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Keywords:

  • indoor mold;
  • ion channel;
  • mitochondriotoxin;
  • synergistically toxic;
  • Trichoderma

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

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.

Database

  • Nucleotide sequence data have been deposited in the GenBank database under accession numbers HQ593512 and HQ593513

.


Abbreviations
∆Ψm,

mitochondrial transmembrane potential

Ac,

acetyl

Aib,

α-aminoisobutyric acid

BLM,

black lipid membrane

EC50,

effective median concentration

FIC,

fractional inhibitory concentration

ITS,

internal transcribed spacer

MEA,

malt extract agar

Pheol,

phenylalaninol

TSA

tryptic soy agar

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

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 [4]. 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 [5]. Possible sources of infection include water-related sites, air, foods and catheters. Based on the extensive review of Kredics et al. [5], 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 [8].

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 [14]. The subgroup comprises Aib-containing peptides carrying a C-terminal 2-amino alcohol residue, which are referred to as peptaibols [17]. The first report of an Aib-containing antibiotic from the genus Trichoderma, compound U-22324 (later renamed as alamethicin), was published in 1967 [18]. Subsequently, it was revealed that the first peptaibol isolated from a Trichoderma sp. was actually suzukacillin from ‘T. viride’ 63 C-I [19]; however, the presence of Aib in the SZ-hydrolysate was confirmed only 6 years later [20]. The producer strain NRRL 3199 originally identified as T. viride was recently reidentified as Trichoderma arundinaceum, a member of the Trichoderma brevicompactum clade [21], 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 [23], longibrachins [24], trichobrachins [25, 26] and trichorovin [25]. However, one of the producer isolates, ‘T. longibrachiatum’ CBS 936.69 was recently reclassified as Trichoderma ghanense [14] 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 [27]. 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.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

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).

Table 1. Fungal strains examined during the present study, origins and ITS sequence used for identification.Collections: CBS, Centraalbureau voor Schimmelcultures, Utrecht NL; CECT, Spanish type culture collection; CNM, mycological collection of the Spanish National Centre for Microbiology; DSM, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany; IMI CABI Bioscience, Egham UK
Strain codes ITS sequence 
Trichoderma longibrachiatum Alternate codes and originGenBank accession numberReference
  1. a

    This strain is mistakenly referred to as CBS110360 by Andersson et al. [47].

ThbMoisture-damaged residence, Finland HQ593512 Present study
ThdMoisture-damaged residence, Finland HQ593513 Present study
SzMCThgMoisture-damaged residence, Finland EU401573 TU Vienna code C.P.K.1698 [6]
CNM-CM 2171C.P.K. 1696, foot skin of premature infant with subcutaneous lesions, fatal, Spain AY920397 [6]
CNM-CM 2277C.P.K. 2277, sputum of tuberculosis patient, Spain AY920398 [6]
IMI 291014C.P.K. 1303; soil, Antarctica EU401560 [6]
CECT 2412C.P.K. 2062; CNM-CM 1698; mushroom compost, Wales UK EU401572 [6]
CECT 20105C.P.K. 1698; IMI 297702; CNM-CM 1698, biocontrol strain, Egypt AY585880 [6]
Reference strains
Trichoderma reesei DSM 768Synonym T. reesei Simmons, T. viride QM6a; Cotton canvas, Solomon Islands, Bouganville.  [2, 3]
Trichoderma harzianum ES39Ceiling of a residence, after renovation of Moisture damage, Helsinki, Finland AY585881 [11]; present study
Acremonium tubakii CBS 110649aReed sandy soil  [47]
Table 2. The toxic activities towards porcine spermatozoa by extracts of the fungus T. longibrachiatum and reference strains. The cell extracts were prepared from mycelial biomass grown on MEA at 22 °C for 5 days. The toxicity endpoints EC50 indicate methanol-soluble substances (μg dry wt·mL−1). Depolarization of mitochondria was recorded by epifluoresence microscopy after staining with the membrane potential sensitive dye JC-1. Exposure time (h)
StrainMotility inhibitionDepolarization of mitochondriaRelaxed permeability barrier of cell membrane to propidium iodide
24 h72 h24 h72 h24 h72 h
EC50 (μg dry wt·mL−1)
  1. a

    Toxicity endpoint of extracts (in parentheses) indicate a situation where strains were grown on MEA at 37 °C for 5 days.

Cell extracts of T. longibrachiatum
From indoor isolates
Thb6 (25)a3 (12)a6363
Thd12 (25)a3 (12)a123123
SzMCthg636363
From clinical isolates
CNM-CM 2171126126126
CNM-CM 2277636363
From environmental isolates
IMI 29101462123123
CECT 24126262126
CECT 201056323123
DSM 768> 100> 50    
Cell extracts of reference strains
Trichoderma harzianum ES39424242
Acremonium tubakii CBS 110649> 10050> 100100> 100100
Reference toxin
Alamethicin0.150.080.150.080.150.08

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 mz 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).

image

Figure 1. HPLC-UV and HPLC-MS analysis of peptaibols produced by Trichoderma longibrachiatum Thb. (A) HPLC-UV (215 nm) chromatograms of methanol extract from strain Thb and methanol solution of alamethicin (B). (C) Doubly-charged sodiated molecular ions at m/z 991, b13 ion at m/z 774 and y7 ion at m/z 1163 of peak A2 from (A). (D–F) The corresponding ions of peaks A2–A5 from (A). (G) Doubly-charged sodiated molecular ion at m/z 1197 and b9 ion at m/z 961 of peak A1 from (A).

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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 [28], it was concluded that amino acid sequence 14–15 was Pro-Vxx.

image

Figure 2. MS/MS fragmentation patterns and amino acid sequences of peptaibols found in methanol extract of T. longibrachiatum Thb. (A) The amino acid sequence of y7 ion at m/z 1163 (Fig. 1C, peak A2). The sequences of b13 ions at m/z 774 (Fig. 1C, peak A2) and 788 (Fig. 1D, peak A3) are shown in (B) and (C), respectively. (D) The sequence of doubly-charged sodiated molecular ions at m/z 992 (Fig. 1C, peak A2). The sequences of doubly-charged sodiated molecules at m/z 611 and b9 ion at m/z 961 (Fig. 1G, peak A1) are shown in (E) and (F), respectively.

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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).

Table 3. The [M+Na]+ and [M+2Na]2+ ions of trilongins BI–BIV and CI–CIV and the diagnostic fragment mass ions of b13 and y7 series ions observed by MS/MS and MS3 analysis
Diagnostic ionsTrilongin
BIBIIBIIIBIVCICIICIIICIV
m/z
[M+Na]+19581972197219861959197319731987
[M+2Na]2+99199899810059929999991006
b1311631163117711771163116311771177
b1210781078109210921078107810921092
b11965965979979965965979979
b10908908922922908908922922
b9823823837837823823837837
b8724724738738724724738738
b7639639653653639639653653
b6511511525525511511525525
b5440440440440440440440440
b4355355355355355355355355
b3284284284284284284284284
b2199199199199199199199199
b1128128128128128128128128
y7774788774788775789775789
y6623637623637624638624638
y5495509495509496510496510
y4367381367381367381367381
y3282282282282282282282282
y2197197197197197197197197

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.

Table 4. The [M+Na]+,[M+2Na]2+ mass ions and the b series mass ions (m/z) obtained from MS/MS analysis of [M+2Na]2+ mass ions of 11-residue peptaibols of T. longibrachiatum strains
   b Series mass ions
11-residue peptaibol[M+Na]+[M+2Na]2+b10b9b8b7b6b5b4b3b2
Trilongin AIV a11555891039941856743644547462363264
Trilongin AIV b1155589 956870757644547462363264
Trilongin AIV c11555891039941856757644547462363264
Trilongin AIII a11695961067969884771 561476377264
Trilongin AIII b11695961053956870757 561476377264
Trilongin AIII c11695961067969884771 561476363264
Trilongin AIII d11695961053956870757 561476363264
Trilongin AII a11836031067969884771 561476363264
Trilongin AII b11836031081983898785 575490377264
Trilongin AII c11836031067969884785672575490377264
Trilongin AII d11836031067969884771672575490377264
Trilongin AII e11836031067969884771 561476377264
Trilongin AI11976101080983898785672575489377264
Trilongin A012116171095998913800687589504391278
Table 5. Amino acid sequences of trilongins A0–AIV, BI–BIV and CI–CIV produced by T. longibrachiatum strains and alamethicin (Alm).Ileol, isoleucinol; Iva, isovaline; Vxx, Val/Iva; Lxx: Leu/Ile; Lxxol, Leuol/Ileol; Vxxol, Valol/Ivaol
PeptaibolSequenceIdentical or positionally isomeric compoundReference
1234567891011121314151617181920
Trilongin AIV aAcAibAsnVxxVxxAibProVxxLxxAibProLxxol         Trichobrachin A-VII j [26]
TV29-11-I d [29]
Trilongin AIV bAcAibAsnVxxVxxAibProLxxLxxAibProVxxol         Trichobrachin A-VII c [26]
Trichobrachin III-9e [29]
TV29-11-I b [29]
Hypojecorin A 1 [30]
Trilongin AIV cAcAibAsnVxxVxxAibProLxxVxxAibProLxxol         Trichobrachin A-VII i [26]
Trilongin AIII aAcAibAsnLxxVxxAibProLxxLxxAibProVxxol         Trichobrachin A-IV [26]
Trichorovin TV-IIb [26]
Trichobrachin III-3b [29]
TV29-11-II a [29]
Hypojecorin A 5 [30]
Trilongin AIII bAcAibAsnLxxVxxAibProVxxLxxAibProLxxol         Trichobrachin A-IVd [26]
TV29-11-II f [29]
Trilongin AIII cAcAibAsnVxxLxxAibProLxxLxxAibProVxxol         Trichobrachin A-III [26]
Trichorovin TV-Ia [26]
Trichobrachin III-2b [29]
TV29-11-II b [29]
Hypojecorin A 3 [30]
Trilongin AIII dAcAibAsnVxxLxxAibProVxxLxxAibProLxxol         Trichobrachin A-IVc [26]
Trilongin A II aAcAibAsnLxxLxxAibProLxxLxxAibProVxxol         Trichobrachin A-VIII a [26]
Trichorovins TV-Vb/VIb [26]
Trichorozin I [26]
Trichobrachins III- 8b/9c [30]
Hypojecorin A 6 [30]
Trilongin AII bAcAibAsnLxxLxxAibProLxxVxxAibProLxxol         Trichobrachin A-VIII d [26]
Trilongin AII cAcAibAsnLxxLxxAibProVxxLxxAibProLxxol         Trichobrachin A-VIII e [26]
Harzianin HB 1 [26]
Trilongin AII dAcAibAsnLxxVxxAibProLxxLxxAibProLxxol         Trichobrachin A-VIII b [26]
Trichorovin TV-VIIa [26]
TV29-11-II a [29]
Trichobrachins III-7b/8a/9a [29]
Hypojecorin A 12 [30]
Trilongin AII eAcAibAsnVxxLxxAibProLxxLxxAibProLxxol         Trichobrachin A-VIII c [26]
Trichorovin TV-Va [26]
Trilongin AIAcAibAsnLxxLxxAibProLxxLxxAibProLxxol         Trichobrachin A-IX [26]
Harzianin HK-VI [26]
Trichorovins TV-XI/XII-a/b [26]
Trichorozin III [26]
Trichobrachins II-Fa/Ga/Gb/Ha [30]
Hypojecorins A 15/16 [30]
Trilongin A0AcAibGlnLxxLxxAibProLxxLxxAibProLxxol         Trichobrachin C-I/C-II [26]
Trichorovin TV-XIII [26]
Trichorozin IV [26]
Hypomurocins A-V/Va [30]
Trichobrachins III-16a/17/18 [29]
TV29-11-V b [29]
Trichobrachins III- I/J [30]
Hypojecorins A 17/18 [30]
Trilongin BIAcAibAlaAibAlaAibAlaGlnAibVxxAibGlyLxxAibProVxxAibAibGlnGlnPheolGliodeliquescin A [31]
Trichoaureocin 3 [32]
Trichobrachins II-5/6 [33]
Longibrachin A I [24]
Trichokonin VI [34]
Trilongin BIIAcAibAlaAibAlaAibAlaGlnAibVxxAibGlyLxxAibProVxxAibVxxGlnGlnPheolTrichoaureocin 4 [32]
Suzukacillin 10a [35]
Trichobrachins II-7/8/9 [33]
Longibrachin A II [24]
Trichokonin VII [34]
Trilongin BIIIAcAibAlaAibAlaAibAibGlnAibVxxAibGlyLxxAibProVxxAibAibGlnGlnPheolTrichoaureocin 5 [32]
Trichosporin B-IVc [36]
Trichobrachin II-10 [33]
Longibrachin A III [24]
Trichokonin VIII [34]
Trilongin BIVAcAibAlaAibAlaAibAibGlnAibVxxAibGlyLxxAibProVxxAibVxxGlnGlnPheolTrichoaureocin 6 [32]
Longibrachin A IV [24]
Trilongin CIAcAibAlaAibAlaAibAlaGlnAibVxxAibGlyLxxAibProVxxAibAibGluGlnPheolLongibrachin B II [24]
Trilongin CIIAcAibAlaAibAlaAibAlaGlnAibVxxAibGlyLxxAibProVxxAibVxxGluGlnPheolLongibrachin B III [24]
Trilongin CIIIAcAibAlaAibAlaAibAibGlnAibVxxAibGlyLxxAibProVxxAibAibGluGlnPheolNewPresentstudy
Trilongin CIVAcAibAlaAibAlaAibAibGlnAibVxxAibGlyLxxAibProVxxAibVxxGluGlnPheolNewPresent study
Alm F50/5AcAibProAibAlaAibAlaGlnAibValAibGlyLeuAibProValAibAibGlnGlnPheol  [37]
Alm F50/6aAcAibProAibAlaAibAlaGlnAibVxxAibGlyLeuAibProVxxAibValGlnGlnPheol  [37]
Alm F50/6b,7,8aAcAibProAibAlaAibAibGlnAibValAibGlyLeuAibProValAibAibGlnGlnPheol  [37]
Alm F50/8bAcAibProAibAibAibAibGlnAibValAibGlyLeuAibProValAibAibGlnGlnPheol  [37]

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 Tlongibrachiatum (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.

image

Figure 3. Total ion chromatogram of the HPLC-MS analysis of the T. longibrachiatum strain CECT 20105 peptaibols. The peak numbers refer to the 11-residue peptaibols (1–5) and 20-residue peptaibols (6–13).

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Table 6. The sequences and retention times of the 11- residue and 20- residue peptaibols of T. longibrachiatum strain CECT 20105.Ac, acetyl; Ileol, isoleucinol; Iva, isovaline; U, aminoisobutyric acid; Vx, Val/Iva; Lx, Leu/Ile; Lxol, Leuol/Ileol; Vxol, Valol/Ivaol; Fol, phenylalaninol
Peptaibol[M+2Na]2+ (m/z)SequenceFractionatR (min)
  1. a

    HPLC peaks in Fig. 1.

11-residue peptaibol
Trilongin A IV a1155 AcU N Vx Vx U P Vx Lx U P Lxol 15.3–6.0
Trilongin AIV b1155 AcU N Vx Vx U P Lx Lx U P Vxol
Trilongin AIV c1155 AcU N Vx Vx U P Lx Vx U P Lxol
Trilongin AIII a1169 AcU N Lx Vx U P Lx Lx U P Vxol 26.5–7.8
Trilongin AIII b1169 AcU N Lx Vx UP Vx Lx U P Lxol
Trilongin AIII c1169 AcU N Vx Lx U P Lx Lx U P Vxol
Trilongin AIII d1169 AcU N Vx Lx UP Vx Lx U P Lxol
Trilongin AII a1183 AcU N Vx Lx U P Lx Lx U P Lxol 38.4–9.3
Trilongin AII b1183 AcU N Lx Lx U P Lx Lx U P Vxol
Trilongin AII c1183 AcU N Lx Lx U P Lx Vx U P Lxol
Trilongin AII d1183 AcU N Lx Lx U P Vx Lx U P Lxol
Trilongin AII e1183 AcU N Lx Vx U P Lx Lx U P Lxol
Trilongin AI1197 AcU N Lx Lx U P Lx Lx U P Lxol 411.8
Trilongin A01211 AcU Q Lx Lx U P Lx Lx U P Lxol 513.1
20-residue peptaibol
Trilongin BI1958 AcU A U A U A Q U Vx U G Lx U P Vx U U Q Q Fol 614.4
Trilongin CI1959 AcU A U A U A Q U Vx U G Lx U P Vx U U E Q Fol 715.6
Trilongin BII1972 AcU A U A U A Q U Vx U G Lx U P Vx U Vx Q Q Fol 817.2
Trilongin CII1973 AcU A U A U A Q U Vx U G Lx U P Vx U Vx E Q Fol 919.1
Trilongin CIII1973 AcU A U A U U Q U Vx U G Lx U P Vx U U E Q Fol 1021.4
Trilongin BIII1972 AcU A U A U U Q U Vx U G Lx U P Vx U U Q Q Fol 1123.6
Trilongin CIV1987 AcU A U A U U Q U Vx U G Lx U P Vx U Vx E Q Fol 1226.2
Trilongin BIV1986 AcU A U A U U Q U Vx U G Lx U P Vx U Vx Q Q Fol 1329.8
Table 7. Molecular masses, characteristic ions and percentages of the 20-residue peptaibols in the methanol extractable metabolomes of different T. longibrachiatum strains. The origins of the strains are shown in Table 1. Values were calculated based on the detected y7 ions
PeptaibolMWCharacteristic ionsTrichoderma longibrachiatum strains
y7 b13ThbCNM-CM 2171CNM-CM 2277IMI 291014CECT 2412CECT 20105
m/z Percentage of total amount of peptaibols 
Trilongin BI1936774116349232055405
Trilongin CI1937775116361431131240
Trilongin BII1950788116316181153
Trilongin CII1951789116328113
Trilongin BIII195077411772136725202
Trilongin CIII19517751177221175527
Trilongin BIV1964788117753472
Trilongin CIV1965789117748

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.

Table 8. Concentrations (mg·mL−1) of 11 and 20-residue peptaibols in the crude methanolic extracts of different T. longibrachiatum strains (10 mg dry weight·mL−1). Amino acid sequences of the peptaibols are shown in Table 5
Strain11-residue20-residue
Trilongin AITrilongins BI–BIVTrilongins CI–CIV
Thb0.080.740.06
CNM-CM21710.020.820.48
CNM-CM22770.020.280.42
IMI 2910140.060.820.18
CECT 24120.020.740.16
CECT 201050.050.060.44

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).

Table 9. Toxicity endpoints for motility inhibition of boar spermatozoa exposed trilongins BI–BIV, AI, a mixture of these two and the calculated synergy effects (∑FIC)
PeptaibolEC50 (μg·mL−1)
30 min1 day2 day
  1. a

    Contains trilongins BI–BIV and AI in a mass ratio of 2 : 1, respectively.

Trilongin AI151.51.5
Trilongins BI–BIV30.60.4
Trilongin AI + trilongins BI–BIVa0.60.20.2
Synergy effect
∑FIC0.20.50.6

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).

image

Figure 4. Toxic responses of boar sperm cells to 20-residue trilongins BI–BIV purified from T. longibrachiatum Thb. The cells were stained with the membrane potential responsive dye JC-1 (A, B, C, top row) or with the live-dead stain calcein AM-propidium iodide (D, E, F, bottom row). (A) Exposed to vehicle only (motile); (B) exposed to 0.4 μg·mL−1 (nonmotile) or (C) to 0.8 μg·mL−1 (nonmotile) of the pooled trilongins BI–BIV. The membrane potential (Δψm) of the mitochondrial sheath, located in the proximal part of the sperm tail, high in (A), is lost in (B) and (C) as a result of exposure to trilongins BI–BIV. (D) Exposed to vehicle only; (E) exposed to 0.4 μg·mL−1 of trilongins BI–BIV; and (F) exposed to 0.8 μg·mL−1 of trilongins BI–BIV. Exposure to the trilongins resulted in a relaxed permeability of the cell membrane towards propidium iodide, visible as nuclei showing red fluorescence (E, F). (E, F) The proximal part of the tail showed green fluorescence, indicating the retention of the fluorescent cleavage products by cellular esterases. These were absent in the distal part of the tail. Magnification, × 400. The size of the sperm head is 4 × 8 × 2 μm; the length of the tail is 55–67 μm.

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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).

image

Figure 5. Currents of single ion channels of the 20-residue trilongins BI–BIV and of the 11-residue trilongin AI. (A) Trilongins BI–BIV in 2 m KCl, V = 260 mV; (B) trilongins BI–BIV in 2 m NaCl, V = 260 mV; (C) trilongin AI in 2 m KCl, V = 230 mV; (D) trilongin AI in 2 m NaCl, V = 240 mV. The peptaibols were added to 2 nm.

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image

Figure 6. Currents of the single ion channels of alamethicin (2 nm) in 2 m KCl, V = 230 mV (A), and in 2 m NaCl, V = 220 mV (B).

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image

Figure 7. Currents of the single ion channels in 2 m KCl, V = 260 mV. The 20-residue trilongins BI–BIV amended with (A) or not amended (B) with the 11-residue trilongin AI. The tested peptaibol solutions were the same as those used in Fig. 5.

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Table 10. The four conductance (pS) levels (O1–O4) generated by trilongins BI–BIV, AI and alamethicin in the BLM experiment. Media: 2 m NaCl or 2 m KCl in 10 mm Tris buffer (pH 7.0)
PeptaibolsMedium/ratioConductance levels
O1 (pS)Na/KO2 (pS)Na/KO3 (pS)Na/KO4 (pS)Na/K
Trilongin AINaCl180 500 1040 1730 
KCl190 700 1550 2440 
  0.95 0.71 0.67 0.71
Trilongins BI–BIVNaCl170 480 1000 1640 
KCl210 740 1600 2500 
  0.81 0.64 0.63 0.66
AlamethicinNaCl140 420 1000 1600 
KCl200 800 1700 2600 
  0.70 0.52 0.59 0.61

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

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 [9]. 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 [39] 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) [40] and to act as a Ca2+ channel agonist in isolated bullfrog cardiac myocytes at 20 μg·mL−1 (10 μm) [41]. Alamethicin (40 μg·mL−1; 20 μm) has been shown to mediate the uptake of Ca2+ ions by bovine adrenal chromaffin cells [42].

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 [45].

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. [46] 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. [26] 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 [26] 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.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

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 [48]. 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 [47]. The sequence of the ITS region was analyzed using trichokey, version 2.0 [49]. 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. [47]. HPLC and HPLC-ESI-IT-MS analyses were peformed as described previously [47], 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 [47].

Functional staining

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 [47].

BLM analysis

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 [50]. 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 [39]. The ∑FIC for long (A) and short (B) peptaibols was calculated using the equation:

  • display math(1)

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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

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.

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  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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