport systems. In conclusion, our results strongly indicated that CaThi triggers apoptosis in C. tropicalis cells, involving a pH signaling mechanism.

Application and bioactive properties of CaTI, a trypsin inhibitor from Capsicum annuum seeds: membrane permeabilization, oxidative stress and intracellular target in phytopathogenic fungi cells.

BACKGROUND: During the last few years, a growing number of antimicrobial peptides have been isolated from plants and particularly from seeds. Recent results from our laboratory have shown the purification of a new trypsin inhibitor, named CaTI, from chilli pepper (Capsicum annuum L.) seeds. This study aims to evaluate the antifungal activity and mechanism of action of CaTI on phytopathogenic fungi and detect the presence of protease inhibitors in other species of this genus. RESULTS: Our results show that CaTI can inhibit the growth of the phytopathogenic fungi Colletotrichum gloeosporioides and C. lindemuthianum. CaTI can also permeabilize the membrane of all tested fungi. When testing the inhibitor on its ability to induce reactive oxygen species, an induction of reactive oxygen species (ROS) and nitric oxide (NO) particularly in Fusarium species was observed. Using CaTI coupled to fluorescein isothiocyanate (FITC), it was possible to determine the presence of the inhibitor inside the hyphae of the Fusarium oxysporum fungus. The search for protease inhibitors in other Capsicum species revealed their presence in all tested species. CONCLUSION: This paper shows the antifungal activity of protease inhibitors such as CaTI against phytopathogenic fungi. Antimicrobial peptides, among which the trypsin protease inhibitor family stands out, are present in different species of the genus Capsicum and are part of the chemical arsenal that plants use to defend themselves against pathogens. © 2017 Society of Chemical Industry.

Antimicrobial activity and mechanism of action of a thionin-like peptide from Capsicum annuum fruits and combinatorial treatment with fluconazole against Fusarium solani.

Many Fusarium species are able to cause severe infections in plants as well as in animals and humans. Therefore, the discovery of new antifungal agents is of paramount importance. CaThi belongs to the thionins, which are cationic peptides with low molecular weights (∼5 kDa) that have toxic effects against various microorganisms. Herein, we study the mechanism of action of CaThi and its combinatory effect with fluconazole (FLC) against Fusarium solani. The mechanism of action of CaThi was studied by growth inhibition, viability, plasma membrane permeabilization, ROS induction, caspase activation, localization, and DNA binding capability, as assessed with Sytox green, DAB, FITC-VAD-FMK, CaThi-FITC, and gel shift assays. The combinatory effect of CaThi and FLC was assessed using a growth inhibition assay. Our results demonstrated that CaThi present a dose dependent activity and at the higher used concentration (50 µg mL-1 ) inhibits 83% of F. solani growth, prevents the formation of hyphae, permeabilizes membranes, induces endogenous H2 O2 , activates caspases, and localizes intracellularly. CaThi combined with FLC, at concentrations that alone do not inhibit F. solani, result in 100% death of F. solani when combined. The data presented in this study demonstrate that CaThi causes death of F. solani via apoptosis; an intracellular target may also be involved. Combined treatment using CaThi and FLC is a strong candidate for studies aimed at improved targeting of F. solani. This strategy is of particular interest because it minimizes selection of resistant microorganisms.

Thionin-like peptide from Capsicum annuum fruits: mechanism of action and synergism with fluconazole against Candida species.

BACKGROUND: Thionins are a family of plant antimicrobial peptides (AMPs), which participate in plant defense system against pathogens. Here we describe some aspects of the CaThi thionin-like action mechanism, previously isolated from Capsicum annuum fruits. Thionin-like peptide was submitted to antimicrobial activity assays against Candida species for IC50 determination and synergism with fluconazole evaluation. Viability and plasma membrane permeabilization assays, induction of intracellular ROS production analysis and CaThi localization in yeast cells were also investigated. RESULTS: CaThi had strong antimicrobial activity against six tested pathogenic Candida species, with IC50 ranging from 10 to 40 µg.mL(-1). CaThi antimicrobial activity on Candida species was candidacidal. Moreover, CaThi caused plasma membrane permeabilization in all yeasts tested and induces oxidative stresses only in Candida tropicalis. CaThi was intracellularly localized in C. albicans and C. tropicalis, however localized in nuclei in C. tropicalis, suggesting a possible nuclear target. CaThi performed synergistically with fluconazole inhibiting all tested yeasts, reaching 100% inhibition in C. parapsilosis. The inhibiting concentrations for the synergic pair ranged from 1.3 to 4.0 times below CaThi IC50 and from zero to 2.0 times below fluconazole IC50. CONCLUSION: The results reported herein may ultimately contribute to future efforts aiming to employ this plant-derived AMP as a new therapeutic substance against yeasts.

Antimicrobial Activity of ILTI, a Kunitz-Type Trypsin Inhibitor from Inga laurina (SW.) Willd.

Over the last few years, a growing number of proteinase inhibitors have been isolated from plants and particularly from seeds and have shown antimicrobial activity. A 20,000 Da serine peptidase inhibitor, named ILTI, was isolated from Inga laurina seeds and showed potent inhibitory enzymatic activity against trypsin. The aim of this study was to determine the effects of ILTI on the growth of pathogenic and non-pathogenic microorganisms. We observed that ILTI strongly inhibited in particular the growth of Candida tropicalis and Candida buinensis, inducing cellular agglomeration. However, it was ineffective against human pathogenic bacteria. We also investigated the potential of ILTI to permeabilize the plasma membrane of yeast cells. C. tropicalis and C. buinensis were incubated for 24 h with the ILTI at different concentrations, which showed that this inhibitor induced changes in the membranes of yeast cells, leading to their permeabilization. Interestingly, ILTI induced the production of reactive oxygen species (ROS) in C. tropicalis and C. buinensis cells. Finally, ILTI was coupled with fluorescein isothiocyanate, and subsequent treatment of C. tropicalis and C. buinensis with DAPI revealed the presence of the labeled protein in the intracellular spaces. In conclusion, our results indicated the ability of peptidase inhibitors to induce microbial inhibition; therefore, they might offer templates for the design of new antifungal agents.

A sunflower lectin with antifungal properties and putative medical mycology applications.

Lectins are carbohydrate-binding proteins with a high specificity for a variety of glycoconjugate sugar motifs. The jacalin-related lectins (JRL) are considered to be a small sub-family composed of galactose- and mannose-specific members. Using a proteomics approach, we have detected a 16 kDa protein (Helja) in sunflower seedlings that were further purified by mannose-agarose affinity chromatography. The aim of this work was to characterize the biological activity of Helja and to explore potential applications for the antifungal activity of this plant lectin against medically important yeasts. To initially assess the agglutination properties of the lectin, Saccharomyces cerevisiae cells were incubated with increasing concentrations of the purified lectin. At a concentration of 120 µg/ml, Helja clearly agglutinated these cells. The ability of different sugars to inhibit S. cerevisiae cell agglutination determined its carbohydrate-specificity. Among the monosaccharides tested, D-mannose had the greatest inhibitory effect, with a minimal concentration of 1.5 mM required to prevent cell agglutination. The antifungal activity was evaluated using pathogenic fungi belonging to the Candida and Pichia genera. We demonstrate that 200 µg/ml of Helja inhibited the growth of all yeasts, and it induced morphological changes, particularly through pseudohyphae formation on Candida tropicalis. Helja alters the membrane permeability of the tested fungi and is also able to induce the production of reactive oxygen species in C. tropicalis cells. We concluded that Helja is a mannose-binding JRL with cell agglutination capabilities and antifungal activity against yeasts. The biological properties of Helja may have practical applications in the control of human pathogens.

Thionin-like peptides from Capsicum annuum fruits with high activity against human pathogenic bacteria and yeasts.

Plants defend themselves against pathogens with production of antimicrobial peptides (AMPs). Herein we describe the discovery of a new antifungal and antibacterial peptide from fruits of Capsicum annuum that showed similarity to an already well characterized family of plant AMPs, thionins. Other fraction composed of two peptides, in which the major peptide also showed similarity to thionins. Among the obtained fractions, fraction 1, which is composed of a single peptide of 7 kDa, was sequenced by Edman method and its comparative sequence analysis in database (nr) showed similarity to thionin-like peptides. Tests against microorganisms, fraction 1 presented inhibitory activity to the cells of yeast Saccharomyces cerevisiae, Candida albicans, and Candida tropicalis and caused growth reduction to the bacteria species Escherichia coli and Pseudomonas aeruginosa. Fraction 3 caused inhibitory activity only for C. albicans and C. tropicalis. This fraction was composed of two peptides of ∼7 and 10 kDa, and the main protein band correspondent to the 7 kDa peptide, also showed similarity to thionins. This plasma membrane permeabilization assay demonstrates that the peptides present in the fractions 1 and 3 induced changes in the membranes of all yeast strains, leading to their permeabilization. Fraction 1 was capable of inhibiting acidification of the medium of glucose-induced S. cerevisiae cells 78% after an incubation time of 30 min, and opposite result was obtained for C. albicans. Experiments demonstrate that the fraction 1 and 3 were toxic and induced changes in the membranes of all yeast strains, leading to their permeabilization.

New small proteinase inhibitors from Capsicum annuum seeds: Characterization, stability, spectroscopic analysis and a cDNA cloning.

Recent results from our laboratory have previously shown the purification of a small serine proteinase inhibitor (PI), named CaTI1, from Capsicum annuum seeds. This work demonstrated the characterization of CaTI now named CaTI1, and the identification of two other small serine PIs, named CaTI2 and CaTI3, also present in these seeds. CaTI1 presented molecular mass of 6 kDa and pI value of ∼9.0. CaTI1 inhibited both trypsin and chymotrypsin with inhibition constants (Ki and Ki') of 14 and 2.8 nM for trypsin and 4.3 and 0.58 nM for chymotrypsin, respectively. Circular dichroism analysis suggested the predominance of both disordered and ß-strands regions in the secondary structure. CaTI1 presented striking physico-chemical stability. In an attempt to get the entire sequence of CaTI1 we found another PI called CaTI2. The discussion of this finding is in the main text. A degenerate primer was designed based on the sequence of trypsin inhibitor CaTI1 in an attempt to achieve the cloning of this PI. Surprisingly, the alignment of the predicted peptide derived from the cDNA with the protein database showed similarity with other C. annuun PIs, and thus it was called CaTI3.

Antifungal and other biological activities of two 2S albumin-homologous proteins against pathogenic fungi.

The aim of this study was to determine whether 2S albumins from Passiflora edulis f. flavicarpa and Capsicum annuum seeds inhibit growth, induce plasma membrane permeabilization and induce endogenous production of nitric oxide in different pathogenic and non-pathogenic yeasts. The 2S albumin from P. flavicarpa (Pf-Alb) inhibited the growth of Kluyveromyces marxiannus, Candida albicans and Candida parapsilosis. The membranes of these yeast strains were permeabilized in the presence of Pf-Alb. The Pf-Alb also inhibited the glucose-stimulated acidification of the medium by Saccharomyces cerevisiae and C. albicans cells, which indicates a probable impairment of fungal metabolism because the inhibition of acidification occurred at various Pf-Alb concentrations and pre-incubation times. The 2S albumin from C. annuum (Ca-Alb) inhibited the growth of the yeasts K. marxiannus, C. tropicalis, C. albicans and S. cerevisiae. These yeast strains exhibited NO induction in the presence of Ca-Alb and displayed cellular agglomeration, elongated cells and the induction of pseudohyphae. Pf-Alb and Ca-Alb at various concentrations also inhibited the glucose-stimulated acidification of the medium by S. cerevisiae cells. Our results indicate that the ability of antimicrobial plant proteins such as 2S albumins to induce microbial inhibition could be an important factor in determining pathogen virulence. Therefore, 2S albumins might be targets for the design of new antifungal drugs.