The basic leucine zipper transcription factor MeaB is critical for biofilm formation, cell wall integrity, and virulence in Aspergillus fumigatus

ABSTRACT The regulation of fungal cell wall biosynthesis is crucial for cell wall integrity maintenance and directly impacts fungal pathogen virulence. Although numerous genes are involved in fungal cell wall polysaccharide biosynthesis through multiple pathways, the underlying regulatory mechanism is still not fully understood. In this study, we identified and functionally characterized a direct downstream target of SomA, the basic-region leucine zipper transcription factor MeaB, playing a certain role in Aspergillus fumigatus cell wall integrity. Loss of meaB reduces hyphal growth, causes severe defects in galactosaminogalactan-mediated biofilm formation, and attenuates virulence in a Galleria mellonella infection model. Furthermore, the meaB null mutant strain exhibited hypersensitivity to cell wall-perturbing agents and significantly alters the cell wall structure. Transcriptional profile analysis revealed that MeaB positively regulates the expression of the galactosaminogalactan biosynthesis and β-1,3-glucanosyltransferase genes uge3, agd3, and sph3 and gel1, gel5, and gel7, respectively, as well as genes involved in amino sugar and nucleotide sugar metabolism. Further study demonstrated that MeaB could respond to cell wall stress and contribute to the proper expression of mitogen-activated protein kinase genes mpkA and mpkC in the presence of different concentrations of congo red. In conclusion, A. fumigatus MeaB plays a critical role in cell wall integrity by governing the expression of genes encoding cell wall-related proteins, thus impacting the virulence of this fungus. IMPORTANCE Aspergillus fumigatus is a common opportunistic mold that causes life-threatening infections in immunosuppressed patients. The fungal cell wall is a complex and dynamic organelle essential for the development of pathogenic fungi. Genes involved in cell wall polysaccharide biosynthesis and remodeling are crucial for fungal pathogen virulence. However, the potential regulatory mechanism for cell wall integrity remains to be fully defined in A. fumigatus. In the present study, we identify basic-region leucine zipper transcription factor MeaB as an important regulator of cell wall galactosaminogalactan biosynthesis and β-1,3-glucan remodeling that consequently impacts stress response and virulence of fungal pathogens. Thus, we illuminate a mechanism of transcriptional control fungal cell wall polysaccharide biosynthesis and stress response. As these cell wall components are promising therapeutic targets for fungal infections, understanding the regulatory mechanism of such polysaccharides will provide new therapeutic opportunities.

appears to have decreased virulence (39).The potential role of MeaB in A. fumigatus needs to be identified.
SomA and its orthologs play a conserved role in fungal virulence attributes (40)(41)(42).The transcriptome sequencing (RNA-seq) and chromatin immunoprecipitation coupled to sequencing (ChIP-Seq) of SomA have been successfully exploited to identify the regulatory mechanisms of fungal virulence-related processes in A. fumigatus (32).In this study, we identified a direct downstream target of SomA, MeaB, which possesses several pathogenicity-related characteristics.The loss of meaB caused severe defects in the biofilm formation, cell wall integrity, and virulence of A. fumigatus.Transcriptional profile analysis revealed that MeaB positively regulates the expression of the GAG biosynthesis and β-1,3-glucanosyltransferase genes uge3, agd3, and sph3 and gel1, gel5, and gel7, respectively, as well as genes involved in amino sugar and nucleotide sugar metabolism.Further study demonstrated that the bZIP transcription factor MeaB is a stress response effector and contributes to the proper expression of mitogen-activated protein kinase genes mpkA and mpkC in the presence of different concentrations of congo red (CR).Thus, we illuminate a mechanism of transcriptional control fungal cell wall polysacchar ide biosynthesis and stress response.

ChIP-seq and transcriptome analysis identified a number of novel SomA target genes that play a role in biofilm formation and cell wall integrity
Previous studies confirmed that SomA is a key transcription factor of A. fumigatus, which controls fungal development, biofilm formation, cell wall stress response, and virulence (32,40).To investigate the transcriptional network connects to these crucial pathways, we identified and functionally characterized a number of transcription factors downstream of SomA based on our previous ChIP-seq and RNA-seq data (32).Among the 426 direct targets bound by SomA in ChIP-seq analyses, there were 29 genes belonging to different family transcription factors (see Table S1).The 29 single mutants from a library of A. fumigatus TF null mutants (43) were selected to evaluate whether they play a critical role in biofilm formation and cell wall integrity.As shown in Fig. S1, the biofilm biomass of hapB, creA, and meaB null mutants was significantly decreased compared to that of the A. fumigatus wild type (WT).Three of the deletion mutants (ΔhapB, ΔcreA, and ΔmeaB) were also hypersensitive to the cell wall-perturbing agent congo red (Fig. S1), suggesting the potential roles in cell wall integrity.Furthermore, the evaluation of the ChIP-seq data suggested that SomA was predominantly occupied in the promoter region of these genes (Fig. 1A).The transcript levels of three indicated transcription factors based on RNA-seq demonstrated a reduced abundance of meaB mRNA, but not creA or hapB, in the somA mutant strain, suggesting a direct regulation of transcription factor MeaB by SomA (Fig. 1A).Consistent with these findings, reverse transcription quantitative polymerase chain reaction (RT-qPCR) confirmed that the expression of meaB was significantly decreased in the somA mutant [Tet-somA strain (OFF)] grown in the AMM compared with that in the WT strain (Fig. 1B).Consequently, the electrophoretic mobility shift assay further confirmed the in vitro binding of SomA to the conserved "G TACTCCGTAC" motif-containing promoter fragments of meaB (Fig. 1C).Considering the regulatory mechanism of A. fumigatus, CreA in biofilm formation and cell wall integrity has been elucidated (44,45).Our results of HapB in cell wall integrity and biofilm formation (Fig. S1 and S2) were also consistent with the previous findings that it is a cell wall stress response factor (34,35) and that it is also a potential regulator of GAG polysaccharide biosynthesis genes based on the hapB mutant RNA-seq data (46), respectively.In the present study, we focus on the regulatory mechanism of MeaB on these fungal pathogenicity-related pathways.
To explore the biological functions of A. fumigatus MeaB, a ΔmeaB null mutant and complemented (meaB com ) strains were generated.The complementation strain meaB com was constructed by the ectopic insertion of the target gene into the genome of the ΔmeaB background strain.As shown in Fig. 1D and E, the ΔmeaB mutant strain showed markedly reduced radial growth on solid rich medium (YG) and minimal medium (AMM) compared to the WT and complemented strains.Moreover, consistent with the decreased radial growth on solid medium, the ΔmeaB mutant exhibited a significantly reduced biomass when submerged into liquid AMM for 24 h (Fig. 1F and G).Collectively, the above results suggest that the bZIP transcription factor MeaB is a direct downstream target of SomA, which regulates A. fumigatus hyphal growth.

MeaB deficiency causes reduced GAG production, adhesion, and attenuated virulence in A. fumigatus
Given the crucial role of SomA in the adhesion of A. fumigatus, we believed that the downstream target of SomA, meaB, may play a role in mediating biofilm formation.The formation of adherent biofilms was visualized by staining with crystal violet.As shown in Fig. 2A and B, in comparison to the parental and complemented strains, the ΔmeaB mutant was markedly impaired in the formation of adherent biofilms on plastic, representing half of the total amount of biofilm biomass produced by the parental strain in liquid AMM.Since GAG functions as the dominant adhesin of A. fumigatus and mediates biofilm formation, we hypothesized that the reduced augmentation of biofilm production might reflect a decrease in GAG production.Consistent with this hypothesis, the immunofluorescence staining of GAG by fluorescein-tagged soybean agglutinin lectin (SBA-FITC) revealed that the distribution of GAG production on the hyphal surface of ΔmeaB mutant was significantly decreased (Fig. 2C and D).RT-qPCR analysis confirmed that the expressions of GAG biosynthesis genes uge3, agd3, and sph3 were dependent on MeaB, suggesting that transcription factor MeaB governs the biofilm formation by regulating the expression of GAG biosynthesis-related genes (Fig. 2E).GAG is an important virulence factor in A. fumigatus, and multiple studies have found that certain regulatory factors that govern GAG biosynthesis are required for full virulence of A. fumigatus (32,40,(47)(48)(49)(50).To determine if MeaB contributes to fungal pathogenesis, the effects of the ΔmeaB mutant were assessed in a Galleria mellonella infection model.Larvae were infected with 1 × 10 6 A. fumigatus conidia per larva of the indicated strains, and survival was monitored at 37°C.Survival analysis revealed that loss of meaB resulted in a significantly improved survival rate (P < 0.01) of larvae compared to the WT strain 6 days post-infection (Fig. 2F).In summary, these data show that MeaB regulates GAG biosynthesis gene expression and contributes to full virulence of A. fumigatus.

MeaB is involved in the maintenance of cell wall integrity
Next, we analyzed the growth of the ΔmeaB mutant strain under different cell wall perturbing conditions.As shown in Fig. 3A through C, the ΔmeaB mutant strain presen ted increased sensitivity to chitin-binding agents on AMM plates, such as CR and calcofluor white (CFW), but did not show any sensitivity to the β-glucan synthase inhibitor CAS.Furthermore, we also observed that loss of meaB resulted in a hypersensi tivity to the detergent sodium dodecyl sulfate (SDS) and high osmotic stress conditions (1.2 M sorbitol).Interestingly, lack of meaB not only causes a high temperature (45°C) sensitivity phenotype but also exhibits increased growth defects under low temperature (28°C) conditions (Fig. 3B and C), indicating that the cell membrane of A. fumigatus may also be affected by the meaB mutant.Besides, it is reported that the bZIP transcription factor MeaB is a fungal nitrogen metabolism regulator, and the growth phenotypes of such mutant will be different on distinct nitrogen source plates.Therefore, we performed the cell wall stress assay on another minimal medium with nitrate as the sole nitrogen source (NMM).In agreement, the A. fumigatus ΔmeaB mutant strain showed significant growth defects on NMM supplemented with CR and CFW, but not on the CAS plate (Fig. S3).Given that the cell wall architecture is crucial for fungal stress response, we per formed transmission electron microscopy assays to observe the influence of MeaB on A. fumigatus hyphal germlings.The thickness of the cell wall in the ΔmeaB mutant strain was found to be 1.4-fold (P < 0.05) thicker than that in the WT or complemented strains (Fig. 3D and E), indicating that MeaB plays a potential role in cell wall architecture maintenance.

MeaB regulates fungal carbohydrate metabolism and cell wall-related polysaccharide biosynthesis
To further elucidate the regulatory mechanism underlying MeaB-mediated cell wall integrity, we measured the global gene expression changes in the ΔmeaB mutant strain compared to the WT strain cultured in liquid AMM at 37°C for 24 h by RNA-seq.RNA-seq identified 819 (333 up-and 486 downregulated) (Fig. S4) differentially expressed genes  S2).The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the DEGs revealed that the significantly enriched pathways were glycine, serine, threonine metabolism, and tyrosine metabolism, as well as amino sugar and nucleotide sugar metabolism (Fig. 4A), a key pathway providing precursors for the biosynthesis of cell wall and GAG polysaccharides.To identify the potential physiological roles of the MeaBdependent genes in specific fungal processes, we subjected these DEGs to Gene Ontology (GO) analysis.Consistent with our KEGG analysis, the significantly enriched genes with functions in the cellular component were associated with the extracellular region, plasma membrane, and fungal-type cell wall (Fig. 4B).Besides, these DEGs were significantly enriched in genes with functions in diverse processes, including transmembrane transport processes, iron ion homeostasis, and cellular response external stimulus processes (Fig. 4B).Of note, among the DEGs dependent on MeaB, we observed that the expression of genes in cell wall polysaccharide metabolism pathways was significantly downregulated, including the beta-glucanase Eng3, the 1,3-beta-glucano syltransferases Gel1/5/7, and the hexokinase AFUB_017510, as well as a kind of major facilitator superfamily sugar transporter (Fig. 4C).Most importantly, we found that GAG polysaccharide biosynthesis cluster genes uge3, agd3, gtb3, and sph3 were significantly downregulated (fold change >2, P < 0.05) (Fig. 4C), which is consistent with the above RT-qPCR analysis.Collectively, these results suggest that MeaB is involved in fungal carbohy drate metabolism and cell wall-related polysaccharide biosynthesis.

MeaB regulates the expression of cell wall glucanosyltransferase genes gel1, gel5, and gel7 and the mitogen-activated protein kinase genes mpkA and mpkC
The RNA-seq GO analysis indicated that MeaB is a potential stress response effector; to do so, we monitored the mRNA levels of meaB in the WT strain when grown in liquid AMM for 24 h and after the addition of 300 µg/mL CR for 0.5, 1, and 2 h.As expected, the expression of meaB was significantly induced by 300 µg/mL CR stress (Fig. 5A).Next, we sought to determine the expression patterns of MeaB at the protein level.We labeled MeaB with a FLAG tag at the C-terminus and expressed the fusion protein under the control of MeaB native promoter in the parental strain.Plate assays confirmed that MeaB-FLAG function well and do not change the sensitivity of A. fumigatus to CR or CFW (Fig. S5).In agreement with RT-qPCR results, western blotting analysis showed that the protein expression of MeaB was improved by 300 µg/mL CR treatment (Fig. 5B).Therefore, we speculate that the transcription factor MeaB regulates the fungal cell wall integrity by increasing its expression level in response to cell wall stress.To further explain the regulatory mechanism of MeaB on cell wall integrity, RT-qPCR was performed to detect the expressions of cell wall chitin-and glucan-related synthases.As shown in Fig. 5C, the expression levels of cell wall glucanosyltransferases gel1, gel5, and gel7 were significantly downregulated in the ΔmeaB mutant strain compared to the WT strain cultured in liquid AMM for 24 h, which also confirms our RNA-seq data.However, we did not observe any difference in the expressions of cell wall β-glucan synthases fks1 and representing two classes of chitinases chsA/G and chsE/F between the ΔmeaB mutant and WT strains, under the current experimental conditions (Fig. 5C).In addition, we also measured the relative expression levels of these cell wall biosynthesis related-genes under the CR stress conditions.Consistently, there was no significant difference in the relative expression levels of these genes between the meaB mutant and WT strain under distinct stress conditions (Fig. S6).pathway, and its role was associated with the response to cell wall disturbing com pounds, while the function of MpkC was related to signaling required for carbon source utilization and osmo-stress response (51-54).As expected, the expression of the mpkA gene was dramatically induced by CR treatment in the WT strain (Fig. 5D).Strikingly, the CR-mediated increase in the expression of mpkA gene that appeared in the WT strain was repressed in the ΔmeaB mutant strain, suggesting the stress response of MpkA partial dependence on MeaB (Fig. 5D).Similar to mpkA, we found that the expression of the mpkC gene was significantly induced for about threefold higher by CR treatment for 0.5, 1, and 2 h, and the deletion of meaB almost abolished the CR stress response of mpkC (Fig. 5E).Collectively, these results suggest that stress response effector MeaB regulates cell wall integrity by governing the expression of cell wall glucanosyltransferase genes gel1, gel5, and gel7 and the mitogen-activated protein kinase genes mpkA and mpkC.

DISCUSSION
The fungal cell wall is a dynamic organelle important for pathogen survival, and its composition and structural organization are frequently reshuffled depending on the environmental conditions (16).While the remodeling of the fungal cell wall structure is a complex and tightly regulated process, the underlying mechanism is still not fully understood.It has been shown that the transcription factor SomA regulates A. fumigatus cell wall architecture and composition by governing the expression of cell wall chitin and β-glucan polysaccharide biosynthesis genes (32).In the present study, we identify and functionally characterize a bZIP transcription factor MeaB, which is a direct transcrip tional target of SomA.We show that the deletion of meaB leads to severe defects in biofilm formation and attenuates virulence in a Galleria mellonella infection model, as well as hypersensitivity to cell wall stress agents, which is similar to the critical functions of SomA in A. fumigatus.However, different from SomA, the major novel findings from the study of MeaB are that MeaB is a stress response effector and contributes to the proper expression of both mpkA and mpkC and that MeaB regulates the expression of cell wall glucanosyltransferase genes gel1, gel5, and gel7, but not chitinase genes or β-glucan synthase fksA.As we know, the GEL family proteins play key roles in splitting and remodeling the β-glucan molecule, resulting in the elongation of the glucan chain (16).In addition, gel1 and gel7 also contribute to the cell wall composition and integrity of A. fumigatus (55,56), which is consistent with the present findings of MeaB.Further more, the deletion of meaB altered the cell wall architecture of A. fumigatus, which may be one of the reasons for the sensitivity of ΔmeaB mutant to CR or CFW.As for MpkA, the protein is a canonical mitogen-activated protein kinase involved in fungal cell wall integrity (51,57).The reduced expression of mpkA in ΔmeaB mutant may be one of the reasons for A. fumigatus decreased resistance to cell wall-perturbing agents.However, further study is needed to determine whether the phosphorylation level of MpkA will be affected by the ΔmeaB mutant.Likewise, we observe that MeaB also seems to play a role in osmotic stress response in the current assay, as the mutant strain exhibited hypersensitivity to 1.2 M sorbitol stress.In agreement with this finding, the RNA-seq and RT-qPCR analysis reveal a regulatory role of MeaB on MpkC, a key osmo-stress response kinase.MpkC also plays an important role in carbon source metabolism and cell wall stress response (53,54), which might also account for the increased susceptibility of A. fumigatus ΔmeaB to the treatment with CR and CFW, underscoring an important role of MeaB in cell wall integrity.
Notably, cell wall organization and GAG production directly impact virulence and host immune recognition in A. fumigatus (58)(59)(60)(61).Based on the aforementioned results, we demonstrate that MeaB contributes to full virulence in a Galleria mellonella infection model of A. fumigatus.An association with virulence traits of MeaB has been previously reported in other fungi.In the F. oxysporum study, loss of meaB resulted in increased virulence in ammonium-supplemented pathogenicity assays (38,62).Whereas there was no evidence of increased virulence of the A. flavus ΔmeaB mutant strain, interestingly, the meaB overexpression strain was reduced in virulence (39).Thus, the virulence-related mechanisms driven by these fungi are largely dependent on the distinct transcriptional regulation of pathways implicated in virulence.For example, MeaB normally inhibits the Ste12 activation of a MAPK signaling pathway in F. oxysporum (62), and loss of this inhibition could explain the enhanced virulence of the ΔmeaB mutant strain.In A. flavus, the authors revealed an opposite regulation of ste12 by meaB (39), suggesting that the contribution of meaB on virulence in A. flavus does not act through the same signaling pathways as F. oxysporum.The involvement of cell wall integrity, biofilm formation, and stress response in fungal pathogenicity and virulence has already been validated.Therefore, the virulence characteristics of A. fumigatus ΔmeaB may originate from the divergence control mechanisms for these distinct networks.
The cell wall and extracellular polysaccharides, including glucans, chitin, galacto mannan, and GAG, share multiple common substrates and intermediates in the biosynthesis of these polymers.Many proteins playing a certain role in cell wall biogenesis are also involved in the regulation of biofilm GAG biosynthesis.Apart from the aforementioned transcription factors SomA and MeaB, a salient case is that of CreA; the carbon catabolite repressor not only regulated cell wall homeostasis (44) but was also closely associated with galactosaminogalactan-mediated biofilm formation (45).Here, our data indicate that loss of the CCAAT-binding transcriptional regulator, HapB, causes severe defects in congo red resistance and GAG detection.These results are consistent with the observation that hapB deletion results in an increased susceptibility to cell wall stress agent caspofungin and decreased expression of GAG biosynthesis-rela ted genes based on the previous RNA-seq data (34,35,46).Similarly, the MADS-Box transcription factor RlmA and the histone acetyltransferase GcnE also seem to be involved in these two pathways (31,63).In summary, these results reveal that the biosynthesis of these distinct cell wall polysaccharides is a complex and interconnected process.The evidence can also be gained from another perspective; the transcription factors SomA (an ortholog in Saccharomyces cerevisiae is Flo8), HapB, and CreA are protein kinase A (PKA)-dependent targets (34,64,65), while the transcription factor RlmA is a down effector of MpkA (31,66).It is worth noting that both PKA and MpkA signal pathways play critical roles in fungal cell wall integrity and that both of the kinase modules contribute to fungal adhesion and biofilm formation (67-71), although biofilm-contained GAG has not yet been found in yeast.In summary, the present study reveals the regulatory network of transcription factors on polysaccharide synthesis in distinct cell wall components.
In conclusion, this study functionally describes a transcription factor MeaB that regulates cell wall integrity and stress response, as well as contributes virulence of A. fumigatus.This reinforces our understanding of the fungal cell wall organization and their regulatory mechanisms in the opportunistic fungal pathogen and provides insights into the fungal virulence mechanisms that may aid in the discovery of novel antifungal drug targets (72).

Strains and growth conditions
All strains used in this study are listed in Table S3 in the supplemental material.A. fumigatus strains were typically grown at 37°C in rich medium (YG; 2% glucose, trace elements, and 0.5% yeast extract), minimal medium containing 1% glucose as carbon sources, 10 mM ammonium tartrate (AMM) or 70 mM sodium nitrate as nitrogen sources (NMM), trace elements, and pH 6.5, and RPMI 1640 (Sigma-Aldrich, USA).Solid MM and YG were the same as those described above except that 2% agar was added.The A. fumigatus A1160::pyrG was used as a wild-type control strain (73).

Genetic modifications
All primers used in this study are listed in Table S4 in the supplemental material.The ΔmeaB null mutant strain was generated using a pyr4 selectable marker strategy, and the fusion PCR method was performed to construct the meaB knockout cassette as previously described (74).Briefly, approximately 1.2 kb of the upstream and downstream flanking DNA segments of the meaB open reading frame (ORF) was amplified with the primer pairs MeaB-P1/-P3 and MeaB-P4/-P6, respectively.The pyr4 gene was amplified from plasmid pAL5 with the primer pair Pyr4-F and -R.The resulting PCR products were used as the templates to generate the meaB knockout cassette with the primers MeaB-P2 and -P5.The fusion product was cloned into the pEASY-Blunt Zero vector (TransGen Biotech) and transformed into the 1160 recipient strain.Transformants were grown on AMM and verified by diagnostic PCR.
The complemented strain meaB com was generated by the ectopic insertion of the target gene into the genome of the ΔmeaB background strain.The full-length ORF and native promoter of meaB were amplified from A. fumigatus 1160 genomic DNA using the primer pair meaB com -F and -R.The resulting fragment was cloned into the hygromycin B resistance gene hph-contained plasmid pAN7-1 and then transformed into the meaB knockout strain, and the transformants were selected on the YG medium supplemented with 200 µg/mL hygromycin B (Sangon Biotech).
The FLAG-tagged MeaB strain was constructed using a similar fusion PCR strategy as described above.Three DNA fragments of flag-pyr4 upstream and downstream flanking sequences of the meaB termination codon were amplified with the primer pairs FlAG-SF/ Pyr4-R, MeaBflag-P1/-P3, and MeaBflag-P4/-P6, respectively.The resulting PCR products were used as templates to generate meaB-flag-pyr4 cassette using the primers MeaBflag-P2 and P5, followed by sequencing, and then transformed into the 1160 recipient strain.Transformants were grown on AMM and verified by diagnostic PCR and western blotting.

Plate assays
To monitor the sensitivity of A. fumigatus strains to various cell wall-perturbing agents, 2 µL of 1 × 10 7 conidia/mL spore suspension was spotted onto minimal medium agar plates that were supplemented with 30 µg/mL CR, 50 µg/mL CFW, 1 µg/mL CAS, 25 µg/mL SDS, or 1.2 M sorbitol.The plates were cultured at 37°C, and images were taken at 48 h.

Biofilm formation assay
A. fumigatus biofilm visualization and quantification were performed as previously described (50), with minor modifications.Briefly, 1 mL of AMM per well containing 2 × 10 5 A. fumigatus conidia was cultured in 24-well polystyrene plates at 37°C for 22 h.Then, the adherent biofilms were washed twice with 500 µL of distilled water and dried at room temperature.Adherent biofilms were stained with 300 µL of 0.1% (wt/vol) crystal violet for 10 min at room temperature.After removing the excess crystal violet solution, the stained biofilms were washed twice with 500 µL of distilled water.The stained adherent biofilms were extracted by adding 1 mL of ethanol.Biofilm biomass was determined by measuring the absorbance of the destained solution at 600 nm.

Galactosaminogalactan characterization
The hypha surface galactosaminogalactan polysaccharide was charactered by immu nofluorescence staining using GAG-specific fluorescein-labeled soybean agglutinin (SBA-FITC) (Vector Labs, USA) (23).In brief, the A. fumigatus strains were cultivated on coverslips in RPMI 1640 (Sigma-Aldrich) for 8 to 10 h, subsequently washed with phosphate-buffered saline (PBS), and stained with SBA-FITC for 2.5 h in the dark.After fixation with paraformaldehyde, samples were washed with PBS and microscopically imaged (Zeiss, Germany).

RT-qPCR and RNA-seq
Fresh A. fumigatus conidia were cultured in liquid AMM in a rotary shaker at 200 rpm at 37°C for 48 h.For RT-qPCR analysis, total RNA was isolated using a spin column total RNA purification kit (Sangon Biotech) according to the manufacturer's instructions.gDNA digestion and cDNA synthesis used the HiScriptII Q RT SuperMix for qRCR kit (Vazyme).The quantitative PCR was carried out using the AceQ qPCR SYBR green master mix kit (Vazyme) on an ABI one-step fast thermocycler (Applied Biosystems).Three independent biological replicates were used, and the gene expression levels were calculated using the 2 −ΔΔCT method (75) and normalized to A. fumigatus tubulin.Except for the supplemen tation with 300 µg/mL CR for 0.5, 1, or 2 h after 24 h of growth of A. fumigatus, the stress response assay was the same as described above.For RNA-seq analysis, all sample purification, library construction, and sequencing were performed by Shanghai Personal Biotechnology (China) based on the Illumina sequencing platform.The threshold value of differentially expressed genes was a fold change of >2 and a P-value of <0.05.All the samples were evaluated using three biological repetitions.

Western blotting analysis
The A. fumigatus conidia were cultured in liquid AMM in a rotary shaker at 200 rpm at 37°C for 48 h, followed by being exposed to 300 µg/mL CR for 0.5, 1, or 2 h.The mycelia were collected and ground in liquid nitrogen with a mortar and pestle.Total protein isolation was carried out in alkaline lysis buffer (0.2 M NaOH and 0.2% β-mercaptoetha nol) as previously described (76).The lysate samples were loaded on 10% SDS-PAGE gel and blotted on a polyvinylidene difluoride membrane (Millipore) and then hybridized with anti-FLAG (Sigma-Aldrich, F3165) and anti-β-actin (ABclonal, AC026).The secondary antibodies were peroxidase-labeled goat anti-mouse (Proteintech, SA00001-1) and goat anti-rabbit (Proteintech, SA00001-2) and detected by an enhanced ECL luminescence detection kit (Vazyme) and then visualized by the imaging system (Bio-Rad).The band intensity was calculated using ImageJ software.

Transmission electron microscopy analysis of the cell wall
The thickness of the cell wall of hyphal germlings was determined by TEM, as previously described (29,32,77).Briefly, fresh A. fumigatus conidia were cultured in liquid AMM at 37°C for 12 h and then fixed overnight in 0.1 M sodium phosphate buffer (pH 7.4) containing 2.5% glutaraldehyde at 4°C.The samples were sequentially embedded in 1% (wt/vol) agar, fixed in 1% OsO 4 , dehydrated in ethanol, embedded in epoxy resin monomer (SPI), and then sliced using an ultra-thin slicing machine (Leica UC7).After staining with uranyl acetate and lead citrate, the samples were imaged using a transmis sion electron microscope (Hitachi HT7700).

Virulence assay
A. fumigatus virulence assays in Galleria mellonella were carried out as previously described (77,78) with minor modifications.Briefly, G. mellonella larvae were injected through the last prolegs with 10 µL of PBS containing 6 × 10 7 conidia of the respective strains.The larvae only injected with 10 µL of PBS served as a control group.All larvae were incubated at 37°C in the dark, and their survival was monitored every 24 h for 7 days.Three biological repetitions were performed, and each group had 20 larvae.

FIG 1
FIG 1 The bZIP transcription factor MeaB is a direct downstream target of SomA.(A) Genome browser images depicting the enrichment and transcript levels of SomA on the hapB, creA, and meaB genes based on ChIP-seq (blue) and RNA-seq (purple).ChIP1 and ChIP2 are two independent repetitions.Scale bar, 1 kb.(B) RT-qPCR analysis of the relative expression levels of hapB, creA, and meaB in somA mutant (Tet-somA OFF).The A. fumigatus strains were cultured in AMM with (Continued on next page)

FIG 1 (
FIG 1 (Continued)(Tet-somA ON, induce the expression of SomA) or without doxycycline for 24 h.Gene expression was normalized to the endogenous reference gene tubA.The results were performed by three independent biological experiments.(C) Electrophoretic mobility shift assay (EMSA) of SomA binding to the promoter fragments of meaB.The specificity of EMSA binding was confirmed by adding specific cold probe competitors (50-fold unlabeled probe).(D) Phenotypes of the WT and ΔmeaB and meaB com strains grown on solid rich medium (YG) or minimal medium (AMM) at 37°C for 48 h.(E) Quantitative analysis of relative colony diameter of the indicated strains.The data are presented as the percentages of the colony diameter of the WT strain, and the results are the means of three repetitions ± standard deviation (SD).(F and G) Phenotypes and quantifications of the WT and ΔmeaB and meaB com strains grown in liquid minimal medium at 37°C for 48 h.* P < 0.05; *** P < 0.001.ns, not significant.

FIG 2 FIG 3
FIG 2 MeaB is involved in biofilm formation and virulence of A. fumigatus.(A) Formation of adherent biofilms by the WT and ΔmeaB and meaB com strains after 24 h of growth on polystyrene plate surfaces in AMM.(B) Biofilms were visualized by staining with crystal violet and quantified by determining the absorbance at 600 nm.(C) Representative images of hyphae of the WT and ΔmeaB and meaB com strains stained with SBA-FITC after growth in RPMI 1640.Scale bar, 10 µm.DIC, differential interference contrast.(D) Quantification of the mean fluorescence intensity (MFI) of A. fumigatus hyphae grown under the same conditions as the ones described above for panel (C).Data are presented as the percentages of the MFI of the WT strain, and the means ± SD are from three independent biological samples, each with five hyphal sections measured ( *** P < 0.001).(E) RT-qPCR analysis of the relative expression levels of the GAG cluster genes in the WT and ΔmeaB and meaB com 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.(F) Survival curves for G. mellonella larvae infected with the WT and ΔmeaB and meaB com strains; PBS-injected larvae were used as a negative control.Statistical analysis between groups used the log-rank test ( ** P < 0.01).

FIG 3 ( 8
FIG3 (Continued)    to control groups of the indicated strain, and the results are the means of three repetitions ± SD. (D) Representative TEM images of hyphae of the WT and ΔmeaB and meaB com strains cultured in MM.Scale bar, 200 nm.(E) Quantification of the mean cell wall thickness of the WT and ΔmeaB and meaB com strains as in panel (A).Data are presented as the means ± SD of three biological samples, with 10 sections measured for each.* P < 0.05; *** P < 0.001.

FIG 4
FIG 4 MeaB regulates fungal cell wall biogenesis.(A) KEGG pathway enrichment of differentially expressed genes in the ΔmeaB mutant strain compared to the WT.False discovery rate values and gene numbers are represented using a gradient of color and bubble size, respectively.(B) Bar charts show the GO enrichment analysis of the DEGs in the meaB mutant.The 30 most significantly enriched GO terms of biological process, cellular components, and molecular function were selected as representations.(C) The heat map shows the selected genes (FC >2 and P < 0.05) putatively involved in the cell wall polysaccharide metabolism pathway.Blank gray cells indicate missing FPKM values, FPKM, fragments per kilobase per million.

FIG 5
FIG 5 The stress response effector MeaB regulates the expression of cell wall glucanosyltransferases and mitogen-activated protein kinases.(A and B) RT-qPCR and western blotting analysis of the relative expression levels of meaB in the WT in the presence of 300 µg/mL CR for 0.5, 1, or 2 h.Gene expression was normalized to the endogenous reference gene tubA.Quantified western blot signal intensity was performed by ImageJ software.(C) RT-qPCR analysis of the relative expression levels of cell wall biosynthesis-related genes in the WT and ΔmeaB and meaB com strains after 24 h of growth in MM. (D and E) RT-qPCR analysis of the relative expression levels of mpkA and mpkC in the WT and ΔmeaB mutant in the presence of 300 µg/mL CR for 0.5, 1, or 2 h.Results represent data from three independent biological experiments.* P < 0.05; ** P < 0.01; *** P < 0.001.