key: cord-0305123-0i1dzlyq authors: de Castro, Patrícia Alves; Moraes, Maísa; Colabardini, Ana Cristina; Horta, Maria Augusta Crivelente; Knowles, Sonja L.; Raja, Huzefa A.; Oberlies, Nicholas H.; Koyama, Yasuji; Ogawa, Masahiro; Gomi, Katsuya; Steenwyk, Jacob L.; Rokas, Antonis; Ries, Laure N.A.; Goldman, Gustavo H. title: Regulation of gliotoxin biosynthesis and protection in Aspergillus species date: 2021-08-16 journal: bioRxiv DOI: 10.1101/2021.08.16.456458 sha: 78aa2b4f816f644f94d2971d05b4878227827c25 doc_id: 305123 cord_uid: 0i1dzlyq 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 Summary A. 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. . 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 ). 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 ). 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 ). 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) . 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 GliZ, a transcriptional regulator of gliotoxin biosynthesis, 848 contributes to Aspergillus fumigatus virulence Trimmomatic: a flexible trimmer for 852 Illumina sequence data BLAST+: architecture and applications A 857 highthroughput gene knockout procedure for Neurospora reveals functions 858 for multiple transcription factors The role of glutathione S-transferase GliG in gliotoxin biosynthesis 862 in Aspergillus fumigatus Resistance is not futile: Gliotoxin 865 biosynthesis, functionality and utility Structural, mechanistic and functional insight into gliotoxin bis-869 thiomethylation in Aspergillus fumigatus Doyle 873 S. Regulation of nonribosomal peptide synthesis: bis-thiomethylation 874 attenuates gliotoxin biosynthesis in Aspergillus fumigatus OrthoFinder: phylogenetic orthology inference 878 for comparative genomics The negative cofactor 2 complex is a 29 884 key regulator of drug resistance in Aspergillus fumigatus Sequencing of Aspergillus nidulans and comparative analysis with A. 896 fumigatus and A. oryzae Hypoxia and 899 fungal pathogenesis: to air or not to air? UFBoot2: Improving the Ultrafast Bootstrap Approximation The MAP kinase MpkA controls cell wall integrity, 906 oxidative stress response Aspergillus fumigatus Identification and characterization of a 911 putative basic helix-loop-helix transcription factor involved in the early 912 stage of conidiophore development in Aspergillus oryzae Factor RgdA Governs Growth, Development, Toxigenesis, and Virulence 917 in Aspergillus fumigatus. mSphere Meiotic and mitotic recombination in Aspergillus and its 920 chromosomal aberrations Aspergillus mycotoxins and their effect on the 922 host MAFFT Multiple Sequence Alignment 925 Software Version 7: Improvements in Performance and Usability Fungal secondary metabolism: regulation, function and drug 928 discovery Aspergillus fumigatus Abrogates Leukotriene B4 Formation through 932 Inhibition of Leukotriene A4 Hydrolase Characterization of Aspergillus fumigatus Isolates from Air and 936 Surfaces of the International Space Station Oberlies 939 NH, Rokas A. Gliotoxin, a Known Virulence Factor in the Major Human 940 Pathogen Aspergillus fumigatus, Is Also Biosynthesized by Its 941 Nonpathogenic Relative Aspergillus fischeri. mBio The Aspergillus fumigatus transcription factor RglT is 1009 important for gliotoxin biosynthesis and self-protection, and virulence. 1010 PLoS Pathog The birth, evolution and death of metabolic 1013 gene clusters in fungi Hertweck 1016 C. Biosynthesis and function of gliotoxin in Aspergillus fumigatus Gliotoxin--bane or boon? Environ 1020 Opposed effects 1023 of enzymatic gliotoxin N-and S-methylations SreA-mediated iron 1032 regulation in Aspergillus fumigatus Self-1036 protection against gliotoxin-a component of the gliotoxin biosynthetic 1037 cluster, gliT, completely protects Aspergillus fumigatus against exogenous 1038 gliotoxin The Role of Zinc in Gliotoxin Biosynthesis of 1041 Ergothioneine biosynthesis and functionality in 1045 the opportunistic fungal pathogen Histone 1048 deacetylase activity regulates chemical diversity in Aspergillus Genomic and Phenotypic 1053 Analysis of COVID-19-Associated Pulmonary Aspergillosis Isolates of 1054 Epub ahead of print Secondary Metabolite Profiles, and Cards of Virulence 1059 Towards 1066 understanding the gliotoxin detoxification mechanism: in vivo 1067 thiomethylation protects yeast from gliotoxin cytotoxicity. Microb Cell S-adenosyl-methionine-dependent methyltransferases: highly versatile 1072 enzymes in biocatalysis, biosynthesis and other biotechnological 1073 applications Use of the 1076 kojA promoter Aspergillus fumigatus: saprophyte or pathogen? Identification and characterization of genes responsible for biosynthesis of 1086 kojic acid, an industrially important compound from Aspergillus oryzae Involvement of Sulfur 1089 in the Biosynthesis of Essential Metabolites in Pathogenic Fungi of 1090 Molecular and Therapeutic 1091 Implications. Front Microbiol Fungal 1094 metabolite gliotoxin inhibits assembly of the human respiratory burst 36 1095 NADPH oxidase Aspergillus 1098 fumigatus morphology and dynamic host interactions The 1102 Transcription Factor ZafA Regulates the Homeostatic and Adaptive 1103 Response to Zinc Starvation in Aspergillus fumigatus GliA in Aspergillus fumigatus is 1108 required for its tolerance to gliotoxin and affects the amount of extracellular 1109 and intracellular gliotoxin Epidithiodioxopiperazines. occurrence, synthesis and 1112 biogenesis 1115 Perturbations in small molecule synthesis uncovers an iron-responsive 1116 secondary metabolite network in Aspergillus fumigatus Systematic Global Analysis of Genes Encoding Protein Phosphatases in 1121 Aspergillus fumigatus. G3 (Bethesda) Using RNA-Seq Data to Evaluate reference genes suitable for 1124 gene expression studies in soybean A 1126 nonribosomal peptide synthetase-derived iron(III) complex from the 1127 pathogenic fungus Aspergillus fumigatus Sake: Production and flavor Ani ΔrglT strains exposed to GT when compared to the Afu or Ani wild-type Table S1 . Genes significantly differentiallly expressed in the A. 1254 fumigatus wild-type strain exposed to GT in comparison to the GT-free, control 1255 condition.1256 Supplementary Table S2 . Genes significantly differentiallly expressed in the A. 1257 fumigatus ΔrglT strain in comparison to the wild-type strain when exposed to GT.1258 Supplementary Table S3 . Genes significantly differentiallly expressed in the A.1259 nidulans wild-type strain exposed to GT in comparison to the GT-free, control 1260 condition. If applicable, the A. fumigatus homologue is also indicated. Supplementary Table S4 . Genes significantly differentiallly expressed in the A. 1262 nidulans ΔrglT strain in comparison to the wild-type strain when exposed to GT. If applicable, the A. fumigatus homologue is also indicated. Also shown are a list 1264 of genes that are dependent or independent on RglT for their expression.