Vacuoles and peroxisomes are involved in Aspergillus fumigatus gliotoxin production and self-protection

Abstract

Aspergillus fumigatus is a saprophytic fungus that can cause a variety of human diseases known as aspergillosis. Mycotoxin gliotoxin (GT) production is important for its virulence and must be tightly regulated to avoid excess production and toxicity to the fungus. GT self-protection by GliT oxidoreductase and GtmA methyltransferase activities is related to the subcellular localization of these enzymes and how GT can be sequestered from the cytoplasm to avoid increased cell damage. Here, we show that GliT:GFP and GtmA:GFP are localized in the cytoplasm and in vacuoles during GT production. Peroxisomes are also required for proper GT production and self-defense. The Mitogen-Activated Protein (MAP) kinase MpkA is essential for GT production and self-protection, interacts physically with GliT and GtmA and it is necessary for their regulation and subsequent presence in the vacuoles. Our work emphasizes the importance of dynamic compartmentalization of cellular events for GT production and self-defense.

Introduction

Fungal secondary metabolites (SMs) are important genetic traits for fungal survival and fitness in specific niche conditions. The genes that encode SMs are generally organized in biosynthetic gene clusters (BGCs) ¹. SMs are used in signaling, in mechanisms of antagonism, and to cause damage and facilitate the infection in animal and plant hosts, protecting the fungus from host immune cells, or mediating the acquisition of essential nutrients ^2–9. One of the best studied fungal SMs is gliotoxin (GT), a sulfur-containing mycotoxin, a member of the epipolythiopiperazines, produced by different fungal species, including Gliocadium fimbriatum (from which it was originally isolated and named accordingly), Aspergillus fumigatus and closely related non-pathogenic species ^{10, 11}, and also by species of Trichoderma and Penicillium ^12–15. GT and other SMs can also cause toxicity to the producer fungus and mechanisms of self-protection and self-defense genes have evolved ¹⁶, such as: (i) the presence of more resistant target genes, like in the A. fumigatus fumagillin BGC, that has at least five copies of the methionine aminopeptidase, two of them present within the BGC and three in other regions of the genome; (i) presence of efflux transporters, such as the major facilitator superfamily transporter GliA, which belongs to the A. fumigatus GT BGC; and (iii) enzymes that modify the SMs, such as the reversible enzymatic activity of the oxidoreductase GliT that attenuates GT production by producing dithiol-GT (dtGT) with its subsequent conversion to bis(methylthio)gliotoxin (bmGT) by the S-adenosylmethionine-dependent gliotoxin bis-thiomethyltransferase GtmA ¹⁶. Removal of these GT self-defense genes by knock-out causes increased susceptibility to GT ^{15, 17–22}.

A fumigatus is a saprophytic fungus that can cause a variety of human and animal diseases known as aspergillosis ²³, produces GT as an important genetic determinant of virulence ^{24, 25}. GT has already been detected in vivo in murine models of invasive aspergillosis (IA), in human cancer patients ²⁴, and produced by isolates derived from patients with COVID-19 associated pulmonary aspergillosis ²⁵. The GT cluster is located on chromosome VI in A. fumigatus Af293 and contains 13 gli genes responsible for GT biosynthesis and secretion ¹⁵. The biosynthesis of GT is exquisitely regulated since there is crosstalk between different cellular pathways, such as sulfur metabolism [cysteine (Cys) and methionine (Met)], oxidative stress defenses [glutathione (GSH) and ergothioneine (EGT)], methylation [S-adenosylmethionine (SAM)], and iron metabolism (Fe–S clusters) ^26–30. Several signal transduction pathways involving transcription factors (TFs), protein kinases, regulators of G-protein signalling as well as chromatin modifying enzymes are involved in the regulation of GT biosynthesis ¹⁵. In the mammalian host, GT has an immunosuppresive role by: (i) interfering with macrophage-mediated phagocytosis through prevention of integrin activation and actin dynamics, resulting in macrophage membrane retraction and failure to phagocytose pathogen targets ³¹; (ii) inhibiting the production of pro-inflammatory cytokines secreted by macrophages and the activation of the NFkB regulatory complex ³²; (iii) disrupting the correct assembly of NADPH oxidase through preventing p47phox phosphorylation and cytoskeletal incorporation as well as membrane translocation of subunits p47phox, p67phox and p40phox ³³; and (iv) suppressing neutrophil chemoattraction by targeting the activity of leukotriene A4 (LTA4) hydrolase, an enzyme that participates in LTA biosynthesis ³⁴. A. fumigatus GT production and regulation must be tightly regulated to avoid excess production and toxicity to the fungus. GliT produces the final toxic form of GT by catalysing disulphide bridge closure of the precursor dithiol gliotoxin (dtGT) ^{15, 20}. However, if there is excess GT production, GliT can reduce GSH and produce dtGT attenuating GT toxicity ^{15, 20}. GtmA, whose gene is not located in the GT BGC, is able to convert dtGT into bisdethiobis(methylthio)-gliotoxin (bmGT) and to attenuate GT production postbiosynthetically ^{17, 18, 21, 22}. It is thought that the primary role of GtmA is a decrease in GT biosynthesis and not a backup for GliT and toxin neutralization ¹⁵ (https://www.biorxiv.org/content/10.1101/2022.09.28.509882v2). Recently, we have identified the TF RglT as the main regulator of GliT and GtmA through directly binding to their promoter regions during GT-producing and self-protection conditions ³⁵. We also screened an A. fumigatus deletion library of 484 TFs for GT sensitivity and identified 15 TFs important for GT self-protection ³⁶. Of these, KojR, which is essential for kojic acid biosynthesis in Aspergillus oryzae, was also crucial for virulence and GT biosynthesis in A. fumigatus, and for GT protection in A. fumigatus.

An important aspect of the A. fumigatus GT production and self-protection by GliT and GtmA is related to the cellular sub-location of these enzymes and how GT can be temporarily sequestered from the cytoplasm to avoid increased cell damage. It has been speculated that reduced dithiol gliotoxin (dtGT) may be sequestered into intracellular vesicles where it is converted to the oxidized form, by an unidentified activity, prior to release from the cell by an exocytotic mechanism complementary to GliA-mediated efflux ²⁰. Therefore, the aim of this work is to elucidate GliT and GtmA subcellular localization under GT production and self-protection. We demonstrate that both proteins are localized in the cytoplasm and enriched in vacuolar structures during GT production while are present in unidentified structures that resemble the intracellular endomembrane network and/or endocytosis/exocytosis vesicles. We observe that GT is not only secreted by GliA but also through a non-canonical vesicle-driven secretion mechanism. To verify the involvement of peroxisomes on GT self-protection, we characterized the PexE^Pex5 and PexG^Pex7 peroxisome receptors, and how they mediate GT self-protection. We demonstrate that both receptors are involved in the regulation of GT production but only A. fumigatus ΔpexE mutant is GT-sensitive and has attenuated virulence in a murine model of invasive pulmonary aspergillosis (IPA). To gain more information about metabolic pathways involved in the GliT and GtmA regulation, we immunoprecipitated these two proteins under GT producing conditions and identified several regulatory proteins as interacting with GliT and GtmA, among them the mitogen-activated protein kinase MpkA. We demonstrated that ΔmpkA is not able to produce either GT or bmGT and is highly sensitive to GT. MpkA controls positively most of the gli genes mRNA accumulation during either GT production or self-protection. MpkA is required for GliT and GtmA protein levels and for their localization into the vacuoles during GT production. Not only MpkA is involved in GT self-protection but other protein kinases since a screening of a collection of 110 non-essential protein kinase deletion mutants revealed six and one mutants as more GT-susceptible and – resistant, respectively. One of these susceptible mutants corresponds to the sensor histidine kinase SlnA^Sln1 and we demonstrate that SlnA is important for GT production but it is influencing MpkA phosphorylation to the same extent than the wild-type, suggesting SlnA is controlling MpkA-independent pathway(s) involved in GT production. However, SlnA is important for the modulation of the MpkA phosphorylation during GT self-protection, suggesting SlnA is one of the possible receptors involved in the MpkA regulation of GT self-protection.

Results

GliT:GFP and GtmA:GFP are localized in vacuoles, vesicles, endoplasmic reticulum, and in the cytoplasm during GT production and self-defense.

As an initial step to characterize the GliT and GtmA regulation, we characterize their localization under producing (24 to 72 h growth in Czapek-Dox liquid medium) and self-protection (germlings previously grown in GT non-producing liquid medium were exposed to GT 3 μg/mL) conditions. Protein localization with longer time points, such as 48 and 72 hs was technically challenging due to hyphal overgrowth, therefore a timepoint of 24h was selected to conduct the microscopy experiments. After 24 hs in GT-production conditions, GliT:GFP and GtmA:GFP were localized in the cytoplasm and had enriched localization in structures that resemble to vacuoles (Figures 1A and 1B).This was confirmed by co-staining with CellTracker Blue CMAC Dye (7-amino-4-chloromethylcoumarin; https://www.thermofisher.com/order/catalog/product/C2110) which accumulates in the vacuoles (Figures 1A and 1B). GliT:GFP and GtmA:GFP germlings were exposed to GT 3 μg/ml for 2 hours and co-stained with either CMAC or FM4–64 Dye [N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl) Hexatrienyl) Pyridinium Dibromide] (Figures 2A to 2D). FM4–64 is recommended for staining vacuolar membranes and for studying the endocytic pathway ³⁷; https://www.thermofisher.com/order/catalog/product/T13320). Upon GT exposure, GliT:GFP accumulates in the cytoplasm, but not in the GT-free control, without any co-localization with CMCA (Figure 2A) but some with FM4–64 (Figure 2B). GtmA:GFP showed no fluorescence signal in the control but when the germlings were exposed to GT, a large punctuated distribution appeared that did not show any co-localization with CMAC but restricted co-localization with FM4–64 (Figures 2C and 2D). Some spots of GliT:GFP and GtmA:GFP showed some network of strands around the peripheral nuclear envelope as shown by Hoechst co-localization, suggesting these two proteins are also localized in the endoplasmic reticulum (Figures 2C and 2F).

Figure 1 – GliT:GFP and GtmA:GFP have enriched vacuolar localization during GT production.

GliT:GFP (A) and GtmA:GFP (B) germlings were grown in liquid Czapek-dox medium for 24 h at 37 °C. Cell tracker Blue CMAC (CellTracker Blue CMAC Dye (7-amino-4-chloromethylcoumarin) was used for vacuolar staining. Bars, 5 μm.

Figure 2 -. GliT:GFP and GtmA:GFP localize in the cytoplasm, membraneous and small vesicle structures.

GliT:GFP (A, B, and C) and GtmA:GFP (D, E, and F) germlings were grow in liquid minimal medium for 24 h at 37 °C and exposed or not to GT 3 μg/mL for 2 hours and co-stained with either CMAC (7-amino-4-chloromethylcoumarin) or FM4–64 dyes [N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl) Hexatrienyl) Pyridinium Dibromide], or Hoechst. Bars, 5 μm.

Upon GT-production conditions, purified secreted vesicles (50 to 400 nm) contained about 12 % of the total secreted GT and 3 % of the total secreted bmGT (Figures 3A to 3C). Categorization of the proteins identified into these vesicles by mass spectrometry revealed an enrichment for protein binding, amino acid metabolism, translation, and sugar, glucoside, polyol and carboxylate catabolism (Figure 3D). We identified proteins that contribute to the biosynthesis of at least nine secondary metabolites, including GliF, GliN, and GliT(Figure 3E; clusters described by ³⁸.

Figure 3 – A. fumigatus secretes GT and bmGT into vesicles.

(A) A. fumigatus was grown at 37 °C for 72 h in liquid Czapek-dox medium. Vesicles were enriched and classified in a nanoparticle tracking analysis, nanosight. Transmission electron microscopy for enriched vesicles. Bars, 1 μm and 500 nm. (B and C) HR-ESI-MS for GT and bmGT from the wild-type supernatants (SUP) and enriched vesicles (VES). (D) Fungifun categorization of the proteins identified into the vesicles. (E) Proteins that participate in secondary metabolite biosynthesis clusters according to the classification described by Lind et al., (2017).

Taken together these results suggest that GliT:GFP and GtmA:GFP have different protein localizations during the early phase of GT production and GT self-defense. Upon GT-production conditions both proteins accumulated either in the cytoplasm or in structures that resembled to vacuoles. Furthermore, when stimulating GT-production, about one-tenth of GT production was in secreted vesicles, suggesting that in addition to GT transport by the MFS transporter GliA, GT and also bmGT can be secreted through nonconventional secretion in vesicles. In contrast, in GT-defense conditions GliT and GtmA showed cytoplasmatic accumulation and some aggregation in structures that could be related to the intracellular endomembrane network, endoplasmic reticulum, and organelles and/or endocytosis/exocytosis vesicles.

Aspergillus spp peroxisome receptors PexE^Pex5 and PexG^Pex7 are important for GT self-protection.

Peroxisomes are important eukaryotic organelles responsible for detoxification, catabolism of linear and branched-chain fatty acid, and removal of H2O2 by catalases ^39–42. Peroxisomal proteins, such as the peroxin receptors Pex5 and Pex7, have the function to recruit, transport, and introducing the peroxisomal matrix proteins into the peroxisomes ^39–42. The matrix proteins contain the peroxisomal targeting signals PTS1 and/or PTS2 that are recognized by the peroxins Pex5 and Pex7, respectively. PTS1 is a tri-peptide with the consensus sequence (S/A/C)(K/H/R)(L/M) located at the extreme C-terminus, whereas PTS2 is a nona-peptide of the consensus sequence (R/K)(L/V/I)X5(H/Q)(L/A) located near the N-terminus of a matrix protein ^39–42. Many SMs are initially synthesized in the peroxisomes, such as aflatoxin and sterigmatocystin, as biosynthetic steps of many other SM pathways, such as paxilline, AK-toxin, penicillin, cephalosporin and some siderophores ^{16, 43}. Although the GliT:GFP and GtmA:GFP subcellular localization at the vacuoles during GT production is unequivocal, there is a diffuse subcellular location to these proteins upon GT-self protection. Since there are some punctuated distribution of GtmA:GFP (see Figures 2D and 2E), we raised the hypothesis that these proteins could be localized in the peroxisomes upon GT-self defense. Initially, we used several lysosome targeting signal predictors [such as psortII (https://psort.hgc.jp), PTS1 prediction IMP bioinformatics group (https://mendel.imp.ac.at/pts1/) and DeepLoc-1.0 (https://services.healthtech.dtu.dk/service.php?DeepLoc-1.0), aiming to predict if GliT and GtmA could be transported to the lysosomes. The results were ambiguous since only DeepLoc-10 predicts both proteins as being lysosomal (GliT=0.6683 and GtmA=0.4158). We reasoned that the molecular characterization of A. fumigatus Pex5 and Pex7 homologs (here called PexE and PexG, respectively) could help us to assign a possible function for peroxisomes in GT production and detoxification. We deleted both homologs in A. fumigatus and extended this analysis for the GT non-producer A. nidulans that has a homolog for GliT but no homologs for GtmA ³⁶. We have previously demonstrated that GliT in both species is under the control of the transcription factor RglT ³⁵. A. fumigatus ΔpexE mutant showed reduced growth and conidiation, and it is very sensitive to GT (Figures 4A and 4B). In contrast, A. fumigatus ΔpexG has a comparable phenotype to the wild-type strain (Figure 4C). Both A. nidulans ΔpexE and ΔpexG are more sensitive to GT than the wild-type but similarly to A. fumigatus, only ΔpexE has a reduced growth and conidiation phenotypes (Figures 4D to 4F). As previously shown ⁴⁴, A. nidulans ΔpexE mutant have a partial biotin deficiency phenotype (Figure 4D), but this partial auxotrophy is not observed in A. fumigatus ΔpexE mutant (Figure 4A).

Figure 4 – Peroxisomes are required for GT production, self-protection, and virulence.

(A) The A. fumigatus wild-type, ΔpexE, and ΔpexG strains were grown for 5 days at 37 °C on MM and MM+biotin. (B and C) The A. fumigatus wild-type, ΔpexE, and ΔpexG strains were grown for 2 days at 37 °C on MM and MM+30 μg/ml of GT. (D) The A. nidulans wild-type, ΔpexE, and ΔpexG strains were grown for 5 days at 37 °C on MM and MM+biotin. (E and F) The A. nidulans wild-type, ΔpexE, and ΔpexG strains were grown for 2 days at 37 °C on YUU and YUU+5 μg/ml of GT. (G and H) GT and bmGT production in the A. fumigatus wild-type, ΔpexE, and ΔpexG strains. The strains were grown in liquid Czapek-dox medium for 3 days at 37 °C. GT and bmGT were extracted from the supernatants and analysed by mass spectrometry. (I and J) Survival curves (n=10 mice/strain) infected with the indicated A. fumigatus strain and percentage of variation in the body weight. Phosphate buffered saline (PBS) was administered in a negative control group (n=10). The indicated p-values are based on the log-rank, Mantel-Cox, and Gehan-Breslow-Wilcoxon tests comparing the ΔpexE, and ΔpexG mutants with the wild-type strain; *, p < 0.05 and **, p < 0.001. In the percentage of body weight variation, ΔpexE mutants are compared to the wild-type while ΔpexG mutants are compared to the PBS negative control.

A. fumigatus ΔpexE mutants have a reduced GT (about 7.5- to 15-fold reduction; Figure 4G, left graph) and bmGT production (about 15- to 3-fold reduction; Figure 4G, right graph) while surprisingly ΔpexG mutants show a dramatic increase in GT production (about 25- to 50-fold compared to the wild-strain, Figure 4H, left graph) and a bmGT production comparable with wild-type (Figure 4H, right panel). Not only the SMs production is altered in the pex mutants but also several other SMs, such as brevianamide F, fumigaclavine C, pseurotin A, and fumagillin have increased or decreased production in these mutants when compared to the wild-type strain (Table 1). The A. fumigatus ΔpexG mutants are more sensitive to oxidative stress caused by menadione but not to t-butyl hydroperoxide, allyl alcohol, and hydrogen peroxide (Supplementary Figure S1), and have no growth deficiencies when grown on cysteine or methionine as single sulfur sources (Supplementary Figure S1). No growth defects in the ΔpexE mutants exposed to the same growth conditions were observed (Supplementary Figure S1). We also investigated if ΔpexE and ΔpexG could impact A. fumigatus virulence in a chemotherapeutic murine model of IPA. All mice infected with the wild-type and ΔpexG strains died between day 6 and 15 post-infection (p.i.), whereas 40 to 70 % of the mice infected with the ΔpexE survived for the duration of the experiment (Figures 4I and 4J).

Table 1 –

Secondary metabolites (SMs) produced by A. fumigatus wild-type, ΔpexE, and ΔpexG

SMs*	Wild-type	ΔpexE	p<0.05	ΔpexG	p<0.05
Brevianamide F	3.1 ± 0.2e7	2.3 ± 1.6e6	0.00	8.3 ± 0.5e6	0.00
Cycloprostatin A	0.00 ± 0.00	3.0 ± 0.8e6	0.01	5.7 ± 1.9e6	0.02
Cyclotryprostatin A	2.9 ± 1.9e6	0.00 ± 0.00	0.02	0.00 ± 0.00	0.02
Cyclotryprostatin C	0.00 ± 0.00	0.00 ± 0.00	ND	2.9 ± 0.9e8	0.01
Demethoxyfumitremorgin C	4.8 ± 2.1e6	2.8 ± 1.1e6	0.12	8.5 ± 0.7e7	0.00
Fumagillin	4.4 ± 1.0e7	0.00 ± 0.00	0.01	8.7 ± 0.9e7	0.00
Fumigaclavine C	0.00 ± 0.00	2.4 ± 0.4e6	0.00	9.3 ± 2.3e6	0.01
Fumiquinazoline A or B	2.3 ± 0.9e6	6.2 ± 4.8e6	0.15	1.5 ± 0.5e8	0.02
Fumiquinazoline E	3.2 ± 1.5e6	1.7 ± 0.7e6	0.12	8.8 ± 3.9e6	0.06
Fumiquinazoline F or G	1.7 ± 0.4e7	3.2 ± 2.8e7	0.23	5.8 ± 0.5e8	0.00
Fumitremorgin A	0.00 ± 0.00	2.1 ± 1.3e5	0.06	0.00 ± 0.00	ND
Fumitremorgin B	0.00 ± 0.00	0.00 ± 0.00	ND	2.3 ± 0.6e6	0.01
Fumitremorgin C	1.4 ± 0.6e7	1.7 ± 1.1e7	0.03	6.6 ± 1.8e8	0.01
Fungisporin	1.3 ± 0.2e7	0.00 ± 0.00	0.00	7.0 ± 04e6	0.05
Pseurotin A	1.5 ± 0.1e7	5.6 ± 5.2e6	0.05	1.8 ± 0.3e7	0.10
Pseurotin D	0.00 ± 0.00	5.7 ± 1.6e6	0.01	0.00 ± 0.00	ND
Pseurotin F2	0.00 ± 0.00	2.4 ± 2.4e6	0.11	3.1 ± 0.7e6	0.01
Pyripyropene A	4.6 ± 2.3e6	1.4 ± 0.5e6	0.06	2.8 ± 1.7e6	0.17
Pyripyropene E	0.00 ± 0.00	1.6 ± 1.3e6	0.08	1.0 ± 0.4e7	0.03
Pyripyropene F	0.00 ± 0.00	0.00 ± 0.00	ND	1.5 ± 0.8e6	0.04
Pyripyropene N	4.8 ± 0.2e6	1.1 ± 0.5e7	0.08	3.3 ± 1.4e6	0.11
Spirotryprostatin A	4.6 ± 1.8e7	2.0 ± 1.1e7	0.07	2.4 ± 0.3e8	0.00
Trypostatin A	1.2 ± 0.4e7	2.6 ± 1.7e6	0.02	9.7 ± 1.1e6	0.26
Trypostatin B	6.2 ± 1.3e6	6.3 ± 6.5e6	0.49	2.9 ± 0.8e8	0.01

^*The results represent the average of the areas of the chromatograms of three independent biological repetitions ± standard deviation. Statistical analysis was performed by using t-test and comparing the mutants versus the wild-type. The red and green colours represent the SMs that have higher and lower SMs production, respectively, than the wild-type. ND=not determined.

These results strongly indicate that PexE and PexG are important for A. fumigatus GT production and self-protection, and virulence, and also affect the production of other SMs.

GliT and GtmA protein interactions during GT production.

As a preliminary step to understand metabolic pathways involved in the GliT and GtmA regulation, GliT:GFP and GtmA:GFP were immunoprecipitated (IP) during 24, 48, and 72 hs in GT production conditions. During 24 to 72 hs GT production, after subtraction of the proteins of the wild-type strain that were non-specifically immunoprecipitated, 133 proteins that interact with GliT:GFP and 203 proteins with GtmA:GFP were identified (Figure 5A). Seventy-one proteins were found to be in association with both GliT:GFP and GtmA:GFP while 62 and 132 were unique for GliT:GFP and GtmA:GFP, respectively (Figure 5A). Functional categorization (FunCat) (https://elbe.hki-jena.de/fungifun/fungifun.php) analyses (P-value < 0.05) of all significantly IP proteins showed enrichment for (i) GliT:GFP in purine nucleotide/nucleoside anabolism, pyridoxal phosphate binding, amino acid metabolism, and stress response; (ii) GtmA:GFP in receptor mediated signaling, purine nucleotide/nucleoside anabolism, NAD/NADP binding, amino acid metabolism, and stress response; and (iii) both GliT:GFP and GtmA:GFP in pyridoxal phosphate binding, purine/nucleoside binding, amino acid metabolism, and stress response (Figures 5B to 5D and Supplementary Table S1).

Figure 5 – Identification of proteins that interact with GliT:GFP and GtmA:GFP during GT production.

A. fumigatus was grown for 24, 48, and 72 h at 37 °C in liquid Czapek-dox medium. Proteins were extracted and immunoprecipitated (IP) with anti-GFP antibody. (A) Venn diagram showing the number of unique and shared IP proteins identified by mass spectrometry as interacting with GliT:GFP and/or GtmA:GFP. (B, C and D) Fungifun categorization of proteins interacting with GliT:GFP, GtmA:GFP, and GliT:GFP and GtmA:GFP. (E and F) Co-IPs for GliT:GFP, GtmA:GFP, GliT:GFP MpkA:3xHA, GtmA:GFP MpkA:3xHA and MpkA:3xHA strains.

Among all the GliT:GFP and GtmA:GFP IP proteins, of particular interest were the other enzymes in the gli pathway, such as GliN and GliG (in either GliT and/or GtmA), and GtmA IP by GliT but not GliT IP by GtmA (Table 2 and Supplementary Table S1). Several protein kinases and phosphatases were also pulled down including including, Mitogen-Activated protein kinases (MAPK), MpkA, MpkB, and SakA, two calcium/calmodulin-dependent protein kinases, and GskA (either IP by GliT:GFP and/or GtmA:GFP), and protein phosphatases, such as PphB and PtcB (IP by GtmA:GFP). Considering the impact of oxidative stress and glutathione metabolism in the conversion from GT to dtGT (and subsequently to bmGT), it is worth noting the IP of glutathione peroxidase, lactoylglutathione lyase, cystathionine beta-lyase (that plays a role in “de novo” L-methionine biosynthesis process), peptide-methionine(S)-S-oxireductase, and FhpA, a flavohemoprotein (Table 2).

Table 2 –

Proteins that interact with A. fumigatus GliT:GFP and GtmA:GFP under GT production.

Accession	Description
	Gliotoxin pathway
Afu6g09740	GliT, gliotoxin sulfhydryl oxidase required for gliotoxin biosynthesis	24, 48, and 72 h, GliT and 48 and 72 h, GtmA
Afu6g09720	GliN, methyltransferase, encoded in the putative gliotoxin biosynthetic gene cluster	48 h, GliT and 48, 72 h GtmA
Afu6g09690	GliG, glutathione S-transferase encoded in the gliotoxin biosynthetic gene cluster	48 h, GtmA
Afu2g11120	GtmA, methyltransferase activity	24, 48, and 72 h GtmA
	Protein kinases
Afu4g13720	MpkA, mitogen-activated protein kinase (MAPK)	48 and 72 h, GliT and 48 h, GtmA
Afu6g12820	MpkB, mitogen-activated protein kinase (MAPK)	72 h, GliT
Afu1g12940	SakA, mitogen-activated protein kinase (MAPK)	24 h, GliT and 24 h and 48 h, GtmA
Afu2g13680	Calcium/calmodulin-dependent protein kinase	48 h, GtmA
Afu2g03490	Calcium/calmodulin-dependent protein kinase	48 h, GliT and 48 h, GtmA
Afu6g05120	GskA, protein serine/threonine kinase activity	48 h, GliT and 48, 72 h GtmA
	Protein phosphatases
Afu6g10830	PphB, protein serine/threonine phosphatase activity	48 h, GtmA
Afu1g09280	PtcB, type 2C protein phosphatase (PP2C) involved in dephosphorylation of SakA MAP kinase	24 and 48 h, GtmA
	Heat-shock proteins
Afu3g14540	hsp30, 30-kilodalton heat shock protein	24 h, GliT and 48 h GtmA
Afu5g13920	Wos2, Hsp90 binding co-chaperone	72 h, GliT
	Glutathione metabolismo
Afu3g12270	Glutathione peroxidase; peroxiredoxin	72 h, GliT
Afu6g07940	Lactoylglutathione lyase	24 h, GliT
	Miscellaneous
Afu2g02780	Ortholog(s) have mRNA binding activity, role in negative regulation of MAPK cascade and cytosol localization	48 h, GliT
Afu2g03140	Peptide-methionine (S)-S-oxide reductase activity	48 h, GtmA
Afu3g09320	Serine hydroxymethyltransferase	48 h, GliT and 48 h, GtmA
Afu4g03410	FhpA, flavohemoprotein; protein induced by heat shock and hipoxia	48 h, GliT
Afu4g03950	Cystathionine beta-lyase activity, role in ‘de novo’ L-methionine biosynthetic process	48 h, GliT and 48 h, GtmA
Afu4g11720	Phosphatidyl synthase; protein levels increase in response to pkaC overexpression	72 h, GliT
Afu6g08360	Thiazole biosynthesis enzyme; hypoxia induced protein; induced by gliotoxin exposure	72 h, GliT and 72 h GtmA

Taken together these results suggest that GliT and GtmA are interacting with several protein kinases and phosphatases and enzymes involved in the glutathione metabolism and oxidative stress response during GT-production.

MAP kinase MpkA is important for GT production and self-defense.

Considering that A. fumigatus MpkA has previously been described as being involved in GT regulation and production ⁴⁵, we decided to validate and investigate the role played by this MAPK in GliT and GtmA regulation. To further support our identification of GliT:GFP and GtmA:GFP as interactors with MpkA, we carried out a co-immunoprecipitation analysis using epitope-tagged forms of these two proteins. A functional 3xHA-tagged form of MpkA was introduced into these strains (Supplementary Figure S2). Isogenic GliT:GFP and GtmA:GFP strains either containing or lacking the MpkA:3xHA allele were grown under GT production conditions or grown overnight and exposed to 5 μg/mL of GT for 30 min, 1 h, and 3 h, native protein extracts were prepared, and the MpkA:3xHA protein was recovered by IP with anti-mouse HA antibody. These anti-HA immunoprecipitations were electrophoresed on SDS-PAGE and then analyzed by Western blotting using anti-HA antibodies (Figure 5E). Only when both the MpkA:3xHA and the GliT:GFP and GtmA:GFP fusions were present was co-immunoprecipitation seen. When GliT:GFP was IP by using anti-GFP and revealed by anti-HA, under GT production conditions, there is surprisingly only seen in 24 h production and under GT self-protection this is seen in all four time points including the control (Figure 5E). When GtmA:GFP was IP by using anti-GFP and revealed by anti-HA, under GT production conditions there is not any band (Figure 5). Under GT self-protection, MpkA:3xHA is seen in all four time points including the control (Figure 5E). Expression of only the GliT:GFP or GtmA:GFP fusion proteins did not show any evidence for nonspecific recovery of this factor by the anti-HA antibody. By using Net-Phos-3.1 (https://services.healthtech.dtu.dk/service.php?NetPhos-3.1), we were able to predict three putative threonine p38MAPK phosphorylation sites at residues 278, 297, and 303 of GliT but no significant phosphorylated residues at GtmA. The functionality of these putative phosphorylation sites remains to be investigated. These data strongly support the view that GliT and MpkA associate in vivo during GT production and GT self-protection while GtmA associates only during GT self-protection.

The ΔmpkA has a strong growth defect and its complementation by mpkA⁺ restores this phenotype to the wild-type growth (Figure 6A). The ΔmpkA mutant does not produce either GT or bmGT (Figure 6B). Since other MAP kinases (like SakA and MpkC, Table 2) were shown to be interacting either with GliT and/or GtmA, we also investigated the GT production in the corresponding null mutants, including another MAPK mutant, ΔmpkB, and the double mutant ΔsakA ΔmpkC. All the mutants showed comparable GT and bmGT production with the wild-type, except for the ΔsakA ΔmpkC mutant that has reduced bmGT production (Figure 6B). The ΔmpkA mutant has reduced growth in liquid medium in the presence of GT (70 μg/mL) when compared to the wild-type and complemented strains as quantified by Alamar blue (Figure 6C). The MpkA phosphorylation is increased during GT production and self-protection (Figure 6D). The GliT:GFP and GtmA:GFP protein accumulation are highly expressed from 24 to 72 h while both proteins have increased expression when the strains are exposed to GT (5 μg/mL) for 3 h; however, the GliT expression is much higher than GtmA:GFP (Figure 6D). The ΔmpkA has reduced mRNA accumulation of most of the gli genes during GT production and self-protection (except for gliF, gliT, gliJ, and gtmA) (Figures 6E and 6F). We have not observed any differences in vacuolar formation in the ΔmpkA when compared to the wild-type strain (Supplementary Figure S3). We hypothesized the MpkA could affect the GliT:GFP and GtmA:GFP protein levels and subsequent translocation to the vacuoles. Functional ΔmpkA GliT:GFP and ΔmpkA GtmA:GFP strains showed reduced GliT:GFP and GtmA:GFP levels under GT production and vacuolar translocation frequencies (Figure 6D and Supplementary Figure S3).

Figure 6 – The mitogen-activated protein kinase (MAPK) MpkA is essential for GT production and self-protection.

(A) The wild-type, ΔmpkA and ΔmpkA::mpkA⁺ strains were grown for 5 days at 37 °C in solid minimal medium. (B) The wild-type and the MAPK mutants ΔsakA, ΔmpkC, ΔsakA ΔmpkC, ΔmpkA, and ΔmpkB were grown for 72 hours at 37 °C in liquid Czapek-dox medium, secondary metabolites of the supernatant extracted and GT and bmGT identified (*, p < 0.05). All the results are the average of three repetitions ± standard deviation. (C) Metabolic activity expressed by Alamar blue of A. fumigatus grown for 48 hours in the absence or presence of different concentrations of GT (*, p < 0.05). All the results are the average of three repetitions ± standard deviation. (D) Western blot analysis for GT production and self-protection. GliT:GFP and GtmA:GFP were identified by anti-GFP antibody, MpkA-P and total MpkA were identified by using P-p44/42 and p44/42 antibodies while actin by anti-actin antibody. For GT production A. fumigatus was grown under the same conditions described in (B) while for GT self-protection A. fumigatus was grow in liquid minimal medium for 24 h at 37 °C and exposed or not to GT 3 μg/mL for 2 hours. (E and F) RTqPCR for genes of the GT pathway and gtmA. (G) Western blot analysis for GT production GliT:GFP and GtmA:GFP were identified by anti-GFP antibody while actin by anti-actin antibody. (H) RTqPCR for rglT and kojR.

Recently, we identified two transcription factors, RglT and KojR, that were demonstrated to be essential for the regulation of GliT and GtmA, and GT production ^{35, 36}. The ΔmpkA has reduced and increased mRNA accumulation of rglT during GT production and self-protection, respectively, while kojR has decreased mRNA accumulation at 72 hours GT production but no differences to the wild-type during GT self-protection (Figure 6G).

Taken together these results indicate that MpkA is essential for GT production and self-protection, positively regulating gliT and gtmA mRNA and protein accumulation during GT production and negatively regulating during GT self-protection. Moreover, MpkA positively and negatively regulates rglT mRNA accumulation during GT-production and self-protection, respectively.

Screening of protein kinases involved in gliotoxin self-defense.

Aiming to identify additional protein kinases (PK) involved in gliotoxin self-protection, we performed a screening looking for differences in growth in the presence of MM+GT compared to MM by quantifying radial growth susceptibility. For this screening, 109 PK mutants ⁴⁶ (Supplementary Table S2) were grown on solid MM and MM+30 μg/mL of GT. In the primary screening we identified 11 PK mutants (5 and 6 with less growth and more growth, respectively) (Figure 7A). Further validation screening was performed by growing these selected PK mutants in liquid MM in the absence and presence of GT 30 μg/mL and the metabolic activity was quantified by using Alamar blue (Figure 7B). Upon Alamar blue validation, we identified four PK mutants with decreased radial growth in the presence of GT (Figure 7B): AFUB_010510 (Δkin1, regulates polarized exocytosis and the Ire1p-mediated unfolded protein response), AFUB_017740 (ΔslnA, transmembrane histidine phosphotransfer kinase and osmosensor; regulates MAP kinase cascade), AFUB_059390 (Δhrk1, implicated in activation of the plasma membrane H(+)-ATPase Pma1p in response to glucose metabolism), and AFUB_070630 (ΔmpkA). Six mutants (AFUB_077790, AFUB_081540, AFUB_030570, AFUB_011380, AFUB_044400, and AFUB_074550) had increased growth in the presence of GT in both screenings but were not validated in the Alamar blue assay and were not considered here for further analysis.

Figure 7 – Screening of the protein kinase null mutant library for GT susceptibility.

(A) Growth screening. (B) Metabolic activity expressed by Alamar blue of A. fumigatus grown for 48 hours in the absence or presence of 30 μg/mL of GT (*, p < 0.05; **, p < 0.01, and ***, p < 0.001). All the results are the average of three repetitions ± standard deviation. (C) GT and bmGT production in the A. fumigatus wild-type and ΔslnA strains. The strains were grown in liquid liquid Czapek-dox medium for 3 days at 37 °C. GT and bmGT were extracted from the supernatants and analysed by LC-MS. (D and E) Western blot analysis for GT production and self-protection. Western blot analysis for GT production and self- protection. GliT:GFP and GtmA:GFP were identified by anti-GFP antibody. MpkA-P and total MpkA were identified by using P-p44/42 and p44/42 antibodies while actin by anti-actin antibody. For GT production A. fumigatus was grown under the same conditions described in (C) while for GT self-protection A. fumigatus was grow in liquid minimal medium for 24 h at 37 °C and exposed or not to GT 3 μg/mL for 0.5 to 3 hours. (F) RTqPCR for gliT and gtmA under GT production and self-protection conditions, as described in (D and E).

We decided to further characterize slnA mutant because we have previously demonstrated that several kinds of stress, such as osmotic, cell wall, and oxidative stresses could impact ΔslnA growth ⁴⁷. The ΔslnA mutant has less GT and similar bmGT production than the wild-type strain, respectively (Figure 7C). MpkA phosphorylation in the ΔslnA mutant has comparable levels to the wild-type during GT production (Figure 7D) but increased levels during GT protection (about 3-fold at 1 h exposure to 5 μg/mL GT, Figure 7E). The ΔslnA mutant has higher gliT and gtmA mRNA accumulation upon GT production than the wild-type (Figure 7F). Upon GT self-protection, gliT mRNA accumulation is lower and higher at 0.5 and 1 h exposure to 5 μ/mL GT than the wild-type strain, respectively (Figure 7F); gtmA mRNA accumulation is also lower in the ΔslnA mutant upon GT self-protection conditions than the wild-type (Figure 7F).

Taken together, these results suggest the SlnA is important for the modulation of MpkA phosphorylation, affecting the GT production levels and GT protection.

Discussion

We can envisage the following main non-exclusive mechanisms of SM self-defense in fungi (adapted from ^{1, 48}): (i) secretion of toxic compounds in the extracellular milieu, (ii) sequestration of toxic compounds into vacuoles, (iii) modification of toxic compounds into inactive forms, and (iv) increased copy number of SM targets. GT is an important SM for A. fumigatus virulence and pathogenicity. GT production induces the modulation of several metabolic pathways, such as sulfur assimilation and transsulfurylation pathways, oxidative stress defenses, methylation, and iron metabolism aiming to cope with GT biosynthesis and self-protection (for reviews, see ^{12, 15, 22, 49}). The two main mechanisms of GT detoxification are canonical transport through the GliA, a MFS transporter and GT modification through the action of two enzymes responsible for GT detoxification, GliT oxidoreductase and GtmA methyltransferase ¹⁵. There was some speculation in the literature if dtGT might be sequestered into intracellular vesicles and converted by an exocytotic mechanism complementary to GliA-mediated efflux ²⁰. It is technically very challenging to identify the subcellular location of either dtGT, bmGT or GT. However, it is possible to assess the subcellular localization of the two major players in the conversion of GT-dtGT-bmGT, GliT and GtmA, in the production of these compounds by constructing functional GFP strains. Our work provided several lines of evidence suggesting that part of the cellular supply of dtGT, bmGT and GT could be stored into vacuoles and/or vesicles during GT production: (i) GliT:GFP and GtmA:GFP are localized into vacuoles and in the cytoplasm; (ii) about 12 and 3 % of the total GT and bmGT, respectively, are secreted noncanonically as vesicles; and (iii) we were able to identify GliF (encoding a cytochrome P450 monooxygenase), GliN (encoding a methyltranferase), and GliT, but not GtmA, in the secreted vesicles. GliF catalyzes the previous biosynthetic intermediate before GliN, and GliN catalyzes this intermediate conversion to dtGT ⁵⁰.

Short GT exposure displayed a more complex and diffuse pattern of GliT:GFP and GtmA:GFP subcellular localization in the cytoplasm, vesicles, endomembraneous compartments, and endoplasmic reticulum. Interestingly, GliT and GtmA are not observed as localized in the vacuoles during self-protection upon short exposure times (like 2 h exposure to 3 μg/mL GT). Longer exposures to the same concentration of GT, such for instance 8 and 16 hours, did not allow us to be assertive about a possible colocalization in the vacuoles since the levels of GliT:GFP and GtmA:GFP expression were high and massively distributed along the whole cytoplasm. It is possible the small vesicles observed populated by GliT:GFP and GtmA:GFP are derived from the endoplasmic reticulum. Since the fungus is exposed to a high GT concentration, part of the free GT is immediately transported to these vesicles, metabolized or not to dtGT and bmGT, and secreted through a noncanonical way. In both cases, production and self-protection, it remains to be determined how GT is transported to the vesicles and/or vacuoles. Recently, we identified two transporter encoding genes (a MFS transporter, AN1472/AFUA_8G04630, and an ATP-binding cassette transporter, AN7879/AFUA_1G10390) that are RglT-dependent and positively modulated at transcriptional level upon A. fumigatus and A. nidulans exposure to GT ³⁶. It remains to be demonstrated if these transporters are located into the vacuole cell membranes and if they could be possible candidates for GT transport to the vacuoles.

Fungal peroxisomes are organelles specialized in both anabolism and catabolism with very important functions for cell protection by detoxifying and sequestering reactive oxygen species ^{43, 51}. Acetyl CoA generation by β-oxidation of fatty acids and the enzymes isocitrate lyase and malate synthase specific to the glyoxylate cycle for the capture of acetyl CoA for gluconeogenesis or to provide intermediates for the TCA cycle within mitochondria are also present in the peroxisomes ^{43, 51}. Several enzymatic steps for diverse fungal SMs, such penicillin, aflatoxin, and sterigmatocystin, occur within the peroxisomes (for a review, see ⁴³). We were not able to demonstrate the colocalization of GliT:GFP and GtmA:GFP with peroxisomal targeted RFP (mRFP:PST1). However, we investigated the influence of peroxisomes on GT production and self-protection by characterizing A. fumigatus pexE and pexG deletion mutants. Interestingly, while ΔpexE produces very low levels of GT and bmGT and is very sensitive to GT, the ΔpexG mutant is as sensitive to GT as the wild-type and has increased production of GT. These results suggest that both receptors play a role in GT production and self-protection. What could be the peroxisomal mechanisms influencing GT production and self-protection? We detected increased sensitivity of ΔpexG to menadione that could suggest peroxisomes are required for the optimal oxireduction cellular environment necessary for GT production and self-protection. We were not able to assign any other phenotype that could impact GT self-protection in these mutants, such as sulfur source utilization. Interestingly, PexE and PexG are also important for self-defense in A. nidulans, a non GT producer. Our results indicate an important role for peroxisomes in GT production and self-defense, and virulence.

Fungal MAPK pathways are essential for the regulation of several cellular processes and different kinds of stress ^{52, 53, 54, 55, 56}. The central module for each MAPK has three protein kinases: a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK), and a MAPK. MAPK cascades are usually stimulated by upstream sensors, such as cell membrane receptors, and its final output is the activation of downstream elements, such as cytoplasmic proteins and transcriptional regulators ^{52, 53, 54, 55}. A. fumigatus has four MAPK: MpkA, which mainly regulates cell wall integrity ⁵⁶; MpkC and SakA, similar to Saccharomyces cerevisiae Hog1, which are involved in the response to oxidative and osmotic stresses ^{57, 58, 59} and MpkB, homologous to yeast Fus3 and involved in melanin production ^{60, 61}. We have demonstrated that GliT and GtmA are physically interacting with the MAPKs SakA, MpkB, and MpkA during GT production and self-protection. The sakA, mpkC, and double sakA mpkC null mutants have comparable GT levels than the wild-type. The ΔmpkA mutant is not able to produce either GT or bmGT, and is more sensitive to GT. Interestingly, MpkA affects the GliT and GtmA RNA and protein levels and its subsequent translocation to the vacuoles. Previously, it has been shown that A. fumigatus MpkA is also involved in the control of the production of several SMs, such as melanin, pseurotin A, siderophores, and GT ^{45, 61}. However, the mechanism of how MpkA regulates these SMs has not been previously investigated. By using co-IPs, we have validated MpkA interaction with GliT and GtmA but it is yet to be investigated if GliT and GtmA are directly interacting with MpkA or through other MpkA-associated proteins and in this case which residues are phosphorylated by MpkA. We also observed GliT and GtmA physical interactions with other protein kinases and phosphatases, such as calcium/calmodulin-dependent protein kinases, GskA kinase, and PphB and the MAPK-dependent PtcB phosphatases. It remains to be determined the roles played by these enzymes for the regulation of GT production and self-protection.

In a screening performed with a genome-wide collection of A. fumigatus non-essential PK mutants, we have also identified other PKs besides MpkA as important for GT self-defense. One of these kinases, the histidine kinase SlnA, was previously extensively characterized in our laboratory and we have not observed striking phenotypes in this mutant apart from a reduction of its radial diameter of about 15 % when compared to the wild-type strain ⁴⁷. SlnA is the homolog of S. cerevisiae Sln1, a two-component system that controls the branch of the yeast HOG pathway ^{57, 62, 63}. The typical organization of a two-component system consists of the following components: (i) a sensor histidine kinase (SHK) that contains an input (or sensor) domain, an HK catalytic domain, and a histidine autophosphorylation site, and (ii) a response regulator (RR) that contains a receiver (REC) domain and an output (or effector) domain ⁶⁴. The sensor domain is modified by a stimulus, a histidine close to the HK domain is phosphorylated (or dephosphorylated), and this phosphoryl group is transferred to the REC domain of the RR ⁶⁴. We observed that SlnA is important for the modulation of the MpkA phosphorylation during GT self-protection but not GT production, and slnA null mutant has lower GT production than the wild-type. These results indicate that SlnA is one of the sensors that activate GT self-protection via MpkA phosphorylation. Since GT production is intimately related to GT self-production it is possible the reduction of GT production in the slnA null mutant is due to a reduction of GliT and GtmA activities, and consequently low GT accumulation.

In summary, our work demonstrates another mechanism of GT self-protection through a possible GT-dtGT-bmGT storage in the vacuolar system while part of GT-bmGT is further directly secreted into vesicles by noncanonical secretory mechanisms. Surprisingly, we observed a function for peroxisomes in the GT production and self-protection. Further work will focus on the mechanisms how GT is transported into vacuoles, GliT and GtmA are partially localized into the vacuoles, and peroxisomes influence GT production and self-protection.

Methods

Strains and media.

All strains used in this study are listed in Supplementary Table S3. Strains were grown at 37 °C. Conidia of A. fumigatus and A. nidulans were grown on complete medium (YG) [2% (w/v) glucose, 0.5% (w/v) yeast extract, trace elements] or minimal media (MM) [1% (w/v) glucose, nitrate salts, trace elements, pH 6.5]. Solid YG and MM were the same as described above with the addition of 2% (w/v) agar. When necessary, uridine and uracil (1.2 g/L) were added. Trace elements, vitamins, and nitrate salt compositions were as described previously ⁶⁵. Gliotoxin production was induced by growing the strains in Czapek-Dox (http://himedialabs.com/TD/M076.pdf) broth. For phenotypic characterization, plates were inoculated with 10⁴ spores per strain and left to grow for 120 h at 37°C. Radial growth experiments were expressed as ratios, dividing colony radial diameter of growth in the stress condition by colony radial diameter in the control (no stress) condition.

Microscopy.

For microscopic analyses of GFP fluorescence under gliotoxin self-protection condition, the strains were grown on coverslips in 4 mL of MM for 14 h at 30°C. After incubation, the coverslips with adherent germlings were left untreated or treated with for gliotoxin (Sigma Aldrich, St. Louis, USA) at a final concentration of 3ug/mL for 2 h. Gliotoxin production was induced by growing the strains in Czapek-Dox broth for 24 h. CMAC (CellTracker Blue CMAC - Molecular Probes) staining of vacuoles was performed by adding 10 μM CMAC dye to the cultures for 15 min, at 30°C; and Hoechst 33342 (Molecular Probes, Eugene, OR, USA) (12μg/mL), 10 min, was used to stain the nuclei. Subsequent to the staining, the coverslips were rinsed with PBS and mounted for examination. Slides were visualized on a Carl Zeiss Observer Z1 fluorescence microscope using the excitation wavelength of 450 to 490 nm, and emission wavelength of 500 to 550 nm. DIC (differential interference contrast) images and fluorescent images were captured with an AxioCam camera (Carl Zeiss) and processed using AxioVision software (version 4.8).

Extracellular vesicles isolation and characterization.

Strains were grown in Czapek-Dox broth for 72 h. The EVs isolation was performed as previously described ^{66, 67}. The supernatant from fungal culture was concentrated using Amicon ultrafiltration system (100kDa cutoff, Millipore, Billerica, MA, USA), and the samples were centrifuged at 15,000 × g for 15 min to remove debris. The supernatant was harvested and ultracentrifuged at 100,000 × g for 1 h. The pellet was resuspended in nuclease free water (Sigma Aldrich, St. Louis, USA) and filtered through 0.45μm syringe filter (Corning, Germany). All isolation process was performed at 4°C. The EVs characterization was performed by Nanoparticule Tracking Analysis (NanoSight NS300 - Malvern Instruments, Malvern, United Kingdom). The EVs samples were prepared as previously described ⁶⁸, and the EVs profile (size, concentration and distribution) were obtained by NanoSight software (version 3.2.16).

Transmission electron microscopy (TEM) analysis of extracellular vesicles.

The isolated extracellular vesicles were immediately fixed in 0.1M sodium phosphate buffer (pH 7.4) containing 2.5% (v/v) of glutaraldehyde and 2% (w/v) of paraformaldehyde for 2 h at 4°C. Samples were encapsulated in agar (2% w/v) and subjected to fixation (1% OsO4), contrasting (1% uranyl acetate), ethanol dehydration, and a two-step infiltration process with Spurr resin (Electron Microscopy Sciences) of 16 h and 3 h at RT. Additional infiltration was provided under vacuum at RT before embedment in BEEM capsules (Electron Microscopy Sciences) and polymerization at 60°C for 72 h. Semithin (0.5-μm) survey sections were stained with toluidine blue to identify the areas of best cell density. Ultrathin sections (60 nm) were prepared and stained again with uranyl acetate (1%) and lead citrate (2%). Transmission electron microscopy (TEM) images were obtained using a Philips CM-120 electron microscope at an acceleration voltage of 120 kV using a MegaView3 camera and iTEM 5.0 software (Olympus Soft Imaging Solutions GmbH).

Gliotoxin and bmGT extraction and analysis by High Performance Liquid Chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS).

The strains ΔmpkA, ΔmpkB, A1163, ΔsakA ΔmpkC, ΔmpkC, ΔsakA, ΔpexE, ΔpexG, GtmA:GFP, GliT:GFP, vesicles of GtmA:GFP, GliT:GFP and WT (1 mL) were grown in Czapek-Dox broth for 72 h as this medium has previously been shown to result in detectable gliotoxin levels in culture supernatants ²⁰. For extraction, the cultures were submitted to liquid-liquid partition with 15 mL, 300 mL and 300 μL of chloroform, respectively, for three times. Organic fractions were washed with a saturated solution of NaCl and dried with anhydrous Na₂SO₄. The suspensions were filtered and concentrated under vacuum. The instrumentation for the LC-MS used was the Shimadzu Nexera XR LC-20AD (Kyoto, Japan) chromatography model, consisting of CBM-20A control, SPD-M20A DAD and ELSD-LTII evaporative light scattering detectors, Nexera SIL-20A auto injector, CTO-20A oven, using reversed phase C18 column (Ascentis, 2.7 μm, 100 × 4.6 mm, 35 °C) with gradient of aqueous (0.1% acetic acid) and acetonitrile (10% to 100% of acetonitrile) for 35 minutes. LabSolutions software (Shimadzu Corporation, Kyoto, Japan) was used for data acquisition and analysis.

Determination of SMs produced by the wild-type and pex mutants.

The samples used in the Liquid Chromatogrophy-High Resolution Mass Spectrometry (LC-HRMS) analyses were prepared from the extracts obtained for the different A. fumigatus strains grown for 5 days at 37 °C in liquid MM. Each sample (supernatant) was prepared from 100 mg of extract, which were dissolved in HPLC grade methanol. SMs were extracted for 90 min in an ultrasonic bath at room temperature. Then, filtration was performed through a 0.22 μm sterile syringe filter and transferred to 2 mL HPLC vials.

Analysis LC-HRMS were performed in an UHPLC-MS/MS - Thermo Q Exactive Orbitrap Mass Spectrometers (MS) - Dionex UltiMate 3000 RSLCnano System, operating in positive mode. LC analyses were performed in a C18 (100 mm × 2.1 mm × 2.6 mm; Thermo) column. The gradient was initiated with 5% B mobile phase (0.1% formic ácid in ACN), which was maintained for 5 min, followed by linearly increasing to 40% B within 5 min, then increased to 45% B in 2 minutes, then up to 98% B in 18 min and held for 2 min. Finally, the phase was changed to 95% A (0.1% formic acid) in 2 minutes and maintained until 24 min. Mass spectrometry analysis was performed in full scan, with a scan range from m/z 100 to 1500 Da. MS/MS fragmentation spectra were acquired from the five most intense ions per scan. The injection volume was 5mL.

Extracts from A. fumigatus mutants were identified using the GNPS database (http://gnps.ucsd.edu). The GNPS Feature-based Molecular Networking (FBMN) analysis was performed following the protocol already established on the website (https://ccms-ucsd.github.io/GNPSDocumentation/featurebasedmolecularnetworking/). MS/MS spectra were selected with only the top five fragmentation ions in the ± 5 ppm window. The mass tolerance of precursor ions and MS/MS fragment ions were adjusted to 0.02 Da in both cases. The spectral libraries used for this study were pre-established according to the features table generated by MZMine 3 (http://mzmine.github.io/). Correspondences between network spectra and libraries were filtered to show values greater than 0.7 (Cosine Score). The results obtained for this study are available at: https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=ea7a4f8507f5433db1a6bc50fb9ca67a. The SMs annotations were classified according to ⁶⁹. Annotation Level I, confirmation of structure by comparing the MS/MS profile with reference standards; Annotation Level II, through the comparison of MS/MS fragments with correspondence spectra present in the GNPS and other databases; Annotation Level III, candidates who presented structural evidence through in silico generated MS and MS/MS fragments; Annotation Level IV, annotated SMs in which it was possible to determine the molecular formula unequivocally through the information contained in the LC-HRMS analyses. In this study the simulated spectra used were generated by SIRIUS 5.6.3 ⁷⁰ and the input data were automatically compared and classified against databases present in SIRIUS 5.6.3 ⁷¹.

Finally, the areas corresponding to the annotated ions were calculated manually, as a way of determining their production in the different mutants of A. fumigatus, using the software Thermo Xcalibur 3.0.63 (Copyright 1988–2013 Thermo Fisher Scientific Inc.).

Generation of A. nidulans and A. fumigatus mutants.

All gene replacement cassettes were constructed by “in vivo” recombination in S. cerevisiae, as previously described by ⁷². For construction of A. nidulans and A. fumigatus pex5 and pex7 null mutants, approximately 1.0 kb from each 5-UTR and 3-UTR flanking region of the targeted ORFs regions were selected for primer design (P1 to P16). The primers gene_pRS426_5UTR_fw and gene_pRS426_3UTR_rv contained a short homologous sequence to the MCS of the plasmid pRS426. Both the 5- and 3-UTR fragments were PCR-amplified from the genomic DNA of A. nidulans AGB551 strain or A. fumigatus CEA17, pyrG^™ strain. The pyrG gene placed within the cassette as a prototrophic marker was amplified from pCDA21 ⁷³ plasmid using the primers P17/P18. The cassette was PCR-amplified from these plasmids utilizing TaKaRa Ex Taq^™ DNA Polymerase (Clontech Takara Bio) and used for A. nidulans and A. fumigatus transformation. Southern blot analysis was performed to confirm the deletions (Supplementary Figure S4). To generate the MpkA:linker-3xHA-trpC-pyrG fusion fragment, a 2.6 Kb portion of DNA consisting of the 5-UTR region and mpkA ORF, along with a 1 Kb segment of DNA consisting of the 3-UTR flanking region were amplified with primers P19/P20 and P21/P22, respectively, from CEA17 gDNA. The 2.7 kb 3xHA - trpC - pyrG fusion was amplified with primers OZG916/OZG964 from the pOB430 plasmid. For the cassettes GtmA:linker-GFP-trpC-prtA and GliT:linker-GFP-trpC-prtA the fragments 5-UTR + ORF and 3-UTR (1Kb) were also PCR amplified from CEA17 gDNA with primers P23 to P30. The linker-GFP-trpC fragment was amplified from the pOB435 plasmid with primers P31/P32, and the prtA gene was amplified from the plasmid pPTRI with primers P33/P34. Cassettes were generated by transforming each fragment along with the plasmid pRS426 cut with BamHI/EcoRI into the S. cerevisiae strain. The DNA plasmid of the transforming bacteria was extracted, cassettes were PCR-amplified from these plasmids utilizing TaKaRa Ex Taq^™ DNA Polymerase, which were subsequently transformed into the background of the CEA17, pyrG⁻ (for construction of the MpkA:HA::pyrG) and MpkA:HA::pyrG (for construction of the double-tagged strains: MpkA:HA::pyrG, GtmA:GFP::prtA or MpkA:HA::pyrG, GliT:GFP::prtA). Mutants were selected on MM or MM supplemented with 1 μg/mL pyrithiamine and confirmed with PCR. Primer sequences are listed in Supplementary Table S4.

Western blot analysis.

Total cellular protein extractions were carried out as described previously ⁷⁴, and quantified using Bradford reagent (Bio-Rad), according to manufacturer’s instructions. Fifty μg of protein from each sample were resolved in a 12% (w/v) SDS–PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore). GFP-tagged strains were detected using 1:5,000 dilution of the mouse monoclonal GFP antibody (Santa Cruz Biotechnology) and secondary antibody anti-mouse IgG HRP conjugate (Cell Signaling Technology), at 1:10,000 dilution. For the HA-tagged proteins detection, a mouse monoclonal anti-HA antibody (Sigma) was used at 1:5,000 dilution as a primary antibody, followed by the same anti-mouse IgG HRP conjugate as a secondary antibody. The phosphorylated fractions of the MAP kinase, MpkA, were examined using anti-phospho p44/42 MAPK antibody (Cell Signaling Technologies) following the manufacturer’s instructions using a 1:2,000 dilution. The primary antibody was detected using an HRP-conjugated secondary antibody raised in rabbit (Sigma). Chemoluminescent detection was achieved using an ECL Prime Western Blot detection kit (GE HealthCare). To detect these signals on blotted membranes, the ECL Prime Western Blotting Detection System (GE Helthcare, Little Chalfont, UK) and LAS1000 (FUJIFILM, Tokyo, Japan) were used.

Murine model of pulmonary aspergillosis.

Wild-type BALB/c female mice, body weight 20 to 22 g, aged 8–9 weeks, were kept in the Animal Facility of the Laboratory of Molecular Biology of the School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo (FCFRP/USP), in a clean and silent environment, under normal conditions of humidity and temperature, and with a 12 h light and dark cycle. The mice were given food and water ad libitum throughout the experiments. The procedures adopted in this study were performed in accordance with the principles of ethics in animal research and were approved by the Committee on Ethics in the Use of Animals (CEUA) of the FCFRP/USP (Permit Number: 08.1.1277.53.6; Studies on the interaction of Aspergillus fumigatus with animals) from the University of São Paulo, Campus of Ribeirão Preto.

Mice were immunosuppressed with cyclophosphamide (150 mg per kg of body weight), which was administered intraperitoneally on days -4, -1, and 2 prior to and post infection. Hydrocortisonacetate (200mg/ kg body weight) was injected subcutaneously on day -3. A. fumigatus strains were grown on YAG for 2 days prior to infection. Fresh conidia were harvested in PBS and filtered through a Miracloth (Calbiochem). Conidial suspensions were spun for 5 min at 3,000 × g, washed three times with PBS, counted using a hemocytometer, and resuspended at a concentration of 5.0 × 10⁶ conidia/ ml. The viability of the administered inoculum was determined by incubating a serial dilution of the conidia on YAG medium, at 37°C. Mice were anesthetized by halothane inhalation and infected by intranasal instillation of 1.0 × 10⁵ conidia in 20 μl of PBS. As a negative control, a group of 10 mice received PBS only. Mice were weighed every 24 h from the day of infection and visually inspected twice daily. The statistical significance of comparative survival values was calculated by Prism statistical analysis package by using Log-rank (Mantel-Cox) Test and Gehan-Brestow-Wilcoxon tests.

GFP or HA-Tag Protein Purification and Identification by LC-MS/MS.

To precipitate GFP/HA-tag labeled strains, protein crude extracts were prepared from cultures grown for 24, 48 and 72 h in Czapek Dox or in MM followed by gliotoxin treatment (5 μg / mL). Crude protein extracts from mycelia were obtained by extraction from ground mycelia with B250 buffer (250 mM NaCl, 100 mM Tris–HCl pH 7.5, 10% glycerol, 1 mM EDTA and 0.1% NP-40) supplemented with 1.5 mL/L 1 M DTT, 2 tablets/100 mL complete-mini protease inhibitor cocktail EDTA-free (Roche), 3 mL/L 0.5 M Benzamidine, 10 ml/L phosphatase inhibitors 100× (10 M NaF, 5 M Na Vanadate, 8 M β- glycerol phosphate), and 10 mL/L 100 mM PMSF. Extracts were centrifuged at 13,000 g for 20 minutes at 4°C, and the supernatant was collected into a new eppendorf. The same amount of protein for each sample was added to 20 μl of Magnetics GFP-trap beads or to Dynabeads Protein A (Thermo Fisher Scientific) previously incubated with monoclonal anti-HA antibody (Sigma). Cell extracts and beads were incubated with shaking at 4 °C for 4 h. After incubation, the magnetics beads were collected using magnetic hack and washed according to manufacturer’s instructions. To release the proteins from the beads, samples were incubated with sample buffer and boiled at 98 °C for 5 min. Western Blot assays was carried out as described above. For the LC-MS/MS identification, the washed beads were re-suspended in 50 mM ammonium bicarbonate solution. Proteins were reduced with 10 mM DTT (DL-Dithiothreitol–Sigma-Aldrich) for 20 min at 56°C, alkylated with 40 mM iodoacetamide (Sigma-Aldrich) for 15 min at room temperature in the dark, and digested with trypsin (Promega) in the ratio 1:50 (μg trypsin/μg protein) at 37°C overnight. The digestion was stopped by addition of trifluoroacetic acid (TFA) to reach a final concentration of 1% and then the sample was desalted with C18 columns (StageTips).

RNA extraction, cDNA synthesis and RT-qPCR.

All experiments were carried out in biological triplicates, and conidia (10⁷) were inoculated in the liquid culture medium [Czapek Dox ou MM (with or without gliotoxin treatment)]. For total RNA isolation, mycelia were ground in liquid nitrogen, and total RNA was extracted using TRIzol (Invitrogen), treated with RQ1 RNase-free DNase I (Promega) and purified using the RNAeasy kit (Qiagen) according to the manufacturer’s instructions. RNA was quantified using a NanoDrop. For RT-qPCR, the RNA was reverse transcribed to cDNA using the ImProm-II reverse transcription system (Promega) according to manufacturer’s instructions, and the synthesized cDNA was used for real-time analysis using the SYBR green PCR master mix kit (Applied Biosystems) in the ABI 7500 Fast real-time PCR system (Applied Biosystems, Foster City, CA, USA). Sybr Primer sequences are listed in Supplementary Table S4. Afu2g02680 (Putative matrix AAA protease) gene was used as normalizers ³⁶.

Data availability statement.

All the data are available as supplementary tables and figures. Proteomic data was deposited at ProteomeExchange (https://www.proteomexchange.org) under the accession number PXD041133.