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, the A. nidulans genome 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. 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 homologs whose expression pattern was similar in both species (43 RglT-independent and 11 RglT-dependent). Based on this approach, we identified a novel RglT-dependent methyltranferase, MtrA, involved in GT protection. Taking into consideration the occurrence of RglT-independent modulated genes, we screened an A. fumigatus deletion library of 484 transcription factors (TFs) for sensitivity to GT and 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 virulence and GT biosynthesis in A. fumigatus, and for GT protection in A. fumigatus, A. nidulans, and A. oryzae. KojR regulates rglT, gliT, gliJ expression and sulfur metabolism in Aspergillus species. Together, this study identified conserved components required for GT protection in Aspergillus species.

. GtmA, whose gene is not located in the 143 GT BGC, is able to convert dtGT into bisdethiobis(methylthio)-gliotoxin (bmGT) 144 and to attenuate GT production postbiosynthetically (Dolan et al., 2014(Dolan et al., , 2017; 145 Smith et al., 2016; Scharf et al., 2014). It is thought that the primary role of GtmA 146 is a decrease in GT biosynthesis and not a back up for GliT and toxin 147 neutralisation (Dolan et al., 2015). 148 Until recently, the TF regulating gliT has remained elusive. The TF RglT 149 was shown to regulate not only GliZ, a Zn(II)2Cys6 transcription factor required 150 for gliotoxin biosynthesis, but several other gli genes, including gliT, which is not GT-producing conditions. Interestingly, RglT and GliT were shown to have similar 154 roles in GT protection in A. nidulans, a fungus that does not produce GT and is 155 distantly related to A. fumigatus (Ries et al., 2020). The genome of A. nidulans 156 does not encode homologues for gliA and gtmA. Therefore, the aim of this work 7 173 the transcriptional control of RglT in the presence of exogenous GT, we 174 performed RNA-sequencing (RNA-seq) of the A. fumigatus wild-type (WT) and 175 ∆rglT strains when exposed to 5 µg/ml GT for 3 h. In these conditions, A. 176 fumigatus is protecting itself from the effects of GT, as has previously been shown that RglT is important for these processes.  Figure 1D).

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These results suggest that RglT is important not only for GT biosynthesis 219 and self-protection, but also for the regulation of the expression of genes involved 220 in the production of other SMs. process and a heterogeneous set of cellular components (p-value < 0.01; Figure   237 2A). GO analysis for the down-regulated genes showed enrichment for a 9 238 heterogeneous set of genes involved in biological processes and cellular 239 components (p-value < 0.01; Figure 2A). When comparing the WT to the ΔrglT 240 strain in the presence of GT, 132 genes were down-regulated and 68 genes were 241 up-regulated (Supplementary Table S4). GO enrichment analyses of the ΔrglT 242 strain showed a transcriptional up-regulation of genes encoding proteins involved 243 in nucleotide binding and cellular components (p-value < 0.01; Figure 2B). GO   Table S3).

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Furthermore, a total of 12 genes were found: six genes were dependent on RglT  Supplementary Figure S1). The expression of these 10 genes showed a high 284 level of correlation with the RNA-seq data (Pearson correlation from 0.896 to 285 0.952; Figure 2E).  Tables S1 and S2).

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Previous studies have shown that GT biosynthesis and self-protection is 306 intimately linked to sulfur metabolism, oxidative stress resistance, as well as iron  Taken together, these results strongly suggest that some of the mutants 331 identified as more sensitive to GT are also involved in pathways related to 332 oxidative stress and iron and zinc metabolism.  between their distributions) also fit the observed data well (Table 2).

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To determine whether KojR is also required for protection from GT in other  and suggest additional complex mechanisms in regulating the processes. 522 We were able to show a conserved mechanism of protection from GT in   harbor high levels of incongruence. These differences concern the placements of was PCR-amplified from these plasmids utilizing TaKaRa