Trachymyrmex septentrionalis ants promote fungus garden hygiene using Trichoderma-derived metabolite cues.

Fungus-growing ants depend on a fungal mutualist that can fall prey to fungal pathogens. This mutualist is cultivated by these ants in structures called fungus gardens. Ants exhibit weeding behaviors that keep their fungus gardens healthy by physically removing compromised pieces. However, how ants detect diseases of their fungus gardens is unknown. Here, we applied the logic of Koch's postulates using environmental fungal community gene sequencing, fungal isolation, and laboratory infection experiments to establish that Trichoderma spp. can act as previously unrecognized pathogens of Trachymyrmex septentrionalis fungus gardens. Our environmental data showed that Trichoderma are the most abundant noncultivar fungi in wild T. septentrionalis fungus gardens. We further determined that metabolites produced by Trichoderma induce an ant weeding response that mirrors their response to live Trichoderma. Combining ant behavioral experiments with bioactivity-guided fractionation and statistical prioritization of metabolites in Trichoderma extracts demonstrated that T. septentrionalis ants weed in response to peptaibols, a specific class of secondary metabolites known to be produced by Trichoderma fungi. Similar assays conducted using purified peptaibols, including the two previously undescribed peptaibols trichokindins VIII and IX, suggested that weeding is likely induced by peptaibols as a class rather than by a single peptaibol metabolite. In addition to their presence in laboratory experiments, we detected peptaibols in wild fungus gardens. Our combination of environmental data and laboratory infection experiments strongly support that peptaibols act as chemical cues of Trichoderma pathogenesis in T. septentrionalis fungus gardens.

Importance of isoleucine residue in ion channel formation ability of 11-residue peptaibols.

Peptaibols are a class of short peptides, typically 7 to 20 amino acids long, characterized by noncanonical amino acid residues such as aminoisobutyric acid (Aib). Although the helix length is shorter than the membrane thickness, the 11-residue peptaibol trichorovin-XII (TV-XII) can form ion channels in membranes. Assuming that a higher proportion of isoleucine (Ile) relative to leucine (Leu) residues is crucial for maintaining the ion channel activity of TV-XII, peptide analogs of TV-XII with varying Ile content were designed, synthesized, and evaluated. The secondary structure of all derivatives under hydrophobic conditions was confirmed by CD measurement as an α-helix-like ß-bend ribbon spiral structure. The most stable ion channel activity was found in compound 4a with maximum Ile. Furthermore, the C-terminal Ile analog showed greater ion channel activity compared to the Leu analog. This suggests that the choice between Leu and Ile can influence the expression of ion channel activity, which will be crucial for the de novo designed functional peptides.

Bergofungin D, a peptaibol template for the introduction of chemical modifications, synthesis of analogs and comparative studies of their structures.

Bergofungin D is a helical peptide of the peptaibol family consisting of 14 amino acids, six of which are the helix inducer aminoisobutyric acid (Aib). In the second third of the sequence, a hydroxyproline causes a bending of the helix and a disruption of the hydrogen bond network, and Aib7 is the only amino acid in this region involved in the hydrogen bond network. Therefore, modification of this residue can serve as a probe to monitor the effect of introducing amino acid substitutions on this more fragile helical turn. To validate this approach, we simplified the original bergofungin D by reducing the number of non-classical amino acids, replacing the (R)-isovaleric acid by its enantiomer or an Aib and the hydroxyproline with a proline, respectively, without affecting its secondary structure. Within the modified structure, we replaced Aib7-Aib8 by its 1,2,3-triazolodipeptide equivalent or Aib7 by a serine or a dehydrobutyrine. We have reported and analyzed five crystal structures, three of which are new, demonstrating the usefulness of the modified bergofungin D as a probe for monitoring the introduction of amino acid substitutions within a helical structure.

Cytotoxic Peptaibols from Trichoderma strigosum.

Five new lipopeptaibols (1-5) and eight new 19-residue peptaibols (8-15) along with two known lipopeptaibols, lipovelutibols C (6) and D (7) were isolated from Trichoderma strigosum. The planar structures of the newly discovered peptaibols (1-5, 8-15) were elucidated using 1D and 2D NMR, and UPLC-MS/MS data. The absolute configurations for new peptaibols (1-5, 8-15) were elucidated using the advanced Marfey's method and GITC (2,3,4,6-tetra-O-acetyl-ß-d-glucopyranosyl isothiocyanate) derivatization. Through analysis of CD spectra, these peptabols were found to have right-handed helical conformations. While most of the new compounds were significantly more active than the positive control, 9, 10, 12, and 15 containing Ser and Leu at positions 10 and 11, respectively, were the most cytotoxic against MDA-MB-231, SNU449, SKOV3, DU145, and HCT116 cancer cell lines, and the 19-residue peptaibols were generally more potent than lipopeptaibols.

Cytotoxic Peptaibols from Trichoderma guizhouense, a Fungus Isolated from an Urban Soil Sample.

Soil sustains human life by nourishing crops, storing food sources, and housing microbes, which may affect the nutrition and biosynthesis of secondary metabolites, some of which are used as drugs. To identify lead compounds for a new class of drugs, we collected soil-derived fungal strains from various environments, including urban areas. As various human pathogens are assumed to influence the biosynthetic pathways of metabolites in soil fungi, leading to the production of novel scaffolds, we focused our work on densely populated urban areas and tourist attractions. A soil-derived fungal extract library was screened against MDA-MB-231 cells to derive their cytotoxic activity. Notably, 10 µg/mL of the extract of Trichoderma guizhouense (DS9-1) was found to exhibit an inhibitory effect of 71%. Fractionation, isolation, and structure elucidation efforts led to the identification of nine new peptaibols, trichoguizaibols A-I (1-9), comprising 14 amino acid residues (14-AA peptaibols), and three new peptaibols, trichoguizaibols J-L (10-12), comprising 18 amino acid residues (18-AA peptaibols). The chemical structures of 1-12 were determined based on their 1D and 2D NMR spectra, HRESIMS, electronic circular dichroism data, and results of the advanced Marfey's method. The 18-AA peptaibols were found to exhibit cytotoxicity against MDA-MB-231, SK-Hep1, SKOV3, DU145, and HCT116 cells greater than that of the 14-AA peptaibols. Among these compounds, 10-12 exhibited potent sub-micromolar IC50 values. These results are expected to shed light on a new direction for developing novel scaffolds as anticancer agents.

Multiple lysine substitutions in the peptaibol trichogin GA IV enhance the antibiotic activity against plant pathogenic Pseudomonas syringae.

Plant diseases caused by Pseudomonas syringae are essentially controlled in the field with the use of copper-based products and antibiotics, raising environmental and safety concerns. Antimicrobial peptides (AMPs) derived from fungi may represent a sustainable alternative to those chemicals. Trichogin GA IV, a non-ribosomal, 11-residue long AMP naturally produced by the fungus Trichoderma longibrachiatum has the ability to insert into phospholipidic membranes and form water-filled pores, thereby perturbing membrane integrity and permeability. In previous studies, peptide analogs modified at the level of specific residues were designed to be water-soluble and active against plant pathogens. Here, we studied the role of glycine-to-lysine substitutions and of the presence of a C-terminal leucine amide on bioactivity against Pseudomonas syringae bacteria. P. syringae diseases affect a wide range of crops worldwide, including tomato and kiwifruit. Our results show that trichogin GA IV analogs containing two or three Gly-to-Lys substitutions are highly effective in vitro against P. syringae pv. tomato (Pst), displaying minimal inhibitory and minimal bactericidal concentrations in the low micromolar range. The same analogs are also able to inhibit in vitro the kiwifruit pathogen P. syringae pv. actinidiae (Psa) biovar 3. When sprayed on tomato plants 24 h before Pst inoculation, only tri-lysine containing analogs were able to significantly reduce bacterial titers and symptom development in infected plants. Our results point to a positive correlation between the number of lysine substitutions and the antibacterial activity. This correlation was supported by microscopy analyses performed with mono-, di- and tri-Lys containing analogs that showed a different degree of interaction with Pst cells and ultrastructural changes that culminated in cell lysis.

Intrinsic Lipid Curvature and Bilayer Elasticity as Regulators of Channel Function: A Comparative Single-Molecule Study.

Perturbations in bilayer material properties (thickness, lipid intrinsic curvature and elastic moduli) modulate the free energy difference between different membrane protein conformations, thereby leading to changes in the conformational preferences of bilayer-spanning proteins. To further explore the relative importance of curvature and elasticity in determining the changes in bilayer properties that underlie the modulation of channel function, we investigated how the micelle-forming amphiphiles Triton X-100, reduced Triton X-100 and the HII lipid phase promoter capsaicin modulate the function of alamethicin and gramicidin channels. Whether the amphiphile-induced changes in intrinsic curvature were negative or positive, amphiphile addition increased gramicidin channel appearance rates and lifetimes and stabilized the higher conductance states in alamethicin channels. When the intrinsic curvature was modulated by altering phospholipid head group interactions, however, maneuvers that promote a negative-going curvature stabilized the higher conductance states in alamethicin channels but destabilized gramicidin channels. Using gramicidin channels of different lengths to probe for changes in bilayer elasticity, we found that amphiphile adsorption increases bilayer elasticity, whereas altering head group interactions does not. We draw the following conclusions: first, confirming previous studies, both alamethicin and gramicidin channels are modulated by changes in lipid bilayer material properties, the changes occurring in parallel yet differing dependent on the property that is being changed; second, isolated, negative-going changes in curvature stabilize the higher current levels in alamethicin channels and destabilize gramicidin channels; third, increases in bilayer elasticity stabilize the higher current levels in alamethicin channels and stabilize gramicidin channels; and fourth, the energetic consequences of changes in elasticity tend to dominate over changes in curvature.

Multi-oligomeric states of alamethicin ion channel: Assemblies and conductance.

Transmembrane assemblies of the peptaibol alamethicin (ALM) are among the most extensively studied ion channels not only because of their antimicrobial activity but also as models for channel structure and aggregation. In this study, several oligomeric states of ALM are investigated with molecular dynamics simulations to establish properties of the channel and obtain free energy profiles for ion transport and the corresponding values of conductance. The hexamer, heptamer, and octamer of ALM in phospholipid membrane are found to be stable but highly dynamic in barrel-stave structures, with calculated conductance equal to 18, 195, and 1270 pS, respectively, in 1 M KCl ion solution. The corresponding free energy profiles, reported for the first time, are reconstructed from simulations at applied voltage of 200 mV with the aid of the electrodiffusion model both with and without the knowledge of diffusivity. The calculated free energy barriers are equal to 2.5, 1.5, and 0.5 kcal/mol for K+ and 4.0, 2.2, and 1.5 kcal/mol for Cl-, for hexamer, heptamer, and octamer, respectively. The calculated conductance and the ratio between conductance in consecutive states are in good agreement with those measured experimentally. This suggests that the hexamer is the lowest conducting state, with measured conductance equal to 19 pS. The selectivity of K+ over Cl- is calculated as 1.5 and 2.3 for the octameric and heptameric channels, close to the selectivity measured for high-conductance states. Selectivity increases to 13 in the hexameric channel in which the narrowest Gln7 site has a pore radius of only ∼1.6 Å, again in accord with experiment. A good agreement found between calculated and measured conductance through a hexamer templated on cyclodextrin lands additional support for the results of our simulations, and the comparison with ALM reveals the dependence of conductance on the nature of phospholipid membrane.

Total synthesis of the natural, medium-length, peptaibol pentadecaibin and study of the chemical features responsible for its membrane activity.

Peptaibols are naturally occurring, antimicrobial peptides endowed with well-defined helical conformations and resistance to proteolysis. Both features stem from the presence in their sequence of several, Cα -tetrasubstituted, α-aminoisobutyric acid (Aib) residues. Peptaibols interact with biological membranes, usually causing their leakage. All of the peptaibol-membrane interaction mechanisms proposed so far begin with peptide aggregation or accumulation. The long-length alamethicin, the most studied peptaibol, acts by forming pores in the membranes. Conversely, the carpet mechanism has been claimed for short-length peptaibols, such as trichogin. The mechanism of medium-length peptaibols is far less studied, and this is partly due to the difficulties of their synthesis. They are believed to perturb membrane permeability in different ways, depending on the membrane properties. The present work focuses on pentadecaibin, a recently discovered, medium-length peptaibol. In contrast to the majority of its family members, its sequence does not comprise hydroxyprolines or prolines, and its helix is not kinked. A reliable and effective synthesis procedure is described that allowed us to produce also two shorter analogs. By a combination of techniques, we were able to establish a 3D-structure-activity relationship. In particular, the membrane activity of pentadecaibin heavily depends on the presence of three consecutive Aib residues that are responsible for the clear, albeit modest, amphiphilic character of its helix. The shortest analog, devoid of two of these three Aib residues, preserves a well-defined helical conformation, but not its amphipathicity, and loses almost completely the ability to cause membrane leakage. We conclude that pentadecaibin amphiphilicity is probably needed for the peptide ability to perturb model membranes.

18-Residue Peptaibols Produced by the Sponge-Derived Trichoderma sp. GXIMD 01001.

Seven new 18-residue peptaibols, trichorzins A-G (1-7), were isolated from the sponge-derived fungus Trichoderma sp. GXIMD 01001. Their structures and configurations were characterized by a comprehensive interpretation of the NMR spectroscopic data, MS/MS fragmentation, Marfey's method, and ECD analysis. The general sequences of trichorzins A-G are as follows: Ac-Aib1-Ala/Ser2-Ala3-Aib/Iva4-Iva5-Gln6-Aib/Iva7-Val/allo-Ile8-Aib9-Gly10-Leu11-Aib12-Pro13-Leu14-Aib15-Aib16-Gln17-Trpol/Pheol18. The obtained compounds were assessed for their potential antiproliferative and antimicrobial activities. All obtained compounds did not show potent antibacterial activity but exhibited significant cytotoxicity, with the lowest IC50 values at 0.46-4.7 µM against four human carcinoma cell lines.

Peptide Analogs of a Trichoderma Peptaibol Effectively Control Downy Mildew in the Vineyard.

Plasmopara viticola, the agent of grapevine downy mildew, causes enormous economic damage, and its control is primarily based on the use of synthetic fungicides. The European Union policies promote reducing reliance on synthetic plant protection products. Biocontrol agents such as Trichoderma spp. constitute a resource for the development of biopesticides. Trichoderma spp. produce secondary metabolites such as peptaibols, but the poor water solubility of peptaibols limits their practical use as agrochemicals. To identify new potential bio-inspired molecules effective against P. viticola, various water-soluble peptide analogs of the peptaibol trichogin were synthesized. In grapevine leaf disk assays, the peptides analogs at a concentration of 50 µM completely prevented P. viticola infection after zoosporangia inoculation. Microscopic observations of one of the most effective peptides showed that it causes membrane lysis and cytoplasmic granulation in both zoosporangia and zoospores. Among the effective peptides, 4r was selected for a 2-year field trial experiment. In the vineyard, the peptide administered at 100 µM (equivalent to 129.3 g/ha) significantly reduced the disease incidence and severity on both leaves and bunches, with protection levels similar to those obtained using a cupric fungicide. In the second-year field trial, reduced dosages of the peptide were also tested, and even at the peptide concentration reduced by 50 or 75%, a significant decrease in the disease incidence and severity was obtained at the end of the trial. The peptide did not show any phytotoxic effect. Previously, peptide 4r had been demonstrated to be active against other fungal pathogens, including the grapevine fungus Botrytis cinerea. Thus, this peptide may be a candidate for a broad-spectrum fungicide whose biological properties deserve further investigation.

Synthesis, Conformational Analysis and Antitumor Activity of the Naturally Occurring Antimicrobial Medium-Length Peptaibol Pentadecaibin and Spin-Labeled Analogs Thereof.

Peptaibols are proteolysis-resistant, membrane-active peptides. Their remarkably stable helical 3D-structures are key for their bioactivity. They can insert themselves into the lipid bilayer as barrel staves, or lay on its surface like carpets, depending on both their length and the thickness of the lipid bilayer. Medium-length peptaibols are of particular interest for studying the peptide-membrane interaction because their length allows them to adopt either orientation as a function of the membrane thickness, which, in turn, might even result in an enhanced selectivity. Electron paramagnetic resonance (EPR) is the election technique used to this aim, but it requires the synthesis of spin-labeled medium-length peptaibols, which, in turn, is hampered by the poor reactivity of the Cα-tetrasubstituted residues featured in their sequences. After several years of trial and error, we are now able to give state-of-the-art advice for a successful synthesis of nitroxide-containing peptaibols, avoiding deleted sequences, side reactions and difficult purification steps. Herein, we describe our strategy and itsapplication to the synthesis of spin-labeled analogs of the recently discovered, natural, medium-length peptaibol pentadecaibin. We studied the antitumor activity of pentadecaibin and its analogs, finding potent cytotoxicity against human triple-negative breast cancer and ovarian cancer. Finally, our analysis of the peptide conformational preferences and membrane interaction proved that pentadecaibinspin-labeling does not alter the biological features of the native sequence and is suitable for further EPR studies. The nitroxide-containing pentadecaibins, and their synthetic strategy described herein, will help to shed light on the mechanism of the peptide-membrane interaction of medium-length peptaibols.

Anticancer and Targeting Activity of Phytopharmaceutical Structural Analogs of a Natural Peptide from Trichoderma longibrachiatum and Related Peptide-Decorated Gold Nanoparticles.

In the large field of bioactive peptides, peptaibols represent a unique class of compounds. They are membrane-active peptides, produced by fungi of the genus Trichoderma and known to elicit plant defenses. Among the short-length peptaibols, trichogin GA IV is nonhemolytic, proteolysis-resistant, antibacterial, and cytotoxic. Several trichogin analogs are endowed with potent activity against phytopathogens, thus representing a sustainable alternative to copper for plant protection. In this work, we tested the activity of trichogin analogs against a breast cancer cell line and a normal cell line of the same derivation. Lys-containing trichogins showed an IC50 below 12 µM, a peptide concentration not significantly affecting the viability of normal cells. Two analogs were found to be membrane-active but noncytotoxic. They were anchored to gold nanoparticles (GNPs) and further investigated for their ability to act as targeting agents. GNP uptake by cancer cells increased with peptide decoration, while it decreased in the corresponding normal epithelial cells. This work highlights the promising biological properties of peptaibol analogs in the field of cancer therapy either as cytotoxic molecules or as active targeting agents in drug delivery.

Peptaibol Analogs Show Potent Antibacterial Activity against Multidrug Resistant Opportunistic Pathogens.

New classes of antibacterial drugs are urgently needed to address the global issue of antibiotic resistance. In this context, peptaibols are promising membrane-active peptides since they are not involved in innate immunity and their antimicrobial activity does not involve specific cellular targets, therefore reducing the chance of bacterial resistance development. Trichogin GA IV is a nonhemolytic, natural, short-length peptaibol active against Gram-positive bacteria and resistant to proteolysis. In this work, we report on the antibacterial activity of cationic trichogin analogs. Several peptides appear non-hemolytic and strongly active against many clinically relevant bacterial species, including antibiotic-resistant clinical isolates, such as Staphylococcus aureus, Acinetobacter baumannii, and extensively drug-resistant Pseudomonas aeruginosa, against which there are only a limited number of antibiotics under development. Our results further highlight how the modification of natural peptides is a valuable strategy for obtaining improved antibacterial agents with potential therapeutic applications.

Tolypocladamide H and the Proposed Tolypocladamide NRPS in Tolypocladium Species.

The genome of entomopathogenic fungus Tolypocladium inflatum Gams encodes 43 putative biosynthetic gene clusters for specialized metabolites, although genotype-phenotype linkages have been reported only for the cyclosporins and fumonisins. T. inflatum was cultured in defined minimal media, supplemented with or without one of nine different amino acids. Acquisition of LC-MS/MS data for molecular networking and manual analysis facilitated annotation of putative known and unknown metabolites. These data led us to target a family of peptaibols and guided the isolation and purification of tolypocladamide H (1), which showed modest antibacterial activity and toxicity to mammalian cells at micromolar concentrations. HRMS/MS, NMR, and advanced Marfey's analysis were used to assign the structure of 1 as a peptaibol containing 4-[(E)-2-butenyl]-4-methyl-l-threonine (Bmt), a hallmark structural motif of the cyclosporins. LC-MS detection of homologous tolypocladamide metabolites and phylogenomic analyses of peptaibol biosynthetic genes in other cultured Tolypocladium species allowed assignment of a putative tolypocladamide nonribosomal peptide synthetase gene.

Tolypocladamides A-G: Cytotoxic Peptaibols from Tolypocladium inflatum.

Seven new peptaibols named tolypocladamides A-G have been isolated from an extract of the fungus Tolypocladium inflatum, which inhibits the interaction between Raf and oncogenic Ras in a cell-based high-throughput screening assay. Each peptaibol contains 11 amino acid residues, an octanoyl or decanoyl fatty acid chain at the N-terminus, and a leucinol moiety at the C-terminus. The peptaibol sequences were elucidated on the basis of 2D NMR and mass spectral fragmentation analyses. Amino acid configurations were determined by advanced Marfey's analyses. Tolypocladamides A-G caused significant inhibition of Ras/Raf interactions with IC50 values ranging from 0.5 to 5.0 µM in a nanobioluminescence resonance energy transfer (NanoBRET) assay; however, no interactions were observed in a surface plasmon resonance assay for binding of the compounds to wild type or G12D mutant Ras constructs or to the Ras binding domain of Raf. NCI 60 cell line testing was also conducted, and little panel selectivity was observed.

Longibrachiamide A, a 20-Residue Peptaibol Isolated from Trichoderma longibrachiatum Rifai DMG-3-1-1.

Longibrachiamide A (1), a new 20-residue peptaibol, along with three known ones (2-4), were isolated from the fungus Trichoderma longibrachiatum Rifai DMG-3-1-1, isolated from a mushroom Clitocybe nebularis (Batsch) P. Kumm, which was collected from coniferous forest of northeast China in our previous work. The structure of longibrachiamide A (1) was determined by its NMR and ESI-MS/MS data, the absolute configuration of 1 was further determined by Marfey's analyses. And the complete NMR data of 2-4 were also reported for the first time. The similar CD spectra of 1-4 showed that they all had mixed 310 -/α-helical conformations. Compounds 1-4 showed strong cytotoxicities against BV2, A549 and MCF-7 cells, and also showed moderate inhibitory effects against the tested Gram-positive bacteria, including MRSA T144 and VRE-10.

Eleven-Residue Peptaibols Isolated from Trichoderma longibrachiatum Rifai DMG-3-1-1and Their Structure-Activity Relationship.

Total 23 eleven-residue peptaibols, including five reported ones (1-5) in our previous work, were isolated from the fungus Trichoderma longibrachiatum Rifai DMG-3-1-1, which was obtained from the mushroom Clitocybe nebularis (Batsch) P. Kumm. The structures of the 13 new peptaibols (6-10 and 12-19) were determined by their NMR and MALDI-MS/MS data, their absolute structures were further determined by Marfey's analyses and their ECD data. Careful comparison of the structures of 1-23 showed that only seven residues varied including the 2nd (Gln2 /Asn2 ), 3rd (Ile3 /Val3 ), 4th (Ile4 /Val4 ), 6th (Pro6 /Hyp6 ), 8th (Leu8 /Val8 ), 10th (Pro10 /Hyp10 ) and 11th (Leuol11 /Ileol11 /Valol11 ) residues. Comparison of the IC50 s against the three tested cell lines of 1-23 indicated that 2nd, 3rd and 4th amino acid residues affected their cytotoxicities powerfully. Compounds 2, 5, 9, 11, 21 and 22 showed moderate antibacterial activities against Staphylococcus aureus MRSA T144, which also showed stronger cytotoxicities against BV2 and MCF-7 cells.

Insight into the Mechanism of Action and Peptide-Membrane Interactions of Aib-Rich Peptides: Multitechnique Experimental and Theoretical Analysis.

The increase in resistant bacterial strains necessitates the identification of new antimicrobial molecules. Antimicrobial peptides (AMPs) are an attractive option because of evidence that bacteria cannot easily develop resistance to AMPs. The peptaibols, a class of naturally occurring AMPs, have shown particular promise as antimicrobial drugs, but their development has been hindered by their mechanism of action not being clearly understood. To explore how peptaibols might interact with membranes, circular dichroism, vibrational circular dichroism, linear dichroism, Raman spectroscopy, Raman optical activity, neutron reflectivity and molecular dynamics simulations have been used to study a small library of peptaibol mimics, the Aib-rich peptides. All the peptides studied quickly partitioned and oriented in membranes, and we found evidence of chiral interactions between the phospholipids and membrane-embedded peptides. The protocols presented in this paper open new ground by showing how chiro-optical spectroscopies can throw light on the mechanism of action of AMPs.

Acremopeptaibols A-F, 16-Residue Peptaibols from the Sponge-Derived Acremonium sp. IMB18-086 Cultivated with Heat-Killed Pseudomonas aeruginosa.

Six new 16-residue peptaibols, acremopeptaibols A-F (1-6), along with five known compounds, were isolated from the cultures of the sponge-associated fungus Acremonium sp. IMB18-086 grown in the presence of the autoclaved bacterium Pseudomonas aeruginosa on solid rice medium. The peptaibol sequences were established based on comprehensive analysis of 1D and 2D NMR spectroscopic data in conjunction with HRESIMS/MS experiments. The configurations of the amino acid residues were determined by advanced Marfey's analysis. Compounds 1-6 feature the lack of the highly conserved Thr6 and Hyp10 residues in comparison with other members of the SF3 subfamily peptaibols. A plausible biosynthetic pathway of compounds 1-6 was proposed on the basis of genomic analysis. Compounds 1, 5, 7, and 10 exhibited significant antimicrobial activity against Staphylococcus aureus, methicillin-resistant S. aureus, Bacillus subtilis, and Candida albicans. Compounds 7-10 showed potent cytotoxicities against the A549 and/or HepG2 cancer cell lines.