20-Residue and 11-residue peptaibols from the fungus Trichoderma longibrachiatum are synergistic in forming Na+/K+ -permeable channels and adverse action towards mammalian cells.

Certain species of the filamentous fungal genus Trichoderma (e.g. Trichoderma longibrachiatum and Trichoderma citrinoviride) are among the emerging clinical pathogens and also the most common species in the indoor space of mould-damaged buildings. The molecules involved in its pathology are not known. In the present study, we report that 0.5-2.6 wt% of the T. longibrachiatum mycelial biomass consisted of thermostable secondary metabolites mitochondriotoxic to mammalian cells. These were identified by LC/MS as one 11-residue and eight 20-residue peptaibols, AcAib-Asn-Leu/Ile-Leu/Ile-Aib-Pro-Leu/Ile-Leu/Ile-Aib-Pro-Leuol/Ileol (1175 Da) and AcAib-Ala-Aib-Ala-Aib-Ala/Aib-Gln-Aib-Val/Iva-Aib-Gly-Leu/Ile-Aib-Pro-Val/Iva-Aib-Val/Iva/Aib-Gln/Glu-Gln-Pheol(1936-1965 Da) (Aib, α-aminoisobutyric acid; Ac, acetyl; Ileol, isoleucinol; Iva, isovaline; Leuol, leucinol; Pheol, phenylalaninol). The toxic effects on boar sperm cells depended on these peptaibols, named trilongins. The trilongins formed voltage dependent, Na(+)/K(+) permeable channels in biomembranes. The permeability ratios for Na(+) ions, relative to K(+), of the 11-residue trilongin channel (0.95 : 1) and the 20-residue trilongin channel (0.8 : 1) were higher than those of alamethicin. The combined 11-residue and 20-residue trilongins generated channels that remained in an open state for a longer time than those formed by either one of the peptaibols alone. Corresponding synergy was observed in toxicokinetics. With 11-residue and 20-residue trilongins combined 1 : 2 w/w, an effective median concentration (EC(50) ) of 0.6 µg·mL(-1) was reached within 30 min, and the EC(50) shifted down to 0.2 µg·mL(-1) upon extended exposure. By contrast, with 11-residue or 20-residue trilonging separately in 30 min of exposure, the EC(50) values were 15 and 3 µg·mL(-1) , respectively, and shifted down to 1.5 and 0.4 µg·mL(-1) upon extended exposure. This is the first report on ion-channel forming peptaibols with synergistic toxicity from T. longibrachiatum strains isolated from clinical samples.

Microbial toxin's effect on mitochondrial survival by increasing K+ uptake.

We studied the effects of toxins, which inhibited the motility of boar spermatozoa, on rat liver mitochondria. The toxins studied were originally from bacteria isolated from moisture-damaged buildings where inhabitants exhibited symptoms, or from food causing poisoning. Some strains of Bacillus cereus and Streptomyces griseus produced potassium ionophoric peptides cereulide and valinomycin (Mikkola, et al., European Journal of Biochemistry 1999; 263: 112-117). Of interest is that channels were formed in black-lipid membranes (BLM) with a selectivity of K(+) > Na(+) at a concentration of 26 nM. Recently, bafilomycin A1--an inhibitor of V-H(+)ATPases--was found also to be a K(+)-specific ionophore active at nanomolar concentrations (Teplova, et al., J Bioenerg Biomembr 2007; 39: 321-329), while B. amyloliquefaciens produced amylosin, a cation channel-forming peptide with a higher selectivity for K(+) over Na(+) at around 200 nM concentrations (Mikkola, et al., Toxicon 2007; 49: 1158-1171). Of interest is that channels were formed in BLM with a selectivity of K(+) > Na(+) at a concentration of 26 nM. The ionophores and the channel-forming amylosin caused swelling of energized mitochondria due to uptake of K(+), loss of membrane potential, inhibition of maximal respiration rates due to loss of pyridine nucleotides, and inhibition of ATP synthesis. Various cell types may have different sensitivities to the effects of the ionophores. Thus, the mitochondrial membrane potential in neuronal cells was more sensitive to cereulide than in differentiated Paju cells (Teplova, et al., Acta Biochimica Polonica 2004; 51: 539-544). Swelling causes release of proapoptotic factors from mitochondria, which explains that undifferentiated neuronal cells were sensitive, while differentiated Paju cells were resistant, which probably is due to them having an increased expression of the antiapoptotic protein Bcl-2 and the neuroprotective stanniocalcin.

Bafilomycin A1 is a potassium ionophore that impairs mitochondrial functions.

Novel activities of bafilomycin A1, a macrolide antibiotic known as an inhibitor of V-ATPases, were discovered. Bafilomycin A1 induced uptake of potassium ions by energized mitochondria and caused mitochondrial swelling, loss of membrane potential, uncoupling of oxidative phosphorylation, inhibition of the maximal respiration rates, and induced pyridine nucleotide oxidation. The mitochondrial effects provoked by nanomolar concentrations of bafilomycin A1 were connected to its activity as a potent, K(+)-specific ionophore. The K(+) ionophoric activity of bafilomycin A1 was observed also in black lipid membranes, indicating that it was an inherent property of the bafilomycin A1 molecule. It was found that bafilomycin A1 is a K(+) carrier but not a channel former. Bafilomycin A1 is the first and currently unique macrolide antibiotic with K(+) ionophoric properties. The novel properties of bafilomycin A1 may explain some of the biological effects of this plecomacrolide antibiotic, independent of V-ATPase inhibition.

Oral secretions from herbivorous lepidopteran larvae exhibit ion channel-forming activities.

Feeding insects introduce oral secretions (OS) into the wounded tissue of the attacked plant. Various OS-derived molecules must be involved in subsequent processes including the induction of plant defence reactions. Using the planar lipid bilayer membrane technique, isolated OS were analyzed with respect to their membrane activities. Transmembrane ion fluxes were generated by OS of eight different lepidopteran larvae, all of which form comparable ion channels in artificial membranes. Currents were characterized by long lasting open times and conductivities from 250pS up to 1100pS. Channels formed by Spodoptera exigua secretions showed a preference for cations over anions. OS also induced a transient increase of the cytosolic calcium concentration in soybean cells, determined by the aequorin technique. Known compounds of the OS, fatty-acid-glutamine conjugates, also interfered with the membrane but were unable to form stable channels. Since ion fluxes and depolarization are early responses upon insect feeding, OS-derived components may be involved in the elicitation process by direct interaction with the plant membranes.

Biological effects of Trichoderma harzianum peptaibols on mammalian cells.

Trichoderma species isolated from water-damaged buildings were screened for toxicity by using boar sperm cells as indicator cells. The crude methanolic cell extract from Trichoderma harzianum strain ES39 inhibited the boar sperm cell motility at a low exposure concentration (50% effective concentration, 1 to 5 microg [dry weight] ml of extended boar semen(-1)). The same exposure concentration depleted the boar sperm cells of NADH(2). Inspection of the exposed boar sperm cells by transmission electron microscopy revealed damage to the plasma membrane. By using the black lipid membrane technique, it was shown that the semipurified metabolites (eluted from a SepPak C(18) cartridge) of T. harzianum strain ES39 induced voltage-dependent conductivity. The high-performance liquid chromatography-purified metabolites of T. harzianum strain ES39 dissipated the mitochondrial membrane potential (Deltapsi(m)) of human lung epithelial carcinoma cells (cell line A549). The semipurified metabolites (eluted from a SepPak C(18) cartridge) of T. harzianum strain ES39 were analyzed by mass spectrometry (MS). Matrix-assisted laser desorption ionization and nanoflow electrospray ionization MS revealed five major peptaibols, each of which contained 18 residues and had a mass ranging from 1,719 to 1,775 Da. Their partial amino acid sequences were determined by collision-induced dissociation tandem MS.

Differences in membrane pore formation by peptaibols.

The efficiencies of membrane pore formation by 14 naturally occurring peptaibols and two structurally modified ampullosporins were compared using an artificial bilayer membrane model. Major differences were found in the dependence on peptide sequences and the constituting amino acids. Alamethicin F-30, chrysospermins C/D, paracelsin and texenomycin A displayed higher activity by several orders of magnitude in comparison with smaller peptaibols containing < 17 amino acids such as ampullosporins, trichofumins. bergofungins and cephaibols. Biological activities such as the induction of pigment formation by the fungus Phoma destructiva and long acting hypothermia and depression of locomotor activity in mice were correlated with moderate membrane permeabilization. No or weak membrane activities corresponded with biological inactivity. Highly membrane-active structures such as alamethicin F-30, chrysospermin C, texenomycin A and paracelsin A displayed antibiotic effects against the fungus and toxicity in mice.

Isolation, structure elucidation and biological activities of trichofumins A, B, C and D, new 11 and 13mer peptaibols from Trichoderma sp. HKI 0276W.

Trichofumins A-D were isolated from cultures of Trichoderma sp. HKI 0276 as new 11 and 13mer peptaibols. Similar to 15mer peptaibols they promote morphogenesis of the fungus Phoma destructiva and cause hypothermia in mice as a characteristic of neuroleptic activity. Membrane measurements using a synthetic BLM model showed that A, B, C and D increased membrane permeability for cations in a similar manner as was shown for larger peptaibols but with comparably lower efficiency.