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 (EC50) of 0.6 μg·mL−1 was reached within 30 min, and the EC50 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 EC50 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.
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mitochondrial transmembrane potential
black lipid membrane
effective median concentration
fractional inhibitory concentration
internal transcribed spacer
malt extract agar
tryptic soy agar
Filamentous fungi from the genus Trichoderma (Ascomycota, Hypocreales) are well known as producers of industrial enzymes, especially cellulases [1-3]. Certain members of the genus are included among the promising biocontrol agents as a result of their antagonistic activities against plant pathogenic fungi . In addition, Trichoderma strains are also known rarely to cause opportunistic infections in humans, varying from localized to fatal disseminated diseases in particular risk populations, including patients undergoing peritoneal dialysis, transplant recipients and patients with haematological malignancies . Possible sources of infection include water-related sites, air, foods and catheters. Based on the extensive review of Kredics et al. , nine species from the genus Trichoderma (Trichoderma longibrachiatum, Trichoderma citrinoviride, Trichoderma pseudokoningii, Trichoderma reesei, Trichoderma harzianum, Trichoderma koningii, Trichoderma atroviride, Trichoderma viride) have been previously reported from clinical cases. However, several clinical isolates originally identified based on their morphological characters were recently reidentified by sequence-based molecular techniques as T. longibrachiatum, which thus proved to be the most frequently occurring, almost exclusive clinical aetiological agent within the genus Trichoderma [6, 7]. Therefore, it was suggested that the biotechnological and agricultural application of T. longibrachiatum should be avoided or at least carefully monitored to minimize possible health risks.
Trichoderma species were reported to be among the dominating microfungi in the indoor environments of water-damaged buildings [8, 9]. Their possible association with building-related symptoms of ill health has been suggested [10-12]; however, because a causal relationship could not be established, their actual degree of contribution is yet unknown. The Trichoderma species detected in such environments include the clinically relevant species T. longibrachiatum, which, along with the closely-related species T. citrinoviride, may represent almost half of the Trichoderma isolates from building materials .
Peptaibiotics represent a constantly growing group of peptide antibiotics with increased interest as a result of their unique bioactivities and conformations [13-17]. They are defined as linear or cyclic polypeptide antibiotics of 4–21 amino acid residues that are characterized by a molecular masses in the range between 500 and 2200 Da, a high α-aminoisobutyric acid (Aib) content, the presence of other non-proteinogenic amino- or lipoamino acids, an acylated N-terminus, and (if linear) a C-terminal residue mostly consisting of a free or acetylated amide-bonded 2-amino alcohol . The subgroup comprises Aib-containing peptides carrying a C-terminal 2-amino alcohol residue, which are referred to as peptaibols . The first report of an Aib-containing antibiotic from the genus Trichoderma, compound U-22324 (later renamed as alamethicin), was published in 1967 . Subsequently, it was revealed that the first peptaibol isolated from a Trichoderma sp. was actually suzukacillin from ‘T. viride’ 63 C-I ; however, the presence of Aib in the SZ-hydrolysate was confirmed only 6 years later . The producer strain NRRL 3199 originally identified as T. viride was recently reidentified as Trichoderma arundinaceum, a member of the Trichoderma brevicompactum clade , and all other alamethicin-producing Trichoderma species (T. brevicompactum, Trichoderma protrudens, Trichoderma turrialbense) also belong to the so-called ‘Brevicompactum clade’ [14, 22]. The occurrence of several peptaibol compounds has been reported also from Trichoderma strains belonging to the clinically relevant species T. longibrachiatum. These included tricholongins , longibrachins , trichobrachins [25, 26] and trichorovin . However, one of the producer isolates, ‘T. longibrachiatum’ CBS 936.69 was recently reclassified as Trichoderma ghanense  and, until now, only the identities of trichobrachin- and trichorovin-producing T. longibrachiatum strains were confirmed by phylogenetic data.
Crude extracts of various T. longibrachiatum isolates have been reported to contain thermostable substances that inhibited motility of boar spermatozoa and quenched the mitochondrial transmembrane potential (∆Ψm) of the sperm cells at low exposure concentrations . In the present study, we describe the isolation, structure, toxicity and ion channel-forming activities, as well as synergistic properties, of two different sizes of peptaibols produced by T. longibrachiatum isolates originating from agricultural and clinical samples, and also from an indoor environment where serious building-related symptoms of ill health were claimed.
Cell free extracts of T. longibrachiatum strains were toxic to porcine sperm cells
Cell extracts of T. longibrachiatum isolates (Table 1) originating from clinical (n = 2), terrestrial (n = 3) and sick building samples (n = 3) were assayed for the presence of substances toxic to mammalian cells. Internal transcribed spacer (ITS) sequences confirmed the identity of the strains as T. longibrachiatum (Table 1). Boar sperm cells were used as toxicity indicator cells. The cell free extracts (prepared by heating in methanol at 100 °C) destroyed several cellular functions of boar sperm cells: motility, inner ΔΨm and cell membrane permeability barrier to propidium iodide (Table 2). The effective median concentration (EC50) was 3–6 μg of the methanol-soluble substance·mL−1. Corresponding extracts from T. longibrachiatum DSM 768 or from Acremonium tubakii (strain CBS 110649) showed no toxicity up to concentrations ten-fold higher. The eight toxic T. longibrachiatum strains were cultivated on tryptic soy agar (TSA), brain heart infusion and malt extract agar (MEA) at 22 °C and at 37 °C to optimize growth and toxin production. The growth for all strains was optimal on MEA at 22 °C and at 37 °C, although the production of toxin was higher at room temperature 22 °C. Toxicity of the extracts of Thb and Thd decreased by a factor two to four when the extracted biomass was cultivated at 37 °C (Table 2). The toxicity of the extracts increased (by factor of four) when incubation was extended from 5 to 15 days. The toxic substances of T. longibrachiatum strains were resistant to heat (10 min at 100 °C).
|Strain codes||ITS sequence|
|Trichoderma longibrachiatum||Alternate codes and origin||GenBank accession number||Reference|
|Thb||Moisture-damaged residence, Finland||HQ593512||Present study|
|Thd||Moisture-damaged residence, Finland||HQ593513||Present study|
|SzMCThg||Moisture-damaged residence, Finland||EU401573||TU Vienna code C.P.K.1698 |
|CNM-CM 2171||C.P.K. 1696, foot skin of premature infant with subcutaneous lesions, fatal, Spain||AY920397|||
|CNM-CM 2277||C.P.K. 2277, sputum of tuberculosis patient, Spain||AY920398|||
|IMI 291014||C.P.K. 1303; soil, Antarctica||EU401560|||
|CECT 2412||C.P.K. 2062; CNM-CM 1698; mushroom compost, Wales UK||EU401572|||
|CECT 20105||C.P.K. 1698; IMI 297702; CNM-CM 1698, biocontrol strain, Egypt||AY585880|||
|Trichoderma reesei DSM 768||Synonym T. reesei Simmons, T. viride QM6a; Cotton canvas, Solomon Islands, Bouganville.||[2, 3]|
|Trichoderma harzianum ES39||Ceiling of a residence, after renovation of Moisture damage, Helsinki, Finland||AY585881||; present study|
|Acremonium tubakii CBS 110649a||Reed sandy soil|||
|Strain||Motility inhibition||Depolarization of mitochondria||Relaxed permeability barrier of cell membrane to propidium iodide|
|24 h||72 h||24 h||72 h||24 h||72 h|
|EC50 (μg dry wt·mL−1)|
|Cell extracts of T. longibrachiatum|
|From indoor isolates|
|Thb||6 (25)a||3 (12)a||6||3||6||3|
|Thd||12 (25)a||3 (12)a||12||3||12||3|
|From clinical isolates|
|From environmental isolates|
|DSM 768||> 100||> 50|
|Cell extracts of reference strains|
|Trichoderma harzianum ES39||4||2||4||2||4||2|
|Acremonium tubakii CBS 110649||> 100||50||> 100||100||> 100||100|
The toxic substances of T. longibrachiatum were 20-residue and 11-residue peptaibols
Toxic cell extract of T. longibrachiatum strain Thb was fractionated with HPLC. Five peaks in the HPLC elution profile (215 nm) of the Thb extract inhibited the motility of boar spermatozoa (labelled A1–A5 in Fig. 1A). Similarly, fractionated alamethicin (A4665) consisted of four alamethicin F50 peptaibols, with molecular masses of 1962, 1976, 1976 and 1990 Da (labelled B1–B4 in Fig. 1B). HPLC-MS analysis of the toxic fractions A2–A5 of strain Thb extract (Fig. 1A) showed the doubly-charged cationized molecules [M+2Na]2+ at m⁄z 991.5 (16.6 min; peak A2), 998.6 (18.8 min; peak A3), 998.5 (22.1 min; peak A4) and 1005.6 (25.8 min; peak A5) and the corresponding triply-charged cationized molecules [M+3Na]3+ at m/z 668.9, 673.6, 673.6 and 678.1 (Fig. 1C–F). Negatively-charged unprotonated molecules [M-2H]2− at m/z 967.6, 974.7, 974.7 and 981.5 were observed in peaks A2–A5, respectively. These experimental values fitted the calculated monoisotopic masses of 1936.1 Da (peak A2), 1950.1 Da (peak A3), 1950.1 Da (peak A4) and 1964.2 Da (peak A5).
MS/MS analysis of y7 ions at m/z 788 and m/z 774 and the MS3 analysis of the mass ion m/z 624 (y6) produced y-series fragments revealing residues 16–20 and showed that the C-terminus contained phenylalaninol (Pheol) (Fig. 2B,C). MS/MS analysis of b13 ion at m/z 1163 of peak A2 (Fig. 2A) and the MS3 analysis of the mass ion at m/z 440 (b5) produced b-series fragments showing that the N-terminus contained an acetyl group (Ac) and the revealed residues 1–13. MS/MS analysis of the doubly-charged [M+2Na]2+ ion at m/z 991 confirmed residues 16–20 and 4–13 (Fig. 2D). Because the fragment ion 196 Da (sequence between 14 and 15) matched with the cleavage of Pro-Vxx and, knowing that the bond between the complementary ion pairs Aib and Pro is weak , it was concluded that amino acid sequence 14–15 was Pro-Vxx.
The diagnostic fragment ions of the above MS analysis of peptaibols are reported in Table 3. The conclusion based on the results presented above is that the compounds eluting as peaks A2–A5 in Fig. 1A were 20-residue peptaibols with an acetylated α-aminoisobutyric acid at the N-terminus and Pheol at the C-terminus. We named these peptaibols trilongins BI, BII, BIII and BIV, respectively. Their sequences were closely similar to one another, with differences only being only at position 6 (Ala or Aib) and at position 17 (Vxx) or Aib).
Peak A1 (13 min) was also toxic to boar sperm cells. It contained a compound that formed doubly-charged cationized molecules [M+2Na]2+ at m/z 610.6 and a single-charged [M+Na]+ at m/z 1197.9, corresponding to the molecular mass of 1174.9 Da shown in Fig. 1G. MS/MS analysis using m/z 610.6 as the precursor ion revealed the sequence Lxx-Lxx-Aib-(Pro-Lxx)-Lxx-Aib-Pro-Lxxol (Fig. 2E). The remaining mass ion at m/z 264 matched the sodium adduct of the residue AcAib-Asn. In MS/MS analysis of the mass ion m/z 962 (Fig. 1G), corresponding to the acylium ion b9, the sequence of Lxx-Aib-Pro-Lxx-Lxx-Aib was found (Fig. 2F). The deduced amino acid sequence showed that this compound was a peptaibol containing 11 residues with an acetylated N-terminus and Lxxol as the C-terminus. HPLC-MS analysis showed that T. longibrachiatum strains also contained another 11-residue peptaibols with sodiated mass ions at m/z 1155, 1169, 1183 and 1211. The HPLC fractions containing these peptaibols showed no toxicity in the boar sperm assay. The sequences of these 11-residue peptaibols were determined by LC-MS/MS analysis using the double-charged [M+2Na]2+ ions as precursor ions. MS/MS analysis of the precursor ions that gave the b ion series is shown in Table 4. The conclusion based on the above MS data is that the toxic peak A1 of Fig. 1A was a peptaibol with a mean molecular weight of 1175.5 Da and an amino acid sequence of AcAib-Asn-Lxx-Lxx-Aib-Pro-Lxx-Lxx-Aib-Pro-Lxxol. It was named trilongin AI (Table 5). The sequences and the identical or positionally isomeric compounds of the 11-residue peptaibols (named trilongins A0–AIV) are shown in Table 5.
|b Series mass ions|
|Trilongin AIV a||1155||589||1039||941||856||743||644||547||462||363||264|
|Trilongin AIV b||1155||589||–||956||870||757||644||547||462||363||264|
|Trilongin AIV c||1155||589||1039||941||856||757||644||547||462||363||264|
|Trilongin AIII a||1169||596||1067||969||884||771||–||561||476||377||264|
|Trilongin AIII b||1169||596||1053||956||870||757||–||561||476||377||264|
|Trilongin AIII c||1169||596||1067||969||884||771||–||561||476||363||264|
|Trilongin AIII d||1169||596||1053||956||870||757||–||561||476||363||264|
|Trilongin AII a||1183||603||1067||969||884||771||–||561||476||363||264|
|Trilongin AII b||1183||603||1081||983||898||785||–||575||490||377||264|
|Trilongin AII c||1183||603||1067||969||884||785||672||575||490||377||264|
|Trilongin AII d||1183||603||1067||969||884||771||672||575||490||377||264|
|Trilongin AII e||1183||603||1067||969||884||771||–||561||476||377||264|
|Peptaibol||Sequence||Identical or positionally isomeric compound||Reference|
|Trilongin AIV a||AcAib||Asn||Vxx||Vxx||Aib||Pro||Vxx||Lxx||Aib||Pro||Lxxol||Trichobrachin A-VII j|||
|Trilongin AIV b||AcAib||Asn||Vxx||Vxx||Aib||Pro||Lxx||Lxx||Aib||Pro||Vxxol||Trichobrachin A-VII c|||
|Hypojecorin A 1|||
|Trilongin AIV c||AcAib||Asn||Vxx||Vxx||Aib||Pro||Lxx||Vxx||Aib||Pro||Lxxol||Trichobrachin A-VII i|||
|Trilongin AIII a||AcAib||Asn||Lxx||Vxx||Aib||Pro||Lxx||Lxx||Aib||Pro||Vxxol||Trichobrachin A-IV|||
|Hypojecorin A 5|||
|Trilongin AIII b||AcAib||Asn||Lxx||Vxx||Aib||Pro||Vxx||Lxx||Aib||Pro||Lxxol||Trichobrachin A-IVd|||
|Trilongin AIII c||AcAib||Asn||Vxx||Lxx||Aib||Pro||Lxx||Lxx||Aib||Pro||Vxxol||Trichobrachin A-III|||
|Hypojecorin A 3|||
|Trilongin AIII d||AcAib||Asn||Vxx||Lxx||Aib||Pro||Vxx||Lxx||Aib||Pro||Lxxol||Trichobrachin A-IVc|||
|Trilongin A II a||AcAib||Asn||Lxx||Lxx||Aib||Pro||Lxx||Lxx||Aib||Pro||Vxxol||Trichobrachin A-VIII a|||
|Trichobrachins III- 8b/9c|||
|Hypojecorin A 6|||
|Trilongin AII b||AcAib||Asn||Lxx||Lxx||Aib||Pro||Lxx||Vxx||Aib||Pro||Lxxol||Trichobrachin A-VIII d|||
|Trilongin AII c||AcAib||Asn||Lxx||Lxx||Aib||Pro||Vxx||Lxx||Aib||Pro||Lxxol||Trichobrachin A-VIII e|||
|Harzianin HB 1|||
|Trilongin AII d||AcAib||Asn||Lxx||Vxx||Aib||Pro||Lxx||Lxx||Aib||Pro||Lxxol||Trichobrachin A-VIII b|||
|Hypojecorin A 12|||
|Trilongin AII e||AcAib||Asn||Vxx||Lxx||Aib||Pro||Lxx||Lxx||Aib||Pro||Lxxol||Trichobrachin A-VIII c|||
|Trilongin AI||AcAib||Asn||Lxx||Lxx||Aib||Pro||Lxx||Lxx||Aib||Pro||Lxxol||Trichobrachin A-IX|||
|Hypojecorins A 15/16|||
|Trilongin A0||AcAib||Gln||Lxx||Lxx||Aib||Pro||Lxx||Lxx||Aib||Pro||Lxxol||Trichobrachin C-I/C-II|||
|Trichobrachins III- I/J|||
|Hypojecorins A 17/18|||
|Trilongin BI||AcAib||Ala||Aib||Ala||Aib||Ala||Gln||Aib||Vxx||Aib||Gly||Lxx||Aib||Pro||Vxx||Aib||Aib||Gln||Gln||Pheol||Gliodeliquescin A|||
|Longibrachin A I|||
|Trilongin BII||AcAib||Ala||Aib||Ala||Aib||Ala||Gln||Aib||Vxx||Aib||Gly||Lxx||Aib||Pro||Vxx||Aib||Vxx||Gln||Gln||Pheol||Trichoaureocin 4|||
|Longibrachin A II|||
|Trilongin BIII||AcAib||Ala||Aib||Ala||Aib||Aib||Gln||Aib||Vxx||Aib||Gly||Lxx||Aib||Pro||Vxx||Aib||Aib||Gln||Gln||Pheol||Trichoaureocin 5|||
|Longibrachin A III|||
|Trilongin BIV||AcAib||Ala||Aib||Ala||Aib||Aib||Gln||Aib||Vxx||Aib||Gly||Lxx||Aib||Pro||Vxx||Aib||Vxx||Gln||Gln||Pheol||Trichoaureocin 6|||
|Longibrachin A IV|||
|Trilongin CI||AcAib||Ala||Aib||Ala||Aib||Ala||Gln||Aib||Vxx||Aib||Gly||Lxx||Aib||Pro||Vxx||Aib||Aib||Glu||Gln||Pheol||Longibrachin B II|||
|Trilongin CII||AcAib||Ala||Aib||Ala||Aib||Ala||Gln||Aib||Vxx||Aib||Gly||Lxx||Aib||Pro||Vxx||Aib||Vxx||Glu||Gln||Pheol||Longibrachin B III|||
|Trilongin CIV||AcAib||Ala||Aib||Ala||Aib||Aib||Gln||Aib||Vxx||Aib||Gly||Lxx||Aib||Pro||Vxx||Aib||Vxx||Glu||Gln||Pheol||New||Present study|
Diversity of peptaibols among the toxigenic T. longibrachiatum strains
The three toxigenic indoor T. longibrachiatum isolates, Thb, Thd and SzMCThg (Table 1), produced the same 11-residue and 20-residue trilongins A0–AIV and BI–BIV. When the clinical and environmental isolates of T. longibrachiatum (IMI 291014, CECT 20105, CNM–CM 2277, CECT 2412 and CNM–CM; Table 1) were analyzed with LC/MS, four additional 20-residue peptaibols were found. These new peptaibols contained y7 ions 1 Da higher, m/z 775 and 789 than the corresponding y7 ions (m/z 774 and 788) of trilongins BI–BIV. These were named trilongins CI, CII, CIII and CIV. MS/MS analysis of y7 ions of the 20-residue peptaibols CI–CIV (Table 3) revealed amino acid sequences resembling those of the y7 ions of trilongins BI–BIV, except from position 18 where Glu was substituted with Gln (Table 5). Trilongins CI–CIV also varied also at cposition 6 (Ala or Aib) and at position 17 (Vxx or Aib), similar to trilongins BI–BIV (Table 5). MS/MS analysis of b13 ions of trilongins CI–CII at m/z 1163 and CIII–CIV at m/z 1177 showed that the fragmentions were identical to the corresponding fragmentations of trilongins BI–BII (at m/z 1163) and BIII–BIV (at m/z 1177) (Table 3). The deduced amino acid sequences of trilongins BI–BIV and CI–CIV (Table 5) are based on the MS/MS analyses using y7 ions, b13 ions and doubly-charged [M+2Na]2+ sodiated molecules as the precursor ions. Trilongins CIII and CIV show the new sequences (Table 5). The HPLC-MS elution profile of the peptaibols observed in the methanol extract of T. longibrachiatum strain CECT 20105 is shown in Fig. 3. The sequences and retention times of the 11- and 20-residue peptaibols found are provided in Table 6. Table 7 compiles the contributions of the different 20-residue trilongins, BI–BIV and CI–CIV, in the T. longibrachiatum strains.
|Peptaibol||[M+2Na]2+ (m/z)||Sequence||Fractiona||tR (min)|
|Trilongin A IV a||1155||AcU N Vx Vx U P Vx Lx U P Lxol||1||5.3–6.0|
|Trilongin AIV b||1155||AcU N Vx Vx U P Lx Lx U P Vxol|
|Trilongin AIV c||1155||AcU N Vx Vx U P Lx Vx U P Lxol|
|Trilongin AIII a||1169||AcU N Lx Vx U P Lx Lx U P Vxol||2||6.5–7.8|
|Trilongin AIII b||1169||AcU N Lx Vx UP Vx Lx U P Lxol|
|Trilongin AIII c||1169||AcU N Vx Lx U P Lx Lx U P Vxol|
|Trilongin AIII d||1169||AcU N Vx Lx UP Vx Lx U P Lxol|
|Trilongin AII a||1183||AcU N Vx Lx U P Lx Lx U P Lxol||3||8.4–9.3|
|Trilongin AII b||1183||AcU N Lx Lx U P Lx Lx U P Vxol|
|Trilongin AII c||1183||AcU N Lx Lx U P Lx Vx U P Lxol|
|Trilongin AII d||1183||AcU N Lx Lx U P Vx Lx U P Lxol|
|Trilongin AII e||1183||AcU N Lx Vx U P Lx Lx U P Lxol|
|Trilongin AI||1197||AcU N Lx Lx U P Lx Lx U P Lxol||4||11.8|
|Trilongin A0||1211||AcU Q Lx Lx U P Lx Lx U P Lxol||5||13.1|
|Trilongin BI||1958||AcU A U A U A Q U Vx U G Lx U P Vx U U Q Q Fol||6||14.4|
|Trilongin CI||1959||AcU A U A U A Q U Vx U G Lx U P Vx U U E Q Fol||7||15.6|
|Trilongin BII||1972||AcU A U A U A Q U Vx U G Lx U P Vx U Vx Q Q Fol||8||17.2|
|Trilongin CII||1973||AcU A U A U A Q U Vx U G Lx U P Vx U Vx E Q Fol||9||19.1|
|Trilongin CIII||1973||AcU A U A U U Q U Vx U G Lx U P Vx U U E Q Fol||10||21.4|
|Trilongin BIII||1972||AcU A U A U U Q U Vx U G Lx U P Vx U U Q Q Fol||11||23.6|
|Trilongin CIV||1987||AcU A U A U U Q U Vx U G Lx U P Vx U Vx E Q Fol||12||26.2|
|Trilongin BIV||1986||AcU A U A U U Q U Vx U G Lx U P Vx U Vx Q Q Fol||13||29.8|
|Peptaibol||MW||Characteristic ions||Trichoderma longibrachiatum strains|
|y7||b13||Thb||CNM-CM 2171||CNM-CM 2277||IMI 291014||CECT 2412||CECT 20105|
|m/z||Percentage of total amount of peptaibols|
Quantification of peptaibols
The fragmentation patterns of alamethicin F50 were similar to those of trilongins BI–BIV and CI–CIV and contained y7 ion at m/z 774. Therefore, y7 ion of alamethicin at m/z 774 and the corresponding y7 ions, m/z 774, 775, 788 and 789 of the 20-residue trilongins BI–BIV and CI–CIV were used for the quantifications. The quantification of trilongin AI was performed by monitoring A215 and alamethicin as a reference.
The concentrations of the eight 20-residue trilongins BI–BIV and CI–CIV and the 11-residue trilongin AI in the methanol extracts of T. longibrachiatum strains are shown in Table 8. Of the total harvested biomass, 10–20% (w/w) was methanol-soluble. The 20-residue peptaibols in the different strains contributed to 5–13 wt% of the methanol-soluble matter and the 11-residue peptaibol contributed to 0.2–0.8 wt%. The toxic peptaibols thus made up 0.5–2.6 wt% of the harvested mycelial biomass (320 ± 20 mg per Petri dish of diameter 90 mm) of the investigated T. longibrachiatum isolates. One fully grown culture dish thus contained 1500–8800 μg of the toxic peptaibols.
|Trilongin AI||Trilongins BI–BIV||Trilongins CI–CIV|
Toxicity of the purified 20-residue and 11-residue trilongins
Toxicities were measured using boar spermatozoa motility inhibition as the toxicity indicator, separate from the 20-residue trilongins BI–BIV, 11-residue trilongin AI and a combination of trilongins (BI–BIV plus AI) in a mass ratio of 2 : 1. As shown in Table 7, the EC50 of 20-residue trilongins BI–BIV decreased from 3 to 0.4 μg·mL−1 upon extended exposure, whereas the EC50 of 11-residue trilongin AI decreased from 15 to 1.5 μg mL−1. The EC50 of trilongins (BI–BIV plus AI) decreased from 0.6 to 0.2 μg·mL−1 upon extended exposure and the mixture of trilongins was a stronger motility inhibitor than the trilongins alone (Table 9) or any of the crude extracts (Table 2). The calculated synergy effect based on the sum of fractional inhibitory concentrations (∑FIC) was < 1 for all exposure times and the lowest ∑FIC (0.2) was observed after 30 min of exposure (Table 9).
|30 min||1 day||2 day|
|Trilongin AI + trilongins BI–BIVa||0.6||0.2||0.2|
Figure 4A–C shows that ∆Ψm decreased (yellow fluorescence changed to green) upon exposure to trilongins BI–BIV at a concentration of 0.4 μg·mL−1 (Fig. 4B). This exposure relaxed the plasma membrane permeability barrier towards propidium iodide (red fluorescence) (Fig. 4E). Interestingly, the dual pattern of staining (calcein-AM with propidium iodide) in Fig. 4E showed green fluorescence in the proximal part of the sperm tail, which is absent in the distal part of the tail, indicating that the mitochondrial inner membrane retained the calcein-AM cleavage products (green fluorescence). The results provided in Table 9 also show that the T. longibrachiatum peptaibols were as similarly sperm toxic as the well-known peptaibol alamethicin (EC50 0.15 μg·mL−1, exposure time of 1 day; Table 2).
Peptaibols from T. longibrachiatum form K+/Na+ permeable channels in lipid membranes
Single-channel recordings of voltage-dependent channels formed in 2 m KCl and in 2 m NaCl by trilongins BI–BIV and trilongin AI are shown in Fig. 5, as well as in Fig. 6 for alamethicin. For each type of channel, at least four levels of conductance through the single channels were resolved. The single channel conductances provoked by the peptaibols of T. longibrachiatum Thb and by alamethicin in NaCl and in KCl are listed in Table 10. The ratios of Na+ relative to K+ were higher for the trilongins at each of the four conductance levels (O1–O4) compared to the reference substance alamethicin F50 (Table 10). When tested individually, the 11-residue trilongin AI displayed channels with higher relative conductance ratios (Na+ : K+) than the channels formed by the 20-residue trilongins BI–BIV. Compared to alamethicin F50 at level O1, the benefit of Na+ versus K+ was 1.35-fold higher for trilongin AI and 1.16-fold higher for trilongins BI–BIV and, at level O2, the peptaibols values were 1.36- and 1.20-fold higher, respectively, than those of alamethicin F50. The single ion channels remained in an open state for a longer time in the case of the combination of the long peptaibols (trilongins BI–BIV) and the short peptaibol (trilongin AI) (Fig. 7A) than for the long peptaibols alone (Fig. 7B).
|O1 (pS)||Na/K||O2 (pS)||Na/K||O3 (pS)||Na/K||O4 (pS)||Na/K|
In the present study, we show that the fungus T. longibrachiatum produced large quantities (1–2 wt% of the mycelial biomass) of thermostable secondary metabolites identified as members of the families of 20-residue (1936–1965 Da, five to eight isoforms per strain) and 11-residue (1175 Da) peptaibols. These peptaibols were mitochondriotoxic toward porcine sperm cells at submicromolar exposure concentrations. The metabolites named trilongins BI–BIV and trilongin AI formed voltage-dependent, Na+/K+ conductive channels in biomembranes. T. longibrachiatum is an emerging human pathogen and the main pathogen in the fungal genus Trichoderma [5, 27, 38]. This species is also the most common species colonizing mould troubled indoor space . The molecules involved in the pathology associated with this species have remained unknown to date.
A novel finding described in the present study was the toxic synergy between the 11-residue and the 20-residue trilongins of T. longibrachiatum. Synergy was visible as a potentiated toxic action on porcine sperm cells, as well as an extended duration (lifetime) of the ion conducting channels generated in artificial phospholipid membranes [black lipid membrane (BLM)]. The synergistic toxicity of different size classes of peptaibols does not appear to have been reported previously. The toxicokinetics of the combined 11-residue trilongin AI and 20-residue trilongins BI–BIV differed from those of the one-sized peptaibol: when tested singly on boar sperm cells, it took 1–3 days of exposure for the 11-residue trilongin AI and for the 20-residue trilongins BI–BIV to reach EC50 values of 1.5 and 0.4 μg·mL−1, respectively. When combined 1 : 2 w/w, the mixture was highly toxic within 30 min; EC50 was 0.6 μg·mL−1 and shifted down to 0.2 μg·mL−1 upon extended exposure. In that exposure time, the ∑FIC  had lowest value (0.2), indicating a clearly toxic synergy effect (Table 9). It appears that the generation of the (pathological) ion conductive channels was speeded up and stabilized by the simultaneous presence of the two different sizes of trilongins compared to channels formed by trilongins of identical size.
Exposure of porcine spermatozoa to purified trilongins (T. longibrachiatum) or to alamethicin (T. arundinaceum) resulted in a loss of motility and the loss of ∆Ψm at low concentration (EC50 of ≤ 0.1–0.2 μm). This mammalian cell toxicity threshold appears the to be lowest reported for Trichoderma peptaibols so far. The amino acid sequence of trichokonin VI is similar to the 20-residue trilongin BI (Table 5). Trichokonin VI produced by T. pseudokoningii MF2 was recently reported to depolarize mitochondria and vacuolize the cytoplasm of hepatocellular cancer cells on exposure to 20 μm (~ 40 μg·mL−1)  and to act as a Ca2+ channel agonist in isolated bullfrog cardiac myocytes at 20 μg·mL−1 (10 μm) . Alamethicin (40 μg·mL−1; 20 μm) has been shown to mediate the uptake of Ca2+ ions by bovine adrenal chromaffin cells .
Multiple Aib residues were shown to be essential for generating ion conductive channels in biomembranes by peptaibols [43, 44]. The T. longibrachiatum 20-residue trilongins contain eight or nine Aib residues, similar to alamethicin, and Ala in position 2 instead of Pro in alamethicin (Table 5). Aib residues were also shown essential for the non-endocytic entry of peptaibols to mammalian cells .
The 11-residue trilongin AI by itself was toxic also to porcine sperm cells with or without contribution of the 20-residue peptaibols, even though 11 amino acids are most likely too short to span across the phospholipid membrane of mammalian cells. Wada et al.  suggested a head-to-tail model for channel formation in BLMs by the 11-residue trichorovin XIIa. A similar observation was reported by Ruiz et al.  for trichobrachin A-IX (a toxic 11-residue peptaibol, also known as trichorovin TV-XIIa) from a marine isolate of T. longibrachiatum MMS 151, with an amino acid sequence identical to that of trilongin AI (Table 5) described in the present study. The other trichobanchins resembling  11-residue trilongins A0 and AII–AIV (Table 5) found in the present study were neither toxic to boar sperm cells, nor active in the BLM experiments.
Cell free extracts prepared from a T. longibrachiatum mycelial biomass of isolates originating from sick building samples (Table 1) contained 10 w% of the toxic trilongins. The toxic trilongins might be related to the higher human pathogenicity of T. longibrachiatum among the species of the genus Trichoderma. However, we do not claim that the bioactive peptaibols described in the present study are solely responsible for the toxicity detected in the clinical and indoor isolates strains, which remains a subject requiring further investigation.
The fungal strains
The strains examined are described in Table 1 [2, 3, 6, 11, 47]. The indoor isolates of T. longibrachiatum, Thb, Thd, Thg originated from Oulu, northern Finland, a moisture-damaged residence of a family of two adults and three children suffering from serious, bulding-associated symptoms of ill health (Table 1). Trichoderma sp. was cultured from insulation material of the bathroom on TSA plates as the principal fungal colonizer. Cell-free extracts were prepared in methanol for 15 separate colonies and tested for toxicity by the rapid boar spermatozoan assay . The toxic colonies were further cultivated to obtain pure cultures on MEA at 22 °C. The isolates were identified based on the sequences of the ITS region. DNA isolation, amplification of the ITS region, amplicon purification and sequencing were performed as described previously . The sequence of the ITS region was analyzed using trichokey, version 2.0 . The ITS sequences were deposited in the GenBank database (Table 1).
Preparation of cell extracts, purification and MS of the toxins
The strains were grown on MEA plates for the indicated times and harvested into methanol. Methanol extracts of the mycelial biomass were processed and analyzed as described by Andersson et al. . HPLC and HPLC-ESI-IT-MS analyses were peformed as described previously , except that the eluents used for the HPLC separations were 0.1% formic acid (A) and methanol (B), using isocratic elution with 80% of B for 25 min at a flow rate of 1 mL·min−1. For detection, the monitoring of A215 was used. Alamethicin was used as a reference compound.
Toxicity assays with porcine sperms as indicator cells
Sperm cells were exposed by dispensing 1–20 μL of the methanolic fungal extract or the pure substance(s) or vehicle only (methanol) into 2.0 mL of extended boar semen (Figen Ltd, Tuomikylä, Finland), which was used as delivered (27 × 106 sperm cells·mL−1).
Toxicity assays were performed in triplicate with the serial (step = 2) dilutions of the test substance, each as three or more parallels with two biological replicates. The results are given as the median unless the range (minimum – maximum) is indicated. The vehicle only (ethanol; 96 vol%) control was prepared for each dilution step. Sperm motility was read by microscopy (on a heated stage, 37 °C) as described previously .
The number of cells with plasma membrane relaxed permeation of propidium iodide and depleted ∆Ψm were recorded by microscopic assessment of cells stained with calcein-AM, propidiumiodide and the membrane potential sensitive dye JC-1. The details of these protocols have been described previously .
The BLM technique was used to measure ion conductivity changes of phospholipid membrane in response to the presence of HPLC-purified peptaibols from the Trichoderma strains. The experiments were performed as described previously . For the single channel conductances, soybean phosphatidylcholine dissolved in heptane (20 mg·mL−1) was used to form a lipid bilayer membrane covering the circular hole (inner diameter 0.3 mm) in the teflon wall separating the aqueous solutions of 2 m KCl or 2 m NaCl in 20 mm Tris-Cl (pH 7.0) at 15 °C.
Synergy effects of peptaibols
Synergy effects of peptaibols were estimated using the FIC method. ∑FIC values < 1, = 1 and > 1 indicate synergy, additivity and antagonism, respectively . The ∑FIC for long (A) and short (B) peptaibols was calculated using the equation:
where the FIC(A) and FIC(B) are EC50 values of separate long (A) and short (B) peptaibols, respectively, and the FIC(A + B) is the EC50 value of the mixture of peptaibols A and B in the motility biotest with boar sperm cells.
Reagents and media
Alamethicin and soybean phosphatidylcholine were obtained from Sigma-Aldrich (St Louis, MO, USA). JC-1, calcein-AM and propidium iodide were obtained from Invitrogen (Carlsbad, CA, USA). All other chemicals were of analytical quality and were obtained from local suppliers.
This research was supported by Finnish Work Environment Fund (Grants 109124 and 111084), the Academy of Finland (CoE Grant 118637) and the Hungarian Scientific Research Fund (Grant OTKA K-105972). The authors thank Riitta Saastamoinen, Mika Kalsi and Arto Nieminen for their skilled technical support, as well as Tuula Suortti, Leena Steininger and Hannele Tukiainen for effective administration. The Viikki Science Library is thanked for providing expert information.