Cytotoxic Lipopeptide Muscotoxin A, Isolated from Soil Cyanobacterium

Article pubs.acs.org/crt

Cytotoxic Lipopeptide Muscotoxin A, Isolated from Soil Cyanobacterium Desmonostoc muscorum, Permeabilizes Phospholipid Membranes by Reducing Their Fluidity Petr Tomek,†,‡ Pavel Hrouzek,*,†,§ Marek Kuzma,∥ Jan Sýkora,⊥ Radovan Fišer,# Jan Č erný,○ Petr Novák,∇,∥ Simona Bártová,◆,∥ Petr Šimek,¶ Martin Hof,⊥ Daniel Kavan,∥ and Jiří Kopecký† †

Department of Phototrophic Microorganisms−Algatech, Institute of Microbiology, Academy of Sciences of the Czech Republic, Opatovický mlýn, 379 81 Třeboň, Czech Republic ‡ Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland, 85 Park Road, 1023 Auckland, New Zealand § Faculty of Science, Institute of Chemistry, University of South Bohemia, Branišovská 1760, 370 05 Č eské Budějovice, Czech Republic ∥ Laboratory of Molecular Structure Characterization, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague, Czech Republic ⊥ Department of Biophysical Chemistry, J. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, Dolejškova 2155/3, 182 23 Prague 8, Czech Republic # Department of Genetics and Microbiology, Faculty of Sciences, Charles University, Viničná 5, 128 44 Prague 2, Czech Republic ○ Department of Cell Biology, Faculty of Sciences, Charles University, Viničná 7, 128 00 Prague 2, Czech Republic ∇ Department of Biochemistry, Faculty of Sciences, Charles University, Hlavova 8, 128 40 Prague, Czech Republic ◆ Department of Analytical Chemistry, Institute of Chemical Technology, Technická 5, 166 28 Dejvice, Prague, Czech Republic ¶ Institute of Entomology, Biology Centre, Academy of Sciences of the Czech Republic, v.v.i., 370 05 Č eské Budějovice, Czech Republic S Supporting Information *

ABSTRACT: There is mounting evidence that cyanobacterial lipopeptides can kill mammalian cells, presenting a hazard to human health. Unfortunately, their mechanism of toxicity is poorly understood. We have isolated new cyclic undecalipopeptides muscotoxin A and B containing unique lipophilic residue 3-amino-2,5-dihydroxydecanoic acid (5-OH Ahdoa). Muscotoxin B was not used for biological studies due to its poor yield. Muscotoxin A was cytotoxic to YAC-1, Sp/2, and HeLa cancer cell lines (LC50 ranged from 9.9 to 13.2 μM after 24 h of exposure), causing membrane damage and influx of calcium ions. Subsequently, we studied this lytic mechanism using synthetic liposomes with encapsulated fluorescent probes. Muscotoxin A permeabilized liposomes composed exclusively of phospholipids, demonstrating that no proteins or carbohydrates present in biomembranes are essential for its activity. Paradoxically, the permeabilization activity of muscotoxin A was mediated by a significant reduction in membrane surface fluidity (stiffening), the opposite of that caused by synthetic detergents and cytolytic lipopeptide puwainaphycin F. At 25 °C, muscotoxin A disrupted liposomes with and without cholesterol/sphingomyelin; however, at 37 °C, it was selective against liposomes with cholesterol/sphingomyelin. It appears that both membrane fluidity and organization can affect the lytic activity of muscotoxin A. Our findings strengthen the evidence that cyanobacterial lipopeptides specifically disrupt mammalian cell membranes and bring new insights into the mechanism of this effect.

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diversity.3−5 The majority of cyanobacterial secondary metabolites is synthesized in a nucleic-acid free environment via

INTRODUCTION

Cyanobacteria are a prolific source of structurally intriguing secondary metabolites with a wide spectrum of bioactivities.1,2 Hundreds of such compounds have been identified in recent decades, which is proposed to be only a small fraction of their © 2015 American Chemical Society

Received: September 17, 2014 Published: January 26, 2015 216

DOI: 10.1021/tx500382b Chem. Res. Toxicol. 2015, 28, 216−224

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Chemical Research in Toxicology

the tested cell lines was comparable, further work was conducted on HeLa cells due to their adherent nature, which is necessary for calcium influx measurements. To understand the kinetics of muscotoxin A toxicity, we measured the reduction rate of resazurin, an indicator of cell viability. Subsequently, we investigated the effect of muscotoxin A on intracellular calcium levels to establish whether a correlation exists between the reduction in cell viability and membrane permeabilization. Then, as a first step in dissecting the mechanism of action of muscotoxin A, we asked whether it could permeabilize liposomes composed exclusively of phosholipids devoid of other biomembrane components. For this experiment, we used liposomes composed of equimolar amounts of dioleoyl-phospahtydilglycerol (DOPG) and dioleoyl-phosphatidylethanolamine (DOPE) due to the feasibility of their preparation. Next, we investigated how muscotoxin A permeabilizes the bilayers and how the physical state and spatial organization of membranes affect its activity. Two liposome formulations were used. Liposomes 1 were designed to turn into a gel phase (stiff) at 25 °C but were liquid phase (fluid) at 37 °C. Liposomes 2 exemplified a common mammalian plasma membrane,29 which exists as a liquid phase at both 25 and 37 °C and contains complex membrane structures like, e.g., lipid rafts. Changes in fluidity and hydration of the bilayer surface were monitored by hydrophilic fluorescence probe Patman. Its two major emission maxima at 425 and 480 nm indicate low and high bilayer fluidity/hydration, respectively. Fluidity of the lipophilic part (acyl tails) of the bilayer was studied by measuring fluorescence anisotropy using hydrophobic fluorescence probe DPH. The fluorescence anisotropy reflects the motional freedom of the dye, i.e., higher values of anisotropy indicate that the motion of the dye is restricted and vice versa. Lastly, we asked whether muscotoxin A could also permeabilize human non-cancerous cells (primary fibroblasts) and examined its effect on cellular organelles. Materials. All solvents for extraction and HPLC analyses were obtained either from Sigma-Aldrich (Germany) or Analytika (Czech Republic) and were of HPLC-grade purity. Microcystin-LR was purchased from Cayman Chemicals (Ann Arbor, MI, USA), and Cyclosporin A was a gift from TEVA Pharmaceuticals (Czech Republic). Lipids were purchased from Avanti Polar Lipids (Alabaster, AL, USA). N,N,N-Trimethyl-2-(methyl(6-palmitoylnaphthalen-2-yl)amino)ethan-1-aminium chloride (Patman) and 1,6-diphenyl-1,3,5hexatriene (DPH) were obtained from Molecular Probes (Eugene, OR, USA). Carboxyfluorescein (CF), L-glutamine, and the chemicals needed for buffer preparation were purchased from Sigma-Aldrich (Germany). Media and chemicals for cell growth were from PAA (Linz, Austria), and alamarBlue was acquired from AbD Serotec (Japan). Fluorescent probes for immunofluorescence microscopy were from Molecular Probes (Invitrogen, Carlsbad, CA, USA). Isolation and Purification of Muscotoxins. The filamentous cyanobacterium Desmonostoc muscorum NIVA-CYA 81730 was grown in custom-made glass cuvette (150 L) in A-D Anabaena medium,31 which was bubbled with CO2-enriched air (2%) at constant temperature 28 °C for 10 days. The cells were harvested, freezedried, and extracted twice with 50% MeOH at a concentration of 200 mg dry biomass/mL solvent. The extract was partitioned between enthyl acetate (EtOAc) and water at a ratio of 2:1:3 (methanolic extract/EtOAc/water), and the water fraction was evaporated to remove residual EtOAc (1 h, 37 °C). Subsequently, the extract was passed through a SPE (solid phase extraction) cartridge (Oasis HLB 150 mg/6 mL, Waters, Prague) and eluted with 100% MeOH (5 mL). The concentrate was evaporated and redissolved in 50% MeOH (2 g extracted biomass/mL solvent). Purification of muscotoxins was carried with a LabAlliance HPLC system (Watrex, Prague, Czech Republic) at 30 °C with detection at 220 nm. Mobile phases containing MeCN were sonicated (15 min, 30 °C) prior to each use. The first separation was carried out on a C18-RP column (Reprosil 100, 250 × 10 mm, 5 μm, Dr. Maisch GmbH, Germany) eluted with a MeOH (A)/H2O (B) gradient for 64 min as follows: 50% A (0−6 min), 50−73% A (6−12 min), 73−76% A (12−50 min), and 76− 100% A (50−51 min); flow rate and injection volume were 2.6 mL/ min and 0.5 mL, respectively. The collected eluate was cooled on ice at all times. Muscotoxins fraction was evaporated and resuspended in

ancient modular multienzyme complexes known as nonribosomal peptide synthases (NRPS) and polyketide synthases (PKS) or their combination. These biosynthetic machineries can produce myriad of chemicals;6,7 however, peptides are the most abundant class of cyanobacterial metabolites. Structures of cyanobacterial peptides are characterized by atypical amino acid residues, e.g., D-amino acids or β-amino acids, as well as by their unusual chemical modifications, such as N- and O-methylations, halogenation, and cyclization, or the presence of imino bonds instead of more typical amino bonds.2 Such features grant them beneficial properties like thermostability or resistance to proteolysis. Cyclic lipopeptides represent an abundant group of cyanobacterial peptides characterized by the presence of lipophilic β-amino acids with up to 14 residues in the cycle. The biosynthetic origin of lipopeptides puwainaphycins via hybrid NRPS/PKS involving the fatty acyl-AMP ligase domain was recently demonstrated,8 and a similar biosynthetic mechanism can be expected for other cyanobacterial lipopeptides. About 20 structural classes have been identified to date that show a broad range of bioactivities, including cytotoxicity (laxaphycins,9 hormothamnin A,10 minutissamides,11 pahayokolides,12 and lyngbyacyclamides13), antifungal, and/or antibacterial activity.14−20 However, attempts to understand their mechanism of action were made only in the cases of puwainaphycins,21 anabaenolysins,22,23 and pseudodesmin.24 Puwainaphycins and anabaenolysins were demonstrated to induce necrotic cell death in mammalian cells by disrupting the cell membrane.21,22 Moreover, anabaenolysins were shown to interact with membranes in a cholesteroldependent manner, suggesting a preference for eukaryotic plasmatic membranes.23 Pseudodesmin A was shown to assemble into a pore-forming oligomer in membranes.24 Similarly, a large number of cyclic lipopeptides isolated from other eubacterial species like Streptomyces (e.g., daptomycin),25 Pseudomonas (e.g., syringomycin),26 or Bacillus (e.g., surfactin)27 have been shown to mediate their effect via membrane disruption, specifically by pore formation.28 However, they are toxic mainly to prokaryotes, in contrast to cyanobacterial lipopeptides, which appear to target eukaryotic cells. This could be due to differences in amino acid composition. While noncyanobacterial lipopeptides tend to contain acidic or basic residues, we noticed that known cyanobacterial lipopeptides consist exclusively of neutral amino acids. Overall, this suggests that mammalian cell membrane permeabilization might be a common mechanism of toxicity of cyanobacterial lipopeptides, representing a potential hazard to human health. Herein, we present the isolation and characterization of two novel cyanobacterial lipopeptides, muscotoxin A/B, which were found to permeabilize mammalian cell membranes and induce necrotic cell death. The effects of muscotoxin A were studied on human cancer cells and fibroblasts cells using immunofluorescence microscopy and various biochemical techniques. Utilizing liposomes embedded with fluorescent probes, we have demonstrated that muscotoxin A affects the surface of the membrane in an unusual way, which is opposite to that of known cytotoxic lipopeptide puwainaphycin F and synthetic detergents.

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EXPERIMENTAL SECTION

Experimental Outline. The toxicity of muscotoxin A was assessed at 24 h exposure time against three mammalian cancer cell lines representing adherent (HeLa), semiadherent (Sp/2), and suspension (YAC-1) cell culture growth. Since the LC50 of muscotoxin A against 217


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Chemical Research in Toxicology MeCN/MeOH/H2O (27:3:70) at a final concentration of 4 g/mL (biomass/solvent) and subjected to a second chromatographic separation, which was performed on a C18-RP column (Reprosil 100, 250 × 25 mm, 5 μm, Dr. Maisch GmbH, Germany) eluted with MeCN/MeOH/H2O (81:9:10) (A)/MeCN/MeOH/H2O (27:3:70) (B) gradient as follows: 0−5% A (0−3 min), 5−20% A (3−28 min), 20% A (28−40 min), 20−29% A (40−60 min), and 29−100% A (60− 62 min); flow rate and injection volume were 17 mL/min and 1 mL, respectively. The yields of muscotoxin A and B were ∼2 and 0.2 mg/1 g of dry biomass, respectively. NMR Analyses. NMR spectra were measured using a Bruker AVANCE III 600 spectrometer (observational frequency, 600.23 MHz for 1H and 150.93 MHz for 13C) in CD3OD (ARMAR Chemicals, Döttingen, Swiss) at 35 °C (concentrations were 8 and 2 mg/mL for muscotoxin A and B, respectively). The residual signal of the solvent served as an internal standard (δH 3.330, δC 49.30). The 1H NMR spectra were supplemented by 2-fold memory points and, prior to Fourier transformation, was multiplied by a scaling function to enhance the resolution (negative exponential plus Gauss function). 13 C NMR spectra were enhanced by artificial line widening (1 Hz) due to an elevated signal-to-noise ratio. Assignment of signals was based on 2D NMR experiments, COSY (correlation spectroscopy), HSQC, HMBC (heteronuclear multiple bond correlation), NOESY (nuclear Overhauser and exchange spectroscopy), and ROESY (rotating frame Overhauser enhancement spectroscopy), carried out on manufacturer supplied software (Bruker BioSpin GmbH, Rheinstetten, Germany). MS Analyses. The stock solution of muscotoxins (10 μg/mL) for MS experiments was prepared in 50% MeOH with addition of 0.1% formic acid. Measurements were performed on a commercial APEXQe FTMS instrument equipped with a 9.4 T superconducting magnet and a Combi ESI/MALDI ion source (Bruker Daltonics, Billerica MA, USA) using electrospray ionization and on Bruker Impact HD highresolution mass spectrometer with electrospray ionization. The flow rate was 1 μL/min, and the temperature of dry gas (nitrogen) was set at 200 °C. The Q front end consists of a quadrupole mass filter followed by a hexapole collision cell. By switching the potentials on the exit lenses appropriately under the control of the data acquisition computer, ions could be accumulated either in the hexapole of the Combi ESI source or in the hexapole collision cell of the Q front end, prior to transfer to the FTMS analyzer cell. Mass spectra were obtained by accumulating ions in the collision hexapole and running the quadrupole mass filter in non-mass-selective (Rf-only) mode so that ions of a broad m/z range (150−2000) were passed to the FTMS analyzer cell. The species of interest were isolated in the gas phase by setting the Q mass filter to pass the m/z for ions of interest within a 3.0 m/z window. After a clean selection of the desired precursor, the ion had been confirmed and fragmentation was induced by dropping the potential of the collision cell (12 V). All MS and MS/MS spectra were acquired in positive ion mode with the acquisition mass range 150− 2000 m/z, and 1 million data points were collected, resulting in 200 000 maximal resolution at 400 m/z. The accumulation time was set at 0.5 s (1.5 s for MS/MS), the cell was opened for 4500 μs, and 8 experiments were collected for one spectrum. The instrument was internally calibrated using triply and doubly charged ions of angiotensin I and quintuple and quadruple charged ions of insulin, resulting in a typical mass accuracy below 1 ppm. After analysis, the spectra were apodized using sin apodization with one zero fill. Interpretation of mass spectra was done using DataAnalysis, version 3.4, software (Bruker Daltonics, Billerica, MA, USA). Chiral Amino Acid Analyses. Amino acids were hydrolyzed by 6 M HCl at 110 °C for 24 h and derivatized with heptafluorobutyl chloroformate.32 The chirality of the released amino acids (as the corresponding N(O,S)-heptafluorobutoxycarbonyl-heptafluorobutyl derivatives) were determined by gas chromatography−mass spectrometry on a 25 m × 0.25 mm i.d. × 0.12 μm Chirasil-L-Val column (Agilent, Santa Clara, CA, USA) using a method described elsewhere.33 Cell Cultivation and Cytotoxicity Assay. Mammalian cancer cell lines HeLa (adherent human cervical adenocarcinoma), Sp/2

(semiadherent murine B-cell hybridoma), and YAC-1 (suspension murine lymphoblastoma induced by Moloney murine leukemia virus) were a gift from Dr. Jan Kopecký from the Institute of Parasitology of the Czech Academy of Sciences (Ceske Budejovice, Czech Republic). Cells were cultivated in RPMI-1640 medium without L-glutamine supplemented with 5% (YAC-1, Sp/2) or 10% (HeLa) bovine fetal serum, freshly dissolved L-glutamine (300 μg/mL), and 1% (v/v) antibiotic−antimycotic solution at 37 °C and 5% CO2. Cytotoxicity of muscotoxin A was evaluated using the alamarBlue (resazurin) cell viability assay. Adherent (HeLa) and suspension/semiadherent (YAC1, Sp/2) cells were seeded 16 h or immediately prior to the experiment, respectively, in 96-well TC-treated plates (Nunc) at 2.5 × 104 and 5 × 104 cells per well, respectively, in 100 uL of cultivation medium. Muscotoxin A was prediluted in cultivation medium and added to the cells (100 μL, 1% final DMSO concentration). (a) For determination of LC50 (concentration of muscotoxin A that induces cell death in 50% of the cells in 24 h), alamarBlue solution was added into each well 20 h after muscotoxin A, and absorbance was measured 1 and 3 h after addition of alamarBlue at 570 and 600 nm (measurement and reference wavelength, respectively; 37 °C) in a microplate reader (Tecan Sunrise, Tecan Austria GmbH). Viability of cells was calculated as ((A24h − A21h of muscotoxin A)/(A24h − A21h of vehicle))*100. (b) For kinetics of resazurin reduction by HeLa cells (Figure 1), alamarBlue was added (10% (v/v) final concentration) together with muscotoxin A, and absorbance was measured every 1 h (0−15 h), 2 h (17−21 h), 1 h (22−24 h), and 2 h (26−28 h). Vehicle control was 1% DMSO (v/v) treated cells. Absorbance of cultivation medium containing 10% (v/v) alamarBlue without cells was subtracted from all measurements. Fluorescence Measurement of Cytosolic Ca2+. HeLa cells (ATCC, VA) grown on glass coverslips were washed in modified HBSS (140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 3 mM MgCl2, 10 mM Hepes-Na, and 10 mM glucose, pH 7.4) and loaded with 3 μM Fura-2 acetoxymethyl ester (Invitrogen, Carlsbad, CA, USA) for 30 min at 25 °C in the dark, rinsed, and allowed to rest in HBSS for 30 min prior to the fluorescence measurements. The ratiometric measurements were performed using a FluoroMax-3 spectrofluorometer, equipped with DataMax software (Jobin Yvon Horriba, France). The observed area of the coverslip was about 10 mm2, corresponding to approximately 104 cells. Liposome Preparation. General Procedures. Lipid solutions were mixed in a glass tube, solvent was evaporated under a stream of nitrogen at 50 °C, and dry lipid film was resuspended in solution A (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) and vortexed for 4 min. Next, the lipid solution was extruded several times through a polycarbonate membrane (100 nm pores; Avestin, Ottawa, Canada) to produce large unilamellar vesicles (100 nm mean diameter), referred to as liposomes in this study. Measurements were carried out in quartz cuvettes (0.3 cm), and the final concentration of lipids was 300 μM in each experiment. The temperature in the cuvette holder was maintained within ±0.1 °C using a water-circulating bath, and the solution was equilibrated at the desired temperature for 10 min prior to each measurement. CF Leakage Experiment. The required volumes of chloroform solutions of dioleoyl-phosphatydilglycerol (DOPG, 50 mol %) and dioleoyl-phosphatydilcholine (DOPC, 50 mol %) were evaporated under a stream of nitrogen at 50 °C and resuspended in solution A (refer to General Procedures for composition of solution A) containing 80 mM CF. After extrusion (described in General Procedures), the solution of liposomes was passed through HiTrap desalting columns (GE Healthcare, USA) and dialyzed against solution A for 12 h to remove CF that was not trapped in the liposomes. The liposomes were transferred into a cuvette and stirred continuously. After addition of the compounds, the evolution of the steady-state fluorescence intensity at excitation and emission wavelengths of 492 and 518 nm, respectively, was observed with time until it became constant. Subsequently, Triton X-100 (1% final concentration, v/v) was added to determine the value of 100% CF release. Measurements were acquired at 25 °C. 218


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Chemical Research in Toxicology Steady-State Spectra of Patman in Liposomes 1 and 2. Liposomes 1 (dimyristoyl-phosphatidylcholine (DMPC, 90 mol %) and dioleoyl-phosphatidylethanolamine (DOPE, 10 mol %)) and liposomes 2 (palmitoyl-oleoyl-phosphatidylcholine (POPC, 60 mol %), palmitoyl-oleoyl-phosphatidylethanolamine (POPE, 10 mol %), cholesterol (CH, 20 mol %), and sphingomyelin (SM, 10 mol %)) containing fluorescence probe Patman (2% (mol %), final concentration) were prepared as described in General Procedures. Steadystate excitation and emission spectra were recorded in 1 nm steps (2 nm bandwidths were chosen for both the excitation and emission monochromators) at 25 and 37 °C on a Fluorolog-3 spectrofluorometer (model FL3-11; HORIBA Jobin Yvon, Edison, NJ, USA) equipped with a Xenon-arc lamp. Emission spectra of the Patman probe were recorded at 370 nm excitation wavelength. Fluorescence Anisotropy Measurements of DPH in Liposomes 2 (Figure 5). The steady-state fluorescence anisotropy of DPH was measured using liposomes 2 (see the previous paragraph for their composition) containing fluorescent probe DPH (1% (mol %), final concentration) at 25 °C with polarizers inserted into the setup at an excitation wavelength of 350 nm and emission wavelength 450 nm with a 4 nm bandwidth. Four intensities of polarized fluorescence (IVV, IVH, IHV, IHH) were recorded, where V and H represent the vertical and horizontal directions, respectively, and the first and second positions in the subscript denote the direction plane of the polarization in the excitation and emission arms, respectively. The steady-state fluorescence anisotropy was calculated as follows: rst = (IVV − GIVH)/(IVV + 2GIVH), in which G is the instrumental correction factor given by the observed ratio, IHV/IHH. Immunofluorescence Microscopy. Human primary skin fibroblasts (tested between 5 and 20 divisions after culturing them starting from a frozen stock (10% DMSO), prepared by Dr. Jan Č erný (Department of Cell Biology, Faculty of Sciences, Charles Univeristy)) were seeded on 12 mm round sterile coverslips mounted in 24-well microtiter plates (NUNC) at 3 × 105 cells per well in 1 mL of growth medium. The next day, cells were treated with compounds as described for the cytotoxicity assay. After the end of the exposure time, cells were fixed in 3.8% formaldehyde (w/v) for 20 min and washed with phosphate buffered saline (PBS), and residual formaldehyde was removed by incubation with ammonium chloride (5 mM in PBS) for 5 min and subsequently washed with PBS again. Afterward, cells were permeabilized by 0.5% Triton X-100 in PBS (v/v) for 1 min, washed with PBS, and incubated with 2% BSA for 10 min. Next, the cells were incubated with the desired fluorescent probe or antibody for 1 h and then thoroughly washed with 2% BSA in PBS. Subsequently, coverslips were mounted onto microscopic slides using DAPI (1 μg/mL) in 10% Mowiol 4-88. Slides were left overnight in a refrigerator for the mounting medium to solidify. All images were acquired using an Olympus Cell system (Olympus IX81 microscope, Olympus, Japan). The images were postprocessed using ImageJ software.34

Scheme 1. Chemical Structure of Muscotoxin A (R = H) and Muscotoxin B (R = CH3)a

a

Abbreviations: D-Gln (D-glutamine), Gly (glycine), L-Pro (L-proline), γ-MePro (γ-methylproline), L-Phe (L-phenylalanine), D-allo-Ile (D-alloisoleucine), L-Ser (L-serine), Dhb (2,3-didehydrobutyric acid), Ahdoa (3-amino-2-hydroxydecanoic acid). Ordinal numbers of the amino acid residues in the sequence are denoted in the superscript following the amino acid’s name.

MeOH and perfectly soluble in DMSO. According to FT-MS measurements, muscotoxin A provided a pseudomolecular ion [M + H]+ m/z value of 1211.6666, and muscotoxin B, 1225.6810 (Δ below 1 ppm), which served to calculate molecular formulas of C58H90N12O16 and C59H92N12O16 for the A and B variants, respectively. The absorbance maximum of both muscotoxins was at 227 nm. The yields of muscotoxin A and B were ∼2 and 0.2 mg/1 g of dry biomass, respectively. Particular amino acids were identified by COSY, TOCSY, and 1H−13C HSQC−TOCSY (Table S1 and Figures S1 and S3). The amino acid composition of muscotoxin A was Gln, Gly, Dhb, 2 × Ile, Phe, 2 × Pro, and 2 × Ser. Furthermore, dihydroxy-amino acid residue 3-amino-2,5-dihydroxydecanoic acid (5-OH Ahdoa) was resolved. The amino acid sequence was determined by HMBC (Figure S5) using the correlations of the carbonyl carbons to the Hα and Hβ protons. The correlations coming from Hβ to carbonyl allowed the carbonyl belonging to a particular amino acid residue to be identified. Determination of the amino acid sequence was then based on the HMBC contact of carba CO to the Hα proton of the other residue. The following sequential HMBC correlations were observed: 1-CO → 2-Hα, 2-CO → 3-Hα, 3-CO → 4-Hα, 5CO → 6-Hα, 9-CO → 10-Hα, 10-CO → 11-Hα, and 11-CO → H-3. The correlations 4-CO → 5-Hα, 6-CO → 7-Hα, and 8CO → 9-Hα were detected, but their signals overlapped considerably (Figure S5). As a result, the obtained sequence was cyclo[5-OH Ahdoa1-Gln2-Gly3-Pro4-Phe5-Ile6-Ser7-Dhb8Ser9-Ile10-Pro11] (Scheme 1). The same approach was applied to elucidate the structure of muscotoxin B. The combination of COSY, TOCSY, and 1 H−13C HSQC−TOCSY revealed the specific amino acids (i.e., Gln, Gly, Dhb, 2 × Ile, Phe, 2 × Ser, Pro, and γ-MePro) and the same dihydroxy-amino acid residue as that in muscotoxin A, 3amino-2,5-dihydroxydecanoic acid (5-OH Ahdoa) (Figures S2 and S4). The detected HMBC correlations for the amino acid sequence are as follows: 2-CO → 3-Hα, 8-CO → 9-Hα, 9-CO → 10-Hα, and 11-CO → H-3. Some expected HMBC

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RESULTS AND DISCUSSION Structural Characterization of Muscotoxin A and B. Activity-guided fractionation of the methanolic extract of Desmonostoc muscorum NIVA-CYA 81730 identified a single fraction that induced rapid cell death in murine lymphoblastoma cell line YAC-1 (51% reduction of viability compared to untreated control) and human cervical adenocarcinoma cell line HeLa (60% reduction of viability compared to untreated control). HPLC-MS analysis showed that this fraction contains two overlapping peaks corresponding to protonated molecules [M + H]+ at m/z of 1211.6 and 1225.6. A comparison of these two molecular masses with those in our internal database of known cyanobacterial metabolites did not return any hits, suggesting that the structures would be novel. Both compounds were purified (see Experimental Section) and characterized using MS and NMR techniques. Muscotoxin A and B (Scheme 1) were isolated as amorphous white powders moderately soluble in aqueous MeCN and pure 219


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phenylalanine and glycine, indicating that the sequence of 1225.68 is cyclo[Ile-Pro-5-OH Ahdoa-Gln-Gly-MePro-Phe-IleSer-Dhb-Ser]. The absolute configuration of amino acid residues was determined using chiral GC-MS. This analysis showed that muscotoxins A/B contain identical enantiomers: D-allo-Ile, LSer, L-Pro, D-Gln, and L-Phe. Muscotoxins consist of 11 amino acid residues and contain a novel variant of a β-amino acid (5-OH Ahdoa) with a short (C10) aliphatic chain, non-proteinogenic amino acid Dhb (dehydrated threonine), two unusual isoforms of standard proteinogenic amino acids D-Gln and D-allo-Ile, and γ-MePro, which was recently found to substitute for proline in some nonribosomally synthesized peptides. The structure of muscotoxins bears striking similarities with those of previously reported lipopeptides schizotrin A,18 pahayokolides,12 lyngbyazothrins,20 and largamide H,16 but it is still distant enough to justify the classification of muscotoxins as a new group. While largamide H carries an identically functionalized β-amino acid as that in muscotoxins (2,5-dihydroxy), the sequence of the amino acids is different. On the contrary, pahayokolides, lyngbyazothrins, and schizotrin A show very close sequence similarity to that of muscotoxins, but their β-amino acid is more functionalized. Remarkably, the sequence of the polarity of residues in muscotoxins, schizotrin A, pahayokolides, and lyngbyazothrins is highly conserved, and residues are identical in 8 out of 11 positions (considering the β-amino acid as an identical residue). The three dissimilar residues are amino acids of comparable polarity. This demonstrates that the distribution of residue polarity might be important for the function of these lipopeptides.36 In particular, two Ser/Thr residues at positions 7 and 9 will make an important contribution toward the amphipatic properties of these lipopeptides. Muscotoxin A as a Membrane-Permeabilizing Agent. Muscotoxin A was cytotoxic to mammalian cancer cell lines HeLa (LC50 = 9.9 μM), YAC-1 (LC50 = 13.2 μM), and Sp/2 (LC50 = 11.3 μM) after 24 h of exposure. The potency of muscotoxin A was comparable to that of known cyanobacterial membrane-disrupting agents anabaenolysin A/B22 (LC50 = 3.7−17 μM against 10 cell types, exposure time unreported) and puwainaphycin F/G21 (LC50 = 2.15−2.19 μM against HeLa cells after 24 h of exposure). Muscotoxin A exhibited comparable potency against a broad range of cell types in a manner similar to that of anabaenolysins A/B, suggesting that it might also target the cell membrane. Interestingly, Jokela et al. suggested that suspension cells might be more sensitive to anabaenolysins than adherent cells due to their higher available membrane area;22 however, this hypothesis is not in accordance with our data, in which adherent HeLa cells were the most susceptible. Muscotoxin A rapidly (during the first hour) decreased the reduction rate of alamarBlue (resazurin) by HeLa cells in a concentration-dependent manner (Figure 1). The linearity of the plots indicates no apparent recovery in metabolic activity of the cells (Figure 1), consistent with induction of necrotic cell death. At 25 μM, muscotoxin rapidly increased the concentration of intracellular Ca2+ ions ([Ca2+]i) in HeLa cells (Figure 2a) in a manner similar to that of a selective calcium ionophore, ionomycin (Figure 2b). Lower concentrations of muscotoxin A exhibited lower permeabilization activity, and at 7 μM, no significant increase of [Ca2+]i was observed. This was in agreement with the observation that muscotoxin A (7 μM) was

correlations were not detected due the fact that muscotoxin B occurred as a minor variant in low concentration in the sample (Table S2 and Figure S6). Therefore, structural determination was partly based on a comparison with the spectra of muscotoxin A. The sequence corresponds to cyclo[5-OH Ahdoa1-Gln2-Gly3-MePro4-Phe5-Ile6-Ser7-Dhb8-Ser9-Ile10-Pro11] (Figure 1), which was further confirmed by mass spectrometry.

Figure 1. Reduction of alamarBlue (resazurin) by HeLa cells treated with muscotoxin A (M denotes concentration in micromolar). Data represent the mean ± SD of technical triplicates.

The NMR results were supported by analysis of MS and MS/ MS spectra. Collision-induced dissociation (CID) spectra were measured from parent masses 1211.67 and 1225.68, and peakto-peak mass differences were evaluated using a Cyclone set of scripts.35 Evaluation was facilitated by NMR determination of cyclopeptide building blocks, therefore reducing the number of various combinations to be precalculated. Thus, we could afford to calculate up to the whole 11-mer and therefore avoid the troublesome nature of combining partial results. From the peak-to-peak mass differences in the 1211.67 precursor spectrum, we were able to designate the sequence as cyclo[Ser-Dhb-Ser-Ile-(Phe|Pro)-Gly-Gln-(5-OH Ahdoa|Pro)Ile], where the particular sequence of pairs in parentheses was not clear. The second step was to calculate the mass of the theoretical ions potentially derivable from each of the four possibilities and to search for their occurrence in the mass list. The most suitable sequence was cyclo[Ser-Dhb-Ser-Ile-Phe-ProGly-Gln-5-OH Ahdoa-Pro-Ile], with 77 masses found to be within 1 ppm mass tolerance (other sequences were with 75, 72, and 71 matches). Because there were several fragments found with a broken bond between Pro and Ile and between Pro and Gly, we can conclude that the N-terminus of these prolines has to be on the side of Ile and Gly; thus, we can estimate the orientation of the rest of the residues. The final sequence, in N → C orientation, is therefore cyclo[Ile-Pro-5OH Ahdoa-Gln-Gly-Pro-Phe-Ile-Ser-Dhb-Ser]. Furthermore, the complete series of ions confirming the proposed sequence can be derived from the dehydrated mucotoxin A molecule occurring in the acquired MS/MS spectrum (Figure S7). The dehydration occurs on the 5-OH Ahdoa chain and is followed by the series of ions that confirms the loss of all amino acid residues, and the final ion at m/z 183.1329 corresponds well to dehydrated 5-OH Ahdoa (Figure S7B). Although slightly worse mass spectra were acquired from the CID of the 1225.68 peak, a number of fragment ion masses were in accordance with the 1211.67 derived fragments. Thorough analysis of these masses revealed the 14.01 Da difference to be only in fragments containing proline between 220


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Figure 2. Fluorescence ratiometric measurement of intracellular calcium levels in HeLa cells treated with (A) muscotoxin A (M denotes concentration in micromolar) and (B) selective calcium ionophore ionomycin (Ion, 500 nM) and Triton X-100 (T denotes concentration in micromolar). Fluorescence intensity measurements (λem = 510 nm) were acquired every 15 s with 3 s integration time at dual excitation wavelengths (λex = 340 and 380 nm). The ratio of the two recorded intensities is shown.

Figure 3. Leakage of carboxyfluorescein (CF) from liposomes composed of DOPC/DOPG (1:1) incubated at 25 °C and treated with (A) muscotoxin A and (B) puwainaphycin F at 25 and 7 μM. Arrows with letters M and P indicate the addition of muscotoxin A and puwainaphycin F, respectively. Data are expressed as the fraction of complete CF release induced by 1% (v/v) Triton X-100 (arrow with letter T). Data points were recorded every 15 s.

non-toxic to HeLa cells (Figure 1). Here, we demonstrated that muscotoxin A can rapidly permeabilize the outer cell membrane to small ions, inducing cell death in HeLa cells, as was shown previously for puwainaphycin F/G.21 Triton X-100 (165 μM, 0.01%) and muscotoxin A (15 μM) showed comparable permeabilization effects (Figure 2b); however, Triton X-100 was inactive at 83 μM (0.005%). The same concentration of Triton X-100 (83 μM) was non-toxic to HeLa cells (data not shown). This shows that muscotoxin A permeabilizes the membrane of HeLa cells more potently (∼11-fold) than does synthetic detergent Triton X-100. To understand how muscotoxin A permeabilizes the membrane, we have utilized synthetic liposomes. First, we asked whether muscotoxin A has the capacity to permeabilize bilayers composed exclusively of phospholipids independent of any additional components present in biomembranes such as proteins or carbohydrates. Liposomes (DOPC/DOPG 1:1) containing a self-quenching concentration of carboxyfluorescein (CF) were treated with test compounds, and the fluorescence of released CF was measured. Muscotoxin A released 75 and 60% of CF at 25 and 7 μM, respectively (Figure 3a). The known cytolytic lipopeptide puwainaphycin F permeabilized liposomes with slightly faster kinetics but comparable potency at 25 μM (78%); however, it showed much lower efficacy at 7 μM (8%) (Figure 3b). This experiment demonstrated that both muscotoxin A and puwainaphycin F permeabilize bilayers independent of any non-phospholipidic biomembrane components. Consequently, we embedded fluorescent probes into either the hydrophilic (Patman) or hydrophobic (DPH) domain of the two different compositions of liposomes and observed changes in their fluorescence characteristics indicative of bilayer fluidity and hydration. See the Experimental Outline section for a detailed explanation. First, we investigated the effect of muscotoxin A on the surface of a bilayer (fluorophore, Patman). As expected, control liposomes 1 showed a peak at 425 nm and a shoulder at 480 nm at 25 °C, indicating that the bilayers are stiff (Figure 4a, vehicle). On the contrary, at 37 °C, the 480 nm peak became prominent, suggesting that the bilayer became more fluid (Figure 4c, vehicle). The interaction of muscotoxin A with liposomes 1 was likewise affected by temperature. At 25 °C,

Figure 4. Steady-state emission spectra of the Patman fluorescent probe (excitation wavelength 370 nm) embedded in 100 nm liposomes treated with 25 μM muscotoxin A (M25), Triton X-100 (T denotes concentration in micromolar), and 25 μM puwainaphycin F (P25) at 25 °C (A, B) or 37 °C (C, D). Liposomes 1 (DMPC/ DOPE 90:10 mol %; left column, A−C), liposomes 2 (POPC/POPE/ CH/SM 60:10:20:10 mol %; right column, B−D). Spectra were acquired in 1 nm steps and were normalized to the largest and smallest value in each data set. Traces T25 and P25 and are present only in panels A and B, respectively.

muscotoxin A (25 μM) decreased the intensity of the 480 nm band, unmasking a peak at 425 nm indicative of the transition of the bilayer into a gel (solid) phase (Figure 4a). In striking contrast, muscotoxin A did not induce any significant change to liposomes 1 at 37 °C (Figure 4c). Triton X-100 (165 μM, 0.01%) displayed the opposite effect as that of muscotoxin A. 221


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Chemical Research in Toxicology At 25 °C, it produced a well-defined peak at 480 nm with a progressively disappearing shoulder at 425 nm (Figure 4a,c), indicating a substantial increase in membrane fluidity and hydration. A concentration of Triton X-100 equivalent to that of muscotoxin A (25 μM, 0.0015%) did not show any effect on liposomes 1 at 25 °C (Figure 4a), consistent with the observation that this concentration of Triton X-100 was nontoxic to HeLa cells and did not permeabilize the cells to Ca2+ (Figure 2b). Remarkably, activity of muscotoxin A was not dependent on temperature in liposomes 2 that more closely reflect a mammalian membrane (Figure 4b,d). One reason for the activity of muscotoxin A against liposomes 2 even at 37 °C might be the spatial organization of mammalian-like liposomes 2. Cholesterol and sphingomyelin are known to form tightly packed islands (rafts) in the membranes,37 which might attract muscotoxin A. Triton X-100 interacted with liposomes 2 in an identical manner as it did with liposomes 1, demonstrating that the permeabilization activity of Triton X-100 does not depend on the membrane type or temperature, in contrast to muscotoxin A. Other synthetic detergents showed a qualitatively identical effect as that of Triton X-100 (Figure S9), confirming the unique interaction of muscotoxin A. Puwainanaphycin F exhibited an effect comparable to that of Triton X-100, but it showed much lower potency (Figure 4b). Two non-ribosomal cyclic peptides, microcystin-LR and cyclosporin A, did not show any effect on liposomes 2, demonstrating that the activity of muscotoxin A and puwainaphycin F is not a common phenomenon of cyclic peptides (Figure S8). Subsequently, the effect of lipopeptides on the hydrophobic domain of bilayers was examined. Both muscotoxin A and puwainaphycin F did not appreciably influence the fluorescence anisotropy of DPH embedded in liposomes 2 at 25 °C (Figure 5). On the contrary, synthetic detergents (Triton X-100 and

To understand the consequences of membrane permeabilization in live cells, human fibroblasts were incubated with muscotoxin A (25 μM) and Triton X-100 (165 μM, 0.01%) for 30 min at 37 °C, and organelles were visualized using immunofluorescent dyes (Figure 6). While muscotoxin A did not induce significant changes to the actin cytoskeleton (Figure 6e), the rod-like mitochondrial network (Figure 6g) transformed into spherical objects (Figure 6h) (determined by confocal microscopy; data not shown). This effect is known to be induced by elevated [Ca2+]i,38 which correlated well with our [Ca2+]i measurements (Figure 2a). Since the mitochondria stain is potential-sensitive, it also indicated that the mitochondrial membrane was not permeabilized by muscotoxin A, as demonstrated previously with cytolytic lipopeptides anabaenolysins.22 In contrast, Triton X-100 caused mitochondrial disruption (Figure 6i). This further corroborated that the permeabilization mechanism of muscotoxin A and Triton X100 is distinct. Muscotoxin A did not induce any prominent changes to the actin cytoskeleton compared to that with puwainaphycin F, demonstrating that their mechanism of action is dissimilar.

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CONCLUSIONS

The novel cyanobacterial lipopeptides muscotoxin A/B consisting of 11 residues and a β-amino acid (5-OH-Ahdoa) have been isolated from soil cyanobacterium D. muscorum. Muscotoxin A induced rapid cell death in human cancer cells by permeabilization of the outer cell membrane, as was recently demonstrated with cyanobacterial lipopeptides puwainaphycin F, anabaenolysins A/B, and pseudodesmin A.21,23,24 We have shown that muscotoxin A disrupted the mammalian cell membrane by a mechanism that was distinct from and opposite that of synthetic detergents and cytolytic lipopeptide puwainaphycin F. Muscotoxin A induced a significant reduction in the fluidity of the membrane’s surface, probably as a result of phospholipid compaction that shattered the membrane. The question remains as to whether this mechanism is comparable to that of previously characterized bacterial lipopeptides. We would like to test this hypothesis in a follow-up study. Interestingly, while Oftedal et al. showed that the lytic activity of anabaenolysin A is specific to membranes containing cholesterol,23 our study demonstrated that muscotoxin A could permeabilize liposomes lacking cholesterol, but it could do so only close to the gel−liquid crystalline phase transition temperature, Tm. When heating the bilayer at 15 °C above Tm (37 °C), muscotoxin A could no longer efficiently interact with a cholesterol-deficient membrane. We propose that the permeabilization activity of muscotoxin A, and likely other cyanobacterial lipopeptides, might depend on the fluidity of the bilayer rather than an interaction with any of its specific components (e.g., cholesterol). Remarkably, muscotoxin A could not permeabilize mitochondria in living cells (incubated at 37 °C), which also lack cholesterol, further solidifying our hypothesis. It appears that muscotoxin A has a higher affinity for gel-phase or tightly packed bilayers and that its binding decreases membrane elasticity and fluidity, ultimately rupturing the membrane. In summary, our study strengthens the evidence that cyanobacterial lipopeptides exclusively disrupt mammalian cell membranes.

Figure 5. Steady-state fluorescence anisotropy of the DPH fluorescent probe embedded in liposomes 2 (POPC/POPE/CH/SM 60:10:20:10 mol %) incubated with lipopeptides muscotoxin A/puwainaphycin F and detergents octyl glucoside/Triton X-100 at 25 °C.

octyl glucoside) induced a considerable decrease in fluorescence anisotropy of DPH, indicating a large increase in the motional freedom of the dye and thus membrane solubilization. This indicated that both lipopeptides do not affect the hydration and viscosity of the hydrophobic domain of the bilayer, unlike that of the tested synthetic detergents. 222


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Figure 6. Immunofluorescence staining of human fibroblasts: untreated (left column, A, D, G), treated with 25 μM muscotoxin A (middle column, B, E, H), and 165 μM (0.01% (v/v)) Triton X-100 (right column, C, F, I) for 30 min. F-actin (green, Alexa Fluor 488 phalloidin), nuclei (blue, DAPI), mitochondria (orange hot, MitoTracker Red CMXRos). Top row (A−C, overlay of all three stains), middle row (F-actin and nuclei), bottom row (mitochondria). Note that the inset in frame H is an 8× enlarged section to show the spherical morphology of the mitochondria.

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Notes

ASSOCIATED CONTENT

The authors declare no competing financial interest.

S Supporting Information *

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Table S1: 1H and 13C NMR data of muscotoxin A1. Table S2: 1 H and 13C NMR data of muscotoxin A2. Figure S1: 1H NMR spectrum of muscotoxin A. Figure S2: 1H NMR spectrum of muscotoxin B. Figure S3: HSQC spectrum of aliphatic region of muscotoxin A. Figure S4: HSQC spectrum of aliphatic region of muscotoxin B. Figure S5: HMBC spectrum of carbonyl carbon correlations used for muscotoxin A amino acid sequence determination. Figure S6: HMBC spectrum of carbonyl carbon correlations used for muscotoxin B amino acid sequence determination. Figure S7: Fragmentation spectrum of muscotoxin A. Figure S8: Steady-state emission spectra of Patman fluorescent probe embedded in liposomes 2 treated with microcystin-LR (MCLR) and cyclosporin A (CsA) at 25 °C. Figure S9. Steady-state emission spectra of Patman fluorescent probe embedded in liposomes 2 treated with 0.1 and 1% synthetic detergents Triton X-100, Octyl glucoside, CHAPS, and Tween20 at 25 °C. This material is available free of charge via the Internet at http://pubs.acs.org.

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ACKNOWLEDGMENTS We would like to thank Dr. Alena Lukešová for providing the cyanobacterial strain and our technician, Lada Samcová, for maintaining the strain.

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ABBREVIATIONS CF, carboxyfluorescein; CH, cholesterol; DOPC, dioleoylphosphatydilcholine; DOPG, dioleoyl-phosphatydilglycerol; DPH, 1,6-diphenyl-1,3,5-hexatriene; LC50, concentration of compound that induces cell death in 50% of the cells; liposomes, large unilamellar vesicles (100 nm mean diameter); Patman, N,N,N-trimethyl-2-(methyl(6-palmitoylnaphthalen-2yl)amino)ethan-1-aminium chloride; POPC, palmitoyl-oleoylphosphatidylcholine; POPE, palmitoyl-oleoyl-phosphatidylethanolamine; RFU, relative fluorescence units; SM, sphingomyelin

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AUTHOR INFORMATION

REFERENCES

(1) Van Wagoner, R. M., Drummond, A. K., and Wright, J. L. C. (2007) Biogenetic diversity of cyanobacterial metabolites, in Advances in Applied Microbiology (Laskin, A. I., Sariaslani, S., and Gadd, G. M., Eds.) pp 89−217, Vol. 61. (2) Welker, M., and von Dohren, H. (2006) Cyanobacterial peptidesNature’s own combinatorial biosynthesis. FEMS Microbiol. Rev. 30, 530−563. (3) Welker, M., Sejnohova, L., Nemethova, D., von Dohren, H., Jarkovsky, J., and Marsalek, B. (2007) Seasonal shifts in chemotype composition of Microcystis sp communities in the pelagial and the sediment of a shallow reservoir. Limnol. Oceanogr. 52, 609−619. (4) Hrouzek, P., Tomek, P., Lukesova, A., Urban, J., Voloshko, L., Pushparaj, B., Ventura, S., Lukavsky, J., Stys, D., and Kopecky, J.

Corresponding Author

*E-mail: [email protected]. Funding

This work was supported by the Czech Grant Agency - Project Cytotoxicity and induction of apoptosis in mammal cells by cyanobacterial secondary metabolites (No. P503/12/P614), by MSMT project LH 11129 and by the Center for Algal Biotechnology Třeboň - ALGATECH (CZ. 1.05/21.00/ 03.0110), Charles University project UNCE 204013 and European Regional Development Fund (BIOCEV CZ.1.05/ 1.1.00/02.0109). 223


Article

Chemical Research in Toxicology (2011) Cytotoxicity and secondary metabolites production in terrestrial Nostoc strains, originating from different climatic/geographic regions and habitats: is their cytotoxicity environmentally dependent? Environ. Toxicol. 26, 345−358. (5) Shih, P. M., Wu, D. Y., Latifi, A., Axen, S. D., Fewer, D. P., Talla, E., Calteau, A., Cai, F., de Marsac, N. T., Rippka, R., Herdman, M., Sivonen, K., Coursin, T., Laurent, T., Goodwin, L., Nolan, M., Davenport, K. W., Han, C. S., Rubin, E. M., Eisen, J. A., Woyke, T., Gugger, M., and Kerfeld, C. A. (2013) Improving the coverage of the cyanobacterial phylum using diversity-driven genome sequencing. Proc. Natl. Acad. Sci. U.S.A. 110, 1053−1058. (6) Dittmann, E., Fewer, D. P., and Neilan, B. A. (2013) Cyanobacterial toxins: biosynthetic routes and evolutionary roots. FEMS Microbiol. Rev. 37, 23−43. (7) Tillett, D., Dittmann, E., Erhard, M., von Dohren, H., Borner, T., and Neilan, B. A. (2000) Structural organization of microcystin biosynthesis in Microcystis aeruginosa PCC7806: an integrated peptide−polyketide synthetase system. Chem. Biol. 7, 753−764. (8) Mares, J., Hajek, J., Urajova, P., Kopecky, J., and Hrouzek, P. (2014) A hybrid non-ribosomal peptide/polyketide synthetase containing fatty-acyl ligase (FAAL) synthesizes the beta-amino fatty acid lipopeptides puwainaphycins in the cyanobacterium Cylindrospermum alatosporum. PLoS One 9, e111904. (9) Frankmolle, W. P., Knubel, G., Moore, R. E., and Patterson, G. M. L. (1992) Antifungal cyclic-peptides from the terrestrial blue-green alga Anabaena laxa. 2. Structures of laxaphycin A laxaphycin B, laxaphycin D and laxaphycin E. J. Antibiot. 45, 1458−1466. (10) Gerwick, W. H., Jiang, Z. D., Agarwal, S. K., and Farmer, B. T. (1992) Total structure of hormothamnin A, a toxic cyclic undecapeptide from the tropical marine cyanobacterium Hormothamnion enteromorphoides. Tetrahedron 48, 2313−2324. (11) Kang, H. S., Sturdy, M., Krunic, A., Kim, H., Shen, Q., Swanson, S. M., and Orjala, J. (2012) Minutissamides E−L, antiproliferative cyclic lipodecapeptides from the cultured freshwater cyanobacterium cf. Anabaena sp. Bioorg. Med. Chem. 20, 6134−6143. (12) An, T. Y., Kumar, T. K. S., Wang, M. L., Liu, L., Lay, J. O., Liyanage, R., Berry, J., Gantar, M., Marks, V., Gawley, R. E., and Rein, K. S. (2007) Structures of pahayokolides A and B, cyclic peptides from a Lyngbya sp. J. Nat. Prod. 70, 730−735. (13) Maru, N., Ohno, O., and Uemura, D. (2010) Lyngbyacyclamides A and B, novel cytotoxic peptides from marine cyanobacteria Lyngbya sp. Tetrahedron Lett. 51, 6384−6387. (14) Horgen, F. D., Yoshida, W. Y., and Scheuer, P. J. (2000) Malevamides A−C, new depsipeptides from the marine cyanobacterium Symploca laete-viridis. J. Nat. Prod. 63, 461−467. (15) Moon, S. S., Chen, J. L., Moore, R. E., and Patterson, G. M. L. (1992) Calophycin, a fungicidal cyclic decapeptide from the terrestrial blue-green alga Calothrix f usca. J. Org. Chem. 57, 1097−1103. (16) Plaza, A., and Bewley, C. A. (2006) Largamides A−H, unusual cyclic peptides from the marine cyanobacterium Oscillatoria sp. J. Org. Chem. 71, 6898−6907. (17) MacMillan, J. B., Ernst-Russell, M. A., de Ropp, J. S., and Molinski, T. F. (2002) Lobocyclamides A−C, lipopeptides from a cryptic cyanobacterial mat containing Lyngbya confervoides. J. Org. Chem. 67, 8210−8215. (18) Pergament, I., and Carmeli, S. (1994) Schizotrin Aa novel antimicrobial cyclic peptide from a cyanobacterium. Tetrahedron Lett. 35, 8473−8476. (19) Helms, G. L., Moore, R. E., Niemczura, W. P., Patterson, G. M. L., Tomer, K. B., and Gross, M. L. (1988) Scytonemin A, a novel calcium antagonist from a blue-green alga. J. Org. Chem. 53, 1298− 1307. (20) Zainuddin, E. N., Jansen, R., Nimtz, M., Wray, V., Preisitsch, M., Lalk, M., and Mundt, S. (2009) Lyngbyazothrins A−D, antimicrobial cyclic un-decapeptides from the cultured cyanobacterium Lyngbya sp. J. Nat. Prod. 72, 1373−1378. (21) Hrouzek, P., Kuzma, M., Cerny, J., Novak, P., Fiser, R., Simek, P., Lukesova, A., and Kopecky, J. (2012) The cyanobacterial cyclic lipopeptides puwainaphycins F/G are inducing necrosis via cell

membrane permeabilization and subsequent unusual actin relocalization. Chem. Res. Toxicol. 25, 1203−1211. (22) Jokela, J., Oftedal, L., Herfindal, L., Permi, P., Wahlsten, M., Doskeland, S. O., and Sivonen, K. (2012) Anabaenolysins, novel cytolytic lipopeptides from benthic Anabaena cyanobacteria. PLoS One 7, e41222. (23) Oftedal, L., Myhren, L., Jokela, J., Gausdal, G., Sivonen, K., Doskeland, S. O., and Herfindal, L. (2012) The lipopeptide toxins anabaenolysin A and B target biological membranes in a cholesteroldependent manner. Biochim. Biophys. Acta, Biomembr. 1818, 3000− 3009. (24) Sinnaeve, D., Hendrickx, P. M. S., Van hemel, J., Peys, E., Kieffer, B., and Martins, J. C. (2009) The solution structure and selfassociation properties of the cyclic lipodepsipeptide pseudodesmin A support its pore-forming potential. Chem.Eur. J. 15, 12653−12662. (25) Hodinka, R. L., Jackwait, K., Wannamaker, N., Walden, T. P., and Gilligan, P. H. (1987) Comparative in vitro activity of LY146032 (daptomycin), a new lipopeptide antimicrobial. Eur. J. Clin. Microbiol. Infect. Dis. 6, 100−103. (26) Backman, P. A., and Devay, J. E. (1971) Studies on the mode of action and biogenesis of the phytotoxin syringomycin. Physiol. Plant. Pathol. 1, 215−233. (27) Arima, K., Kakinuma, A., and Tamura, G. (1968) Surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis: isolation, characterization and its inhibition of fibrin clot formation. Biochem. Biophys. Res. Commun. 31, 488−494. (28) Raaijmakers, J. M., de Bruijn, I., Nybroe, O., and Ongena, M. (2010) Natural functions of lipopeptides from Bacillus and Pseudomonas: more than surfactants and antibiotics. FEMS Microbiol. Rev. 34, 1037−1062. (29) van Meer, G., Voelker, D. R., and Feigenson, G. W. (2008) Membrane lipids: where they are and how they behave. Nat. Rev. Mol. Cell Biol. 9, 112−124. (30) Hrouzek, P., Lukesova, A., Mares, J., and Ventura, S. (2013) Description of the cyanobacterial genus Desmonostoc gen. nov. including D. muscorum comb. nov. as a distinct, phylogenetically coherent taxon related to the genus Nostoc. Fottea 13, 201−213. (31) Allen, M. B., and Arnon, D. I. (1955) Studies on nitrogen-fixing blue-green algae. 1. growth and nitrogen fixation by Anabaena cylindrica lemm. Plant Physiol. 30, 366−372. (32) Simek, P., Husek, P., and Zahradnickova, H. (2008) Gas chromatographic-mass spectrometric analysis of biomarkers related to folate and cobalamin status in human serum after dimercaptopropanesulfonate reduction and heptafluorobutyl chloroformate derivatization. Anal. Chem. 80, 5776−5782. (33) Simek, P., Husek, P., and Zahradnickova, H. (2012) Heptafluorobutyl chloroformate-based sample preparation protocol for chiral and nonchiral amino acid analysis by gas chromatography, in Amino Acid Analysis: Methods and Protocols (Alterman, M. A., and Hunziker, P., Eds.) pp 137−152, Springer, New York. (34) Schneider, C. A., Rasband, W. S., and Eliceiri, K. W. (2012) NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671−675. (35) Kavan, D., Kuzma, M., Lemr, K., Schug, K. A., and Havlicek, V. (2013) CYCLONEa utility for de novo sequencing of microbial cyclic peptides. J. Am. Soc. Mass Spectrom. 24, 1177−1184. (36) Gerwick, W. H., Roberts, M. A., Proteau, P. J., and Chen, J. L. (1994) Screening cultured marine microalgae for anticancer-type activity. J. Appl. Phycol. 6, 143−149. (37) Brown, D. A., and London, E. (2000) Structure and function of sphingolipid- and cholesterol-rich membrane rafts. J. Biol. Chem. 275, 17221−17224. (38) Boustany, N. N., Drezek, R., and Thakor, N. V. (2002) Calciuminduced alterations in mitochondrial morphology quantified in situ with optical scatter imaging. Biophys. J. 83, 1691−1700.

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Cytotoxic Lipopeptide Muscotoxin A, Isolated from Soil Cyanobacterium

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