Fungal mitochondria govern both gliotoxin biosynthesis and self-protection

Gliotoxin (GT) is a potent epipolythiodioxopiperazine toxin produced by the opportunistic pathogen <i>Aspergillus fumigatus</i> that contributes to virulence and inhibits competing microorganisms. However, GT is highly toxic to the producer itself, necessitating robust self-protection mechanisms. Here, we used a comparative transcriptomics approach between <i>A. fumigatus</i> (GT producer) and <i>A. nidulans</i> (non-producer) to identify additional genetic determinants of GT self-protection downstream of the transcription factor RglT. We characterized five RglT-dependent genes: <i>abcC1</i> (ABC transporter), <i>mfsD</i> (major facilitator superfamily transporter), <i>oxrA</i> (oxidoreductase), <i>mtrA</i> (putative methyltransferase), and <i>nmrC</i> (a GATA-type repressor). Deletion mutants in <i>A. fumigatus</i> and <i>A. nidulans</i> revealed that all except <i>oxrA</i> were required for full GT protection, with Δ<i>mtrA</i> and Δ<i>nmrC</i> exhibiting distinct phenotypes in oxidative stress and iron-starvation conditions. Transcriptomic profiling and protein network analysis showed that MtrA and NmrC influence mitochondrial functions, particularly ubiquinone biosynthesis, despite not localizing to mitochondria. Functional assays confirmed that GT exposure disrupts mitochondrial integrity and sensitizes <i>A. fumigatus</i> to mitochondrial inhibitors. Notably, GT-induced cell death was associated with mitochondrial fragmentation but lacked hallmarks of apoptosis-like nuclear damage. Together, our findings reveal new genetic components of GT detoxification and establish a critical role for mitochondrial function in <i>A. fumigatus</i> GT self-protection and production.