Towards understanding the gliotoxin detoxification mechanism: in vivo thiomethylation protects yeast from gliotoxin cytotoxicity

Abstract

Gliotoxin (GT) is a mycotoxin produced by some species of ascomycete fungi including the opportunistic human pathogen Aspergillus fumigatus. In order to produce GT the host organism needs to have evolved a self-protection mechanism. GT contains a redox-cycling disulfide bridge that is important in mediating toxicity. Recently is has been demonstrated that A. fumigatus possesses a novel thiomethyltransferase protein called GtmA that has the ability to thiomethylate GT in vivo, which aids the organism in regulating GT biosynthesis. It has been suggested that thiomethylation of GT and similar sulfur-containing toxins may play a role in providing self-protection in host organisms. In this work we have engineered Saccharomyces cerevisiae, a GT-naïve organism, to express A. fumigatus GtmA. We demonstrate that GtmA can readily thiomethylate GT in yeast, which results in protection of the organism from exogenous GT. Our work has implications for understanding the evolution of GT self-protection mechanisms in organisms that are GT producers and non-producers.

Keywords: Aspergillus fumigatus; Saccharomyces cerevisiae; gliotoxin; oxidoreductase GliT; thio-methyltransferase GtmA.

Figure 1. FIGURE 1: Conversion of reduced GT to BmGT is mediated by GtmA protein.

Reduced GT undergoes bis-thiomethylation via the action of GtmA protein and requires two molecules of SAM for the reaction to reach completion . Consequently two molecules of SAH are produced that re-enter the methyl-methionine cycle .

Figure 2. FIGURE 2: Expression of gtmA in yeast protects against GT cytotoxicity.

(A) RT-PCR demonstrating that gtmA mRNA is stably expressed in yeast. Yeast ERG9 mRNA production is shown as a positive control. (B) Expression of A. fumigatus genes gtmA or gliT in yeast provides protection against GT cytotoxicity.

Figure 3. FIGURE 3: Detection and monitoring of GT and BmGT in yeast using mass spectrometry.

(A) GT is readily detected in supernatants of yeast cells exposed to exogenous GT. (B) BmGT can only be detected in supernatants from yeast cells, which express gtmA, following exposure to exogenous GT.

Figure 4. FIGURE 4: Conversion of GT to BmGT over time in yeast expressing GtmA.

(A) BmGT is not detected in supernatants of yeast cells exposed to exogenous GT over 3 h exposure. (B) BmGT is readily detected in supernatants from yeast cells expressing gtmA. Increased levels of BmGT can be detected over a 3-hour time period and correlate with a diminution GT levels.