Tolypocladamide H and the Proposed Tolypocladamide NRPS in Tolypocladium Species

Abstract

The genome of entomopathogenic fungus Tolypocladium inflatum Gams encodes 43 putative biosynthetic gene clusters for specialized metabolites, although genotype-phenotype linkages have been reported only for the cyclosporins and fumonisins. T. inflatum was cultured in defined minimal media, supplemented with or without one of nine different amino acids. Acquisition of LC-MS/MS data for molecular networking and manual analysis facilitated annotation of putative known and unknown metabolites. These data led us to target a family of peptaibols and guided the isolation and purification of tolypocladamide H (1), which showed modest antibacterial activity and toxicity to mammalian cells at micromolar concentrations. HRMS/MS, NMR, and advanced Marfey's analysis were used to assign the structure of 1 as a peptaibol containing 4-[(E)-2-butenyl]-4-methyl-l-threonine (Bmt), a hallmark structural motif of the cyclosporins. LC-MS detection of homologous tolypocladamide metabolites and phylogenomic analyses of peptaibol biosynthetic genes in other cultured Tolypocladium species allowed assignment of a putative tolypocladamide nonribosomal peptide synthetase gene.

Figure 1

Principal component analysis of extracts of cultured T. inflatum. (A) PCA plot of LC-MS data for extracts of T. inflatum cultured in triplicate on 10 different defined media (SM), each supplemented with one of nine amino acids (e.g., SM-Val, in red) or not (SM–, in orange). (B) Plot of mass feature loadings for principal components 1 and 2, with the [M + 2H]²⁺ ion for tolypocladamide H (1), found in SM-Val isolates, labeled with precursor mass and retention time.

Figure 2

Collision-induced dissociation (CID) qTOF MS-MS spectrum of tolypocladamide H (1), with b- and y-type ions labeled, and the planar structure of 1, with predicted b- and y-type fragments.

Figure 3

Planar structure of tolypocladamide H (1) showing key COSY and HMBC correlations.

Figure 4

Evolution of tolypocladamides in Tolypocladium. (A) Phylogeny of Tolypocladium created from 686 single-copy orthologous clusters. Branches are color-coded to ecology (red: insect-associated, blue: mycoparasitic, brown: soil-dwelling, green: endophytic). (B) Representations of the adenylation domains of TINF05969 and TINF05969 homologues, with amino acid sequences of their observed products. Sequence predictions are based on ions observed in MS/MS spectra (Tables S3–S6). A-Domains are color-coded to their respective clade. (C) Phylogeny of peptaibol adenylation domains, with tolypocladamide clades highlighted, and their annotated amino acids listed. ^aReferences (34), (35). ^bReference (35). ^cThis study.

Figure 5

GNPS molecular network of tolypocladamide mass features. Nodes representing mass features in the tolypocladamide molecular family are color-coded by source species of Tolypocladium. The mass feature (m/z 1361.88) for tolypocladamide H (1) is highlighted in yellow.