Spt20, a Structural Subunit of the SAGA Complex, Regulates Aspergillus fumigatus Biofilm Formation, Asexual Development, and Virulence

ABSTRACT

The exopolysaccharide galactosaminogalactan (GAG) plays an important role in mediating adhesion, biofilm formation, and virulence in the pathogenic fungus Aspergillus fumigatus. Previous work showed that in A. fumigatus, the Lim domain-binding protein PtaB can form a complex with the sequence-specific transcription factor SomA for regulating GAG biosynthesis, biofilm formation, and asexual development. However, transcriptional coactivators required for biofilm formation in A. fumigatus remain uncharacterized. In this study, Spt20, an orthologue of the subunit of the Saccharomyces cerevisiae transcriptional coactivator Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, was identified as a regulator of biofilm formation and asexual development in A. fumigatus. The loss of spt20 caused severe defects in the GAG biosynthesis, biofilm formation, conidiation, and virulence of A. fumigatus. RNA sequence data demonstrated that Spt20 positively regulates the expression of the GAG biosynthesis genes uge3 and agd3, the developmental regulator medA, and genes involved in the conidiation pathway. Moreover, more than 10 subunits of the SAGA complex (known from yeast) could be immunoprecipitated with Spt20, suggesting that Spt20 acts as a structural subunit of the SAGA complex. Furthermore, distinct modules of SAGA regulate GAG biosynthesis, biofilm formation, and asexual development in A. fumigatus to various degrees. In summary, the novel biofilm regulator Spt20 is reported, which plays a crucial role in the regulation of fungal asexual development, GAG biosynthesis, and virulence in A. fumigatus. These findings expand knowledge on the regulatory circuits of the SAGA complex relevant for the biofilm formation and asexual development of A. fumigatus.

IMPORTANCE Eukaryotic transcription is regulated by a large number of proteins, ranging from sequence-specific DNA-binding factors to transcriptional coactivators (chromatin regulators and the general transcription machinery) and their regulators. Previous research indicated that the sequence-specific complex SomA/PtaB regulates the biofilm formation and asexual development of Aspergillus fumigatus. However, transcriptional coactivators working with sequence-specific transcription factors to regulate A. fumigatus biofilm formation remain uncharacterized. In this study, Spt20, an orthologue of the subunit of the Saccharomyces cerevisiae Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, was identified as a novel regulator of biofilm formation and asexual development in A. fumigatus. The loss of spt20 caused severe defects in galactosaminogalactan (GAG) production, conidiation, and virulence. Moreover, nearly all modules of the SAGA complex were required for the biofilm formation and asexual development of A. fumigatus. These results establish the SAGA complex as a transcriptional coactivator required for the biofilm formation and asexual development of A. fumigatus.

INTRODUCTION

Aspergillus fumigatus is an opportunistic mold causing invasive infections in immunosuppressed patients (1). Despite antifungal treatment with the currently available antifungal agents, the mortality rate for invasive aspergillosis (IA) ranges between 50 and 95% (2, 3). One strategy used by A. fumigatus to establish and maintain pulmonary infection is the production of biofilms during invasive infection in immunocompromised individuals and airway infection in patients with chronic lung disease (4, 5). Recently, the key role of the exopolysaccharide galactosaminogalactan (GAG) has been established in both the formation of A. fumigatus biofilms and modulation of the immune response during invasive infection (6–10). GAG is a linear heteropolymer composed of α-1,4-linked galactose, N-acetylgalactosamine (GalNAc), and galactosamine (GalN) that is bound to the outer cell wall and found within the extracellular matrix of biofilms of Aspergillus species (6, 11). A cluster of five genes has been predicted to encode enzymes with carbohydrate-synthetic or -modifying capacity of GAG (12). It has been reported that proteins involved in the regulation of conidiation (including StuA, MedA, SomA, and PtaB) are involved in GAG regulation (9, 13–15). Among these, the Lim-binding protein PtaB can form a complex with the transcription factor SomA to govern the expressions of medA and stuA as well as the expressions of the GAG biosynthesis genes agd3 and uge3 (14, 16). Recently, we reported that the sequence-specific transcription factor SomA could directly bind to a conserved motif on the promoter regions of GAG biosynthesis-related genes to activate transcription (16). Eukaryotic transcription is regulated by a large number of proteins, ranging from sequence-specific DNA-binding factors and chromatin regulators to the general transcription machinery and their regulators (17–21). However, transcriptional coactivators (chromatin regulators or the general transcription machinery) working with sequence-specific transcription factors to regulate GAG production and biofilm formation in A. fumigatus remain uncharacterized.

The Spt-Ada-Gcn5-acetyltransferase (SAGA) complex is a well-known transcriptional coactivator that is highly conserved in all eukaryotes (22, 23). It plays multiple roles in regulating transcription because of the presence of functionally independent subunit modules. The SAGA complex can be organized into at least four modules with distinct activities in yeast. These include the histone acetyltransferase (HAT) module, the histone deubiquitylation (DUB) module, the TATA-binding protein (TBP)-binding module (a module for direct interaction with the transcriptional activator), and a structural module (23–25). Although the general structure and function of these modules are likely conserved, differences may exist in their composition and function among different species.

The structural module of SAGA comprises Spt7, Spt20, and Ada1 (26). Saccharomyces cerevisiae ySpt20 (also called Ada5) was first described as a suppressor of Ty element transposition that alters the initiation of correct transcription (27, 28). ySpt20 was later identified as a bona fide subunit of ySAGA, where it plays a role in the structural integrity of the complex (29). In an spt20 deletion mutant in budding yeast (29) and spt20 knockdown HeLa cells (30), only a partial SAGA complex is formed. The biological function of Spt20 is commonly linked with the transcriptional activity of the SAGA complex. In yeast, Spt20 is required for the transcriptional elongation of PDR5 and the activation of seripauperin genes upon hypoxic stress exposure (31, 32). Human Spt20 participates in the regulation of endoplasmic reticulum (ER) stress-induced genes (30). However, Schizosaccharomyces pombe Spt20 displayed both transcription-dependent and -independent roles in septin ring assembly (33). In Candida albicans, Spt20 is involved in virulence and is essential for hypha and biofilm formation (34). Until now, no A. fumigatus homologue of ySpt20 has been identified, and its putative role in transcriptional coactivation has not been uncovered.

In this study, A. fumigatus Spt20, an orthologue of S. cerevisiae Spt20, was identified as a regulator of biofilm formation through coimmunoprecipitation (Co-IP) experiments using PtaB-green fluorescent protein (GFP) as a bait. RNA sequence (RNA-seq) data showed that Spt20 positively regulates the expression of the GAG biosynthetic genes uge3 and agd3, the developmental regulator medA, and genes involved in the conidiation pathway. Moreover, more than 10 subunits of the SAGA complex could be immunoprecipitated with Spt20, suggesting that in A. fumigatus, Spt20 acts as a structural subunit of the SAGA complex. Furthermore, it could be confirmed that nearly all modules, including the HAT module, the TATA box-binding module, and the core module, of the SAGA complex were involved in the biofilm formation and asexual development of A. fumigatus to various degrees. In summary, this work establishes the roles of the SAGA complex as a coactivator required for biofilm formation in A. fumigatus.

RESULTS

Spt20 is required for biofilm formation and conidiation in A. fumigatus.

Our previous work showed that the Lim domain-binding protein PtaB is involved in the regulation of biofilm formation (15). Lim domain-binding proteins are assumed to provide scaffolds for the interaction between regulatory proteins and transcription factors. To identify novel transcription factors or activators that may work with PtaB to regulate the biofilm formation of A. fumigatus, PtaB-GFP trap assays were performed using wild-type (WT) A. fumigatus as a negative control. The recruited proteins were analyzed by high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteins shared in biofilm growth and/or under planktonic growth conditions, while being absent in negative controls, were assumed to represent hits (see Fig. S1 in the supplemental material). The detailed LC-MS data are shown in Table S1. Furthermore, candidates among which functions are known or predicted to play a role in transcription regulation were prioritized. A total of five transcription factors or activators (Table S1) were obtained, including the transcription factor SomA (AFUB_088810), which has previously been reported to form a complex with PtaB and govern A. fumigatus biofilm formation (14). To explore the roles of the other four candidate genes in biofilm formation, gene deletion mutants were constructed. The loss of spt20 (AFUB_015950), encoding a predicted subunit of the SAGA transcriptional regulatory complex in S. cerevisiae, resulted in serious defects in conidiation and biofilm formation, phenocopying the ptaB and somA mutants (Fig. 1A and B). No apparent differences were found in the colony morphology and biofilm biomass of the ΔAFUB_022210, ΔAFUB_073640, and ΔAFUB_090650 mutants compared with the parental WT strain (Fig. 1A and B). To overcome the defect of conidiation in the spt20 deletion mutant, a Tet-spt20 strain was constructed in which the expression of spt20 could be conditionally controlled by the addition of doxycycline (Dox) to the medium (Fig. 1C). Consistent with the phenotype of the Δspt20 mutant, in the absence of doxycycline, the Tet-spt20 (off) mutant exhibited a fluffy phenotype accompanied by a drastic decrease in growth rates compared with the WT strain (Fig. 1C). In comparison, the addition of 1 μg/mL doxycycline to the medium allowed WT-like radial growth and conidiation of the Tet-spt20 strain (on). In addition, biofilm formation by the Tet-spt20 (off) mutant was significantly decreased compared with the biofilm formation of the WT or Tet-spt20 (on) strain, which was similar to the biofilm defects observed in the ΔptaB and Tet-somA (off) mutants (Fig. 1D).

FIG 1.

Spt20 is required for biofilm formation and conidiation in A. fumigatus. (A) Colony morphology of the wild-type (WT) and ΔAFUB_022210, ΔAFUB_073640, ΔAFUB_090650, Δspt20, Tet-somA, and ΔptaB mutant strains after 2 days of growth at 37°C on minimal medium (MM). (B) Formation of adherent biofilms of the indicated strains on tissue-culture-treated polystyrene surfaces. Biofilms were visualized by staining with crystal violet. (C) Phenotypes of the WT and Tet-spt20 strains cultured on MM supplemented with or without 1 μg/mL doxycycline (Dox). Colony morphology was imaged after 48 h. (D) Quantitative determination of biofilm formation of the WT, Tet-somA, ΔptaB, and Tet-spt20 strains using the crystal violet assay. Results indicate the means and standard deviations from three independent biological experiments, each with six technical repetitions (***, P < 0.001; NS, not significant).

A Co-IP assay was performed to verify whether PtaB and Spt20 are interaction partners, and a strain expressing the PtaB-GFP and Spt20-FLAG fusion proteins was constructed. Unexpectedly, the Co-IP assay failed to prove that PtaB physically interacts with Spt20, which was probably caused by the low expression level of the Spt20-FLAG fusion protein (data not shown). In addition, the results of yeast two-hybrid (Y2H) studies provided evidence of a direct PtaB interaction with SomA; however, direct interactions between PtaB and Spt20 were not detected (Fig. S2). In summary, the above-described results demonstrate that Spt20 is required for the biofilm formation and conidiation of A. fumigatus. However, the precise relationship between Spt20 and PtaB requires further exploration.

Spt20, PtaB, and SomA regulate overlapping and unique targets.

To further explore the regulatory relationships among PtaB, Spt20, and SomA, their downstream targets were investigated by RNA-seq analysis of the WT, Tet-spt20, Tet-somA, and ΔptaB strains. RNA-seq identified 1,704 (755 up- and 949 downregulated), 1,193 (665 up- and 528 downregulated), and 2,279 (1,195 up- and 1,084 downregulated) differentially expressed genes (fold change [FC] of >2; P < 0.05) in the Tet-somA, ΔptaB, and Tet-spt20 mutant strains, respectively (Table S2). Principal-component analysis (PCA) confirmed that each of the four samples showed high reproducibility of the obtained data, and three of the mutants exhibited greater variability in gene expression than the WT (Fig. 2A). Samples of Tet-somA and ΔptaB mutants were relatively tightly clustered, indicating a far higher similarity in gene expression between these two mutants (Fig. 2A). In comparison, higher variability in gene expression was observed between the Tet-spt20 and Tet-somA or ΔptaB mutants under the same conditions. These results suggest that SomA, rather than Spt20, functions closely with PtaB with respect to downstream gene expression.

FIG 2.

Spt20, SomA, and PtaB share common and unique transcriptional profiles. (A) Principal-component analysis was performed to explore the gene expression relationships among samples of the WT, ΔptaB, Tet-somA, and Tet-spt20 strains. (B) Venn diagram showing the distribution of differentially expressed genes (fold change of >2; P < 0.05) in the ΔptaB, Tet-somA, and Tet-spt20 mutant strains. (C) Hierarchical cluster analysis of the common targets shared in the indicated mutant strains. FPKM, fragments per kilobase per million. (D and E) KEGG enrichment tables of upregulated (D) and downregulated (E) genes in the common targets of the ΔptaB, Tet-somA, and Tet-spt20 mutant strains. P values and gene numbers are represented using gradients of color and bubble size, respectively. MAPK, mitogen-activated protein kinase.

Comparative analysis of the Tet-somA, ΔptaB, and Tet-spt20 transcriptome data sets showed that SomA, PtaB, and Spt20 shared 461 differentially expressed genes (Fig. 2B). Among these, 162 genes were upregulated and 239 genes were downregulated in all three mutant strains. Hierarchical clustering analysis identified a subset of SomA/PtaB target genes that are also dependent on Spt20 for WT transcript levels (Fig. 2C). To identify the potential roles of the common transcriptional profiles in specific fungal processes, these genes were subjected to pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG). The results showed that included among the 162 upregulated genes in the Tet-spt20, Tet-somA, and ΔptaB mutants were genes encoding proteins involved in arginine and proline metabolism, phenylalanine metabolism, as well as chloroalkane and chloroalkene degradation (Fig. 2D). In comparison, the 239 downregulated genes in the Tet-spt20, Tet-somA, and ΔptaB mutants encoded proteins involved in amino sugar and nucleotide sugar metabolism, a key pathway providing precursors for the biosynthesis of cell wall and GAG polysaccharides (Fig. 2E). In summary, the above-described data indicate that Spt20, PtaB, and SomA regulate overlapping and distinct targets, and a subset of SomA/PtaB target genes involved in amino acid metabolism and GAG polysaccharide biosynthesis also depends on Spt20 for normal transcription.

Spt20 regulates the expression of GAG biosynthesis-related genes.

The RNA-seq results indicated that Spt20 positively regulates the GAG biosynthesis-related genes uge3 and agd3 (Table S2). Real-time quantitative PCR (RT-qPCR) analysis confirmed that the expressions of these GAG biosynthesis genes were dependent on Spt20 (Fig. 3A). Interestingly, the expressions of gtb3 and ega3 had slightly increased (P < 0.05) in the Tet-spt20 (off) mutant compared with the expression in the WT or Tet-spt20 (on) strain (Fig. 3A). The role of Spt20 in GAG production was confirmed by GAG-specific fluorescein isothiocyanate-tagged soybean agglutinin lectin (SBA-FITC) staining of fungal hyphae (Fig. 3B). Scanning electron microscopy (SEM) of the hyphal surface also showed that the loss of spt20 resulted in a significant decrease in hyphal surface decorations associated with GAG production (Fig. 3C).

FIG 3.

Spt20 governs GAG biosynthesis. (A) RT-qPCR analysis of the relative expression levels of the galactosaminogalactan (GAG) gene cluster in the WT and Tet-spt20 (on and off) strains after 24 h of growth in MM. Gene expression was normalized to the endogenous reference gene tubA. Results represent data from three independent biological experiments (**, P < 0.01). (B) Cell wall-associated GAG production of the indicated strains as visualized by FITC-conjugated soybean agglutinin lectin staining (green) using DRAQ5 as a counterstain (red). Bar, 10 μm. (C) Scanning electron micrographs of hyphae of the indicated strains after 24 h of growth in RPMI 1640. (D) RT-qPCR analysis of the relative expression levels of GAG regulators in the WT and Tet-spt20 (on and off) strains after 24 h of growth in MM. Gene expression was normalized to the endogenous reference gene tubA. Results represent data from three independent biological experiments (**, P < 0.01; NS, not significant). (E) RT-qPCR analysis of the relative expression levels of medA in the indicated strains. (F) Biofilm formation of the WT, Tet-spt20, and OEmedA::Tet-spt20 strains as determined by a crystal violet assay. Results indicate the means and standard deviations from three independent biological experiments, each with six technical repetitions (***, P < 0.001; NS, not significant).

Previous research demonstrated that GAG biosynthesis is regulated by the transcription factors StuA and MedA (9, 13). Therefore, the role of Spt20 in the expression of these GAG regulators was explored. The expression of medA, but not stuA, was significantly reduced in the Tet-spt20 (off) mutant compared with the WT or Tet-spt20 (on) strain (Fig. 3D). To test if the reduced medA expression was the cause of the loss of biofilm formation, a mutant strain was constructed in which medA was overexpressed in the background of the Tet-spt20 mutant. The overexpression of medA completely restored biofilm formation by the Tet-spt20 (off) mutant (Fig. 3E and F), suggesting that Spt20 mediates its effects on biofilm formation by modulating MedA expression. In comparison, RT-qPCR results showed that the expression level of somA or ptaB in the Tet-spt20 (off) mutant was similar to the expression level in the WT or Tet-spt20 (on) strain and vice versa (Fig. 3D and Fig. S3). In summary, these data suggest that Spt20 is a required element for the transcriptional machinery governing GAG biosynthesis.

Spt20 is essential for asexual development of A. fumigatus.

In addition to their role in regulating biofilm formation, Spt20, PtaB, and SomA are required for asexual development. The spt20 deletion mutant exhibited a fluffy phenotype with a complete loss of conidiation (Fig. 4A and B). To explore this phenotype in greater detail, strains were inoculated on minimal medium (MM) agar-coated microscope slides and incubated for 36 or 48 h at 37°C. After 36 h of growth, the WT and spt20-complemented strains showed initiation of conidiophore development and expressed mature conidiophores by 48 h. As reported previously (15), the ΔptaB mutant produced a minimal amount of conidia with poorly developed conidiophores even after 48 h of incubation (Fig. 4A and B). The Δspt20 strain exclusively formed aerial hyphae and was incapable of forming conidiophores over the entire culture period (Fig. 4A). The defect in conidiophore formation of the Δspt20 mutant was similar to that of the Tet-somA (off) strain.

FIG 4.

Spt20 is essential for asexual development of A. fumigatus. (A) Colony and conidiophore morphologies of the WT, Tet-somA (off), ΔptaB, Δspt20, and Δspt20-complemented strains grown on MM. Colony morphology was imaged after 48 h. (B) Total conidial production of the indicated strains from panel A. Results are expressed as average numbers of conidia from three repetitions ± standard deviations (****, P < 0.0001; ND, not detectable). (C) Heat map depicting the relative expression levels of select conidiation-related genes from the RNA-seq results in the Δspt20 mutant and WT strains. Gene expression is reported relative to that of the WT after 24 h of growth in liquid MM. Results represent data from two independent biological experiments.

To further explore the potential downstream targets of Spt20 in conidiation, RNA-seq was performed (Table S3). Both the Δspt20 mutant and the WT strain were pregrown in liquid MM for 12 h and then shifted to solid MM plates for a further 12 h to induce asexual development. As a control, the Δspt20 mutant and the WT strain were also incubated in liquid MM for 24 h (vegetative growth). The central regulatory pathway of conidiation of Aspergillus contains the three key transcription factors BrlA, AbaA, and WetA (35, 36). Transcript analysis showed that the expression of brlA (but not that of abaA or wetA) was decreased in the Δspt20 mutant compared with that of the WT strain during the conidiation stage. In Aspergillus, the upstream regulators FluG and FlbA to -D are essential for the expression of brlA and contribute to the proper progression of conidiation (37, 38). As expected, during conidiation, the expressions of the fluG and flbABCD genes decreased (FC of >2; P < 0.05) in the Δspt20 mutant compared with the WT strain. In addition, the expression of genes encoding the velvet family proteins VosA and VelC, also involved in conidiation (39), were Spt20 dependent. Finally, the expressions of genes encoding conidial hydrophobins (40–42) (i.e., rodABC) were nearly absent in the Δspt20 mutant compared with the WT strain (Fig. 4C). In comparison, during vegetative growth, the expressions of flbA and flbB were downregulated in the Δspt20 mutant strain compared with the WT strain, while most other conidiation-related genes were upregulated (Fig. 4C). In summary, the above-described data suggest that Spt20 regulates conidiation by governing the expression of brlA and the upstream regulators of conidiation.

Spt20 is crucial for the virulence of A. fumigatus.

Previous studies have found that certain regulatory factors that govern GAG biosynthesis are required for full A. fumigatus virulence (13, 14, 16). To determine if Spt20 contributes to fungal pathogenesis, the effects of the Tet-spt20 mutant were assessed in a neutropenic mouse model of invasive aspergillosis. Immunocompromised mice were infected with conidia of the WT or the Tet-spt20 mutant with or without 500 μg/mL doxycycline. Mice infected with the Tet-spt20 strain without doxycycline supplementation exhibited improved survival compared with mice treated with doxycycline as well as mice infected with WT A. fumigatus both with and without doxycycline treatment (Fig. 5A). Histopathological examination of the lungs of mice infected with the Tet-spt20 (off) mutant strain showed a significant difference in the appearance of fungal lesions compared with those of mice infected with the WT or Tet-spt20 (on) strain. Gomori methenamine silver staining of lung sections showed that the lungs of mice infected with the Tet-spt20 (off) mutant strain exhibited fewer fungal lesions, which were largely composed of swollen conidia and short hyphae (Fig. 5B). In contrast, mice infected with the WT or Tet-spt20 (on) strain had more and larger pulmonary lesions containing longer hyphae (Fig. 5B). In summary, these data show that Spt20 is required for the full virulence of the opportunistic fungal pathogen A. fumigatus.

FIG 5.

Spt20 is crucial for virulence of A. fumigatus. (A) Survival of neutropenic BALB/c mice infected with A. fumigatus conidia of the WT or Tet-spt20 strain, with and without Dox treatment (n = 8 mice per group from two independent experiments) (****, P < 0.0001). (B) Gomori methenamine silver-stained lung sections from mice infected and treated as described above for panel A at 36 h postinfection. Red arrows indicate hyphal filaments, while blue arrows indicate swollen conidia and small hyphae.

Distinct modules of SAGA regulate asexual development and GAG production to various degrees.

Spt20 is one of the structural subunits of the SAGA complex in S. cerevisiae, and therefore, we tested if Spt20 could form a complex with other subunits of SAGA in A. fumigatus. Spt20-GFP was used as a probe to immunoprecipitate other subunits that are present in the SAGA complex. Proteins present only in the pulldowns from Spt20-GFP, but absent in negative controls, were considered to represent candidates (Table S4). As expected, gene ontology (GO) analysis demonstrated that Spt20-interacting proteins were most significantly enriched in the SAGA complex (GO:0000124) (Fig. S4). A total of 11 proteins homologous to the SAGA complex subunits in S. cerevisiae were identified, including the transcription association protein Tra1; a subset of core module proteins, Taf6, Taf5, Taf9, and Ada1; several HAT module proteins (i.e., Gcn5, Ada2, and Ada3); the two TBPs Spt3 and Spt8; as well as the DUB module protein Ubp8 (Table S4). These results indicate that Spt20 acts as a structural subunit of the SAGA complex in A. fumigatus.

Given that the transcriptional coactivator SAGA complex contains distinct functional modules, we investigated whether all modules of SAGA function together or whether different modules play distinct roles in the regulation of conidiation and biofilm formation. The subunits Gcn5 and Ada2 (HAT module), Spt3 and Spt8 (TBP module), Ubp8 (DUB module), and Taf6 (core module) were chosen as representative genes for their respective modules. Mutants with deficiencies in each gene were generated, and the effects of gene disruption on the radial growth, asexual development, and biofilm formation of A. fumigatus were quantified. Attempts to disrupt taf6 were unsuccessful, suggesting that this gene may be essential in A. fumigatus. Among other mutants, distinct subunits were found to regulate the radial growth and asexual development of A. fumigatus to various degrees (Fig. 6A). The loss of spt20 and gcn5 yielded a completely nonconidiating phenotype. In comparison, a minimal amount of conidia (less than 5% compared to the WT) was obtained upon the deletion of ada2, spt3, and spt8 (Fig. 6B). Finally, no significant difference was found in total conidial production between the Δubp8 mutant and WT strains. Mutants deficient in ada2, spt3, spt20, and gcn5 exhibited significantly reduced radial growth on minimal medium (Fig. 6C).

FIG 6.

Spt-Ada-Gcn5-acetyltransferase (SAGA) modules regulate asexual development and GAG production to various degrees. (A) Colony morphology of the WT and the Δada2, Δspt3, Δspt8, Δspt20, and Δgcn5 mutant strains after 2 days of growth at 37°C on MM. (B and C) Spore numbers (B) and colony diameters (C) of the strains indicated in panel A. Results are expressed as average numbers of conidia and radial diameters from three repetitions ± standard deviations (**, P < 0.01; ****, P < 0.0001; NS, not significant; ND, not detectable). (D) Formation of adherent biofilms by the indicated strains after 24 h of growth on tissue-culture-treated polystyrene surfaces in MM. Biofilms were washed and visualized by staining with crystal violet. (E) Quantitative determination of biofilm formation of the indicated mutant strains using the crystal violet assay. Results are expressed as the averages and standard deviations from three independent biological experiments, each with six technical repetitions (***, P < 0.001; NS, not significant). (F) Heat map analysis of the relative expression levels of the GAG gene cluster from the RT-qPCR results of the indicated strains after 24 h of growth in MM. Gene expression was normalized to the endogenous reference gene tubA, and all results represent data from three independent biological experiments (**, P < 0.01). (G) Representative images of hyphae of the indicated strains stained with GAG-specific SBA-FITC after 8 h of growth in RPMI 1640. All images are at the same magnification, and the scale bar represents 10 μm. DIC, differential interference contrast. (H) Quantification of the mean fluorescence intensity (MFI) of A. fumigatus hyphae grown under the same conditions as the ones described above for panel G. Data are presented as the percentages of the MFI of the WT strain grown in RPMI 1640, and the averages and standard deviations are from four independent biological samples, each with five hyphal sections measured (*, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant).

Similar differences in biofilm formation were observed among these strains. Deletions of gcn5, ada2, spt3, and spt8 (but not ubp8) were found to be associated with a decrease in the biofilm biomass (Fig. 6D and E). To determine whether the decrease of biofilms is linked to GAG production, the expression levels of GAG cluster genes in these mutants were examined. The expression levels of uge3 and agd3 were significantly (FC of >2; P < 0.05) downregulated in the Δgcn5 and Δspt8 mutants (Fig. 6F), as was the expression of uge3 in the Δada2 mutant and the expression of agd3 in the Δspt3 mutant. The expression of gtb3 was significantly downregulated in both the Δspt3 and Δspt8 mutants. Interestingly, the expression levels of ega3 and sph3 were significantly (FC of >2; P < 0.05) upregulated in both the Δgcn5 and Δada2 mutants (Fig. 6F). The above-described data indicate a disordered expression pattern of GAG cluster genes in the Δgcn5, Δada2, Δspt3, and Δspt8 mutants. As predicted by its biofilm-forming ability, the expression of GAG cluster genes in the Δubp8 mutant strain was not significantly different from that of the WT strain. Consistent with these findings, the loss of gcn5, ada2, spt3, and spt8 (but not ubp8) resulted in decreased production of cell wall-associated GAG as measured by SBA immunofluorescence (Fig. 6G and H). Collectively, these results suggest that all SAGA modules (except for the deubiquitinase module Ubp8) regulate asexual development and GAG production albeit to various degrees.

DISCUSSION

In this study, Spt20, an orthologue of the structural subunit of the S. cerevisiae SAGA complex, was identified as a novel regulator of biofilm formation and conidiation in A. fumigatus. Similar to ptaB- and somA-null mutants, the loss of spt20 caused severe defects related to biofilm formation and asexual development. RNA-seq analysis demonstrated that Spt20 shared overlapping targets with the PtaB/SomA complex, including a subset of genes involved in GAG biosynthesis and conidiation. These results demonstrate that Spt20, PtaB, and SomA are all required for the transcriptional machinery of biofilm formation and asexual development in A. fumigatus. These results expand the current understanding of the regulatory circuits governing biofilm formation and asexual development in A. fumigatus.

Although Spt20 was identified as a regulator of biofilm formation by PtaB-GFP trap assays, this result could not be confirmed by using in vivo Co-IP assays. This may be caused by the low expression level of Spt20, which was below the detection ability of Western blotting. LC-MS/MS achieved a higher sensitivity of detection than Western blotting. In addition, Y2H assays showed that PtaB does not physically interact with Spt20. Considering the roles of the SAGA complex in transcriptional regulation, we suggest that there are two possibilities to explain the relationships between Spt20 and the PtaB/SomA complex. One possibility is that Spt20 does not physically interact with PtaB/SomA. In S. cerevisiae, the activator Gal4p recruits SAGA to the GAL1 upstream activating sequence through its interaction with Tra1p (43, 44), a subunit of the SAGA complex. In addition, the human transcription factor c-Myc similarly functions to recruit SAGA by interacting with TRRAP (the mammalian orthologue of Tra1) during transcription activation (45). Therefore, PtaB/SomA might recruit the SAGA complex by interacting with other SAGA subunits rather than Spt20. Thus, the interaction between PtaB/SomA and Spt20 may not be detected by Y2H assays. The other possibility is that the interaction between PtaB/SomA and Spt20 is a transient process. Studies in yeast indicated that the transcriptional regulation of specific genes by the transcriptional coactivator complex recruited to the promoter region under certain conditions is a transient process (46–49). Thus, although Spt20 was immunopurified with PtaB and both are required for biofilm formation and asexual development, the precise interplay relationships between PtaB/SomA and the SAGA complex require further exploration.

SAGA is a multisubunit transcriptional coactivator complex with defined functions in regulating the global transcriptional status of eukaryotic cells. The function and composition of this conserved complex are known to vary in different species, enabling them to exhibit tissue specificity. Recently, it has been reported that Gcn5 (GcnE), a member of the HAT module, is involved in asexual development and biofilm formation in A. fumigatus; furthermore, Gcn5 regulates the expression of genes related to conidiation and GAG biosynthesis (50). Although HAT, TBP, and core modules of SAGA are all required for GAG biosynthesis and asexual development, different modules of the SAGA complex regulate biofilm formation and conidiation to various degrees.

Multiple observations suggest that Spt20, the structural subunit of SAGA, is the most important component for the transcription of genes related to GAG biosynthesis and asexual development processes in A. fumigatus. Asexual development was completely eliminated in the spt20 mutant. In comparison, the loss of ada2, spt3, spt8, and gcn5 resulted in only partial conidiation defects in A. fumigatus. The deletion of spt20 was associated with a more severe defect in biofilm formation than what was observed in the Δada2, Δspt3, Δspt8, and Δgcn5 mutants. In addition, Spt20 contributes to A. fumigatus virulence, while the loss of gcn5 (HAT module) did not impact virulence in a murine model of invasive aspergillosis (50). These results are consistent with previous biochemical and genetic evidence indicating that Spt20 (which is required for the integrity of SAGA) plays a more important structural and functional role than Gcn5. Genome-wide expression analysis in S. cerevisiae also confirmed that the loss of spt20 had the strongest effect on global gene expression, whereas Δspt3 and Δgcn5 mutations affect smaller and more specific sets of genes (51). Further research examining the interplay relationships between different modules of the SAGA complex is required to establish cooperative and distinctive traits of the SAGA complex in A. fumigatus. Moreover, in addition to SAGA subunits, putative Spt20-interacting proteins identified by the GFP trap assay were also enriched in both the intracellular component (GO:0005622) and the ribosome component (GO:0005840). Further studies are necessary to explore whether Spt20 cooperates with these candidates to regulate biofilm formation and asexual development in A. fumigatus.

MATERIALS AND METHODS

Strains, media, and culture conditions.

All strains used in this study are summarized in Table 1. Unless otherwise stated, A. fumigatus was grown in minimal medium (MM) (1% glucose, original high-nitrate salts, and trace elements [pH 6.5]), YG medium (2% glucose, 0.5% yeast extract, and trace elements), and RPMI 1640 (Sigma-Aldrich, USA) at 37°C. Solid MM and YG were the same as those described above except that 2% agar was added. To induce the expression of spt20 and somA in the Tet-spt20 and Tet-somA mutants, the medium was supplemented with 1 μg/mL doxycycline.

TABLE 1.

Strains used in this study

Strain	Genotype	Source or reference
A1160	Δku80 pyrG	FGSCa
WT	A1160::pyrG	52
Δuge3	Δuge3::hph	9
ΔptaB	Δku80 pyrG ΔptaB::pyr4	15
ptaB::GFP	Δku80 pyrG ptaB::gfp::pyrG	15
Tet-somA	Δku80 pyrG pyr4 tet(p)::somA::ptrA	16
Δspt20	Δku80 pyrG Δspt20::pyr4	This study
ΔAFUB_022210	Δku80 pyrG ΔAFUB_022210::pyr4	This study
ΔAFUB_073640	Δku80 pyrG ΔAFUB_073640::pyr4	This study
ΔAFUB_090650	Δku80 pyrG ΔAFUB_090650::pyr4	This study
Δspt3	Δku80 pyrG Δspt3::pyr4	This study
Δspt8	Δku80 pyrG Δspt8::pyr4	This study
Δada2	Δku80 pyrG Δada2::pyr4	This study
Δgcn5	Δku80 pyrG Δgcn5::pyr4	This study
Δubp8	Δku80 pyrG Δubp8::pyr4	This study
Tet-spt20	Δku80 pyrG Δspt20::pyr4 tet(p)::spt20::ptrA	This study
OEmedA::Tet-spt20	Δku80 pyrG Δspt20::pyr4 tet(p)::spt20::ptrA Pgpd::medA::hph	This study
spt20::GFP	Δku80 pyrG spt20::gfp::pyrG	This study
spt20::Flag-ptaB::GFP	Δku80 pyrG ptaB::gfp::pyrG spt20::flag::hph	This study
Saccharomyces cerevisiae AH109	MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4Δ gal8Δ LYS2::GAL1UAS-GAL1TATA-HIS3 gal4Δ GAL2UAS-GAL2TATA-ADE2 URA3::MEL1UAS-MEL1TATA-lacZ	Clontech

^aFGSC, Fungal Genetics Stock Center.

The S. cerevisiae strain was cultured in liquid YPD (1% yeast extract, 2% dextrose, and 2% peptone) and synthetically defined medium lacking tryptophan, leucine, histidine, and adenine (Clontech, USA) at 30°C.

Construction of genetic mutant strains.

The spt20 deletion mutant strain was constructed by the fusion PCR method as previously described (53). Briefly, approximately 1 kb of the upstream and downstream flanking sequences of the spt20 open reading frame (ORF) was amplified with the primer pairs Spt20P1/P3 and Spt20P4/P6 from the genomic DNA (gDNA) of the A. fumigatus strain, respectively. A total of 2.1 kb of the marker pyr4 was amplified from the pAL5 plasmid with the primer pair Pyr4F/R. The three above-mentioned PCR products were used as the template to create the spt20 knockout cassette by fusion PCR with primer pair Spt20P2/P5. The resulting fusion product was then cloned into the pEASY-Blunt Zero vector (TransGen Biotech, Beijing, China) and used to transform the recipient strain A1160. Transformants were grown on MM and confirmed by diagnostic PCR. The same strategy was employed to generate other knockout mutants.

Tet-spt20 and spt20 complementation strains were generated on the background of the Δspt20 mutant. For Tet-spt20 mutant construction, the conditional doxycycline-inducible Tet-On promoter cassette was amplified from the pCH008 plasmid (54) using primer pair Tet-spt20F1/R1. The full-length cDNA sequence of spt20 was amplified using primer pair Tet-spt20F2/R2. The two purified PCR products were used as the template to generate the Tet-spt20 cassette by fusion PCR using primer pair Tet-spt20F1/R2, cloned into the pEASY-Blunt Zero vector (TransGen Biotech), and used to transform the Δspt20 recipient strain. Transformants were grown on MM supplemented with 0.1 μg/mL pyrithiamine (Sigma-Aldrich) and confirmed by diagnostic PCR. For spt20 complementation strain construction, the native promoter and ORF of spt20 were amplified using primer pair Spt20comF/R from the genomic DNA of A. fumigatus. The sequenced PCR product was cloned into the XbaI and HindIII sites of the pAN7-1 vector and then used to transform the Δspt20 recipient strain. Transformants were grown on MM supplemented with 200 μg/mL hygromycin B (Shanghai Sangon Biotech, China) and verified by diagnostic PCR.

To construct the OEmedA::Tet-spt20 mutant strain, the ORF of medA was amplified from the gDNA of WT A. fumigatus using primer pair OEmedAF/R. The sequenced PCR product was cloned into the EcoRV-linearized plasmid pHPHGPE controlled by a gpdA promoter to generate plasmid pOEmedA (15) and then used to transform the Tet-spt20 recipient strain. Transformants were grown on MM supplemented with 200 μg/mL hygromycin B (Shanghai Sangon Biotech) and verified by diagnostic PCR.

To construct the green fluorescent protein (GFP)-tagged Spt20 fusion protein, the GFP-pyrG cassette sequence was amplified from pFN03 using primer pair GFP-pyrGF/R. Approximately 1 kb of the upstream and downstream flanking sequences of the Spt20 termination codon was amplified from the genomic DNA using primer pairs Spt20GFPP1/P3 and Spt20GFPP4/P6, respectively. The three above-mentioned PCR products were then used as the template to create the Spt20-GFP fusion cassette by fusion PCR using primer pair Spt20GFPP2/P5. The resulting fusion product was then cloned into the pEASY-Blunt Zero vector (TransGen Biotech) and used to transform the recipient strain A1160. Transformants were grown on MM and confirmed by diagnostic PCR. The same strategy was employed to generate the PtaB-GFP-tagged strain.

The Spt20-FLAG::PtaB-GFP strain was generated by FLAG tagging of Spt20 in the PtaB-GFP strain background. The strategy to generate FLAG-tagged Spt20 was similar to the construction of Spt20-GFP, and certain primers are the same. Briefly, approximately 1 kb of the upstream and downstream flanking sequences of the Spt20 termination codon was amplified from genomic DNA using primer pairs Spt20GFPP1/Spt20FlagP3 and Spt20FlagP4/Spt20GFPP6, respectively. The FLAG-hph cassette sequence was amplified from the pFLAGHPH plasmid with the primer pair Flag-F/hph-R. The three above-mentioned PCR products were then used as the template to create the spt20-FLAG fusion cassette by fusion PCR with primer pair Spt20GFPP2/P5. The resulting fusion product was then cloned into the pEASY-Blunt Zero vector (TransGen Biotech) and used to transform the PtaB-GFP recipient strain. Transformants were grown on MM supplemented with 200 μg/mL hygromycin B (Shanghai Sangon Biotech) and verified by diagnostic PCR. All primers used in this study are listed in Table S5 in the supplemental material.

GFP trap assay and LC-MS/MS.

To identify potential proteins that interact with PtaB, the PtaB-GFP strain was cultured in MM either by shaking (planktonic growth) or statically (biofilm growth) for 24 h. WT A. fumigatus was used as a negative control. To determine if Spt20 can form a complex with other subunits of SAGA, Spt20-GFP and gpd-GFP (control) strains were grown in MM for 24 h. Mycelia were then collected, frozen in liquid nitrogen, and ground into a powder with a mortar. Total proteins were extracted with lysis buffer (8 M urea, 10 mM dithiothreitol, 1% Triton X-100, and 1% protease inhibitor cocktail) and then digested with trypsin. Tryptic peptides were fractionated by high-performance liquid chromatography (HPLC) using a Thermo Betasil C₁₈ column and subsequently analyzed with an LC-MS/MS system (ekspert nanoLC, TripleTOF 5600-plus; AB Sciex). The output data were processed with a confidence level of ≥95% and a number of unique peptides of ≥1. The GFP trap assay was carried out using GFP Trap_A resin (ChromoTek, Germany) according to the manufacturer’s instructions. Immunoprecipitated proteins were analyzed by LC-MS/MS, performed by Wuhan GeneCreate Biological Engineering Co., Ltd. (China).

Biofilm formation assay.

A. fumigatus biofilm formation assays were performed as previously described (13, 16), with minor modifications. In brief, 96-well non-tissue-culture-treated plates (Corning) were inoculated with 150 μL of MM per well containing 2 × 10⁵ conidia/mL, followed by incubation at 37°C for 24 h. Adherent biofilms were washed twice with 200 μL of distilled water and then stained with 100 μL of 0.1% (wt/vol) crystal violet for 10 min. The excess crystal violet solution was removed, and stained biofilms were then washed twice and destained by adding 125 μL of ethanol to each well. The quantification of fungal biofilm was performed by measuring the absorbance of 75 μL of the destained solution at 600 nm.

Plate assays.

To test the developmental phenotype of fungal strains, 2 μL of conidial suspensions (1 × 10⁷ conidia/mL) of the indicated strains was spotted onto the indicated medium plates, grown at 37°C for 48 h, and then observed and imaged. The growth diameter and total spores of the indicated strains were determined in three independent biological experiments.

Yeast two-hybrid assay.

For Y2H studies, the full-length cDNAs of somA and spt20 were cloned into the EcoRI and BamHI sites of the prey vector pGADT7, and the ptaB full-length cDNA was cloned into the EcoRI and BamHI sites of the bait vector pGBKT7. All cDNA sequences were confirmed by sequencing. The bait construct pGBKT7-PtaB (BD-PtaB) (where BD means binding domain) and the prey construct pGADT7-SomA (AD-SomA) (where AD means activation domain) or pGADT7-Spt20 (AD-Spt20) were then cotransformed into yeast strain AH109. Strains were cotransformed with the pGBKT7-53 and pGADT7-T plasmids used as the positive controls, while pGBKT7-lam and pGADT7-T served as the negative controls. Transformants were isolated and tested for growth on synthetically defined medium lacking tryptophan, leucine, histidine, and adenine (Clontech) to identify positive interactions.

RNA isolation and RT-qPCR.

RNA isolation and real-time quantitative PCR were performed using previously described procedures (16). In brief, 1 × 10⁷ fungal conidia were grown in MM for 24 h and then collected by filtration, followed by freezing and crushing in liquid nitrogen. Total RNA was purified using a Uniq-10 column total RNA purification kit (Shanghai Sangon Biotech) according to the manufacturer’s instructions. gDNA digestion and cDNA synthesis were performed using the HiScriptII Q RT supermix for qPCR (gDNA wiper) kit (Vazyme, China). RT-qPCRs were run in an ABI one-step fast thermocycler (Applied Biosystems), using AceQ qPCR SYBR green master mix (Vazyme, China). Relative fold changes were then normalized to tubA, and gene expression levels were calculated using the ΔΔC_T method (55).

RNA-seq and data analysis.

To determine the effect of the Δspt20 mutant on the asexual development of A. fumigatus, the WT and Δspt20 strains were grown in liquid MM for 12 h and then shifted to liquid MM or solid MM for 12 h. Two independent biological replicates were performed. To analyze the downstream targets of the Tet-somA, Tet-spt20, and ΔptaB mutant strains, fungal spores were grown in MM for 24 h. Three independent biological replicates were performed for these studies. After appropriate growth, mycelia were collected by filtration, washed with distilled water, and frozen using liquid nitrogen. Total RNA was purified using the mirVana microRNA (miRNA) isolation kit (Ambion, USA) according to the manufacturer’s protocol. cDNA libraries were constructed using the TruSeq stranded mRNA LT sample prep kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Next, samples were sequenced by next-generation sequencing (NGS) based on the Illumina sequencing platform (HiSeq 2500). All libraries were aligned to the A. fumigatus A1163 genome. Differentially expressed genes showing more than 2-fold enrichment with a P value of <0.05 were subjected to enrichment analysis. RNA purification, cDNA synthesis, and sequencing were performed by Shanghai OE Biotechnology Co., Ltd. (China).

GAG polysaccharide characterization.

GAG-specific SBA-FITC was used to determine the GAG polysaccharide on the surface of hyphae. Immunofluorescence staining of hyphae was performed as previously described (9). Briefly, 2 × 10⁵ A. fumigatus conidia were grown on coverslips in RPMI 1640 (Sigma-Aldrich) for 8 to 10 h. Mycelia were washed with phosphate-buffered saline (PBS) and then stained with SBA (Vector Labs, USA) in a dark chamber. After 2.5 h of incubation on ice, samples were washed with PBS and microscopically imaged (Zeiss, Germany).

Scanning electron microscopy analysis of the cell surface.

For hyphal surface characterization, SEM was carried out as previously described (8), with minor modifications. The WT and Tet-spt20 strains were grown statically in RPMI 1640 for 24 h. Critical point drying and SEM photography of samples were performed according to our previously described methods (16).

Mouse model of invasive pulmonary aspergillosis.

The effects of modulating spt20 expression on virulence were assessed using a neutropenic mouse model of invasive pulmonary aspergillosis (56). Briefly, 8- to 10-week-old female BALB/c mice (Charles River, Senneville, QC, Canada) were neutrophil depleted by intraperitoneal injection of 200 μg anti-Ly6G antibody (57, 58) (clone 1A8; BioXcell) every 48 h, beginning 1 day prior to infection. Mice were then infected intratracheally with 1 × 10⁷ A. fumigatus conidia in 50 μL PBS plus 0.01% (vol/vol) Tween 80 (PBS-T) or PBS-T alone for uninfected controls. Doxycycline (Sigma-Aldrich) was administered by supplementation of drinking water (500 μg/mL doxycycline and 5% sucrose) and mouse chow (625 mg/kg of body weight doxycycline; Envigo-Teklad), beginning 3 days prior to infection. Mice were monitored every 12 h for signs of illness, and moribund animals were euthanized by CO₂ overdose. Doxycycline-free control mice received water and mouse chow without doxycycline.

To visualize fungal morphology within infected mouse lungs, mice were euthanized at 36 h postinfection, and their lungs were harvested, fixed overnight in 4% paraformaldehyde (PFA), and embedded in paraffin. Sections of 4 mm were then stained with Gomori methenamine silver, and images were captured and processed using Aperio ImageScope (Leica Biosystems).

All procedures involving mice were approved by the McGill University Animal Care Committee (protocol number AUP-2015-7674) and followed the guidelines established by the Canadian Council on Animal Care.

Data analysis.

Data are presented as means ± standard deviations (SD) obtained from at least three biological replicates unless otherwise stated. Statistical analyses were performed using GraphPad Prism7 software, and multiple comparisons were analyzed by one-way analysis of variance. A P value of 0.05 was considered to indicate statistically significant differences.

Data availability.

The RNA-seq data have been deposited in the NCBI Sequence Read Archive under accession number PRJNA728750. Other relevant data supporting the findings of this study are available within this article and its associated supplemental material.