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Efficacy of short-synthetic antifungal peptides on pathogenic Aspergillus flavus
Abstract
The efficacies of three short synthetic antifungal peptides were tested for their inhibitory action on pathogenic fungi, Aspergillus flavus. The sequences of the short synthetic peptides are PPD1- FRLHF, 66-10-FRLKFH, 77–3- FRLKFHF, respectively. These test peptides inhibited fungal growth and showed a membranolytic activity. The fungal biomass and ergosterol levels were significantly low in peptides treated samples. Further, the fungal cell wall component chitin was also found to be lower in peptides treated samples. Scanning electron microscopic images also showed highly wrinkled fungal mycelia. Significant membrane permeabilisation as well as potassium ion leakage was also observed in fungal samples treated with peptides. To assess the membrane damage, the uptake of Sytox green dye was employed. At tested concentration, peptides induced fungal membrane damage as evidenced by the green fluorescence. Further, these peptides induced an oxidative stress in A.flavus as evidenced by an increase in the ROS production, malondialdehyde levels, increase in the antioxidant enzymes - superoxide dismutase, catalase with concomitant decrease in the reduced glutathione content. Additionally, a growth dependent reduction in aflatoxin levels were also observed in peptides treated samples. Docking studies on the interaction of the peptides with a trans-membrane protein calcium ATPase of A. flavus showed that all the peptides were able to bind to the protein with high z rank score. The activity of the calcium ATPase was significantly decreased in peptides treated fungal samples, thereby validating the docking results. Among all the tested peptides, 77–3 peptide exhibited the maximal membrane damage property.
... 4 According to the data of Figure 2 and Table 1 [26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41]), a number of AMPs with antifungal activity have already been identified, which can be obtained from various natural sources. Due to the possibility of obtaining of SAFPs capable of imitating various natural peptides, such biomimetics can find effective application in practice and are notable objects for current developments and investigations (Table 2 [32,34,37,[42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58]). ...
... Investigation of the effect of four antimicrobial peptides -PPD1 (FRLHF), 66-10 (FRLKFH), 77-3 (FRLKFHF) and D4E1 (FKLRAKIKVRLRAKIKL) on the aflatoxin production by A. flavus and A. parasiticus suggested that AMPs at near minimum inhibitory concentrations (MIC) were effectively inhibiting aflatoxins, without hindering the growth of the fungi. At higher concentrations, these peptides exerted fungicidal action on A. flavus [52,53]. ...
Biomimetics, which are similar to natural compounds that play an important role in the metabolism, manifestation of functional activity and reproduction of various fungi, have a pronounced attrac-tion in the current search for new effective antifungals. Actual trends in the development of this area of research indicate that unnatural amino acids can be used as such biomimetics, including those containing halogen atoms; compounds similar to nitrogenous bases embedded in the nucleic acids synthesized by fungi; peptides imitating fungal analogues; molecules similar to natural sub-strates of numerous fungal enzymes and Quorum Sensing signaling molecules of fungi and yeast; etc. Most part of the review is devoted to the analysis of semi-synthetic and synthetic antifungal peptides and their targets of action. This review is aimed at combining and systematizing the cur-rent scientific information accumulating in this area of research, developing various antifungals with an assessment of the effectiveness of the created biomimetics and the possibility of combining them with other antimicrobial substances to reduce cell resistance and improve antifungal effects.
... Additionally, sections of the germ tubes had a floating appearance, likely due to differential cytosolic leakage. Previously, cytosolic leakage from A. flavus mycelia was detected after exposure to higher concentrations of D4E1 and other synthetic peptides (Devi and Sashidhar 2019;Devi et al. 2021;Moore et al. 2019), and disintegration of nuclear envelopes, nuclei lysis, and deformation of germinated A. flavus conidia were observed after exposure to D4E1 (Rajasekaran et al. 2010(Rajasekaran et al. , 2012. In our study, we observed evidence of possible membrane perforations and cytosol leakage following exposure of F. graminearum and R. stolonifer to GV185, GV187, D4E1, and AGM182 that resulted in a shriveled appearance to spores and germ tubes, similar to that observed in A. flavus mycelia (Devi et al. 2021) and germinating spores (Rajasekaran et al. 2010(Rajasekaran et al. , 2012 exposed to other peptides. ...
... Previously, cytosolic leakage from A. flavus mycelia was detected after exposure to higher concentrations of D4E1 and other synthetic peptides (Devi and Sashidhar 2019;Devi et al. 2021;Moore et al. 2019), and disintegration of nuclear envelopes, nuclei lysis, and deformation of germinated A. flavus conidia were observed after exposure to D4E1 (Rajasekaran et al. 2010(Rajasekaran et al. , 2012. In our study, we observed evidence of possible membrane perforations and cytosol leakage following exposure of F. graminearum and R. stolonifer to GV185, GV187, D4E1, and AGM182 that resulted in a shriveled appearance to spores and germ tubes, similar to that observed in A. flavus mycelia (Devi et al. 2021) and germinating spores (Rajasekaran et al. 2010(Rajasekaran et al. , 2012 exposed to other peptides. These observations suggest that the AMPs used in these two studies have similar modes of action. ...
Optimizing synthetic antimicrobial peptides for safe and enhanced activity against fungal and bacterial pathogens is useful for genetic engineering of plants for resistance to plant pathogens and their associated mycotoxins. Nine synthetic peptides modeled after lytic peptides tachyplesin 1, D4E1 from cecropin A and protegrin 1 were added to germinated spores of fungal species Aspergillus flavus, Rhizopus stolonifer, Fusarium oxysporum f. sp. vasinfectum, F. verticillioides, F. graminearum, Claviceps purpurea, Verticillium dahliae, Thielaviopsis basicola and bacterial cultures of Psuedomonas syringae p.v. tabaci and Xanthomonas campestris p.v. campestris at different doses and inhibitory dose response curves were modeled to assess antimicrobial activity. Peptides GV185 and GV187, modified from tachyplesin 1, had superior abilities to inhibit fungal and bacterial growth (50% inhibitory concentrations or IC50 ranging from 0.1 to 8.7 µM). Rhizopus stolonifer (IC50 = 8.1 µM), A. flavus (IC50 = 3.1 µM) and F. graminearum (IC50 = 2.2 µM) were less inhibited by GV185 and GV187 than all the remaining fungi (IC50 = 1.4 µM) and bacteria (IC50 = 0.1 µM). Of the remaining peptides, GV193, GV195 and GV196 (IC50 range 0.9 to 6.6 µM) inhibited fungal growth of A. flavus, F. verticillioides and F. graminearum less than GV185 and GV187 (IC50 range 0.8 to 3.9 µM), followed by GV197 (IC50 range 0.8 - 9.1 µM) whereas GV190 and GV192 inhibited poorly (IC50 range 28.2 to 36.6 µM and 15.5 to 19.4 µM, respectively) and GV198 stimulated growth. GV185 and GV187 had slightly weaker hydrophobic and cationic residues than other tachyplesin 1 modified peptides, but still had unexpectedly high lytic activity. Germinated fungal spores of R. stolonifer and F. graminearum exposed to these two peptides and D4E1 and AGM182 appeared wrinkled with perforations near potential cytoplasmic leakage, which provided evidence of plasma membrane and cell wall lysis. We conclude that peptides GV185 and GV187 are promising candidates for genetic engineering of crops for resistance to plant pathogenic bacteria and fungi including A. flavus and aflatoxin contamination.
... It is confirmed that A. flavus exhibits high resistance to various antimicrobial agents, towards which other pathogenic strains are more susceptible (Lucky et al., 2017). On the other hand, many short-chain peptides have proven effective in successful inhibition of this pathogen (Cary et al., 2009;Manju Devi, Raj, & Sashidhar, 2021). In general, MIC for the RW4 peptide was lower than for melittin. ...
The aim of the study was to develop novel antimicrobial coatings which could be used for extending the microbiological quality of perishable food products. Seven different antimicrobial peptides (Lfcinβ, PLGG, MBP-1, LL-37, Cys-LL-37, RW4 and melittin) have been investigated as possible agents for implementation into the previously developed chitosan/furcellaran (CHIT/FUR) coatings. The study included several steps of microbiological analysis, starting with minimum inhibitory concentration (MIC) analysis of crude peptide solutions (concentration from 0.78 to 100 μg/mL) against selected pathogens. The analysis showed that only RW4 and melittin proved effective against all studied pathogens in the investigated doses, except for Aspergillus flavus. The second examination included in-vitro analysis regarding antimicrobial activity of developed coatings with peptides at a concentration of 5 μg/mL, against chosen pathogens. This analysis allowed to indicate a complementary effect of antimicrobial activity between all peptides and the CHIT/FUR complex since the coatings inhibited the growth of the tested pathogens. Afterwards, the coatings were used to coat smoked pork ham slices stored for 10 days and analysed for the increase in total viable counts (TVC). The analysis demonstrated a high efficiency of the CHIT/FUR complex, even without the addition of peptides. Only RW4 and LL-37 did not cause a decrease in antimicrobial power of the control coating. Therefore, in the final trial, the effect of different doses (1.25–10 μg/mL) of RW4 and LL-37 peptides on the TVC of cold-stored coated pork loins. The coatings almost completely inhibited the increase of TVC in the pork loins and the peptides were effective at all studied doses. The newly-developed coatings had strong antimicrobial properties and can be used to extend the shelf-life of perishable food products.
... Peptides have numerous advantages, including easy availability, purification and storage, high potential and specificity, good biodegradability and biocompatible properties, and low toxicity (Qi et al., 2018). These favorable characteristics have allowed them to be used for different purposes, including as antifungal, antimicrobial, anticancer, antioxidant, mineral binding, antithrombotic, antihypertensive, and immunomodulatory agents, and even they are used in cosmetics to provide benefits to the skin through the topical application (Xiao et al., 2015;Schagen, 2017;Chen & Lu, 2020;Manju Devi et al., 2021). Peptides have also attracted the attention of research groups for the development of novel gene therapies; the mechanism of action of each treatment varies depending on the function, the purpose, and the nature of the peptide (Lau & Dunn, 2018). ...
Oxidative stress occurs because of an imbalance in the production of reactive oxygen species. Nuts are rich in bioactive compounds, including phenolic compounds, tannins, and phytosterols. Although nuts are widely consumed because of their beneficial effects on nutrition and health, there is limited information about bioactive peptides from nuts. Pine nut and walnut‐derived peptides are the most studied because these nuts contain a higher amount of protein. Different biological activities have been demonstrated for nut peptides; many of them exhibit antioxidant, anticancer, antihypertensive, antidiabetic, immunomodulatory, antiseizure, or neuroprotective activity. Recent studies have focused on increasing the bioactivity of identified bioactive peptides by applying new technologies and chemosynthetic strategies. Research tendency points to the generation of peptides with specific sequences for application in specific diseases. Nut bioactive peptides can become key functional ingredients for food, pharmaceuticals, or cosmetics.
... Quantitative real time polymerase chain reaction (RT-qPCR) analysis of the aflatoxin gene cluster showed that the aflR gene, and the downstream genes were significantly downregulated. The involvement of oxidative stress in the effect mediated by these peptides was also recently analyzed [218]. Results revealed that high peptide concentrations induced oxidative stress in A. flavus, while a complete inhibition of AF production was not observed, even though a four to five-fold reduction occurred. ...
The global challenge to prevent fungal spoilage and mycotoxin contamination on food and feed requires the development of new antifungal strategies. Antimicrobial peptides and proteins (AMPs) with antifungal activity are gaining much interest as natural antifungal compounds due to their properties such as structure diversity and function, antifungal spectrum, mechanism of action, high stability and the availability of biotechnological production methods. Given their multistep mode of action, the development of fungal resistance to AMPs is presumed to be slow or delayed compared to conventional fungicides. Interestingly, AMPs also accomplish important biological functions other than antifungal activity, including anti-mycotoxin biosynthesis activity, which opens novel aspects for their future use in agriculture and food industry to fight mycotoxin contamination. AMPs can reach intracellular targets and exert their activity by mechanisms other than membrane permeabilization. The mechanisms through which AMPs affect mycotoxin production are varied and complex, ranging from oxidative stress to specific inhibition of enzymatic components of mycotoxin biosynthetic pathways. This review presents natural and synthetic antifungal AMPs from different origins which are effective against mycotoxin-producing fungi, and aims at summarizing current knowledge concerning their additional effects on mycotoxin biosynthesis. Antifungal AMPs properties and mechanisms of action are also discussed.
... Generally, cells can alleviate ROS damage to cells through the action of enzymes (superoxide dismutase, catalase). Some small molecules, such as glutathione, also play critical cellular antioxidants (75). In our research, the gene (TR16620_c0_g1) relative to peroxiredoxin activity was upregulated by 3.06-fold. ...
Penicillium digitatum is the most severe pathogen that infects citrus fruits during storage. It can cause fruit rot and bring significant economic losses. The continuous use of fungicides has resulted in the emergence of drug-resistant strains. Consequently, there is a need to develop naturally and efficiently antifungal fungicides. Natural antimicrobial agents such as clove oil, cinnamon oil, and thyme oil can be extracted from different plant parts. They exhibited broad-spectrum antimicrobial properties and have great potential in the food industry. Here, we exploit a novel cinnamaldehyde (CA), eugenol (EUG), or carvacrol (CAR) combination antifungal therapy and formulate it into nanoemulsion form to overcome lower solubility and instability of essential oil. In this study, the antifungal activity evaluation and transcriptional profile of Penicillium digitatum exposed to compound nanoemulsion were evaluated. Results showed that compound nanoemulsion had a striking inhibitory effect on P. digitatum in a dose-dependent manner. According to RNA-seq analysis, there were 2,169 differentially expressed genes (DEGs) between control and nanoemulsion-treated samples, including 1,028 downregulated and 1,141 upregulated genes. Gene Ontology (GO) analysis indicated that the DEGs were mainly involved in intracellular organelle parts of cell component: cellular respiration, proton transmembrane transport of biological process, and guanyl nucleotide-binding molecular function. KEGG analysis revealed that metabolic pathway, biosynthesis of secondary metabolites, and glyoxylate and dicarboxylate metabolism were the most highly enriched pathways for these DEGs. Taken together, we can conclude the promising antifungal activity of nanoemulsion with multiple action sites against P. digitatum. These outcomes would deepen our knowledge of the inhibitory mechanism from molecular aspects and exploit naturally, efficiently, and harmlessly antifungal agents in the citrus postharvest industry.
Biomimetics, which are similar to natural compounds that play an important role in the metabolism, manifestation of functional activity and reproduction of various fungi, have a pronounced attraction in the current search for new effective antifungals. Actual trends in the development of this area of research indicate that unnatural amino acids can be used as such biomimetics, including those containing halogen atoms; compounds similar to nitrogenous bases embedded in the nucleic acids synthesized by fungi; peptides imitating fungal analogs; molecules similar to natural substrates of numerous fungal enzymes and quorum-sensing signaling molecules of fungi and yeast, etc. Most parts of this review are devoted to the analysis of semi-synthetic and synthetic antifungal peptides and their targets of action. This review is aimed at combining and systematizing the current scientific information accumulating in this area of research, developing various antifungals with an assessment of the effectiveness of the created biomimetics and the possibility of combining them with other antimicrobial substances to reduce cell resistance and improve antifungal effects.
Two new CPA-type indole alkaloids (1 and 2) were isolated from the fungus Aspergillus versicolor. Their structures were determined by means of HR-ESI-MS and extensive 1D and 2D NMR spectroscopic studies. Compounds 1 and 2 were tested for their anti-tobacco mosaic virus (anti-TMV) activity. The results showed that compounds 1 and 2 showed potential anti-TMV activity with inhibition rates of 33.6 and 29.4% respectively. These rates are close to that of positive control.
The present study aimed to co-encapsulate binary synergistic formulation of Pimpinella anisum and Coriandrum sativum (PC) essential oils (0.75:0.25) into chitosan nanoemulsion (Nm-PC) with effective inhibition against fungal proliferation, aflatoxin B1 (AFB1) secretion, and lipid peroxidation in stored rice. Physico-chemical characterization of Nm-PC by SEM, FTIR, and XRD confirmed successful encompassment of PC inside the chitosan nanomatrix with efficient interaction by functional groups and reduction in crystallinity. Nm-PC showed superior antifungal, antiaflatoxigenic, and antioxidant activities over unencapsulated PC. Reduction in ergosterol biosynthesis and enhanced leakage of Ca²⁺, K⁺, Mg²⁺ ions and 260, 280 nm absorbing materials by Nm-PC fumigation confirmed irreversible damage of plasma membrane in toxigenic Aspergillus flavus cells. Significant diminution of methylglyoxal in A. flavus cells by Nm-PC fumigation illustrated biochemical mechanism for antiaflatoxigenic activity, suggesting future exploitation for development of aflatoxin resistant rice varieties through green transgenic technology. In silico findings indicated specific stereo-spatial interaction of anethole and linalool with Nor-1 protein, validating molecular mechanism for AFB1 inhibition. In addition, in situ investigation revealed effective protection of stored rice against fungal occurrence, AFB1 biosynthesis, and lipid peroxidation without affecting organoleptic attributes. Moreover, mammalian non-toxicity of chitosan entrapped PC synergistic nanoformulation could provide exciting potential for application as eco-smart safe nano-green food preservative.
Evolved resistance and regulatory deregistration have severely limited farmers' pesticide options. Many potential new pesticide target sites have been elucidated using targeted gene suppression and mutational tools, but few small molecules could be found that inhibit the target enzymes; the targets were considered ‘undruggable’. Some organisms from all biological kingdoms use toxic peptides to ward off or kill enemies, and the agrochemical industry has used a few peptide analogs (glufosinate and bialophos) for field application. Conversely, pharmaceutical scientists have been using three‐dimensional target protein structure to discover and synthesize short peptides that bind tightly to the surfaces of, and inhibit previously undruggable targets. New computational tools to quickly elucidate 3‐D protein structure from amino acid sequence have just emerged. They replace crystallizing target proteins and performing X‐ray crystallography to elucidate 3‐D structure. These new tools allow prediction of peptides that will bind to the target proteins. They have further modified such peptides to enhance penetration, translocation and temperature stability. There is reason to assume that the same pioneering techniques can be used to develop peptide pesticides as well as pesticide synergists that act against undruggable targets and have excellent environmental and toxicological profiles. © 2022 Society of Chemical Industry.
Perillaldehyde (PAE) is a natural compound used to control post-harvest fungal disease. Ethylenediaminetetraacetic acid (EDTA) also has anti-fungal effects, but its synergistic effects on the anti-fungal mechanisms of PAE against Aspergillus flavus (A. flavus) are unexplored. Therefore, we examined A. flavus mycelial growth, conidial formation, and pathogenicity, as well as the mechanisms, with EDTA and PAE treatment. EDTA was found to accelerate PAE-suppressed mycelial growth and conidial formation. These effects coincided with an increase in membrane permeability, autophagy, and apoptosis. Accordingly, the expression of pathogenicity-associated genes was dramatically decreased with PAE + EDTA treatment. In addition, the levels of the antioxidants vitamin C, total thiols, and total phenols, and the total antioxidant capacity and radical-scavenging capacity were increased with EDTA + PAE. Thus, the findings indicate that EDTA enhances PAE-mediated control of post-harvest A. flavus via autophagy, apoptosis, antioxidant, and membrane permeability mechanisms, making it suitable for global application in the food industry to attenuate A. flavus from post-harvest crops and improve antioxidant nutrient accumulation.
More than 3000 antimicrobial peptides (AMPs) have been discovered, seven of which have been approved by the U.S. Food and Drug Administration (FDA). Now commercialized, these seven peptides have mostly been utilized for topical medications, though some have been injected into the body to treat severe bacterial infections. To understand the translational potential for AMPs, we analyzed FDA-approved drugs in the FDA drug database. We examined their physicochemical properties, secondary structures, and mechanisms of action, and compared them with the peptides in the AMP database. All FDA-approved AMPs were discovered in Gram-positive soil bacteria, and 98% of known AMPs also come from natural sources (skin secretions of frogs and toxins from different species). However, AMPs can have undesirable properties as drugs, including instability and toxicity. Thus, the design and construction of effective AMPs require an understanding of the mechanisms of known peptides and their effects on the human body. This review provides an overview to guide the development of AMPs that can potentially be used as antimicrobial drugs.
Aflatoxins produced by the Aspergillus species are highly toxic, carcinogenic, and cause severe contamination to food sources, leading to serious health consequences. Contaminations by aflatoxins have been reported in food and feed, such as groundnuts, millet, sesame seeds, maize, wheat, rice, fig, spices and cocoa due to fungal infection during pre- and post-harvest conditions. Besides these food products, commercial products like peanut butter, cooking oil and cosmetics have also been reported to be contaminated by aflatoxins. Even a low concentration of aflatoxins is hazardous for human and livestock. The identification and quantification of aflatoxins in food and feed is a major challenge to guarantee food safety. Therefore, developing feasible, sensitive and robust analytical methods is paramount for the identification and quantification of aflatoxins present in low concentrations in food and feed. There are various chromatographic and sensor-based methods used for the detection of aflatoxins. The current review provides insight into the sources of contamination, occurrence, detection techniques, and masked mycotoxin, in addition to management strategies of aflatoxins to ensure food safety and security.
To elucidate interactions between the antifungal cyclic lipopeptides iturin A, fengycin, and surfactin produced by Bacillus bacteria and the microtubular protein β-tubulin in plant pathogenic fungi (Fusarium oxysporum, Colletrotrichum gloeosporioides, Alternaria alternata, and Fusarium solani) in molecular docking and molecular dynamics simulations, we retrieved the structure of tubulin co-crystallized with taxol from the Protein Data Bank (PDB) (ID: 1JFF) and the structure of the cyclic lipopeptides from PubChem (Compound CID: 102287549, 100977820, 10129764). Similarity and homology analyses of the retrieved β-tubulin structure with those of the fungi showed that the conserved domains shared 84% similarity, and the root mean square deviation (RMSD) was less than 2 Å. In the molecular docking studies, within the binding pocket, residues Pro274, Thr276, and Glu27 of β-tubulin were responsible for the interaction with the cyclic lipopeptides. In the molecular dynamics analysis, two groups of ligands were formed based on the number of poses analyzed with respect to the RMSD. Group 1 was made up of 10, 100, and 500 poses with distances 0.080 to 0.092 nm and RMSDs of 0.10 to 0.15 nm. For group 2, consisting of 1000 poses, the initial and final distance was 0.1 nm and the RMSDs were in the range of 0.10 to 0.30 nm. These results suggest that iturin A and fengycin bind with higher affinity than surfactin to β-tubulin. These two lipopeptides may be used as lead compounds to develop new antifungal agents or employed directly as biorational products to control plant pathogenic fungi.
In the last 20 years, an increasing number of studies have been reported on membrane active peptides. These peptides exert their biological activity by interacting with the cell membrane, either to disrupt it and lead to cell lysis or to translocate through it to deliver cargos into the cell and reach their target. Membrane active peptides are attractive alternatives to currently used pharmaceuticals and the number of antimicrobial peptides (AMPs) and peptides designed for drug and gene delivery in the drug pipeline is increasing. Here, we focus on two most prominent classes of membrane active peptides; AMPs and cell-penetrating peptides (CPPs). Antimicrobial peptides are a group of membrane active peptides that disrupt the membrane integrity or inhibit the cellular functions of bacteria, virus, and fungi. Cell penetrating peptides are another group of membrane active peptides that mainly function as cargo-carriers even though they may also show antimicrobial activity. Biophysical techniques shed light on peptide–membrane interactions at higher resolution due to the advances in optics, image processing, and computational resources. Structural investigation of membrane active peptides in the presence of the membrane provides important clues on the effect of the membrane environment on peptide conformations. Live imaging techniques allow examination of peptide action at a single cell or single molecule level. In addition to these experimental biophysical techniques, molecular dynamics simulations provide clues on the peptide–lipid interactions and dynamics of the cell entry process at atomic detail. In this review, we summarize the recent advances in experimental and computational investigation of membrane active peptides with particular emphasis on two amphipathic membrane active peptides, the AMP melittin and the CPP pVEC.
Biophysical and structural investigations are presented with a focus on the membrane lipid interactions of cationic linear antibiotic peptides such as magainin, PGLa, LL37, and melittin. Observations made with these peptides are distinct as seen from data obtained with the hydrophobic peptide alamethicin. The cationic amphipathic peptides predominantly adopt membrane alignments parallel to the bilayer surface; thus the distribution of polar and non-polar side chains of the amphipathic helices mirror the environmental changes at the membrane interface. Such a membrane partitioning of an amphipathic helix has been shown to cause considerable disruptions in the lipid packing arrangements, transient openings at low peptide concentration, and membrane disintegration at higher peptide-to-lipid ratios. The manifold supramolecular arrangements adopted by lipids and peptides are represented by the 'soft membranes adapt and respond, also transiently' (SMART) model. Whereas molecular dynamics simulations provide atomistic views on lipid membranes in the presence of antimicrobial peptides, the biophysical investigations reveal interesting details on a molecular and supramolecular level, and recent microscopic imaging experiments delineate interesting sequences of events when bacterial cells are exposed to such peptides. Finally, biophysical studies that aim to reveal the mechanisms of synergistic interactions of magainin 2 and PGLa are presented, including unpublished isothermal titration calorimetry (ITC), circular dichroism (CD) and dynamic light scattering (DLS) measurements that suggest that the peptides are involved in liposome agglutination by mediating intermembrane interactions. A number of structural events are presented in schematic models that relate to the antimicrobial and synergistic mechanism of amphipathic peptides when they are aligned parallel to the membrane surface.
We have identified a series of tetrahydrocarbazoles as novel P-type ATPase inhibitors. Using a set of rationally designed analogues, we have analyzed their structure-activity relationship using functional assays, crystallographic data and computational modeling. We found that tetrahydrocarbazoles inhibit adenosine triphosphate (ATP) hydrolysis of the fungal H⁺-ATPase, depolarize the fungal plasma membrane and exhibit broad-spectrum antifungal activity. Comparative inhibition studies indicate that many tetrahydrocarbazoles also inhibit the mammalian Ca²⁺-ATPase (SERCA) and Na⁺,K⁺-ATPase with an even higher potency than Pma1. We have located the binding site for this compound class by crystallographic structure determination of a SERCA-tetrahydrocarbazole complex to 3.0 Å resolution, finding that the compound binds to a region above the ion inlet channel of the ATPase. A homology model of the Candida albicans H⁺-ATPase based on this crystal structure, indicates that the compounds could bind to the same pocket and identifies pocket extensions that could be exploited for selectivity enhancement. The results of this study will aid further optimization towards selective H⁺-ATPase inhibitors as a new class of antifungal agents.
Novel thin-layer chromatography-digital image-based analytical methods were developed for the quantitation of ergosterol and chitin content in six food matrices (rice, wheat, maize, sorghum, groundnut, and sunflower), artificially infested with Aspergillus flavus (MTCC 6513)/Fusarium verticillioides (MRC 826). For ergosterol, single-step method, based on liquid/liquid extraction, was followed by thin-layer chromatography (TLC). Chitin was solubilized using lithium chloride (5%) in dimethyl acetamide and converted to chitosan using 5 N NaOH and subsequently complexed with calcofluor white dye. The absorption and emission maxima of chitosan-calcofluor complex were recorded at λ 350/230 and 430 nm, respectively. The sensitivity based on the limit of detection (LOD) was found to be 100 ng both for ergosterol and chitin analysis. Based on ergosterol and chitin analysis, groundnut and maize were found to be suitable substrates for A. flavus (p < 0.013 and p < 0.01), while sorghum followed by groundnut and sunflower were found to be ideal for F. verticillioides (p < 0.01 and p < 0.0001) and rice was established as poor substrate as there was no growth on it up to 12 days of incubation. A strong correlation was found between ergosterol and chitin contents with regression (r²) values of 0.974 and 0.997 in food grains inoculated with A. flavus and F. verticillioides, respectively, during the period of infection. The authenticity of the two methods developed was further confirmed by applying them to commercial food grains and flours. Thus, ergosterol in combination with chitin analysis could be successfully used as an index of fungal contamination employing TLC-digital-based analytical methods.
Candida albicans is a commensal organism, commonly inhabiting mucosal surfaces of healthy individuals, as a part of the resident microbiota. However, in susceptible hosts, especially hospitalized and/or immunocompromised patients, it may cause a wide range of infections. The presence of abiotic substrates, such as central venous or urinary catheters, provides an additional niche for Candida attachment and persistence, particularly via biofilm development. Furthermore, Candida biofilm is poorly susceptible to most antifungals, including azoles. Here we investigated the effects of a synthetic killer peptide (KP), known to be active in vitro, ex vivo and/or in vivo against different pathogens, on C. albicans biofilm. Together with a scrambled peptide used as a negative control, KP was tested against Candida biofilm at different stages of development. A reference strain, two fluconazole-resistant and two fluconazole-susceptible C. albicans clinical isolates were used. KP-induced C. albicans oxidative stress response and membrane permeability were also analysed. Moreover, the effect of KP on transcriptional profiles of C. albicans genes involved in different stages of biofilm development, such as cell adhesion, hyphal development and extracellular matrix production, was evaluated. Our results clearly show that the treatment with KP strongly affected the capacity of C. albicans to form biofilm and significantly impairs preformed mature biofilm. KP treatment resulted in an increase in C. albicans oxidative stress response and membrane permeability; also, biofilm-related genes expression was significantly reduced. Comparable inhibitory effects were observed in all the strains employed, irrespective of their resistance or susceptibility to fluconazole. Finally, KP-mediated inhibitory effects were observed also against a catheter-associated C. albicans biofilm. This study provides the first evidence on the KP effectiveness against C. albicans biofilm, suggesting that KP may be considered as a potential novel tool for treatment and prevention of biofilm-related C. albicans infections.
Remarkable interest has risen in the idea that oxidative/nitrosative stress is mediated in the etiology of numerous human diseases. Oxidative/Nitrosative stress is the result of an disequilibrium in oxidant/antioxidant which reveals from continuous increase of Reactive Oxygen and Reactive Nitrogen Species production. The aim of this review is to emphasize with current information the importance of antioxidants which play the role in cellular responce against oxidative/nitrosative stress, which would be helpful in enhancing the knowledge of any biochemist, pathophysiologist, or medical personnel regarding this important issue. Products of lipid peroxidation have commonly been used as biomarkers of oxidative/nitrosative stress damage. Lipid peroxidation generates a variety of relatively stable decomposition end products, mainly α, β-unsaturated reactive aldehydes, such as malondialdehyde, 4-hydroxy-2-nonenal, 2-propenal (acrolein) and isoprostanes, which can be measured in plasma and urine as an indirect index of oxidative/nitrosative stress. Antioxidants are exogenous or endogenous molecules that mitigate any form of oxidative/nitrosative stress or its consequences. They may act from directly scavenging free radicals to increasing antioxidative defences. Antioxidant deficiencies can develop as a result of decreased antioxidant intake, synthesis of endogenous enzymes or increased antioxidant utilization. Antioxidant supplementation has become an increasingly popular practice to maintain optimal body function. However, antoxidants exhibit pro-oxidant activity depending on the specific set of conditions. Of particular importance are their dosage and redox conditions in the cell.
Plant antimicrobial peptides (AMPs) have evolved differently from AMPs from other life forms. They are generally rich in cysteine residues which form multiple disulfides. In turn, the disulfides cross-braced plant AMPs as cystine-rich peptides to confer them with extraordinary high chemical, thermal and proteolytic stability. The cystine-rich or commonly known as cysteine-rich peptides (CRPs) of plant AMPs are classified into families based on their sequence similarity, cysteine motifs that determine their distinctive disulfide bond patterns and tertiary structure fold. Cystine-rich plant AMP families include thionins, defensins, hevein-like peptides, knottin-type peptides (linear and cyclic), lipid transfer proteins, α-hairpinin and snakins family. In addition, there are AMPs which are rich in other amino acids. The ability of plant AMPs to organize into specific families with conserved structural folds that enable sequence variation of non-Cys residues encased in the same scaffold within a particular family to play multiple functions. Furthermore, the ability of plant AMPs to tolerate hypervariable sequences using a conserved scaffold provides diversity to recognize different targets by varying the sequence of the non-cysteine residues. These properties bode well for developing plant AMPs as potential therapeutics and for protection of crops through transgenic methods. This review provides an overview of the major families of plant AMPs, including their structures, functions, and putative mechanisms.
Peptides derived from human and bovine lactoferricin were designed, synthesized, purified, and characterized using RP-HPLC and MALDI-TOF-MS. Specific changes in the sequences were designed as (i) the incorporation of unnatural amino acids in the sequence, the (ii) reduction or (iii) elongation of the peptide chain length, and (iv) synthesis of molecules with different number of branches containing the same sequence. For each peptide, the antibacterial activity against Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212 was evaluated. Our results showed that Peptides I.2 (RWQWRWQWR) and I.4 ((RRWQWR)4K2Ahx2C2) exhibit bigger or similar activity against E. coli (MIC 4-33 μM) and E. faecalis (MIC 10-33 μM) when they were compared with lactoferricin protein (LF) and some of its derivate peptides as II.1 (FKCRRWQWRMKKLGA) and IV.1 (FKCRRWQWRMKKLGAPSITCVRRAE). It should be pointed out that Peptides I.2 and I.4, containing the RWQWR motif, are short and easy to synthesize; our results demonstrate that it is possible to design and obtain synthetic peptides that exhibit enhanced antibacterial activity using a methodology that is fast and low-cost and that allows obtaining products with a high degree of purity and high yield.
Candida spp. are the most common causes of fungal infections worldwide. Among the Candida species, Candida albicans remains the predominant species that causes invasive candidiasis in most countries. In this study, we used two peptides, KABT-AMP and uperin 3.6 as templates to develop novel antifungal peptides. Their anticandidal activity was assessed using a combination of MIC, time-killing assay and biofilm reduction assay. Hybrid peptides, KU2 and KU3 containing a mixed backbone of KABT-AMP and Uperin 3.6 demonstrated the most potent anticandidal activity with MIC values ranging from 8-16 mg/L. The number of Trp residues and the amphipathic structure of peptides probably enhanced the anticandidal activity of peptides. Increasing the cationicity of the uperin 3.6 analogues resulted in reduced MIC from the range of 64-128 mg/L to 16-64 mg/L and this was also correlated with the antibiofilm activity and killing kinetics of the peptides. Peptides showed synergistic effects when used in combination with conventional antifungals. Peptides demonstrated low haemolytic activity but significant toxicity on two normal human epithelial cell lines. This study provides us with a better understanding on the structure-activity relationship and the balance between cationicity and hydrophobicity of the peptides although the therapeutic application of the peptides is limited.
Excessive free radical generation, especially reactive oxygen species (ROS) leading to oxidative stress in the biological system, has been implicated in the pathogenesis and pathological conditions associated with diverse human inflammatory diseases (HIDs). Although inflammation which is considered advantageous is a defensive mechanism in response to xenobiotics and foreign pathogen; as a result of cellular damage arising from oxidative stress, if uncontrolled, it may degenerate to chronic inflammation when the ROS levels exceed the antioxidant capacity. Therefore, in the normal resolution of inflammatory reactions, apoptosis is acknowledged to play a crucial role, while on the other hand, dysregulation in the induction of apoptosis by enhanced ROS production could also result in excessive apoptosis identified in the pathogenesis of HIDs. Apparently, a careful balance must be maintained in this complex environment. Antimicrobial peptides (AMPs) have been proposed in this review as an excellent candidate capable of playing prominent roles in maintaining this balance. Consequently, in novel drug design for the treatment and management of HIDs, AMPs are promising candidates owing to their size and multidimensional properties as well as their wide spectrum of activities and indications of reduced rate of resistance.
A novel strain of Pseudomonas monteilii, PsF84 was isolated from tannery waste soil, Jajmau, Kanpur, India. 16S rRNA gene sequence phylogenetic analysis confirmed the taxonomic affiliation of PsF84 as P. monteilii. An antifungal volatile organic compound (VOC) was active against hyphal growth of Fusarium oxysporum (CIMAP-IMI-357464) in vitro was isolated from strain PsF84 by using chromatographic techniques. The molecular formula of the antifungal VOC was deduced to be C14H22O by EI-MS and 1D and 2D NMR spectral analysis. 2, 4-di-tert-butylphenol was found to be effective against agriculturally important fungi, viz., F. oxysporum in inhibiting spore germination and hyphal growth. Molecular docking analysis of 2, 4-di-tert-butylphenol with β-tubulin further validated the potential of β-tubulin binding in F.oxysporum. Two residues of β-tubulin protein, HIS 118 and THR 117 showed hydrogen binding with ligand. To our knowledge, this is the first report of antifungal VOC (2, 4-di-tert-butylphenol) produced by P. monteilii PsF84 which can be a potent inhibitor of β-tubulin of F. oxysporum.
Pesticides are used throughout the world as mixtures called formulations. They contain adjuvants, which are often kept confidential and are called inerts by the manufacturing companies, plus a declared active principle, which is usually tested alone. We tested the toxicity of 9 pesticides, comparing active principles and their formulations, on three human cell lines (HepG2, HEK293, and JEG3). Glyphosate, isoproturon, fluroxypyr, pirimicarb, imidacloprid, acetamiprid, tebuconazole, epoxiconazole, and prochloraz constitute, respectively, the active principles of 3 major herbicides, 3 insecticides, and 3 fungicides. We measured mitochondrial activities, membrane degradations, and caspases 3/7 activities. Fungicides were the most toxic from concentrations 300-600 times lower than agricultural dilutions, followed by herbicides and then insecticides, with very similar profiles in all cell types. Despite its relatively benign reputation, Roundup was among the most toxic herbicides and insecticides tested. Most importantly, 8 formulations out of 9 were up to one thousand times more toxic than their active principles. Our results challenge the relevance of the acceptable daily intake for pesticides because this norm is calculated from the toxicity of the active principle alone. Chronic tests on pesticides may not reflect relevant environmental exposures if only one ingredient of these mixtures is tested alone.
Antimicrobial peptides are diverse group of biologically active molecules with multidimensional properties. In recent past, a wide variety of AMPs with diverse structures have been reported from different sources such as plants, animals, mammals, and microorganisms. The presence of unusual amino acids and structural motifs in AMPs confers unique structural properties to the peptide that attribute for their specific mode of action. The ability of these active AMPs to act as multifunctional effector molecules such as signalling molecule, immune modulators, mitogen, antitumor, and contraceptive agent makes it an interesting candidate to study every aspect of their structural and biological properties for prophylactic and therapeutic applications. In addition, easy cloning and recombinant expression of AMPs in heterologous plant host systems provided a pipeline for production of disease resistant transgenic plants. Besides these properties, AMPs were also used as drug delivery vectors to deliver cell impermeable drugs to cell interior. The present review focuses on the diversity and broad spectrum antimicrobial activity of AMPs along with its multidimensional properties that could be exploited for the application of these bioactive peptides as a potential and promising drug candidate in pharmaceutical industries.
With the extensive use of antibiotics, multidrug-resistant bacteria emerge frequently. New antimicrobial agents with novel
modes of action are urgently needed. It is now widely accepted that antimicrobial peptides (AMPs) could be promising alternatives
to conventional antibiotics. In this study, we aimed to study the antimicrobial activity and mechanism of action of protonectin,
a cationic peptide from the venom of the neotropical social wasp Agelaia pallipes pallipes. We demonstrated that protonectin exhibits potent antimicrobial activity against a spectrum of bacteria, including multidrug-resistant
strains. To further understand this mechanism, the structural features of protonectin and its analogs were studied by circular
dichroism (CD). The CD spectra demonstrated that protonectin and its natural analog polybia-CP formed a typical α-helical
conformation in the membrane-mimicking environment, while its proline-substituted analog had much lower or even no α-helix
conformation. Molecular dynamics simulations indicated that the α-helical conformation in the membrane is required for the
exhibition of antibacterial activity. In conclusion, protonectin exhibits potent antimicrobial activity by disruption of the
integrity of the bacterial membrane, and its α-helical confirmation in the membrane is essential for this action.
The effect of surfactants (two cationic, one anionic and three non-ionic) at 0.001, 0.01, 0.1 and 1.0 % concentrations on aflatoxin production, ergosterol content and sugar consumption by Aspergillus parasiticus (NRRL 2999) in YES liquid culture medium is reported. At 0.01% concentration, the cationic surfactants, cetyl dimethyl ammonium bromide (CDAB) and dodecyl trimethyl ammonium bromide (DTAB), and the anionic surfactant, sodium dodecyl sulfate (SDS), completely inhibited spore germination, while DTAB also inhibited the production of ergosterol and toxin (p < 0.05). At a concentration of 0.001%, CDAB was found to enhance toxin production, while SDS was found to inhibit it when compared with other surfactants. Non-ionic surfactants, polyoxyethylene sorbitan monolaurate (Tween-20), polyoxyethylene lauryl ether (Brij-35) and ethoxylated p-tert-octylphenol (Triton X-100) delayed the spore germination up to day 5 at all concentrations and inhibited toxin and ergosterol production at 0.001% concentration. The affect was found to be dose-dependent from 0.001% to 1%, for Triton X-100 only. Positive correlation between ergosterol content and toxin production in the presence of different surfactants at various time periods (3, 5, 7, 9 and 12 days) was found. Tween-20 was most effective in inhibiting toxin production on day 7, when aflatoxin production was found to be maximal in control group. Sugar consumption was directly proportional to the ergosterol content, showing a significant correlation with aflatoxin production.
Aspergillus flavus is one of the most common fungal eukaryotes on the planet. It is notorious for production of aflatoxins, for causing aspergillosis in humans and animals, and as an opportunistic pathogen of animals and plants. Its role in marine habitats is unclear. Until now, little phylogeographic structure has been detected for the species, except at very local scales, and it appears to fit the classic dictum of microbial biogeography: Everything is everywhere. Here we use genetic relationships among isolates to determine phylogeographic structure, mating types, and differences in preferences for: marine vs. terrestrial habitats, various substrates, and clinical vs. nonclinical environments. Phylogenetic relationships among isolates were estimated using amplified fragment length polymorphisms (AFLPs) and mating types were determined for a worldwide sample of A. flavus isolates from diverse substrates and geographic locations. All isolates composed a single population, with no significant differentiation of marine vs. terrestrial isolates, clinical vs. environmental isolates, or association with substrate or geographic origin. There was evidence for local dominance of a single clade, probably clonal in origin and short-lived. The proportion of mating types was 1:1, supporting the hypothesis of recombination in natural populations. However, a high proportion of clinical isolates were MAT1-1 (85%), suggesting that a gene linked to the MAT1-1 idiomorph could play a role in pathogenicity. This study suggests that a more appropriate description of the phylogeography of A. flavus is 'everything is everywhere, but not all the time.'
The gene encoding the plasma membrane proton pump (H+-ATPase) of Aspergillus fumigatus, PMA1, was characterized from A. fumigatus strain NIH 5233 and clinical isolate H11-20. An open reading frame of 3,109 nucleotides with two introns near the N terminus
predicts a protein consisting of 989 amino acids with a molecular mass of approximately 108 kDa. The predicted A. fumigatus enzyme is 89 and 51% identical to H+- ATPases of Aspergillus nidulans and Saccharomyces cerevisiae, respectively. The A. fumigatus PMA1 is a typical member of the P-type ATPase family that contains 10 predicted transmembrane segments and conserved sequence
motifs TGES, CSDKTGT, MLTGD, and GDGVN within the catalytic region. The enzyme represents 2% of the total plasma membrane
protein, and it is characteristically inhibited by orthovanadate, with a 50% inhibitory concentration of ∼1.8 μM. H+-ATPases from Aspergillus spp. contain a highly acidic insertion region of 60 amino acids between transmembrane segments 2 and 3, which was confirmed
for the membrane-assembled enzyme with a peptide-derived antibody. An increasing A. fumigatus PMA1 copy number confers enhanced growth in low-pH medium, consistent with its role as a proton pump. These results provide support
for the development of the A. fumigatus H+-ATPase as a potential drug discovery target.
The neuronal calcium sensor (NCS) proteins (e.g. recoverin, neurocalcins, and frequenin) are expressed at highest levels in excitable cells, and some of them regulate desensitization
of G protein-coupled receptors. Here we present NMR analysis and genetic functional studies of an NCS homolog in fission yeast
(Ncs1p). Ncs1p binds three Ca2+ ions at saturation with an apparent affinity of 2 μm and Hill coefficient of 1.9. Analysis of NMR and fluorescence spectra of Ncs1p revealed significant Ca2+-induced protein conformational changes indicative of a Ca2+-myristoyl switch. The amino-terminal myristoyl group is sequestered inside a hydrophobic cavity of the Ca2+-free protein and becomes solvent-exposed in the Ca2+-bound protein. Subcellular fractionation experiments showed that myristoylation and Ca2+ binding by Ncs1p are essential for its translocation from cytoplasm to membranes. The ncs1 deletion mutant (ncs1Δ) showed two distinct phenotypes: nutrition-insensitive sexual development and a growth defect at high levels of extracellular
Ca2+ (0.1 m CaCl2). Analysis of Ncs1p mutants lacking myristoylation (Ncs1pG2A) or deficient in Ca2+ binding (Ncs1pE84Q/E120Q/E168Q) revealed that Ca2+ binding was essential for both phenotypes, while myristoylation was less critical. Exogenous cAMP, a key regulator for sexual
development, suppressed conjugation and sporulation of ncs1Δ, suggesting involvement of Ncs1p in the adenylate cyclase pathway turned on by the glucose-sensing G protein-coupled receptor
Git3p. Starvation-independent sexual development of ncs1Δ was also complemented by retinal recoverin, which controls Ca2+-regulated desensitization of rhodopsin. In contrast, the Ca2+ intolerance of ncs1Δ was not affected by cAMP or recoverin, suggesting that the two ncs1Δ phenotypes are mechanistically independent. We propose that Schizosaccharomyces pombe Ncs1p negatively regulates sporulation perhaps by controlling Ca2+-dependent desensitization of Git3p.
Novel antimicrobial agents with a low propensity to develop resistance by microorganisms have contemporary relevance. In this perspective, the present study reports the green synthesis and characterization of cecropins peptides (D2A21, D2A10, and D4E1) based silver nanocomposites. The effect of pH and concentration of peptides on the formation of nanocomposite material was studied using UV–Vis spectroscopy. The particle size was determined by transmission electron microscopy, which indicated the size in the range of 3 ± 0.4 to 20 ± 5 nm. Fourier-transform infrared spectroscopy studies suggested the involvement of peptides as a capping and reducing agent. Zeta potential analysis suggested that nanocomposite material was more cationic in nature than its native peptides.
Nanocomposite material exhibited significantly enhanced antibacterial activity as compared to native peptides and silver nanoparticles with minimum inhibitory concentration (MIC) ranging from 1 to 3 μg mL⁻¹ against both gram-positive and negative test bacteria; whereas the MICs of native peptides were found to be in the range of 4–6 μg mL⁻¹. The mode of action of P-AgNPs was evaluated using scanning electron microscopy, membrane potential, and membrane integrity studies; wherein the nanocomposite material was found to act at the cell membrane level, causing complete loss of membrane potential and resulting in compromised membrane integrity with irreversible damage to the cell as shown by the rapid loss of viability due to membrane disruption, resulting in lysis. Among the three peptides tested, D2A21-silver nanocomposite had maximal antibacterial activity. Taken together; our experimental findings suggested that the peptide-based-silver nanocomposites can be considered as potential antibacterial agents for various biomedical applications.
Aflatoxins are potent carcinogenic mycotoxins produced as secondary metabolites mainly by the fungi Aspergillus flavus and Aspergillus parasiticus. Control measures to curtail the contamination of aflatoxin in food products is still a challenge. Although there are several reports on the antifungal peptides, there is no specific study on the action of antifungal peptides on aflatoxin synthesis. This work details the effect of four antimicrobial peptides (AMPs) - PPD1 (FRLHF), 66-10 (FRLKFH), 77-3 (FRLKFHF) and D4E1 (FKLRAKIKVRLRAKIKL) on the aflatoxin production by A. flavus and A. parasiticus. Results of the investigations suggests that AMPs at near minimum inhibitory concentrations (MIC) were effectively inhibiting aflatoxins, without hindering the growth of the fungi. These AMPs, at concentrations near MIC, induced membrane permeabilisation, without inducing cellular leakage. The involvement of oxidative stress for the aflatoxin synthesis was reversed by the antioxidant nature of the peptides as evidenced by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) assay, reactive oxygen species production, malondialdehyde and antioxidant enzymes analysis. Quantitative real time polymerase chain reaction (RT-qPCR) analysis of the aflatoxin gene cluster showed that ‘aflR’ and its downstream genes expressions were significantly down regulated. Conidiation of the fungi were negatively influenced by the peptides as evidenced by scanning electron microscopy analysis and RT-qPCR. mRNA levels of Manganese-superoxide dismutase (Mn-SOD) showed a decrease in the expression in RT-qPCR. The effect of these peptides on aflatoxin inhibition provides insight into their use as novel antiaflatoxigenic molecules.
Antibiotic resistance in bacteria has become a serious threat to public health, and therefore there is an urgent need to develop new classes of antimicrobial agents. Nowadays, natural antimicrobial peptides (AMPs) and their synthetic derivatives are considered as promising alternatives to traditional antibiotics. The broad molecular diversity of AMPs, in terms of sequences and structures, suggests that their activity does not depend on specific features of amino acid sequence or peptide conformation. We therefore selected two common properties of AMPs, (high percentage of hydrophobic and cationic amino acids), to develop a novel approach to synthesize random antimicrobial peptide mixtures (RPMs). Instead of incorporating a single amino acid at each coupling step, a mixture of hydrophobic and cationic amino acids in a defined proportion is coupled. This results in a mixture that contains up to 2 ⁿ sequences, where n is the number of the coupling step, of random peptides with a defined composition, stereochemistry, and controlled chain length. We have discovered that RPMs of hydrophobic and cationic α-amino acids, such as phenylalanine and lysine, display strong and broad antimicrobial activity towards Gram-negative, Gram-positive, clinically isolated antibiotic resistant "superbugs", and several plant pathogenic bacteria. This review summarizes our efforts to explore the mode of action of RPMs and their potential as bioactive agents for multiple applications, including the prevention of biofilm formation and degradation of mature biofilm (related to human health), reduction of disease severity in plant bacterial disease models (related to crop protection), and inhibition of bacterial growth in milk (related to food preservation). All our findings illustrate the effectiveness of RPMs and their great potential for various applications.
The biogenic synthesis of silver nanoparticles was achieved by using gum kondagogu (Cochlospermum gossypium), a natural biopolymer (Gk-AgNPs). Synthesised nanoparticles were characterised by using UV-visible spectroscopy, inductively coupled plasma-atomic emission spectrometer, X-ray diffraction, transmission electron microscope techniques. The silver nano particle size determined was found to be 3.6 ± 2.2 nm. The synthesised Gk-AgNPs showed antifungal activity and exhibited minimum inhibitory concentration and minimal fungicidal concentration values ranging from 3.5 to 6.5 μg mL⁻¹ against Aspergillus parasiticus (NRRL-2999) and Aspergillus flavus (NRRL-6513). Scanning electron microscopy-energy dispersive spectroscopy analysis revealed morphological changes including deformation, shrunken and ruptured mycelium of the fungi. At the biochemical level, the mode of action revealed that there was an elevated level of reactive oxygen species, lipid peroxidation, superoxide dismutase, and catalase enzyme activity. Increased oxidative stress led to increased outer membrane damage, which was confirmed by the entry of N-phenyl naphthylamine to the phospholipid layer of outer membrane and higher levels of K⁺ release from the fungi treated with Gk-AgNPs. This study explores the possible application of biogenic silver nanoparticles produced from gum kondagogu as potent antifungal agents. The potent antifungal activity of Gk-AgNPs gives scope for its relevance in biomedical application and as a seed dressing material.
In the study of cell-penetrating and membrane-translocating peptides, a fundamental question occurs as to the contribution arising from fundamental peptide-membrane interactions, relative to the contribution arising from the biology and energy of the cell, mostly occurring in the form of endocytosis and subsequent events. A commonly used approach to begin addressing these mechanistic questions is to measure the degree to which peptides can interact with, and physically disrupt, the integrity of synthetic lipid bilayers. Here, we describe a set of experimental methods that can be used to measure the potency, kinetics, transience, and the effective size of peptide-induced membrane disruption.
An ion-exchange chromatography method was tested for quantification of fungal invasion in alfalfa forage. The concentration of chitin, an N-acetyl-D-glucosamine polymer found in spore and mycelium cell walls, was measured to quantitate the degree of forage fungal invasion. Chitin concentrations ranged from 1.583 to 7.530 g kg−1 DM in the alfalfa forage. Stored hay chitin concentration can be influenced by standing crop and harvesting conditions as well as hay moisture content at the time of stacking. Chitin concentrations at the time of stacking and after 60 days storage were: 6.26 ± 0.12 and 6.85 ± 0.12; 2.69 ± 0.16 and 3.57 ± 0.12; 2.50 ± 0.12 and 3.95 ± 0.12 g kg−1DM for hay baled at 14.8, 21.5 and 29.1% moisture, respectively. High chitin concentrations for hay baled at the lowest moisture level reflected post-cutting fungal and bacterial invasion of forage exposed to precipitation while lying in a swath. Pre-storage fungal invasion for hay baled at the lowest moisture level resulted in a negative correlation between post-storage chitin concentration and visual mold assessment. Alfalfa hay containing 14.8, 21.5 and 29.1% moisture at the time of stacking had 9.6 ± 5.9, 33.7 ± 7.3 and 60.9 ± 5.9% increases in chitin concentration during a 60-day storage period. Change in chitin concentration during storage was positively correlated (P < 0.01) with visual mold scores of hay removed from storage and negatively correlated (P < 0.05) with dry matter retention during storage.
The inhibitory activity of bioactive polyphenols present in six sorghum genotypes—two red (AON 486 and IS 620), two yellow (LPJ and IS 17779) and two white (SPV 86 and SPV 462) varieties—on Aspergillus parasiticus (NRRL 2999) growth and aflatoxin production was evaluated. In the first experiment the production of aflatoxins in the six sorghum genotypes after removal of surface phenolics by acidic methanol treatment was studied and compared with that in untreated grains. Aflatoxin production was found to be fourfold higher in treated grains. The total phenols and bioactive polyphenols extracted by acidic methanol were quantified using the Folin–Denis method and the bovine serum albumin–benzidine conjugate procedure respectively. In the second experiment the effect of extracted sorghum phenolics under in vitro conditions on fungal growth and aflatoxin production was studied at two concentrations (0.01% and 0.1%) of phenolics. Extracted phenolics added to yeast extract sucrose (YES) medium at 0.1% concentration showed an inhibitory effect on aflatoxin production. At 0.01% phenolic concentration, aflatoxin production was minimal on day 3 after infection. At other time points the aflatoxin content was similar to that in the control. At 9 days after infection the fungal biomass in IS 620 was significantly lower than that in the control. At 0.1% phenolic concentration, aflatoxin production was minimal and the red genotype IS 620 showed maximum resistance. Fungal biomass was lowest at all growth stages in IS 620 as compared with the control. Polyphenol oxidase (PPO) activity was not detected in A. parasiticus grown on YES medium (control). PPO activity was not induced in A. parasiticus by the addition of phenolics to the liquid culture medium (no PPO activity was detected in the culture medium). The inhibitory activity of bioactive polyphenols could be attributed to the lack of PPO enzyme in this fungus. Copyright © 2007 Society of Chemical Industry
The autoxidation of pyrogallol was investigated in the presence of EDTA in the pH range 7.9–10.6.
The rate of autoxidation increases with increasing pH. At pH 7.9 the reaction is inhibited to 99% by superoxide dismutase, indicating an almost total dependence on the participation of the superoxide anion radical, O2·−, in the reaction. Up to pH 9.1 the reaction is still inhibited to over 90% by superoxide dismutase, but at higher alkalinity, O2·− -independent mechanisms rapidly become dominant.
Catalase has no effect on the autoxidation but decreases the oxygen consumption by half, showing that H2O2 is the stable product of oxygen and that H2O2 is not involved in the autoxidation mechanism.
A simple and rapid method for the assay of superoxide dismutase is described, based on the ability of the enzyme to inhibit the autoxidation of pyrogallol.
A plausible explanation is given for the non-competitive part of the inhibition of catechol O-methyltransferase brought about by pyrogallol.
Conidial germination in Neurospora crassa was inhibited by the anticalmodulin (CaM) agents calmidazolium (CaMZ) and
trifuoperazine (TFP), indicating the importance of CaM-dependent events during vegetative growth. An increase in intracellular CaM
concentration, observed during conidial germination, was associated with enhanced incorporation of [$H]phenylalanine into CaM and
increased CaM-dependent protein kinase activity. Phosphorylation of two proteins (71 and 25 kDa) during conidial germination, and
of four proteins (71, 47, 38 and 32 kDa) during hyphal elongation was noted to be CaM-dependent since these phosphorylations
were inhibited in vitro by CaMZ. The possible regulatory functions of CaM during conidial germination are discussed in relation to
phosphorylation of speci®c proteins during vegetative growth in N. crassa.
Aspergillus flavus is saprophytic soil fungus that infects and contaminates preharvest and postharvest seed crops with the carcinogenic secondary metabolite aflatoxin. The fungus is also an opportunistic animal and human pathogen causing aspergillosis diseases with incidence increasing in the immunocompromised population. Whole genome sequences of A. flavus have been released and reveal 55 secondary metabolite clusters that are regulated by different environmental regimes and the global secondary metabolite regulators LaeA and VeA. Characteristics of A. flavus associated with pathogenicity and niche specialization include secondary metabolite production, enzyme elaboration, and a sophisticated oxylipin host crosstalk associated with a quorum-like development program. One of the more promising strategies in field control involves the use of atoxic strains of A. flavus in competitive exclusion studies. In this review, we discuss A. flavus as an agricultural and medical threat and summarize recent research advances in genomics, elucidation of parameters of pathogenicity, and control measures.
A family of peptides with broad-spectrum antimicrobial activity has been isolated from the skin of the African clawed frog Xenopus laevis. It consists of two closely related peptides that are each 23 amino acids and differ by two substitutions. These peptides are water soluble, nonhemolytic at their effective antimicrobial concentrations, and potentially amphiphilic. At low concentrations they inhibit growth of numerous species of bacteria and fungi and induce osmotic lysis of protozoa. The sequence of a partial cDNA of the precursor reveals that both peptides derive from a common larger protein. These peptides appear to represent a previously unrecognized class of vertebrate antimicrobial activities.
This laboratory has previously postulated that bromobenzene-induced hepatic necrosis results from the formation of a reactive metabolite that arylates vital cellular macromolecules. Accordingly, the severity of liver necrosis has been compared with the formation of metabolites of bromobenzene and with covalent binding of metabolites in vivo and in vitro after various pretreatment regimens that alter hepatotoxicity. These data provide direct kinetic evidence that 3,4-bromobenzene oxide is the reactive hepatotoxic metabolite. The studies also demonstrate that the hepatotoxic metabolite is preferentially conjugated (detoxified) with glutathione, thereby depleting glutathione from the liver. Liver necrosis and arylation of cellular macromolecules occur only when glutathione is no longer available. Thus, a dose threshold exists for bromobenzene-induced hepatic necrosis.
A photo-induced cyclic peroxidation in isolated chloroplasts is described. In an osmotic buffered medium, chloroplasts upon illumination produce malondialdehyde (MDA)—a decomposition product of tri-unsaturated fatty acid hydroperoxides—bleach endogenous chlorophyll, and consume oxygen. These processes show (a) no reaction in the absence of illumination; (b) an initial lag phase upon illumination of 10–20 minutes duration; (c) a linear phase in which the rate is proportional to the square root of the light intensity; (d) cessation of reaction occurring within 3 minutes after illumination ceases; and (e) a termination phase after several hours of illumination. The kinetics of the above processes fit a cyclic peroxidation equation with velocity coefficients near those for chemical peroxidation.The stoichiometry of MDA/O2 = 0.02, and O2Chlbleached = 6.9 correlates well with MDA production efficiency in other biological systems and with the molar ratio of unsaturated fatty acids to chlorophyll. The energies of activation for the lag and linear phases are 17 and 0 kcal/mole, respectively, the same as that for autoxidation. During the linear phase of oxygen uptake the dependence upon temperature and O2 concentration indicates that during the reaction, oxygen tension at the site of peroxidation is 100-fold lower than in the aqueous phase.It is concluded that isolated chloroplasts upon illumination can undergo a cyclic peroxidation initiated by the light absorbed by chlorophyll. Photoperoxidation results in a destruction of the chlorophyll and tri-unsaturated fatty acids of the chloroplast membranes.
Melittin, a toxin of bee venom, is a cationic polypeptide composed of 26 amino acids. The six residues of the C-terminal end are polar and 19 of the 20 residues of the N-terminal end are hydrophobic. Exposure of the lecithin bilayer to melittin results in the formation of channels that are more permeable to anions that to cations. Unilateral addition of melittin produces a voltage-dependent increase in membrane conductance when the side where the polypeptide is present in made positive but not when it is made negative. At a fixed voltage, the conductance increases with the fourth power of the melittin concentration in the aqueous phase. At a fixed peptide concentration, the conductance increases approximately e-fold per 6-mV increase in the electrical potential difference across the membrane. These results suggest that four melittin monomers are needed to form a channel and, furthermore, that a minimum of four equivalent electronic charges need to be displaced by the electrical field to explain the voltage dependence of the conductance.
An assay for activities of erythrocyte membrane-bound ATPases adapted from the autoanalyzer method of Raess and Vincenzi (1980a) is described in detail. Mg2+ ATPase, Na+/K(+)-ATPase, Ca2+ ATPase, and calmodulin- (CaM) activated Ca2+ ATPases were determined in microtiter plates. Total volume was 100 microL. Ten microliters of 0.75 mg/mL of red blood cells membranes were added to appropriate buffer in microtiter plates. Plates were preincubated at 37 degrees C for 10 min, reactions were started by the addition of ATP, and plates were incubated for an additional 60 min at 37 degrees C. Reactions were stopped by sodium dodecyl sulfate (SDS). Inorganic phosphate (P(i)) was measured by modifications of the method by Fiske and Subbarow, (Fiske and Subbarow, 1925) in the same plate using a plate reader. The P(i) assay range was between 0 and 250 nm/mL. Results obtained for intraassay precision, (n = 7) are as follows: Mg2+ ATPase = 4.39 +/- 0.25 (5.7% CV); Na+/K(+)-ATPase = 7.33 +/- 0.40 (5.4% CV); Ca2+ ATPase = 15.86 +/- 0.76 (4.8% CV); and CaM-activated Ca2+ ATPase = 74.12 +/- 2.34 (3.2% CV) (nmole P(i)/mg protein/min.). This is a rapid, simple, and nonisotopic method for the determination of membrane-bound ATPases activities. All steps are performed in the same microtiter plate, thus reducing handling and associated errors.
Synthetic combinatorial libraries were evaluated with an iterative process to identify a hexapeptide with broadspectrum activity against selected phytopathogenic fungi. A D-amino acid hexapeptide (FRLKFH) and pentapeptide (FRLHF) exhibited activity against Fusarium oxysporum f. sp. lycopersici, Rhizoctonia solani (anastomosis group 1), Ceratocystis fagacearum, and Pythium ultimum. The peptides showed no hemolytic or mutagenic activity. Fluorescent microscopy studies with a membrane impermeant dye indicated that fungal cytoplasmic membranes were compromised rapidly and that the nuclear membrane was also affected.
Cecropin A is a naturally occurring, linear, cationic, 37-residue antimicrobial peptide. The precise mechanism by which it kills bacteria is not known, but its site of action is believed to be the cell membrane. To investigate the nature of its membrane activity, we examined the ability of cecropin A to alter membrane permeability in synthetic lipid vesicles and in Gram-negative bacteria. Cecropin A exerted distinctly different types of membrane activity depending on its concentration. In synthetic lipid vesicles, cecropin A dissipated transmembrane electrochemical ion gradients at relatively low concentrations, but much higher concentrations were required to release an encapsulated fluorescent probe. Cecropin A dissipated ion gradients whether or not the vesicle membranes contained anionic lipid, although the presence of anionic lipid dramatically increased peptide binding, and modestly increased the release of an encapsulated probe. Cholesterol did not prevent the dissipation of ion gradients by low concentrations of peptide, but it did inhibit release of the encapsulated probe by high concentrations of peptide. At the highest concentrations examined, cecropin A remained monomeric in solution, and did not aggregate, lyse, or otherwise alter vesicle size. In Gram-negative bacteria, cecropin A was potently bactericidal at concentrations which dissipated ion gradients in lipid vesicles, but much higher concentrations were required to cause the release of cytoplasmic contents. These findings point to the conclusion that cecropin A kills bacteria by dissipating transmembrane electrochemical ion gradients. They weigh against theories comparing the antimicrobial activity of cecropin A to the release of encapsulated probes from lipid vesicles, and against roles for cholesterol or anionic lipid headgroups in the selectivity of peptide action against bacteria.
Many short antimicrobial peptides (< 18mer) have been identified for the development of therapeutic agents. However, Structure-activity relationship (SAR) studies about short antimicrobial peptides have not been extensively performed. To investigate the relationship between activity and structural parameters such as an alpha-helical structure, a net positive charge and a hydrophobicity, we synthesized and characterized diastereomers, scramble peptides and substituted peptides of the short antimicrobial peptide identified by combinatorial libraries. Circular dichroism (CD) spectra and in vitro activity indicated that an alpha-helical structure correlated with the antimicrobial activity and a beta-sheet structure also satisfied a structural requirement for antimicrobial activity. Most peptides consisting of L-amino acids lost antifungal activity in the presence of heat-inactivated serum, while active diastereomers and a scramble peptide with the beta-sheet structure retained antifungal activity in the same condition.
The relevance of free radical generation and oxidative stress with regard to aflatoxin production was examined by comparing the oxygen requirement and antioxidant status of a toxigenic strain of Aspergillus parasiticus with that of a nontoxigenic strain at early (trophophase) and late logarithmic (idiophase) growth phases. In comparison to the nontoxigenic strain, wherein the oxygen requirements were relatively unaltered at various growth phases, the toxigenic strain exhibited greater oxygen requirements at trophophase coinciding with onset of aflatoxin production. The activities of antioxidant enzymes such as xanthine oxidase, superoxide dismutase, and glutathione peroxidase and the mycelial contents of thiobarbituric acid-reactive substances as well as of reduced glutathione were all enhanced during the progression of toxigenic strain from trophophase to idiophase. The combined results suggest that aflatoxin production by the toxigenic strain may be a consequence of increased oxidative stress leading to enhanced lipid peroxidation and free radical generation.
The ability of dietary carotenoids such as beta-carotene and lycopene to act as antioxidants in biological systems is dependent upon a number of factors. While the structure of carotenoids, especially the conjugated double bond system, gives rise to many of the fundamental properties of these molecules, it also affects how these molecules are incorporated into biological membranes. This, in turn, alters the way these molecules interact with reactive oxygen species, so that the in vivo behavior may be quite different from that seen in solution. The effectiveness of carotenoids as antioxidants is also dependent upon their interaction with other coantioxidants, especially vitamins E and C. Carotenoids may, however, lose their effectiveness as antioxidants at high concentrations or at high partial pressures of oxygen. It is unlikely that carotenoids actually act as prooxidants in biological systems; rather they exhibit a tendency to lose their effectiveness as antioxidants.
The aim of this study was to investigate the effect of ascorbic acid (AA) an alterations in hepatic secretory and microsomal functions during hepatic ischemia and reperfusion.
Rats were subjected to 60 min of hepatic ischemia, and 1 and 5 h of reperfusion. Five minutes prior to ischemia, the animals were administered either vehicle or ascorbic acid (AA) (30, 100, 300, and 1000 mg/kg) intravenously.
The serum aminotransferase level and lipid peroxidation were markedly higher as a result of ischemia/reperfusion. These increases were significantly attenuated by AA doses of 30 and 100 mg/kg but were augmented by dose of 1000 mg/kg. Bile flow and cholate output were markedly decreased by ischemia/reperfusion. AA doses of 30 and 100 mg/kg restored but dose of 1000 mg/kg inhibited their secretion. Both the cytochrome P(450) content and aminopyrine N-demethylase activity were decreased by ischemia/reperfusion, which were prevented by AA doses of 30 and 100 mg/kg but were aggravated by dose of 1000 mg/kg. Aniline p-hydroxylase activity was elevated by ischemia/reperfusion, and this was prevented by AA doses of 100, 300 and 1000 mg/kg.
Ischemia/reperfusion diminishes the hepatic secretory and microsomal functions. AA has both antioxidant and pro-oxidant effects, depending upon the dose.
A comprehensive docking study was performed on 27 distinct protein-protein complexes. For 13 test systems, docking was performed with the unbound X-ray structures of both the receptor and the ligand. For the remaining systems, the unbound X-ray structure of only molecule was available; therefore the bound structure for the other molecule was used. Our method optimizes desolvation, shape complementarity, and electrostatics using a Fast Fourier Transform algorithm. A global search in the rotational and translational space without any knowledge of the binding sites was performed for all proteins except nine antibodies recognizing antigens. For these antibodies, we docked their well-characterized binding site-the complementarity-determining region defined without information of the antigen-to the entire surface of the antigen. For 24 systems, we were able to find near-native ligand orientations (interface C(alpha) root mean square deviation less than 2.5 A from the crystal complex) among the top 2,000 choices. For three systems, our algorithm could identify the correct complex structure unambiguously. For 13 other complexes, we either ranked a near-native structure in the top 20 or obtained 20 or more near-native structures in the top 2,000 or both. The key feature of our algorithm is the use of target functions that are highly tolerant to conformational changes upon binding. If combined with a post-processing method, our algorithm may provide a general solution to the unbound docking problem. Our program, called ZDOCK, is freely available to academic users (http://zlab.bu.edu/~rong/dock/).
Synthetic peptides designed using criteria derived from statistical analyses of beta-barrel membrane proteins form channels in synthetic membranes at very low peptide:lipid ratios.
The fungicidal effect and mechanism of a tryptophan-rich 13-mer peptide, indolicidin derived from granules of bovine neutrophils, were investigated. Indolicidin displayed a strong fungicidal activity against various fungi. In order to understand the fungicidal mechanism(s) of indolicidin, we examined the interaction of indolicidin with the pathogenic fungus Trichosporon beigelii. Fluorescence confocal microscopy and flow cytometry analysis revealed that indolicidin acted rapidly on the plasma membrane of the fungal cells in an energy-independent manner. This interaction is also dependent on the ionic environment. Furthermore, indolicidin caused significant morphological changes when tested for the membrane disrupting activity using liposomes (phosphatidylcholine/cholesterol; 10:1, w/w). The results suggest that indolicidin may exert its fungicidal activity by disrupting the structure of cell membranes, via direct interaction with the lipid bilayers, in a salt-dependent and energy-independent manner.
The antimicrobial activity of silver nanoparticles against E. coli was investigated as a model for Gram-negative bacteria. Bacteriological tests were performed in Luria-Bertani (LB) medium on solid agar plates and in liquid systems supplemented with different concentrations of nanosized silver particles. These particles were shown to be an effective bactericide. Scanning and transmission electron microscopy (SEM and TEM) were used to study the biocidal action of this nanoscale material. The results confirmed that the treated E. coli cells were damaged, showing formation of "pits" in the cell wall of the bacteria, while the silver nanoparticles were found to accumulate in the bacterial membrane. A membrane with such a morphology exhibits a significant increase in permeability, resulting in death of the cell. These nontoxic nanomaterials, which can be prepared in a simple and cost-effective manner, may be suitable for the formulation of new types of bactericidal materials.