The CCAAT‐binding complex mediates azole susceptibility of Aspergillus fumigatus by suppressing SrbA expression and cleavage

Abstract In fungal pathogens, the transcription factor SrbA (a sterol regulatory element‐binding protein, SREBP) and CBC (CCAAT binding complex) have been reported to regulate azole resistance by competitively binding the TR34 region (34 mer) in the promoter of the drug target gene, erg11A. However, current knowledge about how the SrbA and CBC coordinately mediate erg11A expression remains limited. In this study, we uncovered a novel relationship between HapB (a subunit of CBC) and SrbA in which deletion of hapB significantly prolongs the nuclear retention of SrbA by increasing its expression and cleavage under azole treatment conditions, thereby enhancing Erg11A expression for drug resistance. Furthermore, we verified that loss of HapB significantly induces the expression of the rhomboid protease RbdB, Dsc ubiquitin E3 ligase complex, and signal peptide peptidase SppA, which are required for the cleavage of SrbA, suggesting that HapB acts as a repressor for these genes which contribute to the activation of SrbA by proteolytic cleavage. Together, our study reveals that CBC functions not only to compete with SrbA for binding to erg11A promoter region but also to affect SrbA expression, cleavage, and translocation to nuclei for the function, which ultimately regulate Erg11A expression and azole resistance.

Recent studies have proposed that azole antifungals also exert their fungicidal activity by triggering the synthesis of cell wall carbohydrate patches that penetrate the plasma membrane, thereby killing the fungus (Geissel et al., 2018). Investigation of azole-resistant A. fumigatus suggests that the primary genetic alteration responsible for azole resistance is found within the erg11A locus (Burks et al., 2021). Among these azole-resistant isolates, the substitution of leucine 98 for histidine (L98H) in the erg11A gene along with two copies of a specific 34-bp tandem repeat (TR34) in the erg11A promoter (TR34/L98H) resulted in the overexpression of erg11A, which was found to be the predominant resistance mechanism (Chowdhary et al., 2017;Mellado et al., 2007;Snelders et al., 2008;Zhang et al., 2019). Understanding the regulatory mechanisms of erg11A expression can provide important insight into azole resistance mechanisms in fungal pathogens.
The expression of erg11A has been reported to be regulated by several transcription factors, including SrbA, AtrR, CBC (CCAATbinding complex), and NCT complex (negative cofactor two A and B), by directly binding to the promoter region of erg11A and consequently regulating azole susceptibility (Furukawa, Scheven, et al., 2020;Zhang et al., 2019). SrbA, a transcriptional regulator that belongs to the sterol regulatory element-binding protein (SREBP) family, directly binds to the 34 mer of erg11A promoter and positively regulate erg11A expression to resist azoles (Bat-Ochir et al., 2016;Blosser & Cramer, 2012;Willger et al., 2008Willger et al., , 2012. SrbA also binds to its own promoter to autoregulate its expression, as well as the promoters of sterol biosynthesis-related genes in response to hypoxia (Blatzer et al., 2011;Chung et al., 2014). During hypoxia, endoplasmic reticulum (ER)-associated full-length SrbA undergoes protein cleavage involving the rhomboid protease RbdB, Dsc ubiquitin E3 ligase complex (DscA-E), and signal peptide peptidase SppA, resulting in the release of the N-terminal helix-loop-helix (HLH) transcription factor domain into the nucleus to function as a transcription factor (Bat-Ochir et al., 2016;Dhingra et al., 2016;Vaknin et al., 2016;Willger et al., 2012). The nuclear translocation of the N-terminal SrbA also occurs upon azole stress in A. fumigatus (Song et al., 2017). Consistent with the defective phenotypes observed in the srbA deletion mutant under hypoxia and azole conditions, dscA-E null mutants show increased susceptibility to hypoxia and azole drugs (Dhingra et al., 2016;Willger et al., 2012). The Zn 2 -Cys 6 transcription factor AtrR has also been shown to be a positive regulator of erg11A in A. fumigatus. Similar to SrbA, the binding site of AtrR also falls in the 34 mer region of the erg11A promoter (Hagiwara et al., 2017;Paul et al., 2019). CBC, a heterotrimer composed of HapB, HapC, and HapE, is a negative regulatory complex of erg11A in A. fumigatus.
CBC directly binds to the CGAAT motif within the 34 mer of the erg11A promoter, and CBC dysfunction increases A. fumigatus resistance to azoles (Gsaller et al., 2016). Notably, a clinically relevant HapE P88L mutation in A. fumigatus is reported to significantly perturb the binding affinity of CBC to the erg11A promoter, resulting in an azole-resistant phenotype (Camps et al., 2012;Hortschansky et al., 2017). Recently, the subunits of A. fumigatus NCT complex (negative cofactor two A and B), NctA and NctB, have been identified as a key regulator of azole resistance by directly binding to the TATA box-like AT-rich motifs within promoter regions of erg11A, hapC, srbA and atrR and regulating their expressions (Furukawa, van Rhijn, et al., 2020). Several studies have implicated that the molecular mechanism of azole resistance in A. fumigatus is highly complex and tightly regulated by a network of transcriptional activators and repressors.
In this study, we analyzed an isolate 415-2 selected from our previously reported azole-resistant A. fumigatus library (Wei et al., 2017).
Based on a previous study showing that azole tolerance is governed by the opposing actions of SrbA and CBC on erg11A expression, we found that the increased expression of Erg11A and azole resistance induced by loss of HapB is dependent on SrbA. Additionally, we found that the lack of HapB not only increases SrbA expression but also promotes SrbA cleavage and nuclear translocation, which is probably due to the increased expression of RbdB, the Dsc complex, and SppA. These findings broaden our understanding of how SrbA and CBC coordinately regulate Erg11A expression and azole resistance and may provide a potential avenue for overcoming the resistance to azole drugs.

| Strains, media, and culture conditions
All A. fumigatus strains used in this study are listed in Table A1. In general, these strains were grown on solid minimal media (MM), which contained 0.02 g/ml agar, 0.01 g/ml glucose, 1 ml/L trace elements, and 50 ml/L 20× salt solution . The liquid MM recipe does not contain agar. Uridine (5 mM) and uracil (10 mM) are required for uracil and uridine auxotrophic strains. To test the sensitivity of A. fumigatus to azole drugs, ITC and VRC were supplemented in MM or MM plus uridine and uracil (MMUU). For the plate assay, a 2 μl slurry containing 2 × 10 4 spores was spotted onto solid MM at 37°C for 2 or 2.5 days. Longer culture time (4 days) was required for the observation of the growth phenotypes on MM with ITC or VRC.

| Next-generation sequencing analysis sequencing and single-nucleotide polymorphism analysis
The fresh conidial spores of isolate 415-2 were inoculated into liquid MM and shaken for 24 h at 37°C at 200 rpm, and the resulting mycelial pellets were dried and extracted to obtain genome DNA (gDNA). The next-generation sequencing (NGS) experiment was performed at Shanghai OE Biotechnology Co., Ltd., as a commercial service. gDNA of 415-2 was sequenced by using the Illumina HiSeq 2000 platform with 100-bp paired-end reads in a highoutput mode. An average depth of each nucleotide was gained.
Sequence assembly and mapping were referred to the A. fumigatus A1163 genome (http://www.ncbi.nlm.nih.gov/assem bly/ GCA_00015 0145.1). Analysis of mapping quality and SNPs was performed by using a next-generation sequencing data analysis suite, SHORE software.
For deleting srbA, the traditional homologous recombination strategy was employed. All primers are listed in Table A2. A. fumigatus transformation was carried out as previously described Zhang et al., 2016).

| Molecular cloning
The plasmid p-Ama1-P srbA -gfp-srbA for labeling SrbA with GFP was constructed as follows: using primers Ama1-srbA-F and Ama1-srbA-R, the P srbA -gfp-srbA fragment containing the srbA promoter, GFP and srbA ORF was amplified from the gDNA of an N-tagged GFP-SrbA strain and then subcloned into the BamHI site of the plasmid prg3-AMAI-NotI, generating the plasmid p-Ama1-P srbA -gfp-srbA.
The above plasmids were transformed into different background strains, which are listed in data Table A1.

| Quantitative real-time PCR analysis
Fresh A. fumigatus conidia were grown in MM in a rotary shaker at 220 rpm at 37°C for 48 h. For measuring the relative mRNA expression levels of target genes, total RNA of related strains was extracted using the UNlQ-10 Column TRIzol Total RNA Isolation Kit (Sangon Biotech, B511361-0020), following the manufacturer's introduction. Then, cDNA synthesis was performed with the HiScript II Q RT SuperMix for qPCR Kit (Vazyme, R223-01). For detecting the relative srbA gene copy number, the gDNA of A. fumigatus was extracted using Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech, B518259-0050). At least three biological replicates had been performed for each independent assay. The relative transcript levels of target genes and srbA gene copy number were calculated by the comparative threshold cycle (∆CT) and normalized against the expression of tubA mRNA level and tubA gene copy number, respectively. The difference of the relative mRNA expression and srbA gene copy number was determined as 2 −∆∆CT . All the RT-qPCR or qPCR primers and annotations are listed in data Table A2.

| Western blotting
To extract GFP fusion proteins from A. fumigatus mycelia, 10 8 conidia were inoculated into 100 ml of liquid MM under different treatment conditions (see legends) for a set time. Mycelia were collected, frozen in liquid nitrogen, and ground with a mortar and pestle. In general, protein extraction was performed using a previously described alkaline lysis strategy (Nandakumar et al., 2003).
For extracting the nucleoprotein, the commercial nucleoprotein extraction kit (Beyotime, P0027) was used according to the manufacturer's instructions. The GFP fusion protein was detected by using an anti-GFP mouse monoclonal antibody (Roche) at a 1:3000 dilution. Actin mouse monoclonal antibody (Proteintech, 66009-1) at a 1:5000 dilution against actin was used as an internal loading control. Detailed procedures of protein extraction and western blotting were described previously (Nandakumar et al., 2003;Zhang et al., 2018;Zhang et al., 2016).

| Recombinant CBC protein purification and electrophoretic mobility shift assay
To express His-labeled CBC subunits in vitro, the exons of hapB, hapC, and hapE were amplified with three pairs of primers EmsA-hapB-F/ EmsA-hapB-R, EmsA-hapC-F/EmsA-hapC-R, and EmsA-hapE-F/ EmsA-hapE-R, respectively, and then ligated into the pET30a vector, subsequently transformed into BL21(DE3) Competent Cells were grown in LB medium at 37°C to an OD600 between 0.6 and 0.8, followed by addition of 0.1 mM isopropyl β-D-thiogalactoside. Protein purification was performed as previously described using a rapid Ninitrilotriacetic acid (NTA) agarose minicolumn (Huang et al., 2015).
EMSA was carried out according to previously described with minor modifications (Huang et al., 2015;Long et al., 2018). In briefly, each reaction contains consisted of 6 µl of 5× EMSA binding buffer, 1.5 µl of 1 mg/ml salmon sperm DNA (nonspecific competitor), 60 ng Cy5labeled probe (double-stranded DNA), 0.5 µg HapB, 0.8 µg HapC, and 0.6 µg HapE. For competitive testing, a 30-fold nonlabeled DNA probe (1.8 µg) as a competitive cold probe was added to the reaction. To confirm the specific binding of CBC to the binding motif CCAAT, the CCAAT motif within the promoter of the target gene was randomly mutated into a non-CCAAT sequence. The reaction mixtures were incubated at 37°C for 0.5 h and then separated on a 5% polyacrylamide gel in 0.5× Tris-borate EDTA buffer. Subsequently, the Cy5-labeled probes were detected with an Odyssey machine (LI-COR).

| The azole resistance of isolate 415-2 is due to the hapB mutation
Our previous study has obtained a library of azole-resistant strains through a long-term induction of azole treatment, however, the resistance mechanisms for a majority of these isolates have not been experimentally investigated (Wei et al., 2017). From this library, we found isolate 415-2 without the mutations in erg11A showed high resistance to azoles. In comparison to the wild-type strain, isolate 415-2 displayed partial growth defects and high resistance to ITC and VRC (Figure1-a). To identify the potential mutated genes leading to azole resistance, next-generation sequencing (NGS) was implemented on isolate 415-2. After BLAST F I G U R E 1 Dysfunction of the HapB/CBC complex leads to the resistance of 415-2 isolate to azoles. (a) A series of 2 × 10 4 conidia of wildtype (A1160::pyr4/ZC03) 415-2 isolate, 415-2 hapB , ΔhapB/C/E, hapB c, and hapB 165 strains were spotted onto MMUU (MM plus 5 mM uridine and 10 mM uracil) with different concentrations of ITC or VRC cultured at 37°C for 2 or 4 days. (b) The pie chart reflects the mutations in the 415-2 genome by SNPs analysis

| Increased expression of Erg11A and azole resistance induced by loss of HapB is dependent on SrbA
A previous study showed that CBC represses the mRNA expression of erg11A by binding the azole resistance-associated 34 mer in the erg11A promoter (Gsaller et al., 2016). To further explore whether CBC and SrbA coordinately control Erg11A expression at the protein level, we labeled GFP at the C-terminus of Erg11A in the wild-type, ΔhapBΔsrbA compared to that of wild-type strain irrespective of ITC treatment, confirming that SrbA is required for Erg11A protein expression. In line with immunoblotting results, fluorescence observation also showed that Erg11A-GFP exhibited stronger GFP signals with the endoplasmic reticulum (ER)-localized pattern in the ΔhapB strain than that in the wild-type strain, and the GFP signals were barely observed in the ΔsrbA and ΔhapBΔsrbA strains (Figure 2-b).
Moreover, the phenotypic analysis showed that ΔhapBΔsrbA presented a sensitive phenotype on ITC/VRC-amended medium, which is similar to the ΔsrbA strain (Figure 2-c), suggesting that the increased expression of Erg11A and azole resistance induced by loss of HapB is dependent on SrbA.

| The protein expression and localization of SrbA and HapB under azole treatment conditions
Previous studies indicated that ER membrane-bound SrbA can be if SrbA cleavage was associated with its nuclear localization, total protein was extracted from the WT GFP−SrbA strain using alkaline lysis followed by immunoblotting using a GFP antibody. Immunoblotting showed specific bands at approximately 150 kDa with or without ITC treatment, corresponding to the full-length SrbA-GFP fusion protein (hereafter named GFP-SrbA-F; GFP: ~27 kDa, SrbA: ~120 kDa), however, no cleaved SrbA bands were observed (Figure 3-b). In contrast, the nuclear forms of SrbA (the cleaved N-terminus, hereafter named GFP-SrbA-N) were clearly visible by using a specific nucleoprotein extraction kit (Figure 3-b), suggesting that ITC induces SrbA protein cleavage and nuclear translocation. To explore the effects of ITC on HapB protein, the HapB was labeled with an N-terminus GFP tag at its native locus. The GFP-HapB fusion protein did not cause any morphological phenotypes compared to the parental strain ( Figure   A1), indicating that it is fully functional. As shown in Figure 3

| The nuclear form of SrbA confers azole resistance and increases the expression of Erg11A in A. fumigatus
Considering that ITC induces the SrbA cleavage and nuclear translocation, we wondered whether the nuclear form of SrbA was associated with azole resistance. To test this, we constructed a truncated SrbA strain (GFP-SrbA T ) that expresses a putative nuclear form of SrbA (SrbA T , which contains the first 380 aa of SrbA) fused with GFP at its N-terminus under the control of the srbA native promoter in the wild-type background (Figure 4-a). To examine whether the GFP-SrbA T fusion protein localizes to the nucleus, we coexpressed RFP-tagged histone H2A (RFP-H2A) as a nuclear marker. As shown in Figure 4-b, GFP signals of full-length GFP-SrbA were detected in the peripheral areas of RFP-H2A, whereas GFP-SrbA T was colocalized with RFP-H2A in minimal medium, indicating that the mutant with a nuclear form of SrbA was successfully constructed.
To exclude the interference of the GFP tag with the function of SrbA T , we generated a SrbA T strain that expresses SrbA T without a GFP tag in the wild-type background using the AMA1 vector. A SrbA F strain expressing the full-length of srbA was also constructed similarly as a control. To exclude the possibility that the AMA1 plasmids may introduce different srbA gene copies between SrbA F and SrbA T strains, we compared the relative gene copy number of srbA by quantitative real-time PCR (qRT-PCR) analysis. The result showed no significant difference in srbA copy number between SrbA F and SrbA T strains ( Figure A 2-a). The colony phenotype of SrbA T strain was indistinguishable from SrbA F strain on minimal medium but displayed increased resistance to ITC (Figure 4-c), indicating the con-  (d) The indicated strains were grown in MM at 37°C for 24 h, and then 0.2% DMSO or 16 μg/ml ITC was added to the media for 2 h. The control "0" indicates the strains that were grown in MM for 26 h without DMSO or ITC treatment. The protein samples extracted using the alkaline lysis strategy or nucleoprotein extraction kit were examined by immunoblotting. The wild-types strain without GFP tag was used as a control Taken together, these data suggest that the constitutive nucleuslocalized N-terminus of SrbA renders A. fumigatus resistant to azole by upregulating Erg11A.

| DISCUSS ION
In fungi, the transcriptional regulator SrbA and the CBC complex have been reported to transcriptionally regulate the expression of the azole target Erg11A and therefore play critical roles in azole resistance and sterol biosynthesis (Gsaller et al., 2016). It has been reported that both SrbA and CBC can bind to the TR34 region of the erg11A promoter, and they perform opposing actions to govern sterol biosynthesis and azole tolerance. The absence of any of the CBC subunits results in increased tolerance of A. fumigatus to azoles mainly due to the increased mRNA expression of erg11A. In contrast, the increased azole susceptibility in the srbA null mutant strain is the result of erg11A transcript insufficiency. In this study, we identified that the non-erg11A azole-resistant isolate 415-2 harbors a mutation in the hapB gene that leads to the premature transcription termination of hapB. Importantly, we revealed a potential regulatory mechanism by which the CBC negatively regulates SrbA expression by directly binding to srbA promoter, and represses SrbA cleavage by down-regulating the expression of the rhomboid protease RbdB, the Dsc ubiquitin E3 ligase complex, and the signal peptide peptidase SppA.
We found that azole also induces the cleavage of A. fumigatus SrbA and its translocation into the nucleus (Figure 3-a) (Song et al., 2017).
In addition, expression of the putative nuclear forms of SrbA in the wild-type strain increased Erg11A protein expression and tolerance to azole (Figure 4c sites, respectively (Furukawa, Scheven, et al., 2020), however, no typical CCAAT or CGAAT motif was observed in these regions. This may be because the ChIP peaks may result from the indirect binding events via intermediary partners, as the previous study showed that the transcription factor HapX forms a complex with CBC and CBC/ HapX complex recognizes other DNA motifs than CC(G)AAT such as 5′-RWT-3′ and 5′-TKAN-3′ motifs (Furukawa, Scheven, et al., 2020).
Nevertheless, our qRT-PCR and western blotting experiments further showed that loss of CBC resulted in increased expression of SrbA and the majority of its cleavage-related genes. In addition, since azole drugs and CBC deficiency have been reported to change the sterol profile (Gsaller et al., 2016;Shapiro et al., 2011), we therefore cannot rule out the potential role of altered sterol profile in the regulation of SrbA expression and nuclear retention, which needs further investigation and analysis.

| CON CLUS ION
In this study, our findings have revealed another plausible mechanism by which CBC dysfunction causes the upregulation of SrbA, and RbdB, SppA, and the Dsc complex facilitate SrbA activity, which ultimately elevates Erg11A expression and azole tolerance, and provides new insight into the molecular mechanism underlying the regulation of azole resistance. A working model summarizing the findings of this study is depicted in Figure 7.

CO N FLI C T O F I NTE R E S T
None declared.

E TH I C S S TATEM ENT
None required.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available in this published article and its appendices.

O RCI D
Yuanwei Zhang https://orcid.org/0000-0003-0854-6123 Ling Lu https://orcid.org/0000-0002-2891-7326 F I G U R E 7 A proposed model is shown highlighting the mechanistic basis of erg11A expression regulated by CBC and SrbA. By directly interacting with the 34 mer region of the erg11A promoter, CBC and SrbA negatively and positively regulate the expression of erg11A, respectively. Upon azole stress, full-length SrbA (SrbA-F) is cleaved into the nuclear form (SrbA-N) and subsequently transfers from the endoplasmic reticulum (ER) into the nucleus to exert its function. CBC not only represses the expression of srbA but also inhibits SrbA cleavage and nuclear translocation via repressing the expression of SrbA cleavage-associated genes, including rbdB, Dsc complex genes, and sppA. These repressions are liberated in the mutants of CBC. Notably, CBC dysfunction also prolongs the retention time of SrbA-N in the nucleus, which is not represented in the model A PPEN D I X A series of 2 × 10 4 conidia of related strains were spotted onto MM and cultured at 37°C for 2 days F I G U R E A 2 The relative srbA copy number in the related strains constructed by using the AMA1 vector. The related strains were grown in liquid MM with 220 rpm shaking at 37°C for 24 h. The respective genome was extracted from the resulting mycelium. Then qRT-PCR was used to detect the relative srbA copy number, tubA gene was used as an endogenous control. Statistical significance was determined by Student's t-test. ns, not significant. Values are means ± SD from three independent replicates