Profiling of trichorzianines in culture samples of Trichoderma atroviride by liquid chromatography/tandem mass spectrometry

Abstract

Peptaibols are bioactive linear peptides of 5-20 amino acid residues and contain specific non-proteinogenic amino acids such as alpha-aminoisobutyric acid (Aib). They are antibiotic secondary metabolites of moulds belonging predominantly to the genus Trichoderma, some species of which are successfully used as biocontrol organisms to fight against plant diseases. In the present study we developed a profiling method for the relative quantification of 16 trichorzianine peptaibols in culture samples of T. atroviride and the comparison of their expression patterns by liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS). The method is based on selected reaction monitoring (SRM) in a triple-quadrupole tandem mass spectrometer using three SRM transitions per compound. The trichorzianines were enriched by solid-phase extraction (SPE) on C(18) cartridges. SPE recoveries were evaluated for diluted trichorzianine standard solutions and ranged from 72-97%. Suppression of the ionisation of the peptaibols in the ESI source ranged from 67-128% for most of the trichorzianines in culture filtrates of two different strains of T. atroviride and in spiked culture medium. In the case of trichorzianines TA Vb, TA VIa and TA VIb the presence of matrix components in the fungal culture samples caused a reduction of the SRM signal, with intensities between 34% and 56% relative to pure standard solutions. Finally, the profiling method was successfully applied to culture samples of T. atroviride P1 wild-type and two deletion mutants showing different trichorzianine expression patterns characteristic for the investigated fungal strains. This is the first LC-SRM profiling method for peptaibols for the investigation of peptaibol expression patterns in fungal culture samples.

The Production of Multiple Small Peptaibol Families by Single 14‐Module Peptide Synthetases in Trichoderma/Hypocrea

“…Specificity Prediction from Comparative Analysis of Adenylate Domains. In addition, we have correlated the recently analyzed 19-residue trichorzianin sequence from T. atroviride P1 [3] with the 19 adenylation domains of the respective 19-module NRPS identified by genomic sequencing [2]. Amino Acid-Binding-Pocket Analysis of Peptaibol Synthetase Genes.…”

“…Their biosynthesis proceeds on large multimodular enzymes as has been shown for the 18-residue trichovirins in T. virens [1] and the 19-residue trichorzianins in T. atroviride [2] [3]. Introduction.…”

Peptaibiomics: Towards a Myriad of Bioactive Peptides Containing Cα‐Dialkylamino Acids?

“…The trichoatrokontin-producing strain, T. atroviride ATCC 74058, was previously shown to produce 16 trichorzianins [197]. A first, major reason to explain this phenomenon is that the analytical equipment used for screening and structural elucidation of peptide antibiotics is becoming more and more sophisticated, as illustrated by the recently introduced methods of peptaibiomics and IC-MS. For IC-MS, one single loop of conidia or mycelium from a fully grown Petri dish is sufficient; for a standard peptaibiomics approach, the material grown on a single slope or Petri dish is required.…”

How a Mycoparasite Employs G-Protein Signaling: Using the Example ofTrichoderma

“…tga1 knockout mutants showed strongly reduced extracellular chitinase activities and a decreased transcription of the chitinase-encoding genes nag1 (N-acetyl-glucosaminidase-encoding) and ech42 (endochitinase 42-encoding). Investigation of the antifungal activity of the Δ tga1 mutant revealed reduced amounts of the major antifungal metabolite of T. atroviride , 6-pentyl- α -pyrone [21], while elevated amounts of peptaibols, peptides with antibiotic activity, could be detected [60]. These results indicate contrasting functions of Tga1 in regulating the biosynthesis of different antifungal metabolites.…”