Screening the biosphere: the fungicolous fungus Trichoderma phellinicola, a prolific source of hypophellins, new 17-, 18-, 19-, and 20-residue peptaibiotics.

To investigate the significance of antibiotics for the producing organism(s) in the natural habitat, we screened a specimen of the fungicolous fungus Trichoderma phellinicola (syn. Hypocrea phellinicola) growing on its natural host Phellinus ferruginosus. Results revealed that a particular group of non-ribosomal antibiotic polypeptides, peptaibiotics, which contain the non-proteinogenic marker amino acid, α-aminoisobutyric acid, was biosynthesized in the natural habitat by the fungicolous producer and, consequently, released into the host. By means of liquid chromatography coupled to electrospray high-resolution time-of-flight mass spectrometry, we detected ten 20-residue peptaibols in the specimen. Sequences of peptaibiotics found in vivo were independently confirmed by analyzing the peptaibiome of an agar plate culture of T. phellinicola CBS 119283 (ex-type) grown under laboratory conditions. Notably, this strain could be identified as a potent producer of 39 new 17-, 18-, and 19-residue peptaibiotics, which display the same building scheme as the 20-residue peptaibols found in the specimen. Two of the 19-residue peptaibols are tentatively assigned to carry tyrosinol, a novel C-terminal residue, as deduced from high-resolution tandem mass-spectrometry data. For the new peptaibiotics produced by T. phellinicola, the name 'hypophellin(s)', based on the teleomorph name, is introduced.

1. Introduction

1.1. Fungi as a Prolific Source of Bioactive Natural Products

The current estimate of the total number of fungal species ranges between 1.0 and 1.5 million [1], whereas the number of those validly described should now exceed only 98,000 [2]. Of the 33,500 bioactive microbial metabolites known to date, the fungal kingdom contributes ca. 15,600. Approximately 10,000 of them were shown to display anti-infective, antitumor, and/or antiviral activities. Microbial-derived drugs on the market comprise ca. 400–500 active pharmaceutical agents [3], including therapeutically relevant antibiotics of fungal origin such as β-lactams, fusidic acid, and griseofulvin, as well as the two immunosuppressants mycophenolic acid and cyclosporin A [4].

Given that less than 1% of microorganisms visible under the microscope have been cultivated under laboratory conditions so far, microbial diversity provides an enormous, yet underestimated potential for future drug discovery [5] and in the search for new agricultural antibiotics [6].

1.2. The Potential of Trichoderma Species as Biological Control Agents (BCAs)

Species of the ubiquitous fungal genus Trichoderma and its Hypocrea teleomorphs have attracted considerable interest in the past two decades because of the pivotal role of their secondary metabolites in the antagonistic activities of biocontrol species [7-9]. Most of them occur as opportunistic, plant (endo)symbionts [10], some of which exhibit pronounced antimicrobial activity towards economically important plant pathogens. Recent examples include:

the hyperparasite Trichoderma stromaticum (syn. Hypocrea stromatica), the active agent of ‘Tricovab’ a commercial formulation against Crinipellis (syn. Moniliophthora) perniciosa, the Witches’ broom pathogen of cocoa (Theobroma cacao) [11] [12];

T paucisporum and T theobromicola, displaying in vitro-activities against frosty pod rot of cocoa, Moniliophthora roreri [13];

T martiale, which, in small-scale in situ field trials, proved highly effective against black pod rot of cocoa caused by Phytophthora palmivora [14].

The mode of action of phytoprotective Trichoderma species is considered rather complex. Depending on the species or even strains investigated, the following mechanisms may contribute to the antagonistic potential towards plant pathogenic fungi:

i) Competition for nutrients and/or space, ii) growth promotion of plants, especially colonization of roots, resulting in improved root and plant growth, iii) induction of localized and systemic resistance responses in plants, iv) mycoparasitism, v) increase of uptake and concentration of nutrients by the plant, including the production of siderophores, and vi) production of volatile and non-volatile antibiotics [10].

1.3. Peptaibiotics – Non-Ribosomally Biosynthesized Fungal Peptide Antibiotics Containing α,α-Dialkyl-α-amino Acids

During the past two decades, peptaibiotics have regained particular interest because of their unique bioactivities, resulting from their amphipathicity and helical conformations [15]. These are attributed to the presence of high proportions of peptide-bound α-aminoisobutyric acid (Aib), frequently accompanied by d- and/or l-isovaline (Iva) [16], and, in a few sequences, l-α-ethylnorvaline (EtNva), or 1-aminocyclopropane-1-carboxylic acid (Acc) [17]. The presence of these α,α-dialkyl-α-amino acids (Fig. 1,a) has been confirmed in acidic hydrolysates of more than 30 genera of fungi [18].

Fig. 1

a) Structures and configurations of a,a-dialkylamino acids found in peptaibiotics. b) Building scheme of subfamily-1 (SF1) peptaibiotics, produced by Hypocrea phellinicola. Variable positions are underlined. Minor sequence variations are parenthesized. Deletions of certain amino acid positions are highlighted in different shades: C-terminal deletions are highlighted in dark, deletions of Gln in medium, and deletions of [Aib/Ala]6 in light gray. a) Deleted in 17-, 18-, and 19-residues hypophellins. b) Deleted in the 17-residue sequence 29. c) Deleted in 18-residue sequences 11, 12, and 28, and in the 17-residue sequence 29. d) Detected with DTU maXis gradient only. e) Detected with JLU micrOTOF-Q II gradient only.

Peptaibiotics are defined as non-ribosomally biosynthesised, linear or cyclic polypeptide antibiotics of exclusively fungal origin which i) have a molecular weight between 500 and 2,200 Da, thus containing 4–21 residues; ii) show a high content of the marker Aib, as well as further α,α-dialkylamino acids; iii) are characterized by the presence of other non-proteinogenic amino acids and/or lipoamino acids; iv) possess an acylated N-terminus, and v) in the case of linear peptides, have a C-terminal residue that, in most of them, consists of a free or O-acetylated, amide-bonded β-amino alcohol. The C-terminus might also be an amine, amide, sugar alcohol, 2,5-diketopiperazine, a heterocyclic residue, or an amino acid with free carboxy terminus. The majority of Aib-containing peptides carry a C-terminal residue representing a β-amino alcohol. Only this group is referred to as peptaibols sensu stricto, whereas for the others the comprehensive name peptaibiotics is used [17].

1.4. Detection of Peptaibiotics in T. phellinicola Growing on Its Natural Host

The genus Trichoderma, which currently consists of ca. 200 validly described species the number of which increases continually [19-28], is generally recognized as the most prolific source of peptaibiotics [17]. However, reports on the detection of peptaibiotics in samples collected in the natural habitat of the producer(s) are rare. Most of the ca. 1,000 individual sequences of peptaibiotics known to date have been sequenced in extracts of fungal cultures grown under artificial laboratory conditions.

The first example of peptaibiotics isolated from natural specimens were hypelcins A and B obtained from ca. 2 kg of dried, crushed stromata of Hypocrea peltata [29-31]. In 1997 and 1999, three reports were published on the isolation of peptaibiotics from fruiting bodies of Scleroderma texense, Tylopilus neofelleus, and Boletus sp., respectively; all being members of the Boletales [32-34]. However, in 2002, Kiet et al. [35] isolated chrysospermins A–D from the Vietnamese species Xerocomus langbianensis (Boletaceae, Boletales) and attributed the detection of these four 19-residue peptaibols [36] to an unrecognized infection of X. langbianensis with Sepedonium sp. This phenomenon was later commented on by Degenkolb et al. [37] [38]. Finally, Neuhof et al. [39] corroborated the assumption of Kiet et al. [35] by analyzing four fruiting bodies of members of the order Boletales infected by Sepedonium chrysospermum and S. microspermum, respectively. Notably, all samples were screened positive for peptaibiotics of the chrysospermin type. In 2006, Lehr et al. [40] demonstrated that 16-residue peptaibols, the antiamoebins, were solely responsible for antibiosis in herbivore dung naturally colonized by or artificially inoculated with Stilbella fimetaria (syn. S. erythrocephala).

1.5. Bioactivities of Peptaibiotics from Trichoderma

Peptaibiotics are thus assumed to play a key role in the infection process of a host by a fungicolous species because of their unique ability of forming voltage-gated ion channels. This phenomenon is best described by the dipole flip-flop gating model in planar lipid bilayers [41]. Their well-documented membrane activity, however, may also account for other striking bioactivities, such as neurolepsy [42], inhibition of amyloid β-peptide formation [43], inhibition of HIV-1 integrase [44], suppression of tumor cells, targeted calcium-mediated apoptosis, and autophagy in human hepatocellular carcinoma cells [45], as well as induction of defence responses and systemic resistance in tobacco against tobacco mosaic virus [46] and programmed cell death in fungal plant pathogens [47].

1.6. Choice of the Model Organism

Trichoderma phellinicola, a recently described polyporicolous species, which specifically occurs on effused basidiomes of Phellinus spp., was chosen as a model organism. Specimens of H. phellinicola have so far been recorded from Austria, Denmark, Germany [20], and the Czech Republic (see Exper. Part). This species is possibly specific for Phellinus ferruginosus [20].

To confirm the above hypothesis of peptaibiotic production under in vivo conditions, a specimen of Trichoderma phellinicola growing on its host Phellinus ferruginosus, was screened for peptaibiotics. For comparison, the ex-type culture of T. phellinicola, CBS 119283 (= C.P.K. 2137), was investigated. Both morphs were analyzed using a peptaibiomics approach as described in [48-50].

2. Results

2.1. General Considerations

All 17-, 18-, 19-, and 20-residue sequences discussed below were obtained from Trichoderma phellinicola [20]. The name ‘hypophellins’ (HPHs), which covers the entirety of long-chain peptaibiotics (>17 residues) produced by this species, is proposed. We base this name on the teleomorph name Hypocrea phellinicola, which used to be the valid name of the holomorph in dual nomenclature [20]. The introduction of a new name for peptaibiotics from a phylogenetically well-defined species is more favorable than earlier names for many of the 19- and 20-residue peptaibiotics mentioned below, viz. suzukacillins, trichocel-lins, trichokonins, and longibrachins, which were produced by phylogenetically undefined Trichoderma species with thus highly questionable names. The latter issue is further complicated by the fact that many of the peptaibiotic-producing Trichoderma strains reported in the literature have never been deposited in a public culture collection, or deposition was terminated [51].

Hypophellins are numbered consecutively with Arabic numbers as follows: i) sequences produced by the specimen; ii) sequences produced by the culture CBS 119283 grown and analyzed at JLU; iii) sequences produced by the culture CBS 119283 grown and analysed at DTU.

2.2. Peptaibiotic Pattern of the Teleomorph

Notably, the teleomorph of Trichoderma phellinicola proved to be a prolific source of ten 20-residue peptaibols, compounds 1-10, displaying the characteristic building scheme of subfamily 1 (SF1), one of the nine ‘peptaibol subfamilies’ (Fig. 1,b, and and ), as introduced by Chugh and Wallace [52]4).

Table 1

Sequences of 20-Residue Peptaibiotics Detected in the Specimen of Hypocrea phellinicola (micrOTOF-Q II screening)

No.	tR [min]	[M+H]+		Residue
				1	2a)	3	4	5	6	7	8	9	10	11
1	37.8–38.1	1937.1209	Ac	Aib	Ala	Aib	Ala	Aib	Ala	Gln	Aib	Vxx	Aib	Gly
2	37.8–38.1	1938.1068	Ac	Aib	Ala	Aib	Ala	Aib	Ala	Gln	Aib	Vxx	Aib	Gly
3	39.1–39.3	1951.1358	Ac	Aib	Ala	Aib	Ala	Aib	Ala	Gln	Aib	Vxx	Aib	Gly
4	39.8–40.0	1952.1192	Ac	Aib	Ala	Aib	Ala	Aib	Ala	Gln	Aib	Vxx	Aib	Gly
5	40.2–40.4	1951.1416	Ac	Aib	Ala	Aib	Ala	Aib	Aib	Gln	Aib	Vxx	Aib	Gly
6	41.0–41.2	1952.1258	Ac	Aib	Ala	Aib	Ala	Aib	Aib	Gln	Aib	Vxx	Aib	Gly
7	41.3–41.7	1965.1615	Ac	Aib	Ala	Aib	Ala	Aib	Aib	Gln	Aib	Vxx	Aib	Gly
8	42.3–42.5	1966.1354	Ac	Aib	Ala	Aib	Ala	Aib	Aib	Gln	Aib	Vxx	Aib	Gly
9	43.0–43.2	1979.1718	Ac	Aib	Aib	Aib	Ala	Aib	Aib	Gln	Aib	Vxx	Aib	Gly
10	44.0–44.3	1980.1636	Ac	Aib	Aib	Aib	Ala	Aib	Aib	Gln	Aib	Vxx	Aib	Gly

Variable residues are underlined in the table header. Minor sequence variants are underlined in the sequences. This applies to , and .

Table 2

Diagnostic Fragment Ions of 20-Residue Peptaibiotics Detected in the Specimen of Hypocrea phellinicola (micrOTOF-Q II screening)

Diagnostic fragment ions	Peaks [m/z]a)
	1	2	3	4	5	6	7	8	9	10
tR [min]	37.8–38.1	38.6–38.7	39.1–39.3	39.8–40.0	40.2–40.4	41.0–41.2	41.3–41.7	42.3–42.5	43.0–43.2	44.0–44.3
[M + Na]+	1959.1047	1960.0872	1962.1376	n.d.	1973.1212	1974.1064	1987.1372	1988.1245	2001.1535	2002.1445
[M + H]+	1937.1209	1938.1036	1951.1358	1952.1192	1951.1416	1952.1258	1965.1615	1966.1354	1979.1718	1980.1636
a1	100.0808	100.0808	100.0808	100.0806	100.0809	100.0805	100.0808	100.0807	n.d.	n.d.
a2	171.1181	171.1181	171.1197	171.1195	171.1188	171.1185	171.1191	171.1200	185.1315	185.1311
a3	256.1657	256.1657	256.1662	256.1663	256.1666	256.1665	256.1669	256.1671	270.1811	270.1808
a4	327.2121	327.2121	327.2155	327.2142	327.1992	327.2097	327.2102	327.2116	341.2238	341.2180
a5	n.d.	n.d.	412.2739	412.2739	412.2572	412.2720	412.2776	412.2766	n.d.	n.d.
b1	128.0758	128.0758	128.0762	128.0757	128.0763	128.0758	128.0765	128.0760	128.0758	128.0748
b2	199.1102	199.1102	199.1109	199.1107	199.1111	199.1111	199.1113	199.1115	213.1251	213.1251
b3	284.1604	284.1604	284.1615	284.1614	284.1618	284.1615	284.1623	284.1625	298.1845	298.1802
b4	355.1982	355.1982	355.1973	355.1972	355.1981	355.1976	355.1986	355.1988	369.2109	369.2144
b5	440.2479	440.2479	440.2494	440.2492	440.2502	440.1497	440.2508	440.2510	454.2669	454.2699
b6	511.2839	511.2839	511.2850	511.2852	525.3019	525.3015	525.3023	525.3026	539.3231	539.3231
b7	639.3431	639.3431	639.3455	639.3451	653.3661	653.3626	653.3690	653.3681	667.3870	667.3881
b8	724.3937	724.3937	724.3961	724.3957	738.4118	738.4109	738.4130	738.4132	752.4381	752.4367
b9	823.4750	823.4590	823.4611	823.4601	837.4777	837.4772	837.4790	837.4795	851.5058	851.5067
b10	908.5298	908.5095	908.5131	908.5129	922.5302	922.5290	922.5314	922.5317	936.5594	936.5602
b11	965.5490	965.5311	965.5470	965.5316	979.5501	979.5476	979.5508	979.5506	993.5822	993.5819
b12	1078.6366	1078.6151	1078.6340	1078.6138	1092.6478	1092.6289	1092.6325	1092.6326	1106.6642	1106.6710
b13	1163.6824	1163.6642	1163.6810	1163.6662	1177.6994	1177.6816	1177.6853	1177.6859	1191.7215	1191.7196
y7	774.4598	775.4614	788.4742	789.4595	774.4586	775.4436	788.4750	789.4596	788.4750	789.4596
y7 – H2O	756.4445	757.4491	n.d.	771.4507	n.d.	757.4308	n.d.	771.4468	n.d.	771.4468
y7 – AA (20)	623.3556	624.3581	637.3711	638.3573	623.3563	624.3414	637.3722	638.3576	637.3722	638.3576
y7 – AA (20-19)	495.2979	496.2999	509.3124	510.2961	495.2955	496.2793	509.3120	510.2962	509.3120	510.2962
y7 – AA (20-18)	367.2385	367.2350	381.2515	381.2517	367.2364	367.2358	381.2519	381.2514	381.2519	381.2514
y7 – AA (20-17)	282.1900	282.1831	282.1850	282.1820	282.1853	282.1838	282.1839	282.1839	282.1839	282.1839

n.d., Not detected.

Table 5

Sequences of 10-, 11-, 17-, 18-, and 19-Residue Peptaibiotics Detected in the Ex-Type Culture (CBS 119283) of Hypocrea phellinicola (maXis screening)

No.	tR [min]	[M + H]+		Residue
				1	2	3	4	5	6	7	8	9	10	11
28	10.8	1747.0131	Ac	Aib	Ala	Aib	Ala	Aib	–	Gln	Aib	Lxx	Aib	Gly
12	11.2	1761.0273	Ac	Aib	Ala	Aib	Ala	Aib	–	Gln	Aib	Lxx	Aib	Gly
14	12.2	1911.1213	Ac	Aib	Ala	Aib	Ser	Aib	–	Gln	Aib	Lxx	Aib	Gly
29	12.6	1632.9708	Ac	Aib	Ala	Aib	Ala	Aib	–	Gln	Aib	Lxx	Aib	Gly
30	13.0	1880.1000	Ac	Aib	Ala	Aib	Gly	Aib	–	Gln	Aib	Lxx	Aib	Gly
31	13.2	1882.0784	Ac	Aib	Ala	Ser	Ala	Aib	–	Gln	Aib	Lxx	Aib	Gly
19	13.5	1880.1008	Ac	Aib	Ala	Ala	Ala	Aib	–	Gln	Aib	Lxx	Aib	Gly
32	14.1	1896.0964	Ac	Aib	Ala	Ser	Ala	Aib	–	Gln	Aib	Lxx	Aib	Gly
33	14.9	1880.1035	Ac	Aib	Ala	Ala	Ala	Aib	–	Gln	Aib	Lxx	Aib	Gly
34	15.5	1866.0863	Ac	Aib	Ala	Ala	Ala	Aib	–	Gln	Aib	Lxx	Aib	Gly
35	15.9	1880.1012	Ac	Aib	Ala	Aib	Ala	Aib	–	Gln	Aib	Lxx	Aib	Gly
36	16.2	1867.0706	Ac	Aib	Ala	Ala	Ala	Aib	–	Gln	Aib	Lxx	Aib	Gly
37	16.4	1880.1007	Ac	Aib	Ala	Ala	Ala	Aib	–	Gln	Aib	Lxx	Aib	Gly
38	16.7	n.d.	Ac	Aib	Ala	Aib	Ala	Aib	–	Gln	Aib	Lxx	Aib	Gly
39	16.8	1880.1009	Ac	Aib	Ala	Aib	Ala	Aib	–	Gln	Aib	Lxx	Aib	Gly
40	17.0	n.d.	Ac	Aib	Ala	Aib	Ala	Aib	–	Gln	Aib	Lxx	Aib	Gly
41	17.2	1880.0997	Ac	Aib	Ala	Ala	Ala	Aib	–	Gln	Aib	Lxx	Aib	Gly
42	17.5	1894.1210	Ac	Aib	Ala	Aib	Ala	Vxx	–	Gln	Aib	Lxx	Aib	Gly
43	17.7	1895.1007	Ac	Aib	Ala	Aib	Ala	Aib	–	Gln	Aib	Lxx	Aib	Gly
44	18.0	1894.1177	Ac	Aib	Ala	Ala	Ala	Vxx	–	Gln	Aib	Lxx	Aib	Gly
45	18.6	1908.1341	Ac	Aib	Ala	Aib	Ala	Vxx	–	Gln	Aib	Lxx	Aib	Gly
46	20.0	1922.1467	[227]a)			Aib	Ala	Aib	–	Gln	Aib	Lxx	Aib	Gly
47	21.5	1936.1660	[241]b)			Aib	Ala	Aib	–	Gln	Aib	Lxx	Aib	Gly
48	22.0	1009.7031	Occ)	Aib	Gly	Lxx	Aib	–	Gly	Lxx	Aib	Gly	Lxx	Lxxol
49	22.1–22.2	1066.7242	Oc	Aib	Gly	Lxx	Aib	Gly	Gly	Lxx	Aib	Gly	Lxx	Lxxol

The N-terminal sequence of compound 46, which is represented by a mass difference of 227 Da, could not be assigned.

The N-terminal sequence of compound 47, which is represented by a mass difference of 241 Da, could not be assigned.

Oc, Tentatively assigned as n-octanoyl residue.

One Gln residue is found in position 7, and another one towards or at the C-terminus in position 18, whereas position 19 is either occupied by a third Gln or a Glu residue. A highly conserved Pro residue is located in position 14 of the peptide chain. All sequences have a Gly residue in position 11 and terminate in Pheol. At least seven, at most nine, residues are occupied by Aib. Variable amino acid residues are located in positions 2, 6, 17, and 18 (,b).

Most of the peptaibols sequenced resemble previously described compounds (,b, , and such as longibrachins A and B [53], trichobrachins II [57], trichoaureocins [54], trichokonins [55] [62] [63], and suzukacillins A [60].

Base-peak chromatograms (BPCs) of a) the H. phellinicola specimen screened with the micrOTOF-Q II, b) the H. phellinicola ex-type plate culture screened with the micrOTOF-Q II, and c) the H. phellinicola specimen screened with the maXis. †, co-eluting peptaibiotics, not sequenced; ‡, non-peptaibiotic metabolite.

2.3. Peptaibiotic Pattern of the Culture

2.3.1. General Considerations

As observed before [20], ascospores of T phellinicola are unstable and die rapidly after collecting. This might have been the reason why no agar culture could be obtained from our specimen. As a substitute, the ex-type culture of T phellinicola CBS 119283 (= C.P.K. 2137) was provided, and its peptaibiotic pattern was analyzed. Except for the two lipopeptaibols 48 and 49, the remaining compounds 11–47 represent the characteristic building scheme of SF1, resembling the previously described 20-residue peptaibols suzukacillins A, trichosporins B, and trichocellins A [60] [61] [64-67].

2.3.2. micrOTOF-Q II Screening

In contrast to the specimen analyzed, the ex-type plate culture grown and analyzed at the Justus Liebig University of Giessen (JLU) produced two new 18- and fifteen new 19-residue peptaibols, compounds 11–27, which lacked the [Ala/Aib]6 residue of the 20-residue peptaibols found in the specimen ( and , and . The two truncated 18-residue sequences, compounds 11 and 12, terminated in free Gln. Sequences 14 and 16–27 carry a C-terminal Pheol. For compounds 13 and 15, a C-terminal tyrosinol residue (abbreviated as ‘Tyrol’) was tentatively deduced from HR-ESI-MS/MS data ( and .

Sequencing of compounds 13 and 15 ocontaining a new C-terminal residue with a peak at m/z 804.46, tentatively assigned as tyrosinol (Tyrol)

2.3.3. maXis Screening

All SF1 peptaibiotics, compounds 12, 14, 19, 28–47, of the ex-type plate culture grown and analyzed at DTU ( and , and exhibit the characteristic deletion of the Ala/Aib residue in position 6. However, different positional isomers and homologues were found, e.g., the 17-residue deletion sequence 29, lacking the C-terminal dipeptide [Gln18–Pheol19]. In compound 31, a Ser-residue was found in position 3, whereas compound 30 exhibited a Gly residue in position 4. Overall, the structural diversity of peptaibiotics produced by the two cultures was much higher as compared to the specimen: variable amino acid residues were in positions 2,3, 4, 5, 6, 17, 18, and 20 (.

2.4. Lipopeptaibols as Trace Components in the Plate Cultures

Two lipopeptaibols, compounds 48 and 49, were produced as trace components in the DTU plate culture. Compound 49 probably represents trichogin A IV [68] [69] or a positional isomer thereof. The new positionally isomeric compound 48, named ‘lipophellin 1’, is characterized by the deletion of [Gly]5 of compound 49 ( and , and .

3. Discussion

3.1. Hypophellins, Novel Long-Chain Peptaibiotics from T. phellinicola

The most notable result of this investigation is, indeed, the unequivocal confirmation of peptaibiotic biosynthesis in the natural habitat of T phellinicola growing on its host Phellinus ferruginosus, commonly known as the Rusty Porecrust. We here describe for the first time the in vivo detection of non-ribosomal peptide antibiotics5), which may significantly contribute to the complex interaction of a fungicolous ascomycete growing on its basidiomycetous host.

3.2. The Peptaibiome of the Specimen

The teleomorph produced a microheteroge-neous mixture of ten 20-residue HPHs, four of which, 6, 8, 9, and 10, are new (Table 1). Compared to smaller sequences consisting of less than 17 residues, long-chain peptaibiotics display a higher membrane-pore-formation activity by several orders of magnitude [71].

Depending on the individual sequence, seven to nine Aib residues are present, which strongly promote the formation of helical structures. i.e., α- or 310-helices, and even mixed forms [72-74], which is due to the steric constraints imposed by the geminal Me groups of the Cα-atom [75]. All of them exhibit the structurally important features, which are required for the formation of transmembrane ion channels in artificial lipid bilayer membranes, as compiled by Duclohier [76], and Duclohier and Wróblewski [77]. A multitude of bioactivities has been described for 20-residue peptaibols of similar structure, which are compiled in .

3.3. The Peptaibiome of the Ex-Type Plate Culture

In contrast to what has been observed for the specimen, 20-residue peptaibols could not be detected. Instead, fifteen 19-residue peptaibols were detected in the micrOTOF-Q II screening and another eighteen in the maXis screening. Although sequences of 11–47 still exhibit the characteristic building scheme of SF1, they are distinguished from the 20-residue peptaibols of the teleomorph specimen by a deletion of the Aib/Ala residue in position 6 (Δ Ala/Aib6) of the peptide chain. This deletion, however, is predicted not to negatively influence the bioactivity of these long-chain peptaibols, as all important structural features are still present, which comply with the requirements for the formation of transmembrane ion channels in artificial lipid bilayer membranes [76] [77]. The three 18-residue sequences, 11, 12, and 28, exhibit a deletion of the C-terminal amino alcohol, whereas the dipeptide [Gln18–Pheol19] is deleted in 29, a 17-residue sequence. Truncated versions of SF1 peptaibols lacking the C-terminal amino alcohol or even the adjacent Gln residue have been reported before.

The ten 19-residue peptaibiotics, trichobrachins I (TB I), lacking the C-terminal Pheol residue, as well as the two 18-residue trichobrachins II-1 and -2 (TB II), which exhibit a deletion of the C-terminal dipeptide [Gln19–Pheol20], were shown to originate from 20-residue trichobrachins II (TB II) by enzymatic degradation [57]. Two minor desPheol compounds F30, representing 1.3% of the alamethicin (ALM) mixture investigated, have been detected by non-aqueous capillary electrophoresis (NACE) coupled to electrospray mass spectrometry [94].

3.4. l-Phenylalaninol as Constituent of Natural Products

C-Terminal l-Pheol is commonly found in peptaibiotics [17] [18] but has also been infrequently reported as a constituent of other plant and fungal secondary metabolites such as N-benzoyl-l-phenylalaninol from Catharanthus pusillus [95] and Diospyros quaesita [96], O-acetyl-N-(N′-benzoyl-l-phenylalanyl)-l-phenylalaninol from Euphorbia fischeriana and E. kansui [97], and N-benzoyl-O-[N′-benzoyl-l-phenylalanyl]-l-phenylalaninol from Penicillium arenicola (syn. P. canadense) [98].

3.5. l-Tyrosinol as a Constituent of Natural Products

To the best of our knowledge, neither d- nor l-tyrosinol6) has ever been reported as constituent of either linear or cyclic peptides of microbial origin, including peptaibiotics. However, l-tyrosinol is a ‘cryptic’ building block of the following natural products:

farinosone C, an amide from Paecilomyces farinosus RCEF 0101 [99];

cordyceamides A and B from a liquid culture of Cordyceps sinensis [100];

preoxazinin-7, the linear precursor [101], and cyclic oxazinins from the digestive glands of Mytilus galioprovincialis [102] [103].

3.6. The Lifestyle of Trichoderma phellinicola: Findings and Thoughts

Taken these findings together, we dare predict a mycoparasitic lifestyle of the host-specific polyporicolous Trichoderma phellinicola:

It has been demonstrated by in vitro studies that chitinases and β-1,3-glucanases act synergistically with peptaibiotics in inhibiting spore germination and hyphal elongation of Botrytis cinerea. Parallel formation of hydrolytic enzymes and 19-residue antifungal trichorzianins A and B by the potent mycoparasite Trichoderma atroviride7) is triggered in the presence of cell walls of plant-pathogenic fungi [106]. Trichorzianins have previously been shown to form voltage-gated ion channels in planar lipid bilayers [107] and to modify the membrane permeability of liposomes, and they are active against Rhizoctonia solani and Phythophthora cactorum [108]. Based on these findings, a model of how peptaibiotics such as trichorzianins and hydrolases interact synergistically was proposed.

First, the host cell wall is digested enzymatically; thereafter, peptaibiotics will penetrate the cell membrane to form ion channels. Cell leakage reduces the ability of the host to effectively repair its cell wall. Eventually, inhibition of chitin and β-glucan synthesis further amplifies the destructive effect of chitinases and β-1,3-glucanases [108]. These mechanisms, however, may also account for the recently published induction of programmed cell death in plant fungal pathogens [47] caused by the 20-residue peptaibol trichokonin VI (= gliodeliquescin A [56])8), from T. koningii, T. pseudokoningii, and T. deliquescens (syn. Gliocladium deliquescens) [20]. The presence of peptaibiotics was also shown to play a role in the induction of plant defence responses [110].

3.7 Remarks on Non-Ribosomal Biosynthesis and Module Skipping by T. phellinicola

The exclusive production of 20-residue peptaibols by the T. phellinicola teleomorph indicates the presence of a 20-module NRPS. As the culture CBS 119283 has been shown to produce 17-, 18-, and 19-residue peptaibiotics only, it is likely to contain a 19-module NRPS, lacking the 6th module activating Ala or Aib. In addition, modules 3 and 4 show differing substrate specificities, as compared to the teleomorph, thus permitting the incorporation of Ala or Ser in position 3 and of Gly, Ala, or Ser in position 4, respectively. These findings indicate substantial variations in the sequences of the SF1-type peptaibol synthetases of both strains. As has been discussed in the case of SF4-type peptaibols, genes involved in secondary-metabolite products show a much broader sequential variety than housekeeping genes [50]. We here, indeed, find evidence for a significant structural variation within a large gene.

Experimental Part

Chemicals. All solvents used, MeCN (99.9%), MeOH (99.9%), CH2Cl2 (99.8%), and HCOOH (98%), were of LC/MS grade from Sigma-Aldrich (D-Steinheim). Water was purified by a Merck-Millipore Milli-Q Synthesis A10 system (D-Schwalbach/Ts.).

Origin of Specimen. The teleomorphic specimen of Trichoderma phellinicola growing on its host Phellinus ferruginosus was collected in the ‘Národni park Podyjí’ (Czech Republic, Moravia), near Hardegg at the bridge across the River Thaya, just across the border between Austria and the Czech Republic.

Origin of Trichoderma phellinicola CBS 119283 (ex-type). All details concerning this new species were given by Jaklitsch [20].

Extraction of Specimens. The teleomorph was extracted with CH2Cl2/MeOH 1:1 (v/v), the solvent was evaporated in vacuo (Rotavapor R-215, Biichi, D-Essen), and the extract was cleaned up over Sep-Pak Classic C cartridges (Waters, D-Eschborn) as described by Krause et al. [48].

Cultivation and Extraction of Pure Cultures. Cultures of the specimen were grown on potato dextrose agar (PDA; Becton Dickinson, D-Heidelberg) at 23° for 6 d. These subcultures were used for inoculation of the main cultures. After 10 d of cultivation at 23° in the dark, main cultures were extracted as described for the teleomorph.

LC/MS Analysis. Two QTOF systems, both from Bruker Daltonic (D-Bremen) controlled by HyStar v. 3.2 were used. Both instruments were equipped with an orthogonal ESI source and coupled to a Dionex UltiMate 3000 UHPLC (Dionex, D-Idstein).

System 1: high-resolution micrOTOF Q-II mass spectrometer. For separation, an Acclaim 120 C, 3 μm, 2.1 × 150 mm, column (Dionex, D-Idstein) at a flow rate of 0.25 ml/min−1 and a temp. of 35° was used. Eluent A consisted of H2O + 0.1% HCOOH and eluent B of 95% MeCN + 0.1% HCOOH. Subsamples of 10 μl were injected. The column was held at 80% A/20% B for 5 min, then a gradient from 20% B to 100% over 55 min was applied. Thereafter, the column was held at 100% B for 15 min, returned to the start conditions in 1 min, and finally equilibrated for 14 min.

Samples were screened for peptaibiotics in the positive-ion mode using the following three-step routine procedure: first a full scan was recorded from m/z 50 to 3000. In System 1, this was followed by CID measurements from m/z 50 to 2000, recorded at energy of 150 eV. Finally, results of CID-MS were verified by MS/MS experiments on selected precursor ions. For precursors of m/z < 1000, a collision energy of 30 eV was applied, precursor ions in the m/z range from 1000 to 1500 were fragmented at a collision energy of 35 eV and precursor ions of m/z > 1500 at a collision energy of 40 eV. The isolation width for MS/MS experiments was set to ± 1 Da.

System 2: The maXis 3G QTOF mass spectrometer operated at a resolution of 40,000 FWHM. An Acquity BEH300 C,1.7 μm, 2.1 × 150 mm, column (Waters, D-Eschborn) was used for separation, using H2O + 0.1% HCOOH (eluent A) and 100% MeCN + 0.1% HCOOH(eluent B). The flow rate was set to 0.3 ml/min and the temp. to 40°. The gradient started with 90% A/10% B and was changed to 50% A/50% B at 7 min, then to 30% A/70 % B at 25 min, then raised to 100% B at 38 min, and held at 100% B until 41 min before setting to starting conditions from time 42 min to 46 min. Three μl were injected. MS were scanned in the m/z range of 100–2,000. Auto MS with precursor ion-dependent collision energy optimization was used for fragmentation in the range of 10–65 eV.

Data interpretation was performed using the DataAnalysis v. 4.0 software (Bruker Daltonic, D-Bremen). Use of high-resolution (HR)ESI-MS allowed the unequivocal sequencing of fragment-ion series according to the Roepstorff/Fohlman–Biemann nomenclature. In cases where the isomeric amino acids (Leu/Ile and Val/Iva, resp.) or the corresponding amino alcohols (Leuol/Ileol) with the same elemental composition could not be distinguished, the abbreviations Lxx, Vxx, and Lxxol were used instead [48-50].

This study was supported by the Hessian Ministry for Science and Art by a grant from the LOEWE-Schwerpunkt program ‘Insect Biotechnology’ to A. V. DTU acknowledges the grant from the Danish Research Council (FI 2136-08-0023) for the maXis QTOF system, and MYCORED (EC KBBE-2007-222690-2) for supporting A. I. Support by the Austrian Science Fund (FWF; project P22081-B17) is acknowledged by W. M. J. The authors are indebted to Prof. Dr. Hartmut Laatsch (Institute of Organic and Biomolecular Chemistry, University of Göttingen, Germany) for his valuable comments on the occurrence of tyrosinol as a constituent of natural products.

Table 3

Sequences of 18- and 19-Residue Peptaibiotics Detected in the Ex-Type Culture (CBS 119283) of Hypocrea phellinicola (micrOTOF-Q II screening)

No.	tR [min]	[M + H]+		Residue
				1	2	3	4	5	6	7	8	9	10	11
11	30.9–31.1	1747.0135	Ac	Aib	Ala	Aib	Ala	Ala	–	Gln	Aib	Lxx	Aib	Gly
12	31.8–32.0	1761.0324	Ac	Aib	Ala	Aib	Ala	Aib	–	Gln	Aib	Lxx	Aib	Gly
13	32.2–32.6	1896.0995	Ac	Aib	Ala	Ala	Ala	Aib	–	Gln	Aib	Lxx	Aib	Gly
14	32.5–32.7	1910.1131	Ac	Aib	Ala	Aib	Ser	Aib	–	Gln	Aib	Lxx	Aib	Gly
15	32.8–33.1	1910.1140	Ac	Aib	Ala	Aib	Ala	Aib	–	Gln	Aib	Lxx	Aib	Gly
16	35.1–35.3	1896.1035	Ac	Aib	Ala	Ala	Ser	Aib	–	Gln	Aib	Lxx	Aib	Gly
17	37.0–37.2	1866.0928	Ac	Aib	Ala	Ala	Ala	Aib	–	Gln	Aib	Lxx	Aib	Gly
18	37.7–37.9	1880.1095	Ac	Aib	Ala	Aib	Ala	Aib	–	Gln	Aib	Lxx	Aib	Gly
19	38.3–38.4	1880.1136	Ac	Aib	Ala	Ala	Ala	Aib	–	Gln	Aib	Lxx	Aib	Gly
20	38.8–39.2	1894.1331	Ac	Aib	Ala	Aib	Ala	Aib	–	Gln	Aib	Lxx	Aib	Gly
21	39.8–40.1	1895.1278	Ac	Aib	Ala	Aib	Ala	Aib	–	Gln	Aib	Lxx	Aib	Gly
22	40.6–40.9	1908.1474	Ac	Aib	Ala	Aib	Ala	Vxx	–	Gln	Aib	Lxx	Aib	Gly
23	41.5–41.6	1909.1391	Ac	Aib	Ala	Aib	Ala	Vxx	–	Gln	Aib	Lxx	Aib	Gly
24	42.1–42.3	1922.1601	Ac	Aib	[255]		Ala	Aib	–	Gln	Aib	Lxx	Aib	Gly
25	43.4–43.6	1936.1738	Ac	Aib	[269]		Ala	Aib	–	Gln	Aib	Lxx	Aib	Gly
26	44.2–44.4	1936.1750	Ac	Aib	Vxx	Aib	Ala	Vxx	–	Gln	Aib	Lxx	Aib	Gly
27	45.0–45.6	1950.1894	Ac	Aib	Lxx	Aib	Ala	Vxx	–	Gln	Aib	Lxx	Aib	Gly

Table 4

Diagnostic Fragment Ions of 18- and 19-Residue Peptaibiotics Detected in the Ex-Type Culture (CBS 119283) of Hypocrea phellinicola (micrOTOF-Q II screening)

Diagnostic fragment ions	Peaks [m/z]a)
	11	12	13	14	15	16	17	18
tR [min]	30.9–31.1	31.8–32.0	32.2–32.6	32.5–32.7	32.8–33.1	35.1–35.3	37.0–37.2	37.7–37.9
[M + Na]+	1768.9850	1783.0115	1918.0846	1932.0976	1932.1017	1918.0877	1888.0616	1902.0891
[M + H]+	1747.0135	1761.0324	1896.0995	1910.1131	1910.1140	1896.1035	1866.0928	1880.1095
a1	100.0718	n.d.	n.d.	n.d.	n.d.	100.0720	100.0720	n.d.
a3	256.1647	256.1624	242.1508	256.1641	256.1707	242.1511	242.1506	256.1675
a4	n.d.	n.d.	n.d.	n.d.	327.1979	n.d.	n.d.	327.2046
a5	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	398.2312	n.d.
b1	128.0687	128.0658	128.0709	128.0708	128.0835	128.0715	128.0719	128.0721
b2	199.1075	199.1076	199.1109	199.1110	199.1161	199.1118	199.1115	199.1081
b3	284.1611	284.1617	270.1453	284.1622	284.1637	270.1434	270.1471	284.1634
b4	355.1972	355.1977	341.1846	371.1863	355.2023	357.1765	341.1815	355.1988
b4 – H2O	n.d.	n.d.	n.d.	353.1758	n.d.	339.1676	n.d.	n.d.
b5	426.2340	440.2546	426.2354	456.2441	440.2494	442.2277	426.2314	440.2546
b6	554.2840	568.3175	554.2840	584.3226	568.3175	570.2870	554.2989	568.3023
b7	639.3523	653.3691	639.3539	669.3625	653.3679	655.3443	639.3530	653.3685
b8	752.4400	766.4531	752.4386	782.4408	766.4563	768.4296	752.4353	766.4519
b9	837.4860	851.5024	837.4880	867.4961	851.5028	853.4825	837.4896	851.5066
b10	894.5048	908.5271	894.5061	924.5223	908.5250	910.5022	894.5076	908.5242
b11	1007.5856	1021.6063	1007.5967	1037.6039	1027.6073	1023.5862	1007.5917	1021.6085
b12	1092.6441	1106.6573	1092.6442	1122.6523	1106.6575	1108.6413	1092.6474	1106.6629
b12 – H2O	n.d.	n.d.	n.d.	n.d.	n.d.	1090.6265	1074.6077	1088.6332
y6	655.3841	655.3841	–	–	–	–	–	–
y6 – AA (18)	509.3130	509.3130	–	–	–	–	–	–
y6 – AA (18-17)	381.2540	381.2540	–	–	–	–	–	–
y6 – AA (18-16)	282.1709	282.1709	–	–	–	–	–	–
y7	–	–	804.4624	788.4706	804.4669	788.4697	774.4592	774.4593
y7 – H2O	–	–	786.4472	770.4510	n.d.	770.4510	756.4383	756.4383
y7 – AA (19)	–	–	637.3680	637.3708	637.3725	637.3705	623.3566	623.3559
y7 – AA (19-18)	–	–	509.3068	509.3140	509.3085	509.3103	495.2961	495.2962
y7 – AA (19-17)	–	–	381.2489	381.2515	381.2545	381.2513	367.2370	367.2373
y7 – AA (19-16)	–	–	n.d.	n.d.	282.1814	282.1814	282.1815	282.1815

n.d., Not detected.

Table 6

Diagnostic Fragment Ions of 10-, 11-, 17-, 18-, and 19-Residue Peptaibiotics Detected in the Ex-Type Culture (CBS 119283) of Hypocrea phellinicola (maXis screening)

Diagnostic fragment ions	Peaks [m/z]a)
	28	12	14	28	30	31
tR [min]	10.8	11.2	12.2	12.6	13.0	13.2
[M + H]+	1747.0131	1761.0273	1911.1213	1632.9708	1880.1000	1882.0784
b1	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
b2	n.d.	199.1093	n.d.	199.1087	199.1087	199.1123
b3	284.1601	284.1607	284.1613	284.1609	284.1604	286.1389
b4	355.1989	355.1980	371.1938	355.1975	341.1819	357.1760
b4 – H2O	n.d.	n.d.	438.2353	n.d.	412.2541	424.2167
b5	440.2512	440.2509	456.2470	440.2506	426.2347	442.2296
b6	568.3097	568.3098	584.3039	568.3096	554.2926	570.2869
b7	653.3615	653.3626	669.3571	653.3619	639.3458	655.3404
b8	766.4456	766.4471	782.4415	766.4461	752.4294	768.4257
b9	851.5003	851.4996	867.4953	851.4987	837.4826	853.4789
b10	908.5192	908.5208	924.5190	908.5199	894.5026	910.4971
b11	1021.6077	1021.6046	1038.5981	1021.6053	1007.5901	1023.5860
b12	1106.6578	1106.6578	1122.6537	1106.6590	1092.6412	1108.6356
b12 – H2O	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
y5	–	–	–	527.3191	–	–
y5 – AA (17)	–	–	–	381.2497	–	–
y5 – AA (17-16)	–	–	–	282.1814	–	–
y5 – AA (17-15)	–	–	–	197.1287	–	–
y6	641.3626	655.3768	–	–	–	–
y6 – AA (18)	495.2923	509.3095	–	–	–	–
y6 – AA (18-17)	367.2353	381.2500	–	–	–	–
y6 – AA (18-16)	282.1812	282.1816	–	–	–	–
y6 – AA (18-15)	197.1274	197.1288	–	–	–	–
y7	–	–	788.4676	–	788.4676	774.4501
y7 – H2O	–	–	637.3673	–	637.3673	623.3515
y7 – AA (19)	–	–	509.3117	–	509.3117	495.2926
y7 – AA (19-18)	–	–	381.2509	–	381.2509	367.2344
y7 – AA (19-17)	–	–	282.1814	–	282.1814	282.1813
y7 – AA (19-16)	–	–	197.1284	–	197.1284	197.1270

n.d., Not detected.

Table 7

Biological Activities of Selected 20-Residue Peptaibols Structurally Closely Related to Hypophellins

Peptaibols	Bioactivities reported	Ref.
Longibrachins	Ion-channel formation in BLM, antimycoplasmic	[53]
Suzukacillins	Antibacterial, antifungal	[78]
	Ion-channel formation in BLM	[79]
	Haemolysis of human erythrocytes	[80]
Trichoaureocins	Haemolysis of sheep erythrocytes, antibacterial (g+)	[54]
Trichobrachins	Antibacterial (g+ )	[57]
Trichocellins	Induction of Ca2+-dependent catecholamine secretion from bovine adrenal medullary chromaffin cells	[67]
	Ion-channel formation in BLM	[81]
Trichokonins	Agonist towards Ca2+-channels in bullfrog cardiac myocytes	[55] [82]
	Antibacterial (g+ ), antifungal	[83]
	Induction of defense responses and systemic resistance in tobacco against tobacco mosaic virus	[46]
	Induction of apoptotic programmed cell death in fungal plant pathogens	[47]
Trichosporins B	Uncoupling of the respiratory activity of rat liver mitochondria	[64] [84]
	Induction of Ca2+-dependent catecholamine secretion from bovine adrenal medullary chromaffin cells	[85–87]
	Ion-channel formation in BLM	[88]
	Antitrypanosomal	[66]
Paracelsins	Antibacterial (g+ )	[89]
	Increasing digestibility of starch and cellulose in ruminants; haemolysis of human erythrocytes; acutely toxic in mice (LD50 5 mg/kg, i.p.)	[90]
	Mosquitocidal (larvae of Culex pipiens)	[91]
	Toxic against aquatic invertebrates (Daphnia magna, Artemisia salina)	[92] [93]
	Ion-channel formation in BLM	[71]
	Antifungal	[93]