The cyclase-associated protein contributes to antifungal susceptibility and virulence in Aspergillus fumigatus

ABSTRACT

Aspergillus fumigatus is the most prevalent pathogenic mould that contributes to high morbidity and mortality in immunocompromised patients. Here, we characterized the functions of the cyclase-associated protein (CAP) in A. fumigatus. To study the role of CAP in virulence and antifungal susceptibility of A. fumigatus, CAP gene knockout strain (ΔCAP) and complemented strain (R-ΔCAP) were constructed. ΔCAP showed a reduced growth rate, abnormal hyphal development, and increased susceptibility to cell wall-perturbing agents (Congo red, calcofluor white, and SDS), oxidative stress-inducing agents (H₂O₂ and menadione), calcineurin inhibitors (FK506 and CsA), and voriconazole (VRC) and itraconazole. Transcriptome analysis revealed that differentially expressed genes responsible for regulating growth, hyphal development, cell wall synthesis, stress responses and antifungal susceptibility were identified in ΔCAP. To identify CAP-interacting proteins, an A. fumigatus strain expressing the CAP protein fused with a C-terminus 6×his tag was constructed and designated Afcap6his. After extracting Afcap6his and Af293 proteins, actin and adenylate cyclase were identified by coimmunoprecipitation (co-IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Additionally, ΔCAP showed downregulated actin expression, AC-cAMP-PKA pathway activity and efflux pump genes (AfuMDR1, AfuMDR2, AfuMDR3, AfuMDR4, and cdr1B) expression as well as increased calcineurin activity. By using an invasive pulmonary aspergillosis (IPA) murine model, ΔCAP exhibited attenuated virulence and increased VRC therapeutic efficiency. Thus, CAP plays an important role in regulating antifungal susceptibility and virulence of A. fumigatus.

Introduction

In recent decades, the incidence of invasive aspergillosis (IA) has increased significantly due to the growing number of immunocompromised individuals such as those with severe COVID-19, tumours, leukaemia, and those receiving bone marrow transplantation, as well as the prolonged use of corticosteroids, broad-spectrum antibiotics, or immunosuppressive agents [1,2]. IA is a severe life-threatening infection with a high mortality rate of up to 95%. IA is mainly caused by the widespread mould Aspergillus fumigatus. It produces minute (3-4 micron) airborne asexual spores (conidia), which are constantly inhaled by humans [1,2]. The high mortality of IA patients is mainly driven by several factors including host immunodeficiency, high virulence and A. fumigatus resistance to antifungals [3,4].

During the infection process, A. fumigatus conidia can escape ciliary clearance and reach the lower respiratory tract [5,6]. To escape the host immune defense system, the major pathogenic factors of A. fumigatus promote its tolerance to high temperature and reactive oxygen species, enabling conidial germination and eventual tissue penetration [7]. Currently, the approved antifungals used to treat IA include triazoles (voriconazole, itraconazole, posaconazole, and isavuconazole), polyenes (amphotericin B) and echinocandins (caspofungin), among which voriconazole (VRC) is recommended as the first-line therapy [4]. Since the first itraconazole (ITC)-resistant A. fumigatus isolate was described by Denning et al. in 1997, triazole-resistant A. fumigatus isolates have been reported in many countries, posing a serious threat to antifungal therapy and resulting in high mortality rates [8,9]. Therefore, it is essential to investigate the role of virulence and resistance factors in A. fumigatus, which may serve as novel targets for improving antifungal efficacy during treatment of IA [7,10].

In our previous work, an A. fumigatus transformant with a reduced growth rate and increased susceptibility to VRC was unexpectedly obtained during genetic manipulation. Using the plasmid rescue technique, the cyclase-associated protein (CAP) gene was identified as being disrupted in this A. fumigatus transformant. Since previous studies have demonstrated that the CAP homologue is responsible for growth, hyphal development, response to high-temperature stress, and virulence in Candida albicans, Saccharomyces cerevisiae and Magnaporthe oryzae [11–14], we here investigated the role of the CAP gene in antifungal susceptibility and virulence of A. fumigatus in this study.

Materials and methods

Fungal strains, primers, media, and culture conditions

All A. fumigatus strains and the primers used in this study are listed in Table S1 and Table S2. All A. fumigatus strains were cultured on minimal medium (MM) at 37°C unless otherwise specified. A. fumigatus strain Af293 was employed as the wild-type (WT) strain. Af293.1, an uracil/uridine-auxotrophic mutant of Af293, was used to generate the CAP gene replacement strain. Af293 and Af293.1 were both kindly provided by Professor Dimitrios P. Kontoyiannis in the University of Texas MD Anderson Cancer Center, USA [15]. The Escherichia coli strain DH10B (Invitrogen) was used for routine cloning and cultured in Luria–Bertani (LB) broth. The Agrobacterium tumefaciens EHA105 strain was grown in LB broth supplemented with or without kanamycin [16].

Construction of the ΔCAP, R-ΔCAP, and Afcap6his strains

The ΔCAP mutant was constructed by homologous recombination using A. tumefaciens-mediated transformation as described (Figure S1A-B) [16]. A 4.4-kb DNA fusion fragment composed of a 1.4-kb A. nidulans pyrG amplified from pALX223 and 1.5-kb upstream and downstream regions of the CAP gene was cloned and inserted into the binary plasmid pDHt/sk [16]. The resulting plasmid was named pDHt/Δcap::pyrG and transformed into the competent A. tumefaciens strain EHA105 [16]. A. tumefaciens strains containing recombinant plasmid were selected on LB plates containing kanamycin and verified by PCR. The correct A. tumefaciens strain was named A.t-pDHt/Δcap::pyrG and cocultured with Af293.1 as described [17]. The right transformant was identified by PCR-Sanger sequencing (Figure S1D). A 477-bp fragment of CAP amplified with the primers F2/R2 was absent, while a 474-bp fragment of pyrG amplified with primers the F3/R3 was present in ΔCAP (Figure S1D). A 1650-bp fragment containing the 5’-flanking region of CAP gene and a partial sequence of pyrG amplified with primers F4/R4 and a 1650-bp fragment containing the 3’-flanking region of the CAP gene and a partial sequence of pyrG amplified with the primers F5/R5 were both present in ΔCAP, but were absent in Af293.1 (Figure S1D). A 5.2-kb and 4.7-kb fragment amplified with primers F6/R6 were present in Af293.1 and ΔCAP, respectively (Figure S1E). These findings suggested that pyrG insertion into the CAP locus generated the ΔCAP mutant strain.

To ensure that all phenotypes in ΔCAP were due to the specific deletion of CAP, the complemented strain was constructed by reintroducing a 3.8-kb PCR fragment containing the CAP coding gene with its 1.0-kb promoter and downstream region into the recipient ΔCAP strain using the PEG-mediated protoplast transformation method using selection with normal colony size and growth rate (Figure S1C) [18]. The complemented strain was designated R-ΔCAP and verified by PCR-Sanger sequencing. A 477-bp fragment of CAP amplified with the primers F2/R2, and a 474-bp fragment of pyrG amplified with the primers F3/R3 were both present in R-ΔCAP. Two 1650-bp fragments of the CAP gene flanking region and a partial sequence of pyrG amplified with the primers F4/R4 and F5/R5 were both present in R-ΔCAP, respectively (Figure S1D). These results suggested that the CAP::pyrG replacement locus was preserved and CAP was inserted ectopically in R-ΔCAP.

To search for proteins interacting with CAP, an A. fumigatus strain expressing the CAP protein fused with a 6×his tag at its C-terminus was constructed and designated Afcap6his. A 5.9-kb pDHt/sk plasmid containing a 1.5-kb 5’ flanking sequence of the CAP gene, pyrG gene, CAP coding gene tagged with a C-terminal 6×his, and 1.5-kb 3’ flanking sequence of the CAP gene was constructed and designated pDHt/cap6his (Figure S2A). The recombinant pDHt/cap6his plasmid was transformed into the A. tumefaciens strain. The correct A. tumefaciens strain containing the recombinant pDHt/cap6his plasmid was cocultured with Af293.1. One transformant was selected and verified by PCR.

Growth rate measurement

Growth rates were determined using the XTT colorimetric measurement assay [19]. A 100 μl suspension (1 × 10⁶ conidia/ml) were inoculated in a 96-well plate. The XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] solution was prepared as described [20]. A 100 μl XTT solution was added to each well at different time points. The plate was incubated at 37°C for 2 h. Optical density at 490 nm was recorded to generate growth curves.

Gross colony and microscopic observations

A 10 μl conidial suspension (1 × 10⁶ conidia/ml) was inoculated on potato dextrose agar (PDA) plates. The plates were incubated at 37°C for 3 days. To observe the microscopic characteristics of all strains, 1 × 10⁵ conidia resuspended in 100 μl of RPMI 1640 medium were inoculated into a 96-well plate containing 100 μl of RPMI 1640 medium. The plates were incubated at 37°C for 48 h. Following incubation, the hyphae were collected, mounted on slides, and stained with or without CFW.

Antifungal susceptibility testing by broth microdilution, E-test, and disk diffusion assays

In vitro antifungal susceptibility testing for AMB, ITC, VRC, POS, CAS, FK506, and CsA (Harveybio Gene Technology Co. Ltd., Beijing, China) against A. fumigatus was performed using the CLSI M38-A3 broth microdilution method with slight modifications when noted [21]. The E-test assay for VRC, ITC, CAS, and AMB against A. fumigatus as previously described [21]. E-test strips were purchased from Autobio Diagnostics Co., Ltd, China. The disk diffusion assay was performed as described [21]. Sterile 6-mm-diameter paper disks containing 80 μg of VRC, 80 μg of ITC, 10 μg of CAS, 1 μg of FK506, and a combination of 10 μg of CAS and 1 μg of FK506 were placed on the centre of the MM plates [22].

Microdilution checkboard testing

In vitro drug interactions between FK506 and CAS against A. fumigatus were observed using the checkerboard broth microdilution assay as described [23]. FICI was calculated as described. If the FICI was ≤0.5, > 0.5 and ≤ 4, and >4, drug interactions were considered synergistic, additive, and antagonistic, respectively [24].

Spot assay

Serial 10-fold dilutions of a conidial suspension (1 × 10⁷conidia/ml) were prepared. 2 μl of each dilution was spotted onto an MM plate supplemented with 0.3 μg/ml VRC, 0.3 μg/ml ITC, 0.5 μg/ml CAS, 0.3 μg/ml AMB, 0.5 μg/ml FK506, 0.5 μg/ml CsA, 5 μg/ml Congo red, 10 μM CFW, 0.1% SDS, 20 μm hydrogen peroxide (H₂O₂), or 10 μm menadione (VK, Sigma-Aldrich). The plates were incubated at 37°C for 48 h [25,26].

Real-time PCR analysis

A total of 10⁶ conidia were inoculated into 100 ml of liquid MM for 20 h, and treated with 10 μg of VRC or an equal volume of ddH₂O for 4 h in order to measure the expression level of efflux pump genes. The mycelium was harvested by centrifugation and frozen in liquid nitrogen. Total RNA was extracted and reverse transcribed with PrimeScript RT Master Mix (Takara, Japan). Real-time PCR was performed with an Applied Biosystems ViiA7 Real-time PCR system using SYBR Green Master Mix. The primers for efflux pump genes used are listed in Table S2 [27]. The mRNA abundance was normalized to the β-tubulin gene of A. fumigatus. Each biological replicate was performed in triplicate.

Transcriptome sequencing and analysis

A total of 10⁶ conidia were inoculated into 100 ml of liquid MM at 37°C. The mycelium was collected at 10, 12, 14, 16, 18, 20, 24, 28, 32, 36, 40, 44, and 48 h. The growth curve was plotted to determine the adaptive phase, logarithmic phase, and stationary phase by measuring the dry biomass [28,29]. For Af293 and R-ΔCAP, 0–12 h represented the adaptive phase, while 12–48 h corresponded to the logarithmic phase. For ΔCAP, 0–24 h represented the adaptive phase, 24–48 h corresponded to the logarithmic phase. Based on these observation, time points of RNA extraction of each strain for transcriptome sequencing were specifically selected to align with the logarithmic phase initiation: 12 h post-inoculation for Af293 and R-ΔCAP, and 24 h for the ΔCAP. Total RNA was extracted using the Fungal RNA Kit (OMEGA). Transcriptome sequencing analysis was performed as described [30]. The DESeq 2 R package was used to identify DEGs with a strict cutoff threshold of |log2FC| > 2 and an adjusted P <0.01. GO and KEGG pathway enrichment analyses were performed.

Transmission electron microscopy

A total of 1 × 10⁶ conidia were inoculated into 200 ml of liquid MM at 37°C for 18 h. The mycelium was harvested by centrifugation, washed 3 times with PBS, fixed with 2.5% glutaraldehyde at 4°C for 4 h. The mycelium was processed as described [31]. The cell wall thickness was measured using the GeomCaliper software.

Western blotting

Total proteins were extracted using a Filamentous Fungal Protein Extraction Kit (BestBio, China). Equal amounts of protein were loaded in each lane, subjected to 10% SDS-PAGE, and transferred to nitrocellulose (NC) membranes at 65 V for 2 h. The membranes were blocked with TBST containing 5% milk for 1 h and incubated with 1:2000 dilutions of an anti-actin antibody (Abcam, USA) and anti-GAPDH antibody (Abcam, USA) [32]. After 3 washes with TBS, the membranes were incubated with 1:2000 dilutions of HRP-conjugated anti-mouse IgG (Cell Signalling Technology, USA) [32]. Specific protein bands were visualized by chemiluminescence.

Co-IP and LC–MS/MS analysis

A total of 1 × 10⁸ conidia of Af293 and Afcap6his were incubated in 100 ml of liquid MM at 37°C for 24 h, respectively. The proteins were extracted and verified by western blotting (Figure S2B). The cell lysates were subjected to co-IP using a Pierce Crosslink Magnetic IP/Co-IP Kit (Number 88805, Thermo Fisher, USA). The precipitated proteins were analyzed by western blotting (Figure S2C) and separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The bands of interest were excised and subjected to in-gel trypsin digestion as described [33]. LC-MS/MS analysis was carried as described [34]. KEGG analysis was achieved using the ClusterProfiler package with an adjusted value of p < 0.05 [33,34].

Measurement of the cAMP level, PKA activity and calcineurin activity

A total of 1 × 10⁸ conidia were cultured in liquid MM for 24 h. The total protein was assayed for the cAMP level and PKA activity using the Cyclic AMP XP® Assay Kit (Cell Signalling Technology, USA) and PKA Kinase Activity Kit (Enzo, USA) [35].

After being cultivated for 20 h, 1 × 10⁸ conidia were treated for 4 h with 0.25 μg/ml CAS, 0.25 μg/ml Congo red, and 0.3 μg/ml FK506. A Cellular Calcineurin Phosphatase Activity Kit (Abcam, UK) was used to measure the calcineurin activity.

Actin, β-1,3-glucan and chitin staining

A total of 1 × 10⁵ conidia was inoculated in a 24-well plate containing a glass coverslip. The actin, β-1,3-glucan and chitin staining were performed as described [32]. For actin staining, the coverslips were treated with a 1:400 dilution of anti-actin antibody (Abcam, UK) in 2% BSA containing 0.5% NP-40 at room temperature for 1 h. The coverslips were stained with a 1:400 dilution of an Alexa Fluor® 488 goat anti-mouse IgG antibody (Invitrogen, USA) in 2% BSA at room temperature for 1 h. For β−1, 3-glucan staining, the conidia were incubated with an anti-β−1,3-glucan antibody (Invitrogen, USA) at 4°C overnight. The conidia were labelled with FITC-conjugated goat anti-mouse IgG (400 μg/ml) for 1 h in the dark [32]. For chitin staining, the conidia were incubated with 100 μg/mL wheat germ agglutinin-fluorescein isothiocyanate (WGA-FITC) (Sigma) for 15 min at room temperature. All stained coverslips were mounted on the slides and photographed using a Leica TCS-SP8 STED 3X fluorescence microscope [32].

Virulence analysis

All animal studies were approved by the institutional animal care and use committee. Outbred BALB/C female mice, 6–8 weeks old, were housed in vented cages. Cyclophosphamide (150 mg/kg of body weight, Sigma) was administered intraperitoneally on days – 4, – 1, and 2 prior to and post infection [36,37]. Hydrocortisone acetate (200 mg/kg body weight, Sigma) was injected on day -3. Fresh conidia were harvested and resuspended at a concentration of 1.5 × 10⁷ conidia/ml. Four groups of 15 mice were anaesthetized by intraperitoneal injection 0.5% pentobarbital sodium. Three groups of 15 mice were infected by intranasal instillation of 1.5 × 10⁷ conidia in 50 μl of PBS. A group of 15 mice received PBS only as a negative control. The mice were weighed every 24 h and observed for 14 days. Statistical comparisons were conducted with log rank tests. Five mice from each group were sacrificed on day +3. Lung sections were stained with haematoxylin eosin (HE) stain and periodic acid-Schiff (PAS) stain as described [37,38]. Fungal genomic DNA was extracted and Real-time PCR was performed to determine the fungal burden as described [38].

In vivo efficacy of VRC against an IPA murine model

VRC was administered at doses of 10 and 20 mg/kg by oral gavage once daily for 7 consecutive days for survival analysis [39,40]. At 3 days post infection, the fungal burden of each group was determined by Real-time PCR as described [39,40].

Results

The strain with CAP gene knockout (ΔCAP) shows reduced growth rates and abnormal hyphal development

The growth of ΔCAP was severely impaired compared with that of Af293 on MM at 37°C, while the defective growth of ΔCAP was reverted when the CAP gene was complemented (R-ΔCAP) (Figure 1A). Similar results were observed by XTT colorimetric assay (Figure 1B). The hyphae of ΔCAP grown for 24 h were shorter and more branched, while the hyphae of Af293 and R-ΔCAP were long and straight as observed by light and fluorescence microscopy (Figure 1C), indicating that CAP is required for growth and hyphal development in A. fumigatus.

Figure 1.

Growth rate and hyphal microscopic characteristics. (A) Colony size observation. (B) Growth rate detected by XTT assay. (C) Microscopic characteristics of hyphal morphology under white light and fluorescence microscopy following calcofluor white staining (×200 magnification).

ΔCAP exhibits increased susceptibility to cell wall-perturbing agents and oxidative stress-inducing agents due to compromised cell wall integrity

To further study the effect of CAP on the stress response, the susceptibility to cell wall-perturbing agents (Congo red, calcofluor white, and SDS) and oxidative stress-inducing agents (H₂O₂ and menadione) was tested. Compared with Af293, ΔCAP exhibited significantly increased susceptibility to Congo red, calcofluor white (CFW), SDS, H₂O₂ and menadione, while the R-ΔCAP restored the susceptibility to these agents (Figure 2A).

Figure 2.

Susceptibility to cell wall-perturbing agents and oxidative stress-inducing agents and cell wall integrity measurement. (A) Susceptibility determined by spot assay. (B) Cell wall thickness measured by transmission electron microscopy (TEM) (×2,0000 and ×1,5000 magnification). (C) The amount of β−1,3-glucan and (D) chitin detected using anti-β-glucan antibody and WGA-FITC.

To examine the role of CAP in cell wall integrity, potential ultrastructural changes in the cell wall were analyzed using transmission electron microscopy (TEM), and the cell wall thickness was measured. The cell walls of ΔCAP were thinner than those of Af293 and R-ΔCAP (Figure 2B). Additionally, following conidial surface staining with β−1, 3-glucan specific antibodies or WGA (chitin specific lectin) the green fluorescence intensity on the conidial surface of ΔCAP was markedly lower than that of Af293 and R-ΔCAP, indicating that the amount of β−1,3-glucan and chitin in ΔCAP was significantly reduced (Figure 2C,D). These results suggest that ΔCAP increases the susceptibility to cell wall-perturbing agents and oxidative stress-inducing agents due to impaired cell wall integrity.

ΔCAP exhibits increased susceptibility to calcineurin inhibitors, triazoles, and the combination of FK506 and caspofungin

Compared with Af293 and R-ΔCAP, ΔCAP was more susceptible to FK506, CsA, VRC and ITC as determined by broth microdilution assay and spot assay (Table 1, Figure 3A), which was also observed by E-test and disk diffusion assays for VRC and ITC (Figure 3B,C).

Table 1.

In vitro susceptibilities of Af293, ΔCAP, and R-ΔCAP to antifungals using CLSI M38-A3 broth microdilution method.

Strains	MIC/MEC (μg/ml)
	FK506	CsA	VRC	ITC	POS	CAS	AMB
Af293	>16	4	0.5	0.5	0.25	>16	2
ΔCAP	0.5	2	0.25	0.25	0.25	>16	2
R-ΔCAP	>16	4	0.5	0.5	0.25	>16	2

FK506, tacrolimus; CsA, cyclosporin A; VRC, voriconazole; ITC, itraconazole; POS, posaconazole; CAS, caspofungin; AMB, amphotericin B; MIC, minimum inhibitory concentration; MEC, minimum effective concentration.

Figure 3.

Susceptibility to calcineurin inhibitors, triazoles, and the combination of FK506 and CAS. Susceptibility determined by (A) spot assay, (B) disk diffusion, (C) E-test assay. (D) Synergism with FK506 and CAS determined by disk diffusion assay. (E) Microscopic observations of hyphal morphology with CAS, FK506, and the combination of FK506 and CAS (200×magnification).

The fractional inhibitory concentration index (FICI) of ΔCAP was 0.28125, which was lower than 0.375 of Af293 and R-ΔCAP (Table 2) using a checkboard microdilution assay. Disk diffusion showed that the inhibition zone diameters were similar for 1 μg FK506 alone against all strains, while the growth density of ΔCAP was significantly reduced within the inhibition zone (Figure 3D). Additionally, the inhibition zone of ΔCAP was more distinct than Af293 and R-ΔCAP when CAS and FK506 were combined (Figure 3D). Microscopic examination revealed that the hyphae of ΔCAP were more swollen and stunted than those of Af293 and R-ΔCAP in the presence of FK506 alone and in combination (200×magnification) (Figure 3E).

Table 2.

In vitro susceptibilities of Af293, ΔCAP, and R-ΔCAP to the combination of CAS with FK506 using checkboard microdilution method.

Strains	MIC (μg/ml)			FICI	Interaction
	CAS	FK506	CAS + FK506
Af293	>16	>16	8/4	0.375	Synergistic
ΔCAP	>16	0.5	1/0.125	0.28125	Synergistic
R-ΔCAP	>16	>16	8/4	0.375	Synergistic

FK506, tacrolimus; CAS, caspofungin; FICI, fractional inhibitory concentration index, If the FICI was ≤0.5, > 0.5 and ≤4, and >4, drug interactions were considered as synergistic, additive, and antagonistic.

ΔCAP shows abnormal transcript levels of genes involved in regulating virulence and antifungal susceptibility

The differentially expressed genes (DEGs) identified in ΔCAP were compared with Af293 and R-ΔCAP by heatmap analysis (Figure 4A). DEGs were mainly enriched in oxidoreductase activity, monooxygenase activity, secondary metabolic processes, phosphopantetheine binding, flavin adenine dinucleotide binding, and acting on paired donors as determined by GO analysis (Figure 4B,C). Additionally, some of the DEGs observed are responsible for regulating growth, hyphal development, cell wall synthesis and stress responses (Figure 4D). These findings provide explanations for the above results showing that ΔCAP 2exhibited a reduced growth rate, abnormal hyphal development, impaired cell wall synthesis, and increased susceptibility to various stresses.

Figure 4.

Transcriptome analysis. (A) Differentially expressed genes (DEGs) among Af293, ΔCAP and R-ΔCAP detected by heatmap analysis. (B) DEGs between Af293 and ΔCAP detected by GO enrichment analysis. (C) DEGs between ΔCAP and R-ΔCAP detected by GO enrichment analysis. (D) DEGs between Af293 and ΔCAP responsible for regulating virulence and antifungal susceptibility by heatmap analysis.

CAP interacts directly with adenylate cyclase (AC) and actin in A. fumigatus

To identify the proteins interacting with CAP in vivo, co-immunoprecipitation (co-IP) and liquid chromatography – tandem mass spectrometry (LC-MS/MS) were performed by extracting proteins from Afcap6his and Af293. A total of 309 proteins were identified and mainly enriched in the biosynthesis of secondary metabolites, and ribosome, carbon, and amino acid metabolism as determined by KEGG analysis (Figure S3A), which was partly consistent with the transcriptome results described above. Notably, actin and AC were the most dominant with peptide coverages of 45% and 34% (Figure S3B-C), indicating that CAP functions predominantly through interacting with actin and AC.

ΔCAP exhibits downregulated actin expression levels and increased calcineurin activity

Since the interaction between CAP and actin was verified by LC-MS/MS analysis as described above, the effect of CAP on actin was investigated by immunofluorescence and western blotting. Actin was randomly distributed with extremely weak fluorescence in the hyphae of ΔCAP, whereas actin was evenly distributed with strong fluorescence and accumulated at the mycelial tips in the hyphae of Af293 and R-ΔCAP (Figure 5A). The actin expression level in ΔCAP was lower than that in Af293 and R-ΔCAP by western blotting (Figure 5B).

Figure 5.

The effect of CAP on the level of actin and AC-cAMP-PKA pathway. (A) Actin expression levels detected by immunofluorescence and (B) western blotting analysis. (C) Calcineurin activity detected following exposure to CAS, Congo red, and FK506. (D) Intracellular cAMP level and PKA activity measurement. (E) The expression levels of AfuMDR1 AfuMDR2, AfuMDR3, AfuMDR4, and cdr1B genes following 4 h of VRC exposure.

Since transcriptome analysis suggested that the expression level of calcineurin had been increased in ΔCAP, all strains were examined for calcineurin activity, which is required for regulating growth, hyphal development, stress responses and virulence. Compared to Af293 and R-ΔCAP, the calcineurin activity in ΔCAP was significantly higher when exposed to CAS and Congo red for 4 h, and slightly higher when un-exposed (Figure 5C).

ΔCAP exhibits a decrease in AC-cAMP-PKA pathway activity and downregulated expression of efflux pump genes

In light of the fact that the PKA mRNA level in ΔCAP was reduced and the aforementioned results confirmed the interaction between CAP and AC, the effect of CAP on the AC-cAMP-PKA pathway was further investigated. The intracellular cAMP level and PKA activity in ΔCAP were markedly lower than those in Af293 and R-ΔCAP (Figure 5D), demonstrating the impairment of the AC-cAMP-PKA signalling pathway in ΔCAP.

Since the AC-cAMP-PKA pathway is responsible for regulating the expression of multiple downstream genes including efflux pump genes, the expression levels of AfuMDR1, AfuMDR2, AfuMDR3, AfuMDR4, and cdr1B were evaluated by qPCR. The results confirmed the RNAseq expression results and showed that the expression levels of these five genes in ΔCAP was lower than those in Af293 and R-ΔCAP after 4 h of VRC exposure (Figure 5E).

ΔCAP exhibits attenuated virulence and enhanced VRC efficacy in an IPA murine model

Since ΔCAP exhibited reduced growth rates, abnormal hyphal development, and increased susceptibility to cell wall-perturbing and oxidative stress-inducing agents, the role of CAP in virulence was evaluated. The survival rate of mice infected with ΔCAP was significantly higher than those infected with Af293 or R-ΔCAP (P < 0.05) (Figure 6A). Consistent with the survival analysis, the fungal burden of mice infected with ΔCAP was lower than those infected with Af293 or R-ΔCAP on day 3 post infection (Figure 6B). By using histopathological examination, marginal fungal growth and mild inflammatory cell infiltration in the lung sections of the ΔCAP-infected mice were observed while aggressive fungal growth and intense inflammatory cell infiltration were observed in the lung sections of the R-ΔCAP and Af293-infected mice (Figure 6C,D).

Figure 6.

Virulence and VRC efficacy in an invasive pulmonary aspergillosis (IPA) murine model. (A) Survival rate analysis. (B) Fungal burden determined by Real-time PCR method. (C) Histopathological examination using haematoxylin eosin (HE) staining and (D) periodic acid-schiff (PAS) staining (The arrows indicated the invasive hyphae). (E) Survival rate analysis following VRC dosage of 10 mg/day and (F) 20 mg/d (log rank test, P < 0.05). (G) Fungal burden following VRC dosage of 10 mg/day and 20 mg/d (log rank test, P < 0.05).

Additionally, the effect of CAP on the efficacy of VRC treatment in an IPA murine model was also evaluated. After treatment with VRC, the survival rate was significantly improved in ΔCAP-infected mice versus that in Af293-infected mice (Figure 6E,F). The fungal burden was significantly decreased in ΔCAP-infected mice compared to that in R-ΔCAP and Af293-infected mice (Figure 6G).

Taken together, these data indicate that CAP gene knockout results in attenuated virulence and enhanced VRC efficacy in an IPA murine model.

Discussion

In this study, we unexpectedly obtained an A. fumigatus transformant with reduced growth rate and increased susceptibility to VRC and ITC during the genetic manipulation. Using rescue plasmid method, we further confirmed that it was the CAP gene being disrupted in this transformant. To better investigate the function of the CAP gene in antifungal susceptibility and virulence of A. fumigatus, ΔCAP and R-ΔCAP strain were hence constructed.

We observed that ΔCAP showed a reduced growth rate and abnormal hyphae as well as increased susceptibility to cell wall-perturbing agents (Congo red, CFW, and SDS), oxidative stress-inducing agents (H₂O₂ and menadione), FK506, CsA, VRC, ITC, and the synergistic antifungal effect of CAS plus FK506 in vitro. In order to elucidate the mechanisms of the abnormal phenotypes in ΔCAP, we performed a transcriptomic and proteomic analyses. Transcriptomic analysis revealed that some DEGs between ΔCAP and Af293 as well as between R-ΔCAP were involved in regulating growth, hyphal development, cell wall integrity, stress responses, and antifungal susceptibility. Proteomic analysis revealed that CAP-interacting proteins were mainly enriched in several fundamental metabolic pathways including secondary metabolite, ribosome, carbon, and amino acid metabolism. Among the CAP-interacting proteins, actin and AC were the most abundant. To explore the effect of CAP on actin and AC, the expression levels of actin and the activity of the AC-cAMP-PKA pathway were further examined.

Actin is a highly conserved protein found in all eukaryotic cells, existing in two distinct forms including monomeric globular actin (G-actin) and polymerized filamentous actin (F-actin) [41]. Actin is responsible for cell proliferation, hyphal development, protein secretion, and cell wall integrity in fungi [42,43]. Additionally, previous studies reported that actin-binding proteins affect fungal growth, sporulation, stress responses and virulence by regulating the actin cytoskeleton [44,45]. Decreased expression of the actin-binding protein cofilin resulted in reduced growth and attenuated virulence in A. fumigatus [32]. Loss of the actin-binding protein Sac1 led to reduced growth, impaired cell wall integrity, increased susceptibility to cell wall-perturbing agents, and attenuated virulence in C. albicans [44]. Loss of actin-localized protein-encoding Sur7 increased susceptibility to oxidative stress-inducing agents and attenuated virulence in yeast [45]. In S. cerevisiae, C. albicans, and M. oryzae, CAP competitively coupled with ADP-G-actin and promoted the exchange of ADP for ATP, the conversion of G-actin to F-actin and actin filament assembly [46,47]. The CAP protein contains an AC-binding domain, two proline-rich regions P1/P2, and an actin-binding domain [20,48]. The P1/P2 regions of CAP were coupled with SH3 domains of various actin-binding proteins [20]. CAP co-localized with actin plaques in the cell cortex in vivo [48]. In this study, we observed that CAP gene knockout resulted in reduced actin expression. We hypothesize that the reduced actin expression in ΔCAP might result from the reduction in F-actin assembly.

AC catalyzes the conversion of ATP to cAMP, thereby further activating the downstream PKA pathway signalling [49–51]. The AC-cAMP-PKA pathway is highly conserved and plays essential roles in regulating growth, sporulation, carbon metabolism, hyphal morphology, stress responses, and virulence in Aspergillus flavus [50], S. cerevisiae [5], C. albicans [18], and Cryptococcus neoformans [52]. In A. flavus, knockout of AC gene results in the reduced growth and sporulation, and the increased susceptibility to hyperosmotic and oxidative stress-inducing agents [49]. And knockout of PKA gene leads to reduced growth and sporulation, and attenuated virulence in A. fumigatus [53]. In this study, reduced cAMP levels and PKA activity were observed in ΔCAP. Therefore, we conclude that the attenuation of AC-cAMP-PKA pathway contributes to a reduction in virulence of A. fumigatus.

Previous studies have shown that deletion of the genes encoding AC and CAP in C. albicans [18] and S. cerevisiae [17] resulted in increased susceptibility to fluconazole, ITC, miconazole, and terbinafine, whereas the susceptibility to triazoles was restored by the addition of exogenous cAMP. Kontoyiannis et al. also reported that cAMP-PKA pathway was responsible for regulating the susceptibility to fluconazole in S. cerevisiae [54]. Additionally, the expression level of efflux pump gene Cdr1 was lower in AC and CAP mutants than that in the parental strains after ITC exposure, indicating that the cAMP-PKA pathway was responsible for regulating Cdr1 expression in C. albicans [18] and S. cerevisiae [13]. In this study, ΔCAP showed decreased cAMP levels and PKA activity as well as decreased expression levels of AfuMDR1, AfuMDR2, AfuMDR3, AfuMDR4, and cdr1B after VRC exposure. This suggests that ΔCAP is more susceptible to VRC and ITC due to inhibition of efflux pump genes regulated by the cAMP-PKA pathway.

Calcineurin is a Ca²⁺-CaM-regulated serine/threonine protein phosphatase that plays essential roles in cell wall integrity, growth, stress response, and hyphal development [55]. FK506 and CsA, as immunosuppressive calcineurin inhibitors, are active against fungi and have been found to be promising antifungal agents. Several studies have shown that FK506 enhanced the antifungal effect of CAS against Aspergillus spp. [56,57]. Previous studies reported that calcineurin activity was increased 2.6-, 2.9-, and 1.6-fold in Aspergillus oryzae when stimulated by hyperosmotic, hypersaline, and high-temperature stresses, respectively [57]. Deletion of the KRE5 gene for cell wall β−1,6 glucan synthesis resulted in increased susceptibility to FK506 due to endoplasmic reticulum stress, activating the calcineurin pathway to negatively regulate chitin synthesis and maintain cell wall integrity in Candida glabrata [58]. In this study, we found that CAP gene knockout led to an increased susceptibility to FK506 due to the activation of the calcineurin pathway to regulate stress response and maintain cell wall integrity.

Growth, hyphal development, cell wall integrity and stress responses are essential virulence factors of A. fumigatus that play important roles in escaping from host immune responses [13,50,53]. Therefore, the effect of the CAP gene on virulence of A. fumigatus was investigated using in an IPA murine model. We observed a higher survival rate and lower fungal load in ΔCAP-infected mice than in Af293-infected mice with or without VRC treatment, suggesting that CAP gene knockout led to attenuated virulence. The enhanced VRC treatment efficacy may be also associated with increased susceptibility to VRC and attenuated virulence in ΔCAP.

In conclusion, CAP plays the essential role in regulating antifungal susceptibility and virulence in A. fumigatus and may serve as a potential novel therapeutic target for IA.

Funding Statement

This work was supported by National Natural Science Foundation of China: [grant number 81861148028].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplemental Material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/22221751.2025.2506795.