Structure and biosynthesis of attinemicin, a widespread antifungal peptide produced by ant-associated Pseudonocardia

Attine ants comprise a group of ants characterized by the cultivation of fungi that form the basis of the ants’ nutrition. To help prevent nest infection by parasites, especially by the specialized fungal pathogen Escovopsis sp., the ants have evolved a symbiotic relationship with actinobacteria from the genus Pseudonocardia that produce bioactive secondary metabolites. Thus, leading to ecology-based hypotheses that the bacteria role is to protect the fungal garden. These compounds show great structural diversity, however, none of them are widely distributed among ant associated Pseudonocardia. In this work, we investigated ants and their symbiotic Pseudonocardia from around Brazil, including the Amazon Forest, Atlantic Forest and Brazilian Cerrado (Savanna). High-resolution tandem mass spectrometry (MS/MS) revealed that a high number of Pseudonocardia strains produced a shared metabolite and the same compound was detected in situ, in ant and nest samples. Isolation revealed this shared metabolite is attinemicin and its structure was solved using NMR, MS and Marfey’s method. Attinemicin is a novel linear peptide, showing in vivo antifungal activity and siderophore properties. In addition, genome mining resulted in the identification of a candidate biosynthetic gene cluster (BGC). Using information about the BGC we screened all of our Pseudonocardia strains for the presence of key biosynthetic genes and 84% harbor the attinemicin BGC. Similar analysis on other ant-associated Pseudonocardia isolated from Panama and Costa Rica were performed and no evidence for the production of attinemicin was discovered. Interestingly, phylogenetic analyses on all of the Pseudonocardia strains from both Brazil and Panama revealed they form separate clades based on geographical location. In summary, this work shows the widespread distribution of a new antifungal secondary metabolite produced by Pseudonocardia associated with Brazilian Attine ants.