Antibacterial Mode of Action of Ib-AMP1 Against Escherichia coli O157:H7

Continual occurrence of foodborne outbreaks, along with the increase in antibiotic resistance which burdens clinical treatments, has urged scientists to search for other potential promising antimicrobial agents. Antimicrobial peptides are emerging as one of the potential alternatives. The mode of action of a given AMP is critical and essential for future application; however, it is still not completely known for many of these compounds. Ib-AMP1 is a plant-derived AMP, purified from seeds of Impatiens balsamina and has been shown to exert antibacterial and antifungal activity at the micromolar level. A study had shown that the therapeutic index of Ib-AMP1 against eight human pathogens is 23.5. The objective of the present study was to determine the in vivo mode of action of Ib-AMP1 against Escherichia coli O157:H7. A concentration-dependent effect of Ib-AMP1 on the E. coli O157:H7 cell membrane occurred. Ib-AMP1 treatments resulted in efflux of K⁺ and ATP, suggesting pores of sufficient size to allow efflux of large molecules. Ib-AMP1 at sublethal concentrations exerts a greater effect at the intracellular level. In contrast, Ib-AMP1 at a lethal concentration permeabilizes cell membranes and may directly or indirectly inhibit intracellular macromolecule synthesis. Collectively, results of this study suggest Ib-AMP1 is bactericidal interfering within outer and inner membrane integrity permitting efflux of ATP and interfering with intracellular biosynthesis of DNA, RNA and protein.     

Bactericidal properties of the antimicrobial peptide Ib-AMP4 from Impatiens balsamina produced as a recombinant fusion-protein in Escherichia coli

Antimicrobial peptides (AMPs) represent a novel class of powerful natural antimicrobial agents. As AMPs are bactericidal, production of AMPs in recombinant bacteria is far from trivial. We report the production of Impatiens balsamina antimicrobial peptide 4 (Ib-AMP4, originally isolated from Impatiens balsamina) in Escherichia coli as a fusion protein and investigate Ib-AMP4's antimicrobial effects on human pathogens. A plasmid vector pET32a-Trx-Ib-AMP4 was constructed and transferred into E. coli. After induction, a soluble fusion protein was expressed successfully. The Ib-AMP4 peptide was obtained with a purity of over 90% after nickel affinity chromatography, ultrafiltration, enterokinase cleavage and sephadex size exclusion chromatography. For maximum activity, Ib-AMP4, which possesses two disulfide bonds, required activation with 5 μg/mL H₂O₂. Antimicrobial assays showed that Ib-AMP4 could efficiently target clinical multiresistant isolates including methicillin-resistant Staphylococcus aureus and extended-spectrum β-lactamase-producing E. coli. Time kill experiments revealed that Ib-AMP4 is bactericidal within 10 min after application. Haemolysis and cytotoxicity assays implied selectivity towards bacteria, an important prerequisite for clinical applications. Ib-AMP4 might be an interesting candidate for clinical studies involving patients with septicemia or for coating clinical devices, such as catheters. The method described here may be applicable for expression and purification of other AMPs with multiple disulfide bridges.     

Ib-AMP4 insertion causes surface rearrangement in the phospholipid bilayer of biomembranes: Implications from quartz-crystal microbalance with dissipation

Most antimicrobial peptides exert their rapid bactericidal activity through a unique mechanism of bacterial membrane disruption. However, the molecular events that underlie this mechanism remain partly unresolved. In this study, the frequency shift (ΔF) obtained through quartz-crystal microbalance with dissipation (QCM–D) indicated that the initial binding of Ib-AMP4 within the lipid membrane started at a critical Ib-AMP4 concentration that exceeded 100 μg/ml. Circular dichroism measurements provided evidence that Ib-AMP4 occurs in a β-sheet configuration which is adapted for insertion into the lipid membrane. Monolayer experiments and the value of dissipation alteration (ΔD) obtained through QCM–D showed that the pressure increased within the phospholipid bilayer upon peptide insertion, and the increase in pressure subsequently forced the bilayer to wrinkle and form pores. However, D continued to increase, indicating that the membrane surface underwent a dramatic morphological transition: the membrane surface likely became porous and uneven as Ib-AMP4 projected from the external surface of the lipid bilayer. Intensive peptide insertion, however, soon plateaued 1 min after the addition of Ib-AMP4. This behaviour corresponded with the results of bactericidal kinetics and liposome leakage assays. A sudden decrease in D accompanied by a negligible decrease in F occurred after replacing the Ib-AMP4 solution with HEPES buffer. This result implied that the bilayer surface rearranged and that poration and wrinkling decreased without further peptide insertion. Transmission electron microscopy results indicated that pore formation occurred during Ib-AMP4 insertion but eventually subsided. Therefore, the mode of action of AMP in bacterial membranes could be elucidated through QCM–D.     

Antifungal mechanism of a cysteine-rich antimicrobial peptide, Ib-AMP1, from Impatiens balsamina against Candida albicans

The antifungal mechanism of a 20-mer peptide, Ib-AMP1, derived from Impatiens balsamina was investigated. The oxidized (disulfide bridged) Ib-AMP1 showed a 4-fold increase in antifungal activity against Aspergillus flavus and Candida albicans than reduced (non-disulfide bridged) Ib-AMP1. Ib-AMP1 had very low activity for phospholipid disruption when compared with cecropin A(1-8)-magainin 2(1-12), a -helical amphiphatic, antimicrobial peptide. Confocal microscopy showed that Ib-AMP1 binds on cell surface or penetrates into cell membranes. These results suggested that Ib-AMP1 may manifest its antifungal activity against Candida albicans by inhibiting a distinct cellular process rather than ion channel or pore formation in cell membrane.     

Enhancing Antibacterial Activity Against Escherichia coli K-12 of Peptide Ib-AMP4 with Synthetic Analogues

A family of Ib-AMP4 peptide analogues was obtained by solid phase synthesis, modifying the net charge and hydrophobicity of C-terminal domain by replacing certain amino acidic residues by arginine and tryptophan. Additionally, disulfide bonds were eliminated by replacing the cysteine residues by methionine, which resulted in a decrease in the number of synthesis byproducts, and consequently diminished the subsequent purification steps. The obtained peptides were purified by RP-HPLC and their molecular mass was determined by MALDI-TOF mass spectrometry. The peptide analogues (IC₅₀ between 1 and 50 μM) presented a higher antibacterial activity against Escherichia coli K-12 than the native peptide (IC₅₀ > 100 μM). The hemolytic activity of the peptide with the highest antibacterial efficacy presented no degradation of erythrocytes for a concentration of 1 μM that corresponds to its IC₅₀ value. The results show that the synthesized peptides are good candidates for the treatment of diseases caused by E. coli.     

Free uptake of cell-penetrating peptides by fission yeast

An increasing number of peptides translocate the plasma membrane of mammalian cells promising new avenues for drug delivery. However, only a few examples are known to penetrate the fungal cell wall. We compared the capacity of different fluorophore-labelled peptides to translocate into fission yeast and human cells and determined their intracellular distribution. Most of the 20 peptides tested were able to enter human cells, but only one, transportan 10 (TP10), efficiently penetrated fission yeast and was distributed uniformly inside the cells. The results show that the fungal cell wall may reduce, but does not block peptide uptake.     

Isolation and characterization of a novel class of plant antimicrobial peptides form Mirabilis jalapa L. seeds.

We have isolated from seeds of Mirabilis jalapa L. two antimicrobial peptides, designated Mj-AMP1 and Mj-AMP2, respectively. These peptides are highly basic and consist of 37 and 36 residues for Mj-AMP1 and Mj-AMP2, respectively. Both peptides contain three disulfide bridges and differ from one another only by 4 amino acids. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the reduced and unreduced peptides suggests that the peptides associate into dimers in their native form. The Mj-AMPs exhibit a broad spectrum of antifungal activity since they are active against all 13 tested plant pathogenic fungi. Concentrations required for 50% inhibition of fungal growth vary from 6 to 300 micrograms/ml for Mj-AMP1 and from 0.5 to 20 micrograms/ml for Mj-AMP2. These peptides were also active on two tested Gram-positive bacteria but were apparently nontoxic for Gram-negative bacteria and cultured human cells. Although the Mj-AMPs show sequence similarity to mu-agatoxins, a class of insecticidal neurotoxic peptides isolated from the venom of spiders, they do not affect pulse transmission in insect nerves.     

Construction of ethanol-tolerant yeast strains with combinatorial library-selected peptides

Combinatorial yeast libraries were constructed by transformation of expression plasmids containing artificially synthesized random sequences into Saccharomyces cerevisiae MT8-1 and IFO10150. Approximately 200 yeast strains with enhanced ethanol tolerance were obtained from yeast libraries by incubation in 10% ethanol for 24 h. Following separate evaluation of their ethanol tolerance, the 10 clones with the highest values were selected. After 3 h incubation in 12.5% ethanol, whereas most of the control cells died, the clone with the highest tolerance from the MT8-1 library, M-1, showed approximately 40% cell viability, and the corresponding clone from the IFO10150 library, I-12, 48% viability. The half-life of M-1 cells was 20 times greater than that of control cells. Three of the library-selected peptides endowing with ethanol tolerance were identified as Gly-Thr-Arg-Leu-His pentapeptides. Four seemed to be extremely hydrophobic, and three of these were predicted to be transmembrane peptides. The three other peptides seemed to be more hydrophilic than standard yeast proteins. The results of the study show that yeast strains with fairly high ethanol tolerance can be successfully constructed by directed selection from yeast libraries expressing combinatorial peptides.     

Antimicrobial properties and death-inducing mechanisms of saccharomycin,a biocide secreted by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

We recently found that Saccharomyces cerevisiae (strain CCMI 885) secretes antimicrobial peptides (AMPs) derived from the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) that are active against various wine-related yeast and bacteria. Here, we show that several other S. cerevisiae strains also secrete natural biocide fractions during alcoholic fermentation, although at different levels, which correlates with the antagonistic effect exerted against non-Saccharomyces yeasts. We, therefore, term this biocide saccharomycin. The native AMPs were purified by gel-filtration chromatography and its antimicrobial activity was compared to that exhibited by chemically synthesized analogues (AMP1 and AMP2/3). Results show that the antimicrobial activity of the native AMPs is significantly higher than that of the synthetic analogues (AMP1 and AMP2/3), but a conjugated action of the two synthetic peptides is observed. Moreover, while the natural AMPs are active at pH 3.5, the synthetic peptides are not, since they are anionic and cannot dissolve at this acidic pH. These findings suggest that the molecular structure of the native biocide probably involves the formation of aggregates of several peptides that render them soluble under acidic conditions. The death mechanisms induced by the AMPs were also evaluated by means of epifluorescence microscopy-based methods. Sensitive yeast cells treated with the synthetic AMPs show cell membrane disruption, apoptotic molecular markers, and internalization of the AMPs. In conclusion, our work shows that saccharomycin is a natural biocide secreted by S. cerevisiae whose activity depends on the conjugated action of GAPDH-derived peptides. This study also reveals that S. cerevisiae secretes GAPDH-derived peptides as a strategy to combat other microbial species during alcoholic fermentations.     

Energy coupling of membrane transport and efficiency of sucrose dissimilation in yeast

Proton coupled transport of α-glucosides via Mal11 into Saccharomyces cerevisiae costs one ATP per imported molecule. Targeted mutation of all three acidic residues in the active site resulted in sugar uniport, but expression of these mutant transporters in yeast did not enable growth on sucrose. We then isolated six unique transporter variants of these mutants by directed evolution of yeast for growth on sucrose. In three variants, new acidic residues emerged near the active site that restored proton-coupled sucrose transport, whereas the other evolved transporters still catalysed sucrose uniport. The localization of mutations and transport properties of the mutants enabled us to propose a mechanistic model of proton-coupled sugar transport by Mal11. Cultivation of yeast strains expressing one of the sucrose uniporters in anaerobic, sucrose-limited chemostat cultures indicated an increase in the efficiency of sucrose dissimilation by 21% when additional changes in strain physiology were taken into account. We thus show that a combination of directed and evolutionary engineering results in more energy efficient sucrose transport, as a starting point to engineer yeast strains with increased yields for industrially relevant products.     

Antibacterial peptides isolated from insects.

Insects are amazingly resistant to bacterial infections. To combat pathogens, insects rely on cellular and humoral mechanisms, innate immunity being dominant in the latter category. Upon detection of bacteria, a complex genetic cascade is activated, which ultimately results in the synthesis of a battery of antibacterial peptides and their release into the haemolymph. The peptides are usually basic in character and are composed of 20-40 amino acid residues, although some smaller proteins are also included in the antimicrobial repertoire. While the proline-rich peptides and the glycine-rich peptides are predominantly active against Gram-negative strains, the defensins selectively kill Gram-positive bacteria and the cecropins are active against both types. The insect antibacterial peptides are very potent: their IC50 (50% of the bacterial growth inhibition) hovers in the submicromolar or low micromolar range. The majority of the peptides act through disintegrating the bacterial membrane or interfering with membrane assembly, with the exception of drosocin, apidaecin and pyrrhocoricin which appear to deactivate a bacterial protein in a stereospecific manner. In accordance with their biological function, the membrane-active peptides form ordered structures, e.g. alpha-helices or beta-pleated sheets and often cast permeable ion-pores. Their cytotoxic properties were exploited in in vivo studies targeting tumour progression. Although the native peptides degrade quickly in biological fluids other than insect haemolymph, structural modifications render the peptides resistant against proteases without sacrificing biological activity. Indeed, a pyrrhocoricin analogue shows lack of toxicity in vitro and in vivo and protects mice against experimental Escherichia coli infection. Careful selection of lead molecules based on the insect antibacterial peptides may extend their utility and produce viable alternatives to the conventional antimicrobial compounds for mammalian therapy.     

Interactions of lactoferricin-derived peptides with LPS and antimicrobial activity

Synthetic peptides derived from human and bovine lactoferricin, as well as tritrpticin sequences, were assayed for antimicrobial activity against wild-type Escherichia coli and LPS mutant strains. Antimicrobial activity was only obtained with peptides derived from the bovine lactoferricin sequence and peptides corresponding to chimeras of human and bovine sequences. None of the peptides corresponding to different regions of native human lactoferricin showed any antimicrobial activity. The results underline the importance of the content of tryptophan and arginine residues, and the relative location of these residues for antimicrobial activity. Results obtained for the same assays performed with LPS mutants suggest that lipid A is not the main binding site for lactoferricin which interacts first with the negative charges present in the inner core. Computer modelling of the most active peptides led to a model in which positively charged residues of the cationic peptide interact with negative charges carried by the LPS to disorganise the structure of the outer membrane and facilitate the approach of tryptophan residues to the lipid A in order to promote hydrophobic interactions.     

Modification of yeast characteristics by soy peptides: cultivation with soy peptides represses the formation of lipid bodies

We have previously reported that the cultivation of yeast cells with soy peptides can improve the tolerance of yeast to freeze–thaw stress (Izawa et al. Appl Microbiol Biotechnol 75:533–538, 2007), indicating that soy peptides can modify the characteristics of yeast cells. To gain a greater understanding of the potencies of soy peptides, we further investigated the effects of cultivation with soy peptides on yeast physiology and found that soy peptides repress the formation of lipid bodies (also called lipid droplets or lipid particles), in which neutral lipids are accumulated. Compared with casein peptone, bacto peptone, yeast nitrogen base, and free amino acid mixtures having the same amino acid composition as soy peptides, cultivation with soy peptides caused decreased levels of mRNAs of neutral lipid synthesis-related genes, such as DGA1, and repressed the formation of lipid bodies and accumulation of triacylglycerol. These results indicate that soy peptides affect the lipid metabolism in yeast cells, and also demonstrate a potentiality of edible natural ingredients as modifiers of the characteristics of food microorganisms.     

Isolation and identification of the products of Streptococcus faecalis inhibiting mycelial transformation of Candida albicans

An attempt was made to isolate and identify Streptococcus faecalis products responsible for the inhibition of mycelial transformation of Candida albicans. Five of streptococcal strains which 48 h broth culture supernatants run at 37 degrees C inhibited the most transformation of Candida albicans from yeast phase to mycelial phage. The strains were cultivated for 48 h in Tryptic Soy Broth at 37 degrees C, centrifuged and culture supernatants sterilised by means of filtration on millipore membranes of 0.4 micron diameter. After multistep purification of supernatants filtration on Diaflo PM 10 ultrafiltration membranes, Sephadex G 25, polyacrylamide gel electrophoresis) a homogenous, active fraction was obtained containing peptides of molecular weight around 6,000 Da. The peptides lost ability to induce mycelial transformation of C. albicans after heating at 100 degrees C for 10 min. Significant inhibition of morphological transformation of fungal cells was seen at the preparation concentration of 0.12 microgram/ml.     

The fluorescent protein iLOV as a reporter for screening of high-yield production of antimicrobial peptides in Pichia pastoris

The methylotrophic yeast Pichia pastoris is commonly used for the production of recombinant proteins at scale. The identification of an optimally overexpressing strain following transformation can be time and reagent consuming. Fluorescent reporters like GFP have been used to assist identification of superior producers, but their relatively big size, maturation requirements and narrow temperature range restrict their applications. Here, we introduce the use of iLOV, a flavin-based fluorescent protein, as a fluorescent marker to identify P. pastoris high-yielding strains easily and rapidly. The use of this fluorescent protein as a fusion partner is exemplified by the production of the antimicrobial peptide NI01, a difficult target to overexpress in its native form. iLOV fluorescence correlated well with protein expression level and copy number of the chromosomally integrated gene. An easy and simple medium-throughput plate-based screen directly following transformation is demonstrated for low complexity screening, while a high-throughput method using fluorescence-activated cell sorting (FACS) allowed for comprehensive library screening. Both codon optimization of the iLOV_NI01 fusion cassettes and different integration strategies into the P. pastoris genome were tested to produce and isolate a high-yielding strain. Checking the genetic stability, process reproducibility and following the purification of the active native peptide are eased by visualization of and efficient cleavage from the iLOV reporter. We show that this system can be used for expression and screening of several different antimicrobial peptides recombinantly produced in P. pastoris.     

An overview of antifungal peptides derived from insect

Fungi are not classified as plants or animals. They resemble plants in many ways but do not produce chlorophyll or make their own food photosynthetically like plants. Fungi are useful for the production of beer, bread, medicine, etc. More complex than viruses or bacteria; fungi can be destructive human pathogens responsible for various diseases in humans. Most people have a strong natural immunity against fungal infection. However, fungi can cause diseases when this immunity breaks down. In the last few years, fungal infection has increased strikingly and has been accompanied by a rise in the number of deaths of cancer patients, transplant recipients, and acquired immunodeficiency syndrome (AIDS) patients owing to fungal infections. The growth rate of fungi is very slow and quite difficult to identify. A series of molecules with antifungal activity against different strains of fungi have been found in insects, which can be of great importance to tackle human diseases. Insects secrete such compounds, which can be peptides, as a part of their immune defense reactions. Active antifungal peptides developed by insects to rapidly eliminate infectious pathogens are considered a component of the defense munitions. This review focuses on naturally occurring antifungal peptides from insects and their challenges to be used as armaments against human diseases.     

Antifungal activity of Latarcin 1 derived cell-penetrating peptides against Fusarium solani

Cell-penetrating peptides and antimicrobial peptides share physicochemical characteristics and mechanisms of interaction with biological membranes, hence, termed as membrane active peptides. The present study aims at evaluating AMP activity of CPPs. LDP-NLS and LDP are Latarcin 1 derived cell-penetrating peptides and in the current study we have evaluated antifungal and cell-penetrating properties of these CPPs in Fusarium solani. We observed that LDP-NLS and LDP exhibited excellent antifungal activity against the fungus. Cellular uptake experiments with LDP-NLS and LDP showed that LDP-NLS acted as a CPP but LDP uptake into fungal spores and hyphae was negligible. CPP and AMP activity of mutated version of LDP-NLS was also evaluated and it was observed that both the activities of the peptide were compromised, signifying the importance of arginines and lysines present in LDP-NLS for initial interaction of membrane active peptides with biological membranes. Dextrans and Propidium Iodide uptake studies revealed that the mode of entry of LDP-NLS into fungal hyphae is through pore formation. Also, both LDP-NLS and LDP showed no cytotoxicity when infiltered into leaf tissues. Overall, our results suggest that LDP-NLS and LDP are selectively cytotoxic to F. solani and can be a potent peptide based antifungal agents.     

A cumulative experience examining the effect of natural and synthetic antimicrobial peptides vs. Chlamydia trachomatis.

We tested the activity of 48 structurally diverse antimicrobial peptides against Chlamydia trachomatis, serovar L2. The peptides' activity against C. trachomatis, serovar L2 was measured in 48-h McCoy cell shell vial assays. Peptides of 16-20 amino acids were more active than larger peptides, such as defensins. Beta-sheet protegrins, as well as alpha-helical peptides such as novispirin (G-10) were equally active. Enantiomers were as active as native structures. Moderate-sized circular mini-defensins were less effective against C. trachomatis. Moderate-sized cationic peptides may be useful in microbicide preparations designed to prevent chlamydial infection.     

Recombinant expression and range of activity of penaeidins, antimicrobial peptides from penaeid shrimp.

Penaeidins are 5.5- to 6.6-kDa antimicrobial peptides recently isolated from the plasma and haemocytes of the tropical shrimp Penaeus vannamei. These molecules differ from the other classes of antimicrobial peptides in that they are composed of a proline-rich N-terminus and of a C-terminus containing six cysteine residues engaged in three disulfide bridges. In order to gain information on their antimicrobial activity, two penaeidins (Pen-2 and Pen-3a) were expressed in Saccharomyces cerevisiae. The recombinant Pen-2 and -3a were characterized in terms of primary structure by Edman degradation, mass spectrometry and gas chromatography. A protocol was then established to purify the amount of penaeidins required for the determination of their activity spectrum. We demonstrate in this study that expression in yeast is appropriate for the large-scale production of functional penaeidins, whose activities are almost indistinguishable from those of the native molecules. Data on Pen-2 and -3a activity demonstrate that penaeidins have a broad spectrum of antifungal properties associated with a fungicidal activity, and that their antibacterial activities are essentially directed against Gram-positive bacteria, with a strain-specific inhibition mechanism. Despite a better efficiency of Pen-3a on most of the tested strains, similar activity spectra and inhibition mechanisms were observed for both Pen-2 and -3a. Finally, no synergistic effect could be observed between the two molecules.     

All D-amino acid hexapeptide inhibitors of melittin's cytolytic activity derived from synthetic combinatorial libraries

The identification of peptides that inhibit the biological functions of proteins was used as a means to explore protein/ligand interactions involved in molecular recognition processes. This approach is based on the use of synthetic combinatorial libraries (SCLs) for the rapid identification of individual peptides that block the interaction of proteins with their biological targets. Thus, each peptide mixture of an all-D -amino acid hexapeptide SCL in a positional scanning format was screened for its ability to inhibit the hemolytic activity of melittin, a model self-assembling protein. The potent inhibitory activity of the identified individual peptides suggests that protein-like complexes are able to specifically bind to peptides having an all-D configuration. These results also show that SCLs are useful for the identification of short, non-hydrolysable sequences having potential intracellular inhibitory activities.