The Antimicrobial Peptide γ-Thionin from Habanero Chile (Capsicum chinense) Induces Caspase-Independent Apoptosis on Human K562 Chronic Myeloid Leukemia Cells and Regulates Epigenetic Marks.

Cancer is a relevant health problem worldwide. In 2020, leukemias represented the 13th most commonly reported cancer cases worldwide but the 10th most likely to cause deaths. There has been a progressive increase in the efficacy of treatments for leukemias; however, these still generate important side effects, so it is imperative to search for new alternatives. Defensins are a group of antimicrobial peptides with activity against cancer cells. However, the cytotoxic mechanism of these peptides has been described mainly for animal defensins. This study shows that defensin γ-thionin (Capsicum chinense) is cytotoxic to the K562 leukemia cells with an IC50 = 290 µg/mL (50.26 µM) but not for human peripheral blood mononuclear cells. Results showed that γ-thionin did not affect the membrane potential; however, the peptide modified the mitochondrial membrane potential (ΔΨm) and the intracellular calcium release. In addition, γ-thionin induced apoptosis in K562 cells, but the activation of caspases 8 and 9 was not detected. Moreover, the activation of calpains was detected at one hour of treatment, suggesting that γ-thionin activates the caspase-independent apoptosis. Furthermore, the γ-thionin induced epigenetic modifications on histone 3 in K562 cells, increased global acetylation (~2-fold), and specific acetylation marks at lysine 9 (H3K9Ac) (~1.5-fold). In addition, γ-thionin increased the lysine 9 methylation (H3K9me) and dimethylation marks (H3K9me2) (~2-fold), as well as the trimethylation mark (H3K9me3) (~2-fold). To our knowledge, this is the first report of a defensin that triggers caspase-independent apoptosis in cancer cells via calpains and regulating chromatin remodelation, a novel property for a plant defensin.

The plant defensins PaDef and γ-thionin inhibit the endothelial cell response to VEGF.

Angiogenesis is involved in wound repair and tissue maintenance but is associated with diverse diseases. Pro-angiogenic factors such as vascular endothelial growth factor (VEGF) regulate this process. Therefore, searching for treatments to inhibit or promote angiogenesis is attractive. Reports from our group showed that plant antimicrobial peptides (PAPs) PaDef from avocado and γ-thionin from habanero pepper are cytotoxic on cancer cells. However, their functions as angiogenic regulators are unknown. In this work, we evaluate the effect of PaDef and γ-thionin on the angiogenic processes of two different endothelial cell lines: bovine endothelial cells (BUVEC) and the human endothelial cell line EA.hy926. The results showed that VEGF (10 ng/mL) stimulated the BUVEC (40 ± 7 %) and EA.hy926 cell proliferation (30 ± 9 %); however, peptides (5-500 ng/mL) reverted this effect. Besides, VEGF increased the migration of BUVEC (20 ± 8 %) and EA.hy926 cells (50 ± 6 %), but both PAPs (5 ng/mL) inhibited the VEGF stimulus (100 %). Furthermore, DMOG 50 µM (an inhibitor of HIF-hydroxylase) was used in BUVEC and EA.hy926 cells to determine the effect of hypoxia on VEGF and peptide activities. The DMOG reverted the inhibitory action of both peptides (100 %), indicating that peptides act through a HIF-independent pathway. Also, the PAPs do not affect the tube formation but decrease it in EA.hy926 cells stimulated with VEGF (100 %). Additionally, docking assays showed a possible interaction between PAPs and the VEGF receptor. These results suggest that plant defensins PaDef and γ-thionin are potential angiogenic modulators of the VEGF activity on endothelial cells.

Pharmacokinetic profile of sarcin and thionin from Aspergillus giganteus and in vitro validation against human fungal pathogen.

Fungal infections are more predominant in agricultural and clinical fields. Aspergillosis caused by Aspergillus fumigatus leads to respiratory failure in patients along with various illnesses. Due to the limitation of antifungal therapy and antifungal drugs, there is an emergence to develop efficient antifungal compounds (AFCs) from natural sources to cure and prevent fungal infections. The present study deals with the investigation of the mechanism of the active compounds from Aspergillus giganteus against aspergillosis. Primarily, the bioavailability and toxicological properties of antifungal proteins such as, sarcin, thionin, chitinase and their derivatives have proved the efficiency of pharmacokinetic properties of selected compounds. Molecular interactions of selected compounds from A. giganteus with the virulence proteins of A. fumigatus (UDP-N-acetylglucosamine pyrophosphorylase, N-myristoyl transferase and Chitinase) have exhibited a good glide score and druggable nature of the AFCs. The antagonistic potential of AFCs on the pathogen was confirmed by SEM analysis where the shrunken and damaged spores of AFCs treated pathogen were observed. The integrity of A. fumigatus cell membrane and nuclear membrane treated with AFCs were analyzed by determining the release of cellular materials. The effective concentration of AFCs was found to be 250 µg/ml (P<0.0001). The GC-MS profiling has revealed the volatile bioactive metabolites present in A. giganteus. Further, interaction studies might provide more information on the synergism activity with the non-volatile metabolites which leads to the development of novel drugs for the treatment of aspergillosis.

Comprehensive Structural and Functional Characterization of a Seed γ-thionin as a Potent Bioactive Molecule Against Fungal Pathogens and Insect Pests.

BACKGROUND: Fungi and insect pests ruin stored crop grain, which results in millions of dollars of damage, presenting an ongoing challenge for farmers in addition to diminishing the safety of stored food. A wide-range defensive system against pathogens is needed to reduce or even eliminate the dependence of the crop yield upon the use of pesticides. Plant defensins (γ-thionins) are antimicrobial peptides (AMPs) that are a component of the host defense system. They are known to interact with cell membranes to exhibit antifungal and insecticidal activity. They exhibit a broad range of activities against fungi and insects and are effective at low concentrations. Thionins act on membranes, greatly reducing the development of pathogen resistance. OBJECTIVE: The aim of this study is to investigate a bioactive molecule that acts against fungal pathogens and stored grain insect pests. METHODS: γ-thionin protein was extracted from Brassica oleracea L. var. capitata f. alba (white cabbage) seed powder in phosphate buffer (100 mM, pH 7.0) and was identified by MALDI-TOF/TOF. The crude extract was subjected to 70% ammonium sulfate saturation followed by gel filtration chromatography. The disc diffusion assay along with a microtiter bioassay was used to determine the antifungal activity of the protein against phytopathogenic fungi. The insecticidal efficacy was evaluated by feeding insect pests with food contaminated with the purified protein. Additionally, an in silico molecular structure prediction study of the protein was performed using Auto Dock Vina for molecular docking of the protein with either fungal membrane moieties or α-amylase from Tenebrio molitor L. MD simulations of protein-ligand complexes were conducted using Schrodinger's Desmond module. RESULTS: γ-Thionin (BoT) was purified from white cabbage seeds and showed 100% homology with thionin (Brassica oleracea L. var. viridis) and 80% homology with defensin-like protein 1 (Raphanus sativus L.), respectively. BoT significantly inhibited the mycelial growth of Aspergillus niger van Tieghem and Aspergillus flavus Link at a concentration of 2 µM. Similarly, 0.12 µM BoT treatment resulted in significant mortality of Tribolium castaneum Herbst and Sitophilus oryzae L. Molecular docking and MD simulation of BoT confirmed the strong binding affinity with fungal membrane moieties (phosphatidylinositol 4,5-bisphosphate and phosphatidic acid), which causes disruption of the cell membrane and leakage of the cellular contents, leading to cell death. BoT blocked the active site of α-amylase, and as a result of the inactivation of this gut enzyme, the digestive systems of insects were disturbed, resulting in their deaths. CONCLUSION: This study revealed that γ-thionin is a good antifungal and insecticidal agent that could be used as an alternate to fungicides and insecticides.

In vitro and in silico studies on membrane interactions of diverse Capsicum annuum flower γ-thionin peptides.

Thionins are small, cysteine-rich peptides that play an important role in plant defense, primarily through their interactions with membranes. Eight novel γ-thionin peptides (CanThio1-8) were isolated from the flower of Capsicum annuum. Sequence analysis revealed that the peptides cluster into three groups. A representative peptide from each group (CanThio1, 2, and 3) was used for experimental characterization. Interestingly, peptides were found to possess some cytotoxic activity against normal human embryonic kidney cell line but higher cytotoxicity against cancer cell line MCF-7. CanThio3 peptide was chosen as a representative peptide to study the molecular mechanism of action on membranes. Microsecond timescale atomistic simulations of CanThio3 were performed in the presence of a POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) lipid bilayer. Simulations revealed that CanThio3 interacts with the bilayer and causes lipid thinning in the vicinity. Nonpolar amino acids specific to the α-core region of CanThio3 along with nonpolar residues in the γ-core region are seen to interact with the lipid tails. The differences in the amino acid sequence of CanThio peptides in these regions explain the variability in cytotoxic activities. In summary, our results demonstrate the membrane-mediated activity of a novel series of γ-thionin peptides from C. annuum.

Isolation and characterisation of the antifungal activity of the cowpea defensin Cp-thionin II.

As a result of the rapidly growing human population, reducing post-harvest crop losses of cereals due to microbial pests has major importance. Plant defensins have the potential to fulfil these demands, being highly specific and efficient antimicrobial agents. Hence, this study aimed to extract and characterise a peptide from cowpea seeds and investigate its antifungal performance. After extraction and partial purification, N-terminal sequencing was used to identify the primary peptide in the extract as cowpea-thionin II. Antifungal activity in vitro was found against Fusarium culmorum (MIC = 50 µg/mL), but Aspergillus niger and Penecillium expansum showed an MIC > 500 µg/mL. The extract was resistant against heat treatment (100 °C, 15 min) but lost its antifungal activity in presence of cations (Na+, K+, Ca2+ and Mg2+, respectively). Membrane permeabilization of fungal hyphae was evident at 25 µg/mL, while induction of oxidative stress only had minor contribution to the antifungal performance. The extract did not induce haemolysis at all concentrations tested (up to 200 µg/mL). Finally, it was successfully used to protect stored wheat grains from fungal spoilage (determined via ergosterol content) when applied at 100 µg/mL. In conclusion, the defensin Cp-thionin II showed the potential for future application as food bio-preservative.

Antifungal activity of synthetic cowpea defensin Cp-thionin II and its application in dough.

Plant defensins are small, cysteine-rich antimicrobial peptides of the immune system found in several organs during plant development. A synthetic peptide, KT43C, a linear analogue of the native Cp-thionin II found in cowpea seeds, was evaluated for its antifungal potential. It was found that KT43C displayed antifungal activity against Fusarium culmorum, Penicillium expansum and Aspergillus niger. Like native plant defensins, KT43C showed thermostability up to 100 °C and cation sensitivity. The synthetic peptide decreased the fungal growth without inducing morphogenic changes in the fungal hyphae. Non-inhibitory concentrations of the peptide induced permeabilization of the fungal membrane. In addition, high concentrations of KT43C induced the production of reactive oxygen species in the granulated cytoplasm. To investigate potential applications, the peptide was used as an additive in the preparation of dough which did not contain yeast. This peptide delayed the development of fungal growth in the dough by 2 days. Furthermore, KT43C did not induce red blood cell lysis up to a concentration of 200 µg.ml-1. These results highlight the potential for the use of synthetic antimicrobial defensins for shelf-life extension of food products.

Barley transcriptome analyses upon interaction with different aphid species identify thionins contributing to resistance.

Aphids are phloem-feeding insects that cause yield loss on a wide range of crops, including cereals such as barley. Whilst most aphid species are limited to one or few host species, some are able to reproduce on many plants belonging to different families. Interestingly, aphid probing behaviour can be observed on both host and non-host species, indicating that interactions take place at the molecular level that may impact host range. Here, we aimed to gain insight into the interaction of barley with aphid species differing in their ability to infest this crop by analysing transcriptional responses. Firstly, we determined colonization efficiency, settlement and probing behaviour for the aphid species Rhopalosiphum padi, Myzus persicae and Myzus cerasi, which defined host, poor-host and non-host interactions, respectively. Analyses of barley transcriptional responses revealed gene sets differentially regulated upon the different barley-aphid interactions and showed that the poor-host interaction with M. persicae resulted in the strongest regulation of genes. Interestingly, we identified several thionin genes strongly up-regulated upon interaction with M. persicae, and to a lesser extent upon R. padi interaction. Ectopic expression of two of these genes in Nicotiana benthamiana reduced host susceptibility to M. persicae, indicating that thionins contribute to defences against aphids.

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.

Defensin γ-thionin from Capsicum chinense has immunomodulatory effects on bovine mammary epithelial cells during Staphylococcus aureus internalization.

ß-Defensins are members of the antimicrobial peptide superfamily that are produced in various species from different kingdoms, including plants. Plant defensins exhibit primarily antifungal activities, unlike those from animals that exhibit a broad-spectrum antimicrobial action. Recently, immunomodulatory roles of mammal ß-defensins have been observed to regulate inflammation and activate the immune system. Similar roles for plant ß-defensins remain unknown. In addition, the regulation of the immune system by mammalian ß-defensins has been studied in humans and mice models, particularly in immune cells, but few studies have investigated these peptides in epithelial cells, which are in intimate contact with pathogens. The aim of this work was to evaluate the effect of the chemically synthesized ß-defensin γ-thionin from Capsicum chinense on the innate immune response of bovine mammary epithelial cells (bMECs) infected with Staphylococcus aureus, the primary pathogen responsible for bovine mastitis, which is capable of living within bMECs. Our results indicate that γ-thionin at 0.1 µg/ml was able to reduce the internalization of S. aureus into bMECs (∼50%), and it also modulates the innate immune response of these cells by inducing the mRNA expression (∼5-fold) and membrane abundance (∼3-fold) of Toll-like receptor 2 (TLR2), as well as by inducing genes coding for the pro-inflammatory cytokines TNF-α and IL-1ß (∼14 and 8-fold, respectively) before and after the bacterial infection. γ-Thionin also induces the expression of the mRNA of anti-inflammatory cytokine IL-10 (∼12-fold). Interestingly, the reduction in bacterial internalization coincides with the production of other antimicrobial products by bMECs, such as NO before infection, and the secretion into the medium of the endogenous antimicrobial peptide DEFB1 after infection. The results from this work support the potential use of ß-defensins from plants as immunomodulators of the mammalian innate immune response.

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.

Helleborus purpurascens-Amino Acid and Peptide Analysis Linked to the Chemical and Antiproliferative Properties of the Extracted Compounds.

There is a strong drive worldwide to discover and exploit the therapeutic potential of a large variety of plants. In this work, an alcoholic extract of Helleborus purpurascens (family Ranunculaceae) was investigated for the identification of amino acids and peptides with putative antiproliferative effects. In our work, a separation strategy was developed using solvents of different polarity in order to obtain active compounds. Biochemical components were characterized through spectroscopic (mass spectroscopy) and chromatographic techniques (RP-HPLC and GC-MS). The biological activity of the obtained fractions was investigated in terms of their antiproliferative effects on HeLa cells. Through this study, we report an efficient separation of bioactive compounds (amino acids and peptides) from a plant extract dependent on solvent polarity, affording fractions with unaffected antiproliferative activities. Moreover, the two biologically tested fractions exerted a major antiproliferative effect, thereby suggesting potential anticancer therapeutic activity.

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.

Double staining cytologic samples with quantitative Feulgen-thionin and anti-Ki-67 immunocytochemistry as a method of distinguishing cells with abnormal DNA content from normal cycling cells.

OBJECTIVE: To improve ploidy analysis in the detection of high-grade cervical dysplasias by combining it with anti-Ki-67 immunocytochemistry in a double staining procedure to distinguish between cells with abnormal DNA content and normal cycling cells. STUDY DESIGN: Cervical cytology specimens from 49 patients with various diagnoses, mostly dysplasias, from a previous study were used. Samples were double stained with Feulgen-thionin and anti-Ki-67 immunocytochemistry. Ki-67-negative cells were noncycling, so nondiploid Ki-67-negative cells were likely truly abnormal cells. RESULTS: The area under the receiver operating characteristic curve for the ability to identify high-grade dysplasias was 0.73 for double staining and 0.74 for thionin-only ploidy analysis on Cytospin specimens. At 90% specificity, sensitivities for double staining and thionin alone were 45% and 32%, respectively, but the difference was not statistically significant. CONCLUSION: Double staining with Feulgen-thionin and anti-Ki-67 immunocytochemistry does not improve the ability of ploidy analysis of cervical cytology specimens to separate high-grade and low-grade dysplasias, but our insights into the technical aspects of double staining, especially the effects of antigen retrieval, give hope that this technique could be applied to other immunocytochemical stains that would have a greater ability to improve ploidy analysis.

Microbial electricity generation of diversified carbonaceous electrodes under variable mediators.

To evaluate a suitable electrode material for the efficient green energy generation of a bio-fuel cell, carbonaceous based carbon cloth, carbon paper, and carbon felt electrodes were investigated under different mediators. The larger surface area, low resistance, and open network of interwoven fibers of the carbon felt electrode facilitated higher electron transfer from the microbial organisms to the electrode surface than that of other carbonaceous electrodes. Carbon paper electrode exhibited lower fuel cell performances due to its lower roughness and high tortuous nature. The green power generation experiments were also carried out under different mediators such as 2-hydroxy-l,4-naphthoquinone and thionin. The electrons mitigation and power generation was augmented by 2-hydroxy-l,4-naphthoquinone than thionin due to its high solubility, stability, and minimal adsorption characteristic to the electrodes. By the combined efforts of extended electrons generation and transportation, bio-fuel cell performances were extended and endorsed its doable applications in bio-fuel cells.

Leishmania donovani: thionins, plant antimicrobial peptides with leishmanicidal activity.

The leishmanicidal activity of plant antibiotic peptides (PAPs) from the principal families, such wheat thionins, a barley lipid transfer protein and potato defensins and snakins were tested in vitro against Leishmania donovani. Only thionins and defensins were active against this human pathogen at a low micromolar range of concentrations. Thionins resulted as the most active peptides tested until now. They collapsed ionic and pH gradients across the parasite plasma membrane together with a rapid depletion of intracellular ATP without affecting mitochondrial potential. Hence the lethal effect of thionins was mostly associated to permeabilization of the plasma membrane leading to an immediate death of the parasite. The present work is the first evidence for leishmanicidal activity in plant peptides. Future prospects for their development as new antiparasite agents on human diseases are considered.