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1 Running head: Integrated systems in Aspergillus fumigatus. Interplay between Gliotoxin Resistance, Secretion and the Methyl/Methionine Cycle in Aspergillus fumigatus. Rebecca A. Owens 1, Grainne O Keeffe 1, Elizabeth B. Smith 1, Stephen K. Dolan 1, Stephen Hammel 1, Kevin J. Sheridan 1, David A. Fitzpatrick 1, Thomas M. Keane 2, Gary W. Jones 1,* and Sean Doyle 1,*. 1 Department of Biology, Maynooth University, Maynooth, Co. Kildare, Ireland. 2 The Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK. *Joint Corresponding Authors Professor Sean Doyle & Dr Gary W. Jones, Department of Biology, Maynooth University, Maynooth, Co. Kildare, Ireland. Tel: ; Fax: ; sean.doyle@nuim.ie Tel: ; Fax: ; gary.jones@nuim.ie Web: 1

2 Table S1. Aspergillus fumigatus strains used in the study. Strain Reference A. fumigatus ATCC A. fumigatus ATCC A. fumigatus Af293 Nierman et al. (1) A. fumigatus gliz Af293.1 Bok et al. (2) A. fumigatus metr AfS167 Amich et al. (3) A. fumigatus glit ATCC26933 Schrettl et al. (4) A. fumigatus glit ATCC46645 Schrettl et al. (4) A. fumigatus glik ATCC26933 Gallagher et al. (5) A. fumigatus glik ATCC46645 Gallagher et al. (5) A. fumigatus glig ATCC26933 Davis et al. (6) A. fumigatus gtma ATCC26933 Dolan et al. (7) A. fumigatus gtma:gtma ATCC26933 (gtma C ) Dolan et al. (7) A. fumigatus glia ATCC26933 This work A. fumigatus glia:glia ATCC26933 (glia C ) This work A. fumigatus gliz: glit Af293.1 This work A. fumigatus gliz: glit:: gliz Af293.1 This work 2

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Figure S1 A. DIG ATCC26933 ΔgliA Ladder ATCC26933 Figure S1: A. Schematic representation of glia deletion and complementation strategy. B. PCR confirmation of glia presence (1629 bp) in A.

8 Figure S1 A. DIG ATCC26933 ΔgliA Ladder ATCC26933 Figure S1: A. Schematic representation of glia deletion and complementation strategy. B. PCR confirmation of glia presence (1629 bp) in A. fumigatus gliac. Expression analysis via RT-PCR of glia expression from cdna derived from wild-type, glia and gliac. C. glit expression is significantly elevated in A. fumigatus glia comapred to both wild-type and gliac. D. Generation of A. fumigatus ΔgliZ:ΔgliT and confirmation of glit deletion in A. fumigatus ΔgliZ293 (35) by Southern blot analysis. E. Reconstitution of gliz in A. fumigatus ΔgliZ:ΔgliT and confirmation by Southern blot analysis. F. RT-PCR confirms that gliz expression is restored in A. fumigatus ΔgliZ:ΔgliT::gliZ. G. Gliotoxin sensitivity of A. fumigatus AF293, gliz, glit, gliz:glit and gliz: glit::gliz. gliz complementation in A. fumigatus ΔgliZ:ΔgliT resulted 8 in wild-type like resistance to gliotoxin, possibly due to unexpected trans effects of this Zn(2)Cys(6) binuclear transcription factor.

9 Figure S1 B. (i) PCR (ii) RT-PCR L glia C L ΔgliA WT glia C 1629 bp 9

10 Figure S1 C. glit expression 10

11 Figure S1 D. DIG Ladder AF293 ΔgliZ ΔgliT ΔgliZΔgliT bp bp

12 Figure S1 E. DIG Ladder AF293 ΔgliZ ΔgliZΔgliT ΔgliZΔgliT::gliZ bp 3584 bp 2898 bp

13 Figure S1 F. AF293 ΔgliZ ΔgliZΔgliT ΔgliZΔgliT::gliZ Neg. ctrl gdna 186 bp AF293 ΔgliZ ΔgliZΔgliT ΔgliZΔgliT::gliZ Neg. ctrl gdna 617 bp 348 bp 13

14 Figure S1 G. [Gliotoxin] (µg/ml) Key: AF293 ΔgliZ ΔgliT::gliZ ΔgliZ ΔgliT ΔgliZ ΔgliT 14

Figure S2 A. ATCC46645 0 0.05 ΔmetR 1 5 mm Met Gliotoxin (0 µg/ml) Gliotoxin (20 µg/ml) Figure S2: A. Phenotypic analysis for A.

15 Figure S2 A. ATCC ΔmetR 1 5 mm Met Gliotoxin (0 µg/ml) Gliotoxin (20 µg/ml) Figure S2: A. Phenotypic analysis for A. fumigatus ΔmetR sensitivity to gliotoxin (0-20 µg/ml) on AMM at 37 C for 72 h. A. fumigatus ΔmetR was supplemented with Met ( mm). Compared to wild-type, A. fumigatus ΔmetR was significant sensitivity to gliotoxin at concentrations > 10 µg/ml, regardless of Met concentration. B. qrt-pcr analysis of glit expression in A. fumigatus ATCC46645 and ΔmetR following 3 h gliotoxin or MeOH exposure. Significantly attenuated expression (p < 0.001) of glit in A. fumigatus ΔmetR is observed in response to gliotoxin. C. Phenotypic analysis of sensitivity to simultaneous gliotoxin and cystathionine exposure in ΔmetR grown on AMM at 37 C for 72 h. Gliotoxin (10 µg/ml) significantly sensitizes A. fumigatus ΔmetR to cystathionine (1.5 mm). 15

16 Figure S2 A. % Growth * * 100 ATCC metr (0.05 mm Met) metr (1 mm Met) metr (5 mm Met) Gliotoxin (µg/ml) 16

17 17 with MeOH with Gliotoxin Figure S2 B *** ATCC46645 metr Relative Ratio

18 Figure S2 30 ** ATCC46645 metr C. Radial Growth (mm) Cystathionine (mm) with 10 g/ml gliotoxin Cystathionine (mm)/gliotoxin (µg/ml) 0/10 0.5/10 1.0/10 1.5/10 ATCC46645 ΔmetR 18

19 Figure S3 GT CH6 GT CH2 gliz CH 3 gtma MtrA CH 3 -THF Hcy SahA 2 SAH CH 3 CH 2 -THF Methyl Cycle MetH Methionine Cycle GtmA NADH, CO 2, NH 3 GdcA THF NAD +, Gly Met ATP SasA 2 SAM PPi+Pi GliT NADP / O 2 ( ) Gliotoxin Biosynthetic Pathway Cell membrane/wall Intracellular [R]? GliA Extracellular 19 CH 3 CH 3

20 Figure S3. Integration of gliotoxin biosynthesis, self-protection and regulatory mechanisms into primary metabolism via gliotoxin bis-thiomethyltransferase (GtmA) activity. Exogenous, and endogenous, gliotoxin induces gli cluster (CH6; chromosome 6) and gtma (AFUA_2G11120 (CH2)) expression, and de novo gliotoxin biosynthesis (2, 4, 7, 8). Gliotoxin biosynthesis involves GliG-mediated bis-glutathionylation of an acyl imine intermediate (6) to form a bis-glutathionylated compound, which is subsequently processed via GliK, and additional enzymes to yield GT-(SH) 2. GliT oxidizes GT-(SH) 2 to gliotoxin followed by GliA-mediated secretion, or alternatively to attenuate gliotoxin biosynthesis, GtmA converts GT-(SH) 2 to BmGT, with concomitant consumption of 2 SAM molecules. Resultant SAH must be re-converted to SAM via the action of the methyl/methionine cycle. Absence of GliT activity, results in GT-(SH) 2 flux via GtmA to BmGT, which results in both SAM depletion and SAH overproduction. Thus, the negative regulation of gliotoxin biosynthesis via GtmA must be countered-balanced by GliT activity to avoid dysregulation of methyl/methionine cycle, SAM depletion and trans consequences of same on global cellular biochemistry in A. fumigatus. The differential proteomic impact of exogenous gliotoxin exposure on the GliT-mediated self-protection system of A. fumigatus in both A. fumigatus glig (no affect) and glik (attenuated), respectively, suggests that either BmGT presence, or interference with GSH homeostasis, also plays a role in enabling gliotoxin biosynthesis or self-regulation. 20

21 References 1. Nierman W.C., Pain A., Anderson M.J., Wortman J.R., Kim H.S. et al Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature 438: Bok J.W., Chung D., Balajee S.A., Marr K.A., Andes D. et al GliZ, a transcriptional regulator of gliotoxin biosynthesis, contributes to Aspergillus fumigatus virulence. Infect Immun 74: Amich J., Schafferer L., Haas H., Krappmann S Regulation of sulphur assimilation is essential for virulence and affects iron homeostasis of the human-pathogenic mould Aspergillus fumigatus. PLoS Pathog 9: e Schrettl M., Carberry S., Kavanagh K., Haas H., Jones G.W. et al Self-protection against gliotoxin-a component of the gliotoxin biosynthetic cluster, GliT, completely protects Aspergillus fumigatus against exogenous gliotoxin. PLoS Pathog 6:e Gallagher L., Owens R.A., O Keeffe G., Dolan S.K., Schrettl M. et al The Aspergillus fumigatus Protein GliK Protects Against Oxidative Stress and is Essential for Gliotoxin Biosynthesis. Eukaryot Cell 11: Davis C., Carberry S., Schrettl M., Singh I., Stephens J.C. et al The role of glutathione S- transferase GliG in gliotoxin biosynthesis in Aspergillus fumigatus. Chem Biol 18: Dolan S.K., Owens R.A., O Keeffe G., Hammel S., Fitzpatrick D.A. et al Regulation of Nonribosomal Peptide Synthesis: Bis-thiomethylation Attenuates Gliotoxin Biosynthesis in Aspergillus fumigatus. Chem Biol 21: O' Keeffe G., Hammel S., Owens R. A., Keane T. M., Fitzpatrick D. A., Jones G. W., Doyle S RNA-seq Reveals the Pan-Transcriptomic Impact of Attenuating the Gliotoxin Self-Protection Mechanism in Aspergillus fumigatus. BMC Genomics 15:

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