Common Mechanism of Cross-Resistance Development in Pathogenic Bacteria Bacillus cereus Against Alamethicin and Pediocin Involves Alteration in Lipid Composition.

To understand the mechanism of development of cross-resistance in food pathogen Bacillus cereus against an antimicrobial peptide pediocin and antibiotic alamethicin, the present study was designed. Pediococcus pentosaceus was taken as a source of pediocin, and it was purified by ammonium sulphate precipitation followed by cation exchange chromatography with 14.01-fold purity and 14.4 % recovery. B. cereus strains alamethicin-resistant strains (IC50 3.23 µg/ml) were selected from sensitive population with IC50 2.37 µg/ml. The development of resistance in B. cereus against alamethicin was associated with decrease in alamethicin-membrane interaction observed by in vitro assay. Resistant strain of B. cereus was found to harbour one additional general lipid as compared to sensitive strain, one amino group lacking phospholipid and one amino group containing phospholipid (ACP). In addition, ACP content was increased in resistant mutant (29.7 %) as compared to sensitive strain (14.56 %). The alamethicin-resistant mutant B. cereus also showed increased IC50 (58.8 AU/ml) for pediocin as compared to sensitive strain (IC50 47.8 AU/ml). Cross-resistance to pediocin and alamethicin in resistant mutant of B. cereus suggested a common mechanism of resistance. Therefore, this understanding could result in the development of peptide which will be effective against the resistant strains that share same mechanism of resistance.

Solid-state fermentation for Trichokonins production from Trichoderma koningii SMF2 and preparative purification of Trichokonin VI by a simple protocol.

Trichokonins are peptaibols produced by Trichoderma koningii SMF2. The main isoforms are Trichokonin VI, Trichokonin VII and Trichokonin VIII. The solid-state fermentation (SSF) was applied for the production of Trichokonin VI. The fermentation factors, which included inoculum size, incubation temperature, initial moisture content and initial pH, were investigated and optimized by response surface methodology. The maximum Trichokonin VI production (4.07mg/g dry substrate) was achieved by employing inoculum size of 18%, incubation temperature at 24.3 degrees C, initial moisture content of 77.5% and initial pH at 5.0. Furthermore, gel filtration and preparative HPLC were used for separation of Trichokonin VI from a crude extract of the T. koningii SMF2 culture. With this preparative purification protocol under optimized fermentation conditions, 146.20mg Trichokonin VI was obtained from 1kg solid cultures. It has been shown that the obtained Trichokonin VI is more than 95% in purity. This is the first report on optimization of peptaibols production in SSF with high content. An efficient method for the preparative purification of Trichokonin VI is also proposed.

Determination of peptaibol trace amounts in marine sediments by liquid chromatography/electrospray ionization-ion trap-mass spectrometry.

Extraction followed by reverse phase liquid chromatography (LC)/electrospray ionization-ion trap-mass spectrometry (ESI-IT-MS) analysis has been successfully developed for the determination of peptaibols, fungal toxic metabolites, in marine sediments. Spiking experiments showed that the mean recovery of target compounds exceeded 85% at a spiking level of 10 ng/g of sediment (wet weight). Detection and quantification limits were 250 and 830 pg/g of sediment, respectively. The method developed constituted the first sensitive assay for quantification of peptaibol trace amounts in a natural environment. A concentration of 5 ng/g in sediment samples collected from Fier d'Ars was found.

Broad-spectrum antimicrobial activity and high stability of Trichokonins from Trichoderma koningii SMF2 against plant pathogens.

Antimicrobial metabolites produced by Trichoderma koningii SMF2 exhibited antimicrobial activity against a range of Gram-positive bacterial and fungal phytopathogens. Purification of these metabolites was achieved using combinations of gel filtration and high-performance liquid chromatography. Identified by liquid chromatography electrospray ionization tandem mass spectrometry, the active metabolites proved to be three known peptaibols: Trichokonin VI, VII and VIII. The Trichokonins were stable and remained biological active over a wide pH range and at every temperature tested, showing no loss of activity even after autoclaving. Trichokonins were insensitive to proteolytic enzymes. Trichokonin VI takes on typical helical structure and the structure changes only slightly at different temperatures and pH values. The present study presented the potential of Trichokonins to be used as biological control agents.

The ion-channel activity of longibrachins LGA I and LGB II: effects of pro-2/Ala and gln-18/Glu substitutions on the alamethicin voltage-gated membrane channels.

Longibrachins LGA I (Ac Aib Ala Aib Ala Aib(5) Ala Gln Aib Val Aib(10) Gly Leu Aib Pro Val(15) Aib Aib Gln Gln Pheol(20), with Aib: alpha-aminoisobutyric acid, pheol: phenylalaninol) and LGB II are two homologous 20-residue long-sequence peptaibols isolated from the fungus Trichoderma longibrachiatum that differ between them by a Gln-18/Glu substitution. They distinguish from alamethicin by a Pro-2 for Ala replacement, which allowed to examine for the first time with natural Aib-containing analogues, the effect of Pro-2 on the ion-channel properties exhibited by alamethicin. The influence of these structural modifications on the voltage-gated ion-channel forming activity of the peptides in planar lipid bilayers were analysed. The general 'barrel-stave' model of ion-channel activity, already described for alamethicin, was preserved with both longibrachins. The negatively charged LGB II promoted higher oligomerisation levels, which could presumably dilute the repulsive effect of the negative Glu ring near the entrance of the channel and resulted in lower lifetimes of the substates, confirming the strong anchor of the peptide C-terminus at the cis-interface. Reduction of the channel lifetimes was observed for the longibrachins, compared to alamethicin. This argues for a better stabilisation of the channels formed by peptaibols having a proline at position 2, which results in better anchoring of the peptide monomer N-terminus at the trans-bilayer interface. Qualitative assays of the temperature dependence on the neutral longibrachin channel properties demonstrated a high increase of channel lifetimes and a markedly reduced voltage-sensitivity when the temperature was decreased, showing that such conditions may allow to study the channel-forming properties of peptides leading to fast current fluctuations.

In vitro and in vivo antitrypanosomal activities of three peptide antibiotics: leucinostatin A and B, alamethicin I and tsushimycin.

In the course of our screening for antitrypanosomal compounds from soil microorganisms, as well as from the antibiotics library of the Kitasato Institute for Life Sciences, we found three peptide antibiotics, leucinostatin (A and B), alamethicin I and tsushimycin, which exhibited potent or moderate antitrypanosomal activity. We report here the in vitro and in vivo antitrypanosomal properties and cytotoxicities of leucinostatin A and B, alamethicin I and tsushimycin compared with suramin. We also discuss their possible mode of action. This is the first report of in vitro and in vivo trypanocidal activity of leucinostatin A and B, alamethicin I and tsushimycin.

Analysis of the lipophilic peptaibol alamethicin by nonaqueous capillary electrophoresis-electrospray ionization-mass spectrometry.

The microheterogeneous peptaibol alamethicin F30 isolated from the culture broth of Trichoderma viride was analyzed by nonaqueous CE-electrospray-MS using an IT and a TOF mass analyzer. Compared to aqueous buffers, higher separation selectivity was observed for methanolic BGE allowing the detection of more minor components. The low electrophoretic mobility observed for neutral analytes under nonaqueous conditions may be explained by ion-dipole interactions between the peptide analytes and electrolyte ions. The amino acid sequences of the individual components were derived from MS(n) using the doubly or triply charged pseudomolecular ions as well as characteristic fragments as precursor ions. The exchange of Ala by alpha-aminoisobutyric acid (Aib) which is frequently observed for peptaibols was detected for several components. Additional variations included the exchange of Gln to Glu, and the loss of the C-terminal amino alcohol or of the first six amino acids from the N-terminus with concomitant formation of pyroglutamyl residues. In most cases comigration of the Aib peptaibols with the respective Ala component was observed as the mass difference of 14 Da as the result of the amino acid exchange was not sufficient to translate into an electrophoretic separation under the conditions applied. However, proper selection of the precursor ions allowed the unequivocal analysis of the components. Additional TOF-MS measurements were performed in order to resolve the ammonium adducts from comigrating compounds (i.e., Aib-Ala exchange) and to confirm the amino acid composition of the individual components. Except for neutral compounds migrating close to the EOF the mass accuracy was better than 4 ppm for the doubly charged pseudomolecular ions and better than 2 ppm for triply charged ions.

Fungal metabolites. XV. Primary structures of antibiotic peptides, hypelcins B-I, B-II, B-III, B-IV and B-V, from Hypocrea peltata. Application of electrospray mass spectrometry and electrospray mass spectrometry/mass spectrometry.

Hypelcin B is a mixture of antibiotic peptides produced by Hypocrea peltata. Hypelcins B-I, B-II, B-III, B-IV and B-V are components of this mixture purified by reversed-phase high-performance liquid chromatography. The amino acid sequences of these peptides were determined by electrospray mass spectrometry and electrospray mass spectrometry/mass spectrometry. The molecular weights of these peptides were all ca. 2000 and the structures were very similar.

The production of alamethicins by Trichoderma spp.

The production of polypeptides containing a high percentage of 2-methylalanine residues by a number of isolates of Trichoderma spp. has been examined. It has been shown that good yields (0.5-1.0 g L-1) can be achieved on synthetic media provided an insoluble carbohydrate is included and provided single-spore isolates that have this production ability are selected from time to time. Such yields could not be obtained on any single nitrogen source investigated, but a mixture of potassium nitrate, glutamine, and 2-methylalanine was effective. It was shown that at least eight polypeptides were produced in shake-flask or tank fermentation and that the proportions of these metabolites depended on the fermentation temperature, its pH, age, and aeration. Fermentation conditions for enhancing the production (independently) of two of the metabolites at the expense of the others are given. These two metabolites have been obtained in crystalline form and details of some of their physical and chemical properties are given.

Sequences of alamethicins F30 and F50 reconsidered and reconciled.

From the culture broth of the mould Trichoderma viride, strain NRRL 3199, a microheterogeneous mixture of the membrane active 20-residue peptaibol alamethicin (ALM) could be isolated. ALMs were isolated by XAD-2 column chromatography and separated by silica gel chromatography and trichloromethane/MeOH gradient elution into an acidic and neutral group of peptides, named ALM F30 and ALM F50, respectively, according to their 100 Rf on TLC. Peptides ALM F50 were separated by semi-preparative and analytical HPLC and subjected to ESI-MS. Ten sequences of ALM F30 and their relative quantities could be determined. The major peptides ALM F30/3 (46%) and ALM F30/7 (40%), distinguished by Aib/Ala exchange in position 6, correspond to sequences described as ALM I and II occurring in the original alamethicin from Upjohn Company. Analogously, 13 sequences of the neutral peptide mixture named ALM F50 could be determined. The major peptide ALM F50/5 (75%) and the minor peptide ALM F50/7 (10%) are distinguished from ALM F30/3 and ALM F30/7 by having Gln17 in place of Glu17, the latter occurring in the F30 group. Notably. currently commercially available alamethicins (Fluka, Sigma) represent microheterogeneous mixtures of the neutral ALM F50 peptides with trace amounts of acidic ALM F30 peptides.

Studies on metabolites of mycoparasitic fungi. II. Metabolites of Trichoderma koningii.

Four peptaibols, named trichokonins (TKs) V, VI, VII, and VIII, were isolated from the culture broth of Trichoderma koningii Oudemans. Primary structures of these peptaibols were elucidated by electrospray ionization mass spectrometry (ESI-MS), FAB-MS, and collision-induced dissociation (CID) techniques along with nuclear Overhauser enhancement spectroscopy (NOESY).