Regulatory and self-protection implications of gliotoxin bisthiomethylation in Aspergillus fumigatus

Gliotoxin is an epipolythiodioxopiperazine (ETP) produced by the opportunistic fungal pathogen Aspergillus fumigatus. It contains an intact disulphide bridge, which mediates its toxic effects via redox cycling. Like many other ETPs, a bisthiomethylated form is also produced. In the case of gliotoxin, bis(methyl)gliotoxin is formed for unknown reasons by a cryptic enzyme. The work presented uncovered the S-adenosylmethionine dependent gliotoxin bis-thiomethyltransferase (GtmA), which converts dithiol gliotoxin to bis(methyl)gliotoxin. Disruption of this non-gli cluster encoded methyltransferase completely abrogated organismal ability to biosynthesize and secrete bis(methyl)gliotoxin, while gliotoxin production and secretion were increased (p=0.0056). Label-free quantitative (LFQ) proteomics of A. fumigatus wild-type, ΔgtmA, and gtmAC strains cultured in Czapek-Dox media revealed an elevated abundance of gli cluster encoded enzymes in the ΔgtmA mutant strain. Phylogenetic analysis of this enzyme revealed that there are 124 GtmA homologs within the Ascomycota phylum. Furthermore, through the characterisation of changes in SAM homoeostasis within A. fumigatus, this work also illuminates a direct link between gliotoxin biosynthesis and primary metabolic processes. Recombinant GtmA was shown to bismethylate dithiol GT using S-adenosyl methionine (SAM) as a methyl donor. Additionally, the crystal structure of GtmA was solved by multiple-wavelength anomalous diffraction to 2.3 Å resolution. This information was used to identify GtmA structural homologs, to uncover residues in involved in GtmA substrate binding and to facilitate targeted engineering of this enzyme. We now propose that the purpose of GtmA mediated bis(methyl)gliotoxin formation primarily serves to attenuate gliotoxin biosynthesis. This appears to be the first example of postbiosynthetic regulation of nonribosomal peptide synthesis in any organism.

Name dc:type.qualificationname

phd

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

National University of Ireland Maynooth

Year dc:date.issued

2015

Author dc:creator

• Dolan, Stephen

Language dc:language

en