Regulation of gliotoxin biosynthesis and protection in Aspergillus species

Aspergillus fumigatus causes a range of human and animal diseases collectively known as aspergillosis. A. fumigatus possesses and expresses a range of genetic determinants of virulence, which facilitate colonisation and disease progression, including the secretion of mycotoxins. Gliotoxin (GT) is the best studied A. fumigatus mycotoxin with a wide range of known toxic effects that impair human immune cell function. GT is also highly toxic to A. fumigatus and this fungus has evolved self-protection mechanisms that include (i) the GT efflux pump GliA, (ii) the GT neutralising enzyme GliT, and (iii) the negative regulation of GT biosynthesis by the bis-thiomethyltransferase GtmA. The transcription factor (TF) RglT is the main regulator of GliT and this GT protection mechanism also occurs in the non-GT producing fungus A. nidulans. However, A. nidulans does not encode GtmA and GliA. This work aimed at analysing the transcriptional response to exogenous GT in A. fumigatus and A. nidulans, two distantly related Aspergillus species, and to identify additional components required for GT protection in Aspergillus species. RNA-sequencing shows a highly different transcriptional response to exogenous GT with the RglT-dependent regulon also significantly differing between A. fumigatus and A. nidulans. However, we were able to observe homologues whose expression pattern was similar in both species (43 RglT-independent and 12 RglT-dependent). Screening of an A. fumigatus deletion library of 484 transcription factors (TFs) for sensitivity to GT identified 15 TFs important for GT self-protection. Of these, the TF KojR, which is essential for kojic acid biosynthesis in Aspergillus oryzae, was also essential for GT biosynthesis in A. fumigatus and for GT protection in A. fumigatus, A. nidulans, and A. oryzae. KojR regulates rglT and gliT expression in Aspergillus spp. Together, this study identified conserved components required for GT protection in Aspergillus species. Author SummaryA. fumigatus secretes mycotoxins that are essential for its virulence and pathogenicity. Gliotoxin (GT) is a sulfur-containing mycotoxin, which is known to impair several aspects of the human immune response. GT is also toxic to different fungal species, which have evolved several GT protection strategies. To further decipher these responses, we used transcriptional profiling aiming to compare the response to GT in the GT producer A. fumigatus and the GT non- producer A. nidulans. This analysis allowed us to identify additional genes with a potential role in GT protection. We also identified A. fumigatus 15 transcription factors (TFs) that are important for conferring resistance to exogenous gliotoxin. One of these TFs, KojR, which is essential for A. oryzae kojic acid production, is also important for GT protection in A. fumigatus, A. nidulans and A. oryzae. KojR regulates the expression of another TF and an oxidoreductase, previously shown to be essential for GT protection. Together, this work identified conserved components required for gliotoxin protection in Aspergillus species.