Rational Design of Amphipathic Antimicrobial Peptides with Alternating L-/D-Amino Acids That Form Helical Structures.

Antimicrobial peptides (AMPs) are promising therapeutic agents against bacteria. We have previously reported an amphipathic AMP Stripe composed of cationic L-Lys and hydrophobic L-Leu/L-Ala residues, and Stripe exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria. Gramicidin A (GA), composed of repeating sequences of L- and D-amino acids, has a unique ß6.3-helix structure and exhibits broad antimicrobial activity. Inspired by the structural properties and antimicrobial activities of LD-alternating peptides such as GA, in this study, we designed Stripe derivatives with LD-alternating sequences. We found that simply alternating L- and D-amino acids in the Stripe sequence to give StripeLD caused a reduction in antimicrobial activity. In contrast, AltStripeLD, with cationic and hydrophobic amino acids rearranged to yield an amphipathic distribution when the peptide adopts a ß6.3-helix, displayed higher antimicrobial activity than AltStripe. These results suggest that alternating L-/D-cationic and L-/D-hydrophobic amino acids in accordance with the helical structure of an AMP may be a useful way to improve antimicrobial activity and develop new AMP drugs.

Proton Logic Gate Based on a Gramicidin-ATP Synthase Integrated Biotransducer.

Ion channels are membrane proteins that allow ionic signals to pass through channel pores for biofunctional modulations. However, biodevices that integrate bidirectional biological signal transmission between a device and biological converter through supported lipid bilayers (SLBs) while simultaneously controlling the process are lacking. Therefore, in this study, we aimed to develop a hybrid biotransducer composed of ATP synthase and proton channel gramicidin A (gA), controlled by a sulfonated polyaniline (SPA) conducting polymer layer deposited on a microelectrode, and to simulate a model circuit for this system. We controlled proton transport across the gA channel using both electrical and chemical input signals by applying voltage to the SPA or introducing calcium ions (inhibitor) and ethylenediaminetetraacetic acid molecules (inhibitor remover). The insertion of gA and ATP synthase into SLBs on microelectrodes resulted in an integrated biotransducer, in which the proton current was controlled by the flux of adenosine diphosphate molecules and calcium ions. Lastly, we created an XOR logic gate as an enzymatic logic system where the output proton current was controlled by Input A (ATP synthase) and Input B (calcium ions), making use of the unidirectional and bidirectional transmission of protons in ATP synthase and gA, respectively. We combined gA, ATP synthase, and SPA as a hybrid bioiontronics system to control bidirectional or unidirectional ion transport across SLBs in biotransducers. Thus, our findings are potentially relevant for a range of advanced biological and medical applications.

Gramicidin, a Bactericidal Antibiotic, Is an Antiproliferative Agent for Ovarian Cancer Cells.

Background and Objectives: Gramicidin, a bactericidal antibiotic used in dermatology and ophthalmology, has recently garnered attention for its inhibitory actions against cancer cell growth. However, the effects of gramicidin on ovarian cancer cells and the underlying mechanisms are still poorly understood. We aimed to elucidate the anticancer efficacy of gramicidin against ovarian cancer cells. Materials and Methods: The anticancer effect of gramicidin was investigated through an in vitro experiment. We analyzed cell proliferation, DNA fragmentation, cell cycle arrest and apoptosis in ovarian cancer cells using WST-1 assay, terminal deoxynucleotidyl transferase dUTP nick and labeling (TUNEL), DNA agarose gel electrophoresis, flow cytometry and western blot. Results: Gramicidin treatment induces dose- and time-dependent decreases in OVCAR8, SKOV3, and A2780 ovarian cancer cell proliferation. TUNEL assay and DNA agarose gel electrophoresis showed that gramicidin caused DNA fragmentation in ovarian cancer cells. Flow cytometry demonstrated that gramicidin induced cell cycle arrest. Furthermore, we confirmed via Western blot that gramicidin triggered apoptosis in ovarian cancer cells. Conclusions: Our results strongly suggest that gramicidin exerts its inhibitory effect on cancer cell growth by triggering apoptosis. Conclusively, this study provides new insights into the previously unexplored anticancer properties of gramicidin against ovarian cancer cells.

Analysing Megasynthetase Mutants at High Throughput Using Droplet Microfluidics.

Nonribosomal peptide synthetases (NRPSs) are giant enzymatic assembly lines that deliver many pharmaceutically valuable natural products, including antibiotics. As the search for new antibiotics motivates attempts to redesign nonribosomal metabolic pathways, more robust and rapid sorting and screening platforms are needed. Here, we establish a microfluidic platform that reliably detects production of the model nonribosomal peptide gramicidin S. The detection is based on calcein-filled sensor liposomes yielding increased fluorescence upon permeabilization. From a library of NRPS mutants, the sorting platform enriches the gramicidin S producer 14.5-fold, decreases internal stop codons 250-fold, and generates enrichment factors correlating with enzyme activity. Screening for NRPS activity with a reliable non-binary sensor will enable more sophisticated structure-activity studies and new engineering applications in the future.

Biosynthetic diversification of non-ribosomal peptides through activity-based protein profiling of adenylation domains.

We describe activity-based protein profiling for analyzing the adenylation domains of non-ribosomal peptide synthetases (ABPP-NRPS) in bacterial proteomes. Using a range of non-proteoinogenic amino acid sulfamoyladenosines, the competitive format of ABPP-NRPS provided substrate tolerance toward non-proteinogenic amino acids. When coupled with precursor-directed biosynthesis, a non-proteinogenic amino acid (O-allyl-L-serine) was successfully incorporated into gramicidin S.

Smart Wearable Patches Using Light-Controlled Activation and Delivery of Photoswitchable Antimicrobial Peptides.

A novel strategy to treat Staphylococcus aureus (S. aureus) skin infections is presented, where UV light is used to facilitate concomitant light-controlled activation and delivery of an antimicrobial therapeutic agent. Specifically, a new photoswitchable gramicidin S analogue was immobilized onto a polymeric wearable patch via a photocleavable linker that undergoes photolysis at the same wavelength of light required for activation of the peptide. Unlike toxic gramicidin S, the liberated active photoswitchable peptide exhibits antimicrobial activity against S. aureus while being ostensibly non-haemolytic to red blood cells. Moreover, irradiation with visible light switches off the antimicrobial properties of the peptide within seconds, presenting an ideal strategy to regulate antibiotic activity for localized bacterial infections with the potential to mitigate resistance.

ß-Turn editing in Gramicidin S: Activity impact on replacing proline α-carbon with stereodynamic nitrogen.

Gramicidin S, natural antimicrobial peptide is used commercially in medicinal lozenges for sore throat and Gram-negative and Gram-positive bacterial infections. However, its clinical potential is limited to topical applications because of its high red blood cells (RBC) cytotoxicity. Given the importance of developing potential antibiotics and inspired by the cyclic structure and druggable features of Gramicidin S, we edited proline α-carbon with stereodynamic nitrogen to examine the direct impact on biological activity and cytotoxicity with respect to prolyl counterpart. Natural Gramicidin S (12), proline-edited peptides 13-16 and wild-type d-Phe-d-Pro ß-turn mimetics (17 and 18) were synthesized using solid phase peptide synthesis and investigated their activity against clinically relevant bacterial pathogens. Interestingly, mono-proline edited analogous peptide 13 showed moderate improvement in antimicrobial activity against E. coli ATCC 25922 and K.pneumoniae BAA 1705 as compared to Gramicidin S. Furthermore, proline edited peptide 13 exhibited equipotent antimicrobial effect against MDR S. aureus and Enterococcus spp. Analysis of cytotoxicity against VERO cells and RBC, reveals that proline edited peptides showed two-fivefold lesser cytotoxicity than the counterpart Gramicidin S. Our study suggests that introducing single azPro/Pro mutation in Gramicidin S marginally improved the activity and lessens the cytotoxicity as compared with the parent peptide.

Couette flow fluorescence detected linear dichroism for analytes in lipid bilayers.

Membranes are important sites of intermolecular interactions in biological systems. However, they present significant analytical challenges as they contain multiple analytes and are dynamic in nature. In this work, we show how a Jasco J-1500 circular dichroism spectropolarimeter can be used with a microvolume Couette flow cell and appropriate cut-off filters to measure excitation fluorescence detected linear dichroism (FDLD) of fluorophores embedded in liposomal membranes. The result is a spectrum that selectively probes the fluorophore(s) and eliminates the scattering that is apparent in the corresponding flow linear dichroism (LD) spectrum. The FDLD spectrum is opposite in sign from the LD spectrum with relative magnitudes modified by the quantum yields of the transitions. FDLD thus enables analyte orientations to be identified in a membrane. Data for a membrane peptide, gramicidin, and two aromatic analytes, anthracene and pyrene, are presented. Issues with the "leakage" of photons by the long pass filters used is also discussed.

Surrogate Based Genetic Algorithm Method for Efficient Identification of Low-Energy Peptide Structures.

Identification of the most stable structure(s) of a system is a prerequisite for the calculation of any of its properties from first-principles. However, even for relatively small molecules, exhaustive explorations of the potential energy surface (PES) are severely hampered by the dimensionality bottleneck. In this work, we address the challenging task of efficiently sampling realistic low-lying peptide coordinates by resorting to a surrogate based genetic algorithm (GA)/density functional theory (DFT) approach (sGADFT) in which promising candidates provided by the GA are ultimately optimized with DFT. We provide a benchmark of several computational methods (GAFF, AMOEBApro13, PM6, PM7, DFTB3-D3(BJ)) as possible prescanning surrogates and apply sGADFT to two test case systems that are (i) two isomer families of the protonated Gly-Pro-Gly-Gly tetrapeptide (Masson, A.; J. Am. Soc. Mass Spectrom.2015, 26, 1444-1454) and (ii) the doubly protonated cyclic decapeptide gramicidin S (Nagornova, N. S.; J. Am. Chem. Soc.2010, 132, 4040-4041). We show that our GA procedure can correctly identify low-energy minima in as little as a few hours. Subsequent refinement of surrogate low-energy structures within a given energy threshold (≤10 kcal/mol (i), ≤5 kcal/mol (ii)) via DFT relaxation invariably led to the identification of the most stable structures as determined from high-resolution infrared (IR) spectroscopy at low temperature. The sGADFT method therefore constitutes a highly efficient route for the screening of realistic low-lying peptide structures in the gas phase as needed for instance for the interpretation and assignment of experimental IR spectra.

New role of gramicidin A in RIG-I-like receptors-mediated IFN signalling.

The pattern recognition receptors (PRRs) sense exogenous molecular patterns most commonly derived from invading pathogens, to active the interferon (IFN) signalling. In the cytoplasm, the viral double-stranded RNAs (dsRNAs) are sensed by retinoic acid-inducible gene I (RIG-I) or melanoma differentiation-associated protein 5 (MDA5), depending on the length and chemical properties. Through the binding and oligomerizing onto the RNAs, they form filament to initiate the signalling cascade. Regulation of these receptors' activities are essential for manipulating the strength of IFN signalling. Here, through the virtual screening of chemical reagents using the published MDA5-dsRNA complex structure (PDB: 4GL2), we identified an antibiotic, gramicidin A as a stimulator that enhanced MDA5-mediated IFN signalling. Cytotoxic assay and IFN signalling assay suggested that disruption of lipid membrane, which is a well-defined mechanism of gramicidin A to perform its action, was dispensable in this process. Sucrose gradient ultracentrifugation assay showed that the gramicidin A treatment enhanced MDA5 oligomerization status in the presence of dsRNA. Our work implicated a new role of gramicidin A in innate immunity and presented a new tool to manipulate MDA5 activity.

Transport of acylated anthocyanins by the Arabidopsis ATP-binding cassette transporters AtABCC1, AtABCC2, and AtABCC14.

Anthocyanins are a group of pigments that have various roles in plants including attracting pollinators and seed dispersers and protecting against various types of stress. In vegetative tissue, these anthocyanins are sequestered in the vacuole following biosynthesis in the cytoplasm, though there remain questions as to the events leading to the vacuolar sequestration. In this study, we were able to show that the uptake of acylated anthocyanins by vacuolar membrane-enriched vesicles isolated from Arabidopsis was stimulated by the addition of MgATP and was inhibited by both vanadate and glybenclamide, but not by gramicidin D or bafilomycin A1 , suggesting that uptake involves an ATP-binding cassette (ABC) transporter and not an H+ -antiporter. Membrane vesicles isolated from yeast expressing the ABC transporters designated AtABCC1, AtABCC2, and AtABCC14 are capable of MgATP-dependent uptake of acylated anthocyanins. This uptake was not dependent on glutathione as seen previously for anthocyanidin 3-O-monoglucosides. Compared to the wild-type, the transport of acylated anthocyanins was lower in vacuolar membrane-enriched vesicles isolated from atabcc1 cell cultures providing evidence that AtABCC1 may be the predominant transporter of these compounds in vivo. In addition, the pattern of anthocyanin accumulation differed between the atabcc1, atabcc2, and atabcc14 mutants and the wild-type seedlings under anthocyanin inductive conditions. We suggest that AtABCC1, AtABCC2, and AtABCC14 are involved in the vacuolar transport of acylated anthocyanins produced in the vegetative tissue of Arabidopsis and that the pattern of anthocyanin accumulation can be altered depending on the presence or absence of a specific vacuolar ABC transporter.

A Note of Caution: Gramicidin Affects Signaling Pathways Independently of Its Effects on Plasma Membrane Conductance.

Gramicidin is a thoroughly studied cation ionophore widely used to experimentally manipulate the plasma membrane potential (PMP). In addition, it has been established that the drug, due to its hydrophobic nature, is capable of affecting the organization of membrane lipids. We have previously shown that modifications in the plasma membrane potential of epithelial cells in culture determine reorganizations of the cytoskeleton. To elucidate the molecular mechanisms involved, we explored the effects of PMP depolarization on some putative signaling intermediates. In the course of these studies, we came across some results that could not be interpreted in terms of the properties of gramicidin as an ionic channel. The purpose of the present work is to communicate these results and, in general, to draw attention to the fact that gramicidin effects can be misleadingly attributed to its ionic or electrical properties. In addition, this work also contributes with some novel findings of the modifications provoked on the signaling intermediates by PMP depolarization and hyperpolarization.

Peptide-induced membrane elastic deformations decelerate gramicidin dimer-monomer equilibration.

Gramicidin A (gA) is a hydrophobic pentadecapeptide readily incorporating into a planar bilayer lipid membrane (BLM), thereby inducing a large macroscopic current across the BLM. This current results from ion-channel formation due to head-to-head transbilayer dimerization of gA monomers with rapidly established monomer-dimer equilibrium. Any disturbance of the equilibrium, e.g., by sensitized photoinactivation of a portion of gA monomers, causes relaxation toward a new equilibrium state. According to previous studies, the characteristic relaxation time of the gA-mediated electric current decreases as the current increases upon elevating the gA concentration in the membrane. Here, we report data on the current relaxation kinetics for gA analogs with N-terminal valine replaced by glycine or tyrosine. Surprisingly, the relaxation time increased rather than decreased upon elevation of the total membrane conductance induced by these gA analogs, thus contradicting the classical kinetic scheme. We developed a general theoretical model that accounts for lateral interaction of monomers and dimers mediated by membrane elastic deformations. The modified kinetic scheme of the gramicidin dimerization predicts the reverse dependence of the relaxation time on membrane conductance for gA analogs, with a decreased dimerization constant that is in a good agreement with our experimental data. The equilibration process may be also modulated by incorporation of other peptides ("impurities") into the lipid membrane.

Gramicidin A-based unimolecular channel: cancer cell-targeting behavior and ion transport-induced apoptosis.

A series of glycoside-peptide conjugates were prepared by engineering at the N-terminus of the natural peptide gramicidin A. The conjugate containing galactose moiety formed a unimolecular transmembrane channel and mediated ion transport to induce apoptosis of cancer cells. More importantly, it exhibited liver cancer cell-targeting behavior due to the galactose-asialoglycoprotein receptor recognition.

Characterization of extracellular and cell bound biosurfactants produced by Aneurinibacillus aneurinilyticus isolated from commercial corn steep liquor.

The presence of biosurfactants produced by a Bacillus strain in corn steep liquor (CSL), a wastewater stream of the corn milling process, has been recently discovered. However, the species responsible for their production has not been identified at the moment. Therefore, in this work, the Bacillus strain isolated from CSL, with capacity to produce biosurfactants, was subjected to amplification and sequence analysis of the 16S rRNA, being identified as Aneurinibacillus aneurinilyticus. This strain has been proved to be endospore forming and thermophile, what would explain its presence in the commercial CSL. It was observed that the strain under evaluation has the ability to produce both cell-bound and extracellular biosurfactant extracts, which were characterized in this work. The electrospray ionization mass spectrometry (ESI) analysis of the biosurfactant extracts revealed that the extracellular biosurfactant produced by Aneurinibacillus aneurinilyticus is composed by a mixture of lipopeptides, containing C16 and C18 fatty acids and amino acids, including valine, phenylalanine, proline, cysteine, histidine, aspartic acid/asparagine, alanine, glycine, leucine/isoleucine, with biomarkers between 1025-458 m/z. Conversely, the cell-bound biosurfactant extract produced by Aneurinibacillus aneurinilyticus was composed by the cyclic decapeptide gramicidin S, with a characteristic peak at 571 m/z, and lipopeptides with characteristic peaks between 1034-705 m/z, containing alanine, glycine, cysteine, serine, proline, aspartic acid/asparagine, similarly to the amino acid sequence of the extracellular biosurfactant extract.

Discovery of gramicidin A analogues with altered activities by multidimensional screening of a one-bead-one-compound library.

Gramicidin A (1) is a peptide antibiotic that disrupts the transmembrane ion concentration gradient by forming an ion channel in a lipid bilayer. Although long used clinically, it is limited to topical application because of its strong hemolytic activity and mammalian cytotoxicity, likely arising from the common ion transport mechanism. Here we report an integrated high-throughput strategy for discovering analogues of 1 with altered biological activity profiles. The 4096 analogue structures are designed to maintain the charge-neutral, hydrophobic, and channel forming properties of 1. Synthesis of the analogues, tandem mass spectrometry sequencing, and 3 microscale screenings enable us to identify 10 representative analogues. Re-synthesis and detailed functional evaluations find that all 10 analogues share a similar ion channel function, but have different cytotoxic, hemolytic, and antibacterial activities. Our large-scale structure-activity relationship studies reveal the feasibility of developing analogues of 1 that selectively induce toxicity toward target organisms.

Antimicrobial Peptides as New Combination Agents in Cancer Therapeutics: A Promising Protocol against HT-29 Tumoral Spheroids.

Antimicrobial peptides are molecules synthetized by a large variety of organisms as an innate defense against pathogens. These natural compounds have been identified as promising alternatives to widely used molecules to treat infections and cancer cells. Antimicrobial peptides could be viewed as future chemotherapeutic alternatives, having the advantage of low propensity to drug resistance. In this study, we evaluated the efficiency of the antimicrobial peptide gramicidin A (GA) and the anticancer drug, doxorubicin (Doxo) against the spheroids from colorectal cancer cells (HT-29). The two drugs were applied separately against HT-29 spheroids as well as together to determine if they can act synergistically. The spheroid evolution, cell viability, and ATP levels were monitored at 24 and 48 h after the applied treatments. The results show significant drops in cell viability and cellular ATP levels for all the experimental treatments. The simultaneous use of the two compounds (GA and Doxo) seems to cause a synergistic effect against the spheroids.

Monolithic Poly(styrene-co-divinylbenzene) Columns for Supercritical Fluid Chromatography-Mass Spectrometry Analysis of Polypeptide.

The organic polymer-based monolithic columns have been evaluated as the separation media for analysis of peptides using supercritical fluid chromatography-mass spectrometry (SFC-MS). We demonstrate for the first time the SFC-MS separation of a mixture of polypeptides carried out using poly(styrene-co-divinylbenzene) monolithic columns and carbon dioxide/methanol mobile phase. A gradient from 2 to 40% methanol modifier containing 0.1% TFA as an acidic additive was applied for the optimized elution and the separation was achieved in less than 3 min. Selected ion monitoring enabled detection of selected masses characteristic of three ionophoric pentadecapeptide antibiotics gramicidin A, B, and C and their two corresponding isoforms. Furthermore, their identity was confirmed through determination of their [M + 2H]2+, [M + 2Na]2+, and [M + H + Na]2+ ions acquired by positive-ion electrospray ionization-mass spectrometry (ESI-MS).

Real-time monitoring of heat transfer between gold nanoparticles and tethered bilayer lipid membranes.

Plasmon resonance frequency irradiated gold nanoparticles (GNPs) have gained interest as a laser-targeted treatment for infections, tumors and for the controlled release of drugs in situ. Questions still remain, however, as to the efficiency of heat delivery within biological tissues and how this can be reliably determined. Here, we demonstrate how a nanomaterial-electrode interface that mimics cell membranes can detect the localized heat transfer characteristics arising from plasmon resonance frequency-matched laser excitation of GNPs. We demonstrate that the lipid bilayer membrane can be affected by conjugated GNP induced hyperthermia when irradiated with a laser power output as low as 135 nW/µm2. This is four orders of magnitude lower power than previously reported. By restricting the lateral movement of the lipids in the bilayer membrane, it was shown that the change in membrane conductance as a result of the heat transfer was due to the creation of transient lipidic toroidal pores within the membrane. We further demonstrate that the heat transfer from the GNPs alters diffusion rates of monomers of the gramicidin-A peptide within the lipid leaflets. This work highlights how targeted low laser power GNP hyperthermia treatments, in vivo, could play a dual role of interfering with both cell membrane morphology and dynamics, along with membrane protein function.

Fluorometric Na+ Evaluation in Single Cells Using Flow Cytometry: Comparison with Flame Emission Assay.

BACKGROUND/AIMS: Sodium is a key player in the fundamental cell functions. Fluorescent probes are indispensable tools for monitoring intracellular sodium levels in single living cells. Since the fluorescence of sodium-sensitive dyes in cells is significantly different from that in an aqueous solution, the fluorescence signal is calibrated in situ indirectly using ionophores for equalizing external and intracellular ion concentration. Attempts to compare data obtained using fluorescent probes and by direct flame emission analysis are sparse and results are inaccurate. METHODS: We determined the intracellular sodium concentration in U937 cells by flow cytometry using the Na+-sensitive probe Asante Natrium Green-2 (ANG), and by standard flame emission photometry combined with the cellular water determination by cell density in Percoll gradient. The intracellular Na+ concentrations was modified using known ionophores or, alternatively, by blocking the sodium pump with ouabain or by causing cell apoptosis with staurosporine. RESULTS: It is revealed that both methods are comparable when intracellular sodium concentration was modified by ouabain-mediated blockage of the sodium pump or staurosporine-induced apoptosis. The ANG fluorescence of cells treated with ionophores is approximately two times lower than that in cells with the same Na+ concentration but not treated with ionophores. Although the mechanism is still unknown, this effect should be taken into account when a quantitative assessment of the concentration of intracellular sodium is required. CONCLUSION: The sodium sensitive dye ANG-2 is a sensitive and useful probe for determination changes in Na+ content and concentration both in single cells and subcellular microparticles. The ANG fluorescence determined in the studied cells in the absence of ionophores, cannot be used as a measure of the real intracellular concentration of Na+ if calibration was carried out in the presence of ionophores.