Oxidoreductase gene fabG contributes to fungal development, cell wall integrity, and virulence in Aspergillus fumigatus
Heng Zhang, Hua Ni, Xinyi Tao, Tian Chen, Yi Zhang, Xiaolei Zhu, Yinping Chen, Mengqi Peng, Yi Sun
TL;DR
The fabG gene in Aspergillus fumigatus is crucial for growth, stress adaptation, and virulence, making it a potential target for antifungal treatments.
Contribution
This study identifies fabG as a novel regulator of cell wall integrity and redox homeostasis in A. fumigatus.
Findings
fabG knockout strains show reduced growth, virulence, and caspofungin sensitivity.
ΔfabG leads to increased ROS, decreased SOD activity, and heightened macrophage-induced killing.
fabG modulates cell wall thickening and stress tolerance, but not in response to caspofungin or rapamycin.
Abstract
Aspergillus fumigatus, a major cause of invasive aspergillosis, relies on oxidoreductases for stress adaptation. The role of the oxidoreductase gene fabG in fungal physiology and virulence remains unclear. This study aims to investigate the function of fabG in regulating A. fumigatus growth, virulence, redox homeostasis, and cell wall integrity (CWI). fabG knockout (ΔfabG) and complemented strains were constructed via homologous recombination. Phenotypic assays evaluated hyphal growth, virulence in Galleria mellonella, and antifungal susceptibility. Transcriptomic profiling, cell wall composition analysis (chitin/β-glucan), biochemical assays (superoxide dismutase [SOD] activity, reactive oxygen species [ROS] levels, and stress tolerance), and macrophage co-culture experiments (phagocytosis/killing) were performed. CWI-related gene expression was assessed under caspofungin/rapamycin…
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Fig 9| Antifungal susceptibility | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Strain | MICs or MEC (μg/mL) | ||||||||
| POS | ITR | VOR | CAS | ISA | CFW | CR | MD | H2O2 | |
| WT | 0.125 | 0.5 | 0.125 | 0.125 | 0.25 | 25 | 25 | 6.25 | 0.03125% |
| Δ | 0.125 | 0.5 | 0.25 | 0.5 | 0.25 | 200 | 400 | 50 | 0.25% |
| Δ | 0.125 | 0.5 | 0.125 | 0.125 | 0.25 | 25 | 25 | 12.5 | 0.0625% |
- —Jingzhou Science and Technology Plan Project
- —Yangtze University Science and Technology Aid to Tibet Medical Talent Training Program Project
- —Key Research and Development Plan of Hubei Province
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Taxonomy
TopicsAntifungal resistance and susceptibility · Fungal and yeast genetics research · Fungal Infections and Studies
INTRODUCTION
The genus Aspergillus, comprising filamentous fungi ubiquitously distributed in soil, decaying organic matter, and air, includes Aspergillus fumigatus as one of the most virulent human pathogens (1, 2). This species thrives due to its unique biological traits, such as thermotolerance, robust oxidative stress adaptation, and efficient spore dispersal (3–8).
The rising prevalence of immunosuppressive therapies and organ transplantation has dramatically expanded the population at risk for invasive aspergillosis (IA). Epidemiological data indicate that approximately 30 million individuals worldwide are threatened by IA, with over 1 million new cases annually and mortality rates reaching 30%–90% (9, 10). Chronic pulmonary aspergillosis further underscores its clinical burden, exhibiting a 5-year survival rate of only 62% (11, 12). With a growing at-risk population, these infections are of critical concern, resulting in A. fumigatus being listed by the WHO as a critical priority fungal pathogen.
The pathogenicity of A. fumigatus hinges on its ability to counteract host microenvironmental stresses. During host-pathogen interactions, the fungus must neutralize reactive oxygen species (ROS) generated by immune cells (e.g., neutrophils and macrophages) while maintaining intracellular redox homeostasis (13, 14). Oxidoreductases play pivotal roles in this process: they scavenge ROS (e.g., superoxide dismutase [SOD] and catalase [CAT]) to mitigate oxidative damage and regulate critical metabolic pathways (e.g., ergosterol biosynthesis) and virulence factor expression (15). Concurrently, the cell wall integrity (CWI) pathway dynamically modulates the synthesis and repair of cell wall components (e.g., β-1,3-glucan and chitin). For instance, CYP51 (14-α sterol demethylase), the target of azole antifungals, is involved in membrane integrity by catalyzing sterol precursor demethylation; its inhibition triggers ROS accumulation and fungal death (16). However, the molecular interplay between redox equilibrium and antifungal efficacy remains poorly understood, and evolving drug resistance further complicates clinical management.
Notably, A. fumigatus survival and virulence require not only redox homeostasis but also coordinated activation of the CWI pathway to withstand external pressures. The CWI pathway senses cell wall damage and activates downstream signaling cascades (e.g., mitogen-activated protein kinase [MAPK] such as Mkk2), thereby regulating β-1,3-glucan synthesis and repair (17). Echinocandins (e.g., caspofungin [CAS]) disrupt fungal cell wall structure by inhibiting β-1,3-glucan synthase. Although their efficacy may be partially limited by compensatory activation of the CWI pathway, they are still commonly used as part of combination therapy or as a second-line option in the treatment of IA (18, 19). Emerging studies suggest that in various species, there is crosstalk between the redox system and the CWI signaling pathway: elevated ROS levels can activate the CWI pathway, initiating the repair of cell wall defects caused by oxidative damage (20–24). Therefore, it is reasonable to speculate that aberrant activation of the CWI pathway may, in turn, regulate the expression of antioxidant enzymes (such as SOD), forming a complex adaptive regulatory network. Nevertheless, how oxidoreductases (e.g., the Oxidoreductase family) collaborate with the CWI pathway to regulate pathogenicity and drug responses in A. fumigatus remains unresolved. Although knowledge of genes involved in cell wall (CW) biosynthesis is expanding, over 30% of the A. fumigatus genome still encodes genes with uncharacterized functions (25).
In this preliminary study, we employed homologous recombination to replace the fabG gene with a pyrG marker, generating a fabG knockout strain (ΔfabG) (26). This model was used to investigate the impact of fabG deletion on fungal growth, drug susceptibility, and virulence, as well as its role in oxidative stress and CWI signaling. Our findings unveil novel functions of fabG in A. fumigatus, providing critical insights into its adaptive mechanisms and therapeutic potential.
MATERIALS AND METHODS
A. fumigatus strains, media, and growth conditions
In this study, the A. fumigatus KU80 strain (a uracil auxotroph strain lacking pyrG and akuB genes, Fungal Genetics Stock Center) was used as the wild-type genetic background to construct the fabG gene deletion strain (ΔfabG) and its complemented strain (ΔfabG::fabG^+^) (27, 28).
The media used in this study included SAB, CZA, LB, and RPMI 1640. All liquid media were prepared from the corresponding solid media without agar. Fresh A. fumigatus conidia were harvested from solid SAB plates with sterile water and adjusted to the required concentration for each experiment using a hemocytometer. Unless otherwise specified, strains were incubated at 37°C.
Phylogenetic tree construction
The sequences of fabG orthologs were downloaded from the NCBI (https://www.ncbi.nlm.nih.gov) and FungiDB (http://fungidb.org/fungidb) websites. A phylogenetic tree was constructed using the neighbor-joining method with MEGA 10 software, and sequence alignment was conducted using Cluster W. Conserved domain analysis of proteins was performed using the Conserved Domain Database website on the NCBI. ESPript (https://espript.ibcp.fr/ESPript/ESPript) was used for multiple sequence alignment.
Generation of mutant strains
A. fumigatus KU80 was used as the parental isolate to generate knockout mutants. Knockout cassettes were generated using the protocol from Zhao et al. (26) (Fig. S1). Briefly, 1.2-kb flanking regions of the gene of interest were PCR amplified and fused to a pyrG cassette via additional fusion PCR. The A. fumigatus KU80 strain was cultured overnight at 37°C and 120 rpm in CZA liquid medium supplemented with uracil, followed by a 5-hour protoplasting treatment in Sabouraud agar + protoplasting solution (pectinase + freshly filtered 0.6 M KCl, citric acid). Protoplasts were filtered through Miracloth (Sigma, USA), washed twice in 0.6 M KCl, and resuspended in 0.6 M KCl + 200 mM CaCl_2_. Fusion PCR product was added to 1 × 10^5^ protoplasts, followed by addition of PEG. This was incubated on ice for 30 minutes. In total, 200 µL of PEG was added, and the mixture was then incubated at room temperature for 10 minutes. The transformation mixture was plated on CZA agar. Transformants were purified twice on CZA agar and PCR validated (Fig. S2). All primers used in this study are listed in Table S1.
Construction of the fabG complemented strain
The fabG gene was amplified from the genomic DNA of A. fumigatus using primers Aim-Re-F and Aim-Re-R in a PCR experiment. The amplified fabG gene was subsequently subcloned into the Nael and KpnI sites of the plasmid PCT74, resulting in the recombinant plasmid p-hph-fabG. The constructed plasmid was transformed into the ΔfabG deletion strain to generate the ΔfabG complemented strain (ΔfabG::fabG^+^) (Fig. S1). All primers used in this experiment are listed in Table S1.
E-TEST
A. fumigatus conidia suspension (2 × 10⁵ cfu/mL) was evenly spread on the surface of RPMI 1640 agar plates (containing 2% agar, pH 7.0). The E-TEST strips were then gently placed on the agar surface, ensuring full contact with the medium. Plates were incubated at 37°C for 48 hours. After incubation, the lowest drug concentration at the intersection of the inhibition zone (elliptical area) with the E-TEST strip was recorded as the minimum effective concentration (MEC).
M38-A3 broth microdilution method
The broth microdilution method was performed according to the M38-A3 standard established by the Clinical Laboratory Standard Institute, using RPMI 1640 liquid medium (29). Antifungal drugs (voriconazole [VOR], itraconazole [ITR], posaconazole [POS], and CAS) were prepared in eight twofold serial dilutions, with the highest concentration of 8 µg/mL and a working concentration range of 0.25–8 µg/mL. A 96-well microplate was used, with 100 µL of each drug dilution added to each well, followed by 100 µL of fungal suspension to achieve a final fungal concentration of 2 × 10⁴ cfu/mL. Wells without drugs were included as growth control. The plates were incubated at 35°C for 48 hours, and the results were recorded after incubation. To ensure accuracy, the experiment was performed in triplicate. Candida parapsilosis ATCC22019 and Aspergillus flavus ATCC204304 were used as the quality control strain. In addition, the same method was used to test cell wall stressors (calcofluor white [CFW] and Congo red [CR]) and oxidative stress-inducing agents (menadione and H_2_O_2_). These agents were prepared in 12 twofold serial dilutions. The maximum concentration of hydrogen peroxide was 10%, with a working concentration range of 0.002%–10%, while the maximum concentrations of the other agents were 800 µg/mL, with a working concentration range of 0.25–800 µg/mL.
Radial growth germination rate analysis
Referring to Michael J. Bromley’s method (28), 500 µL of 5 × 10⁵ cfu/mL suspension was inoculated into RPMI-1640 medium with 2.0% glucose and 165 mM MOPS buffer (pH 7.0) in a 24-well glass-bottom plate. The culture was incubated at 37°C, and the optical density at 600 nm was measured using a multifunctional microplate reader (Allsheng, China).
Spore quantification assay
A layer of cellophane was placed on solid SAB medium, and 1 µL of spore suspension (5 × 10⁵ cfu/mL) was inoculated in the center. After drying, it was incubated at 37°C for 3 days, with spore production measured daily. Following incubation, the cellophane was carefully removed, and sporangiophores were scraped off completely with a swab. Conidia were observed under a bright field microscope and counted with a hemocytometer.
Sensitivity assays for oxidative stress
Congo red (200 µg/mL) and calcofluor white (100 µg/mL) were used to assess CWI; mannitol (2 M), sorbitol (1 M), and NaCl (1 M) were used for osmotic stress sensitivity testing; hydrogen peroxide H_₂_O_₂_ (5 M) and menadione (200 µM) were used to evaluate oxidative stress responses. All plates were prepared using SAB medium as the base. A 2 µL conidial suspension (1 × 10⁶ cfu/mL) was spotted onto the center of each plate. Plates were incubated at 37°C, and colony growth was photographed after 48 hours. Each experiment was performed in triplicate with biological replicates. The inhibition rate was calculated as follows: inhibition rate = (colony diameter without stressors − colony diameter with stressors)/colony diameter without stressors × 100%. “Colony diameter without stressors” specifically refers to the colony diameter of strains cultured on solid SAB medium at 37°C for 48 hours, serving as a unified baseline for inhibition rate calculation across all stress-related assays.
Cell wall thickness analysis
One milliliter of conidia suspension of fresh strains at a concentration of 10^8^ cfu/mL was inoculated into 100 mL of liquid SAB medium and incubated at 37°C and 220 rpm for 24 hours. A portion of the mycelium was taken and soaked in glutaraldehyde and sent to the Wuhan Service Biotechnology, China, for scanning transmission electron microscopy. Cell wall thickness was determined using ImageJ 1.54 software, with measurements taken at three random locations on the cell wall and averaged to obtain the final thickness value (30).
β-Glucan assay
The β-glucan assay was performed following methods previously described (31, 32). Briefly, 1 × 10^7^ conidia of each strain were grown overnight in 25 mL of SAB broth at 37°C. After 16 hours of growing, hyphae were collected by filtration through Miracloth (Sigma, USA) and washed using 0.1 M NaOH solution. Washed fungal hyphae were lyophilized for 24 hours. Five milligrams of dry hyphae were disrupted in a bead-beater three times (1 minute each) with 1 minute of ice incubation between each. Hyphal powder was resuspended to a final concentration of 20 mg/mL in 1 M NaOH, and the solution was incubated at 52°C for 30 minutes. Fifty microliters of each sample was mixed with 185 µL of aniline blue staining solution (183 mM glycine, 229 mM NaOH, 130 mM HCl, and 618 mg/L aniline blue, pH 9.9) into a 96-well masked fluorescence plate (Shenggong, China). The sample-containing plates were incubated at 52°C for 30 minutes, followed by a cool-down period of 30 minutes at room temperature. Fluorescence readings were performed using an excitation/emission wavelength of 405/460 nm, respectively (Allsheng, China). All the experiments were performed in triplicate using three independent A. fumigatus cultures, and the results were represented as relative quantification versus the WT strain.
RNA-seq analysis and real-time quantitative PCR
Fresh A. fumigatus conidia were grown in liquid SAB in a rotary shaker at 220 rpm at 37°C for 48 hours. For RNA sequencing (RNA-seq) analysis, mycelial pellets were collected and quickly frozen in liquid nitrogen. After mRNA extraction, purification, and library construction, sequencing was performed by next-generation sequencing based on the Illumina sequencing platform. Genes were considered differentially expressed if they met both criteria: false discovery rate (FDR) < 0.01 and absolute log₂ fold change ≥ 1.0. The detailed procedures were performed by Beijing Biomaker Biotechnology Co., Ltd. (China). Each sample was analyzed using three biological repetitions.
For RT-qPCR analysis, total RNA was extracted with the TRIeasy Total RNA Extraction Reagent (Yeasen, China) according to the manufacturer’s directions. The Hifair III 1st Strand cDNA Synthesis SuperMix for qPCR (Yeasen, China) was used to synthesize cDNA. Independent assays were performed with three replicates, and transcript levels were calculated by the comparative threshold cycle (ΔCT) and normalized against the mRNA expression of tubA in A. fumigatus. The 2^−ΔΔCT^ method was used to determine the changes in mRNA expression (33). All the RT-qPCR primers are given in Table S2.
SOD and CAT activity measurement
To measure total SOD and CAT activity, 2 × 10⁷ spores were inoculated into 100 mL of liquid SAB medium and shaken at 220 rpm and 37°C for 16 hours. After centrifugation at 12,000 g for 3 minutes, the pellet was washed twice with PBS. Small glass beads were added, and the mixture was homogenized in a high-speed oscillator (Benchmark, USA). The supernatant, collected after centrifugation at 8,000 g for 3 minutes, was used as the sample. Protein concentration was determined with a BCA Protein Quantification Kit (Yeasen, China). SOD activity was measured using a Total Superoxide Dismutase Activity Colorimetric Assay Kit II (Yeasen, China), and CAT activity was assessed with a Catalase Assay Kit (Beyotime, China) (15, 34).
Morphological examination
Following the previously described method (35, 36), slide cultures were prepared and incubated at 37°C for 24 hours. Slide cultures were prepared and incubated at 37°C for 24 hours. Samples were then stained with CFW (Sigma, USA) and observed under a Leica DMiL LED fluorescence microscope (Leica, Germany).
2,7-dichlorofluorescin diacetate staining
Fresh conidia collected from SAB agar plates after 3 days of growth were transferred into SAB liquid medium and incubated at 37°C with 130 rpm shaking for 36 hours. 2,7-dichlorofluorescin diacetate (Yeasen, China; 10 µM) was then added to the sample at a volume of 0.1%. The mixture was incubated at 37°C for 30 minutes, followed by centrifugation at 4,000 rpm for 20 minutes (37). Flow cytometry data were generated using a Beckman Cytomics FC 500 BD FACSCanto II and analyzed with FlowJo version 10 software. The excitation wavelength was 488 nm, and the emission wavelength was 525 nm.
Galleria mellonella virulence assay
The G. mellonella virulence assay was performed using a method described previously (38). Fresh conidia of the corresponding strains were harvested and adjusted to 1 × 10^8^ cfu/mL. Ten microliters of conidia suspension was then injected into the G. mellonella larvae through the left prolegs. The control group was injected with a sterile PBS solution. G. mellonella larvae were then incubated in darkness at 37°C for 7 days, and the number of larval deaths was recorded every 24 hours to calculate the survival rate. The log-rank (Mantel-Cox) test was used to compare survival curves. A P value of < 0.05 was considered statistically significant. Each experiment was replicated three times independently, and each replication contained 20 larvae per strain.
Phagocytosis and fungal killing assay of A. fumigatus conidia by RAW 264.7 macrophage cells
RAW 264.7 macrophage cell line (Procell Life Science Co., Ltd., China) was used for the experiments. The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 0.05 mM β-mercaptoethanol, and 1% penicillin (10,000 U/mL) and streptomycin (10,000 mg/mL) at 37°C in a humidified incubator with 5% CO_₂_. After adherence, RAW 264.7 cells were co-incubated with A. fumigatus conidia at 37°C and 5% CO_₂_ for 2 hours. Unphagocytosed conidia were gently washed away with PBS three times, and fresh medium was added. The cells were returned to the incubator and further incubated for 4 hours to assess fungal killing, or for 16 hours for morphological observation of the conidia. After 4 hours of incubation, the medium was removed, and the cells were collected and lysed by vigorous vortexing with small glass beads to release the internalized conidia. The resulting conidial suspension was serially diluted and immediately plated on SAB agar plates. After incubation under appropriate conditions, colony-forming units were counted, and the fungal killing rate by macrophages was calculated based on CFU data to evaluate the antifungal activity of RAW 264.7 cells. Meanwhile, total RNA was extracted from the cell suspension using Trizol reagent, and the RNA samples were stored at −80°C for further analysis. For microscopic observation of phagocytosed conidia, the PBS used for washing was supplemented with CFW (final concentration: 5 µg/mL), and the samples were incubated at 37°C for 10 minutes. The stained conidia were then observed under a fluorescence microscope using a hemocytometer.
Statistical analysis
All the assays were done in triplicate on three independent days. All the statistical analyses were carried out using GraphPad Prism version 10.1.2 (GraphPad Software Inc., San Diego, CA, USA) for Windows. In each assay, at least three biological replicates were done to measure each parameter in each condition, avoiding biased results. Error bars included in all the graphs represent the standard deviation. ANOVA or t-test was used depending on whether we did multiple comparisons or compared punctual data, respectively, after ensuring that the data sets followed a normal distribution.
RESULTS
Identification of fabG in A. fumigatus
The FabG protein (corresponding to AFUA_8g01550) of A. fumigatus is conserved with respect to homologs in other species of the genus Aspergillus (Fig. 1A and C). The fabG gene is 808 bp in length and encodes a predicted protein of 250 amino acids. The protein contains a domain from positions 4 to 247: NAD(P)-dependent dehydrogenase, short-chain alcohol, dehydrogenase family, region_name as fabG (Fig. 1B). It belongs to the short-chain dehydrogenases/reductases family, a diverse family of oxidoreductases with a single domain. These enzymes catalyze a wide range of activities, including the metabolism of steroids, cofactors, carbohydrates, lipids, aromatic compounds, and amino acids, and are involved in redox sensing. BLAST analysis showed that the closest homolog to A. fumigatus FabG was found in Aspergillus uvarum (Fig. 1A).
fabG is conserved among common pathogenic filamentous fungi. (A) Phylogenetic tree of fabG homologs from different fungal species. Construction of a phylogenetic tree was carried out using MEGA version 11.0. The tree was generated with the maximum likelihood model with a bootstrap value of 1,000 (http://megasoftware.net). Colletotrichum costaricense, GenPept accession no. XP_060318467.1; Colletotrichum tabaci, GenPept accession no. KAK6207696.1; Colletotrichum karsti, GenPept accession no. XP_038747456.1; Dactylella cylindrospora, GenPept accession no. KAF3931206.1; Arthrobotrys megalospora, GenPept accession no. KAK6516023.1; Aspergillus wentii, GenPept accession no. XP_040683636.1; Lipommyces kononenkoae, GenPept accession no. KAK9365068; Lipomyces doorenjongi, GenPept accession no. KAK9352250.1; Hortaea wernecki, GenPept accession no. KA7548169.1; Lipomyces tetrasporus, GenPept accession no. XP_056041121.1; Aspergillus fumigatus Af293, GenPept accession no. XP_747051.1; Aspergillus uvarum, GenPept accession no. XP_025496014.1; Lasiodiplodia hormozganensis, GenPept accession no. KAK0609791.1; Fusarium oxysporum, GenPept accession no. KAJ4137906.1; Fusarium oxysporum, GenPept accession no. RKL46478.1; Wallemia mellicola, GenPept accession no. XP_006958562.1; Penicillium canescens, GenPept accession no. XP_058371582.1; Aspergillus tamarii, GenPept accession no. KAE8167722.1; Aspergillus parasiticus, GenPept accession no. KAB8202943.1; Aspergillus transmontanensis, GenPept accession no. KAE8316012.1. (B) Display of the amino acid domains of FabG. (C) Sequence alignment of FabG using an online alignment tool (https://espript.ibcp.fr/ESPript/ESPript).
Loss of fabG causes severe growth inhibition
In both solid media (CZA and SAB Agar) and liquid culture systems (RPMI-1640 and SAB broth), the ΔfabG mutant displayed drastically reduced conidiation and hyphal development compared to the WT and ΔfabG::fabG^+^ (Fig. 2A through D). Specifically, after cultivation in CZA for 24, 48, and 72 hours, the colony diameters of ΔfabG were reduced by 55.7%, 59.0%, and 52.9%, respectively, compared to WT (Fig. 2E). In SAB media, the reduction was 61.6%, 70.8%, and 73.4% (Fig. 2F). Similar results were observed in ΔfabG::fabG^+^ (Fig. 2F). In addition, ΔfabG produced significantly fewer spores than WT and ΔfabG::fabG^+^, with no conidia after 24 hours (Fig. 2G). The germination rate assay showed delayed spore germination in ΔfabG (Fig. 2H). Under optical microscopy, after 48 hours, WT and ΔfabG::fabG^+^ strains developed numerous conidiophores and began to produce conidia. However, after 72 hours, only hyphal tip swelling was observed in ΔfabG, and the conidiophores had not matured (Fig. 2I). This indicates that the deletion of fabG severely impairs hyphal growth and conidiation ability. Together, these results suggested that fabG is necessary for hyphal growth and conidiation in A. fumigatus.
*A. fumigatus fabG is required for vegetative growth and conidiation. Representative growth phenotypes of the WT, ΔfabG, and ΔfabG::fabG+ on CZA (A and E), SAB (B and F) medium, RPMI-1640 (C), and SAB broth (D) at 37°C for 24, 48, and 72 hours. Diameters between strains were compared at each time point using two-way ANOVA with Tukey’s test for multiple comparisons. (G) Spore production numbers after growth on SAB medium for 1, 2, and 3 days. Statistical analyses were performed by two-way ANOVA with Tukey’s test for multiple comparisons. (H) Radial growth germination rate analysis. P-value was calculated by repeated measures two-way ANOVA with Sidak's correction. (I) Mycelial morphology under optical microscopy, showing that ΔfabG lacks obvious conidiophores, particularly after 72 hours of cultivation. In contrast, the WT and ΔfabG::fabG+ strains exhibit prominent conidiophores and conidia production. Scale bar = 50 µm. ns, P > 0.05; ***P < 0.001; and ***P < 0.0001.
Antifungal drug sensitivity
Azoles and echinocandins are commonly used to treat IA, and the sensitivity of A. fumigatus to these antifungal agents is often a critical determinant of treatment efficacy. To further investigate the sensitivity of the ΔfabG mutant to these antifungal drugs, we evaluated its response to multiple antifungal agents. The results showed that, compared to the wild-type strain, ΔfabG exhibited reduced sensitivity only to CAS, while no significant changes in sensitivity to azoles were observed. Since CAS targets fungal cell wall synthesis, and ΔfabG also exhibited higher resistance to cell wall stressors (CFW and CR), this suggests that FabG may be associated with genes involved in cell wall biosynthesis. Notably, ΔfabG also displayed reduced sensitivity to oxidative stress-inducing agents such as menadione and H_₂_O_₂_ (Table 1; Fig. 3).
Antifungal susceptibility using E-TEST gradient strips for caspofungin.
fabG downregulates genes related to oxidoreductases and the MAPK pathway
To explore the mechanism by which fabG mediates hyphal growth and redox balance, we conducted RNA-seq analysis of WT and ΔfabG strains grown in SAB medium. A total of 1,145 genes were significantly differentially expressed (log2 fold change ≥ 1.0 or ≤−1.0, FDR < 0.01) in the ΔfabG mutant compared to the WT, with 358 genes upregulated and 787 genes downregulated (Fig. 4A). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of all differentially expressed genes in the ΔfabG mutant revealed that the most significant pathways included Peroxisome, Mitogen-Activated Protein Kinase signaling pathway, and Biosynthesis of amino acids (Fig. 4B). Notably, within the altered Peroxisome-related genes, the expression of SOD-encoding gene (AFUA_5G09240) and Catalase-encoding gene (AFUA_2G18030) was significantly decreased. Additionally, the expression of the key regulatory gene of the CWI pathway, MKK2 (AFUA_1G05800), also showed a significant change (Fig. 4C). These results suggest that the altered expression profiles may be associated with the phenotypic defects of the ΔfabG mutant. To validate the RNA-seq results, we analyzed selected genes by quantitative real-time PCR (qRT-PCR). The qRT-PCR results showed that the expression levels of these genes were largely consistent with the RNA-seq data (Fig. 4D). In summary, fabG may play a role in regulating the redox reaction and cell wall synthesis pathways by altering the Peroxisome and MAPK signaling pathways.
*Loss of fabG leads to changes in peroxisome and MAPK pathways. (A) The volcano plot showing the differentially expressed genes between the WT and ΔfabG. (B) KEGG pathway enrichment analysis of differentially expressed genes between the WT and ΔfabG. (C) A heatmap of the expression of peroxisome and MAPK pathway-related genes between the WT and ΔfabG. (D) The RT-qPCR analysis of selected genes in the WT, ΔfabG, and ΔfabG::fabG+. The mRNA levels were normalized to those of the reference gene tubA. Statistical analysis was performed using two-way ANOVA with Tukey’s test for multiple comparisons. **P < 0.01; ***P < 0.0001.
Impact on oxidoreductases
In the ΔfabG mutant, SOD levels were significantly lower than in the WT (Fig. 5A), but CAT activity remained unchanged (Fig. 5B). Furthermore, since enzymes like SOD play a key role in scavenging intracellular ROS, further analysis of ROS levels revealed a significant increase in ROS content in the ΔfabG strain (Fig. 5C and D). Overall, these results indicate that the loss of fabG leads to a reduction in the activity of related oxidoreductases, including SOD, thereby impairing the ability to control oxidative stress.
*Loss of fabG leads to decreased SOD activities and increased ROS levels. (A–C) Detection results for SOD activity, CAT activity, and ROS content. (D) Visualization of ROS levels using FlowJo version 10 software. Statistical analysis was performed using two-way ANOVA with Tukey’s test for multiple comparisons. ns, P > 0.05; **P < 0.01; ***P < 0.001; and ***P < 0.0001.
FabG is required for the maintenance of cell wall integrity in A. fumigatus
To assess the role of fabG in regulating CWI in A. fumigatus, CFW fluorescence staining was employed to quantify chitin content. The ΔfabG mutant exhibited a significant increase in chitin levels compared to the WT strain (Fig. 6A and B). Concurrently, β-glucan content was also markedly elevated (Fig. 6C). The susceptibility of ΔfabG to cell wall-perturbing agents was further evaluated. The mutant displayed reduced sensitivity to CR (which specifically binds β-glucan) and CFW (which targets chitin), whereas the complemented strain (ΔfabG::fabG^+^) displayed phenotypic restoration to WT levels under these stressors (Fig. 6D and E). Transmission electron microscopy revealed a pronounced thickening of the cell wall in ΔfabG (Fig. 6F and G). These findings collectively demonstrate that fabG plays a critical role in maintaining cell wall architecture and orchestrating the CWI pathway.
*fabG is required for the maintenance of cell wall integrity in A. fumigatus. (A) The distribution of the chitin content in the WT and ΔfabG mutants was visualized through fluorescence microscopy with CFW staining. Scale bar = 30 µm. (B) Fluorescence intensity analysis of CFW staining using ImageJ software, normalized to the WT. (C) β-glucan content. (D and E) Colony diameter and inhibition rate analysis after 48 hours of incubation on 100 µg/mL calcofluor white and 200 µg/mL Congo red media. The inhibition rate was calculated as follows: inhibition rate = (colony diameter without stressors − colony diameter with stressors)/colony diameter without stressors × 100%. The “colony diameter without stressors” refers to the colony diameter formed by strains cultured on non-supplemented solid SAB medium at 37°C for 48 hours and is labeled as “None” in the figure. (F) Quantification of cell wall thickness of the WT and ΔfabG mutants. (G) Representative transmission electron microscopy images of the WT and ΔfabG mutants. Scale bar = 200 nm. Statistical analysis in panels (B–D and F) was performed by two-way ANOVA with Tukey’s test for multiple comparisons. ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; and ***P < 0.0001.
Enhanced tolerance to osmotic pressure and oxidative stress in the fabG mutant
The ΔfabG mutant exhibits significantly enhanced tolerance to both osmotic and oxidative stress. Under oxidative stress conditions (H_₂_O_₂_, menadione) and high osmotic stress (NaCl, sorbitol, and mannitol), the growth inhibition of ΔfabG was only 14.47%, compared to 45.73% in WT and 46.81% in the ΔfabG::fabG^+^ (Fig. 7). Although the baseline growth rate of ΔfabG is somewhat reduced, its stability under stress conditions is significantly better than that of the control strains. The CWI pathway, which regulates stress tolerance, plays a critical role here, suggesting that the FabG protein may influence the fungal environmental adaptability through the regulation of the CWI pathway.
*Loss of fabG enhances adaptation to oxidative stress and osmotic pressure. (A and B) Relative hyphal growth inhibition of the indicated strains grown on solid SAB medium at 37°C for 48 hours. Statistical analyses were performed by two-way ANOVA with Tukey’s test for multiple comparisons. (C) Colony morphology of the WT, ΔfabG, and complementation strains grown on solid SAB medium in the presence of 1 M NaCl, 1 M sorbitol, and 2 M mannitol at 37°C for 48 hours. (D) Colony morphology grown on solid SAB medium in the presence of 200 µM menadione and 5 M H2O2 at 37°C for 48 hours. The inhibition rate was calculated as follows: inhibition rate = (colony diameter without stressors − colony diameter with stressors)/colony diameter without stressors × 100%. The “colony diameter without stressors” refers to the colony diameter formed by strains cultured on non-supplemented solid SAB medium at 37°C for 48 hours and is labeled as “None” in the figure. ***P < 0.001;***P < 0.0001.
The expression of CWI pathway genes under caspofungin/rapamycin treatment
As previously mentioned, the fabG gene plays a crucial role in regulating the integrity and thickness of the cell wall in A. fumigatus. The CWI pathway is a key regulatory pathway for fungal cell wall remodeling and synthesis, and fabG may influence the cell wall by participating in this process.
In this study, RNA sequencing analysis revealed that many differentially expressed genes were enriched in the MAPK pathway, with particular changes observed in the expression of the MKK2 gene. This result was further validated by RT-qPCR. To explore this phenomenon in more detail, we treated the strains with CAS and rapamycin (Rapa) and analyzed the expression of key genes involved in the CWI pathway, including chitin synthase genes (chsA, chsB, chsC, chsG, and csmB), α-1,3-glucan synthase genes (agsA and agsC), β-1,3-glucan synthase genes (fksA), 1-3-β-glucan glucosyltransferase genes (gelA and gelB), and the CWI transcription factor (rlmA).
The results showed that, after CAS treatment, the transcription levels of CWI pathway target genes (chsA, chsC, chsG, fksA, gelA, rlmA, and rlmB) were significantly decreased in the WT strain, except for gelB, which showed reduced expression. In contrast, in the ΔfabG strain, only chsG expression decreased. Subsequently, after Rapa treatment, as expected, the expression levels of chsA, chsC, fksA, gelA, rlmA, csmB, agsA, and* agsC* were all increased, while chsB expression decreased. In the ΔfabG strain, however, only chsG expression increased.
Overall, whether co-cultured with CAS or Rapa, the downstream genes of the CWI pathway in the WT strain exhibited relatively strong changes in expression. For example, rlmA expression increased by 11-fold after Rapa treatment, while fksA expression decreased by 33-fold after CAS treatment. In contrast, the expression levels of the tested genes in the ΔfabG strain remained relatively stable, with changes generally within a twofold range. These results indicate that, in the presence of cell wall stressors, the CWI pathway signaling in ΔfabG remains stable, while the signaling in the WT strain undergoes significant changes.
Taken together, these findings suggest that the fabG gene may be a key effector gene in the CWI pathway. Its deletion likely causes a disruption in certain aspects of the CWI signaling, which in turn affects the regulation of cell wall synthesis and repair. This provides new insights into the role of fabG in the CWI pathway and highlights its importance in the maintenance of cell wall homeostasis.
FabG enhances the macrophage killing activity against A. fumigatus conidia
Macrophages play a crucial role in antifungal immunity, such as phagocytosing and killing fungal spores (39). Therefore, we investigated the impact of FabG using an in vitro macrophage model. The results showed that ΔfabG conidia were more susceptible to phagocytosis and killing by macrophages. After 2 hours of incubation, a higher number of ΔfabG conidia were internalized (Fig. 8A and B). Following 4 hours of phagocytic killing, the remaining ΔfabG conidia were significantly fewer than those of the WT and complemented strains (Fig. 8C). Notably, after an extended 16-hour incubation, nearly all ΔfabG conidia were eradicated, whereas the WT and complemented strains exhibited visible hyphal growth and even conidial head formation (Fig. 8F).
*FabG enhances the macrophage killing activity against A. fumigatus conidia. (A) CFW staining of non-phagocytosed conidia after 2 hours of co-culture with macrophages. Scale bar = 20 µm. (B) Phagocytosis index is increased in the ΔfabG (average ± standard deviation, P ≤ 0.01 compared to the WT and complemented strains). (C) Fungal killing assay: conidia were co-incubated with macrophages for 2 hours, after which non-phagocytosed conidia were removed. The cells were then further incubated for 4 hours before being plated onto SAB solid medium to assess conidial survival and calculate the killing rate. (D) Expression of inflammatory factors. Statistical analyses were performed by two-way ANOVA with Tukey’s test for multiple comparisons. (E) Survival curves for G. mellonella larvae infected with the WT, ΔfabG, and ΔfabG::fabG+. Each experiment was replicated three times independently. Statistical analysis was performed using the log-rank test. (F) Microscopic observation of conidia after 16 hours of co-culture with macrophages. Scale bar = 20 µm. **P < 0.01; ***P < 0.001; and ***P < 0.0001.
TNF-α, IL-1β, and IL-6 are key inflammatory mediators secreted by macrophages upon exposure to A. fumigatus (40, 41). Thus, we also measured the expression levels of these cytokines. As expected, TNF-α, IL-1β, and IL-6 expression was significantly elevated in ΔfabG, particularly TNF-α, which showed a 38-fold increase compared to the WT (Fig. 8D). These findings suggest that ΔfabG is more easily recognized and eliminated by macrophages. To investigate whether fabG influences the virulence of A. fumigatus, we used the G. mellonella wax moth infection model to assess the virulence potential of the WT strain, ΔfabG, and ΔfabG::fabG^+^. The ΔfabG exhibited a significantly reduced mortality rate of larvae compared to the WT, whereas the complementary strain showed similar mortality rates as the WT (Fig. 8E).
DISCUSSION
Virulence is closely related to growth, which is particularly evident in A. fumigatus, where its growth rate often determines the degree of invasiveness (42–45). A. fumigatus can evade host immune surveillance through various mechanisms, such as modifying cell wall components to suppress host immune cell functions, thereby avoiding clearance by the immune system (14, 46–48). Oxidoreductases play a key role in eliminating intracellular ROS, thus protecting cells from oxidative damage (14, 49). These enzymes are considered the frontline defense in the cellular defense system (14). This study found that deletion of the fabG gene in A. fumigatus led to defects in colony growth and reduced SOD enzyme activity. In addition, the content of chitin and β-glucan in the cell wall increased, resulting in thicker cell walls, which contributed to enhanced stability of the strain under external stress. Moreover, the G. mellonella virulence model indicated that deletion of fabG may reduce the virulence and infectivity of A. fumigatus.
The fungal oxidative stress response involves enzymatic reactions from the thioredoxin and glutathione systems, including SOD and CAT (15, 50). SOD eliminates ROS by catalyzing the conversion of superoxide anions (O_₂_⁻) into H_₂_O_₂_ and O_₂_, while CAT further degrades H_₂_O_₂_ into H_₂_O. fabG, sodA, and catA all belong to the oxidoreductase family and may be involved in oxidative stress defense in A. fumigatus, with fabG potentially regulating antioxidant enzyme activity indirectly by maintaining the NAD(P)H/NAD(P)^+^ balance (14). Specifically, sodA and catA target ROS molecules without directly participating in cell wall metabolism, and deletion of their encoding genes is associated with sensitivity to oxidative stress. The putative SOD-encoding gene sodD is essential for survival; deletion of sodA or sodB leads to hypersensitivity to oxidative stress, while the sodC mutant only exhibits mild growth defects under heat stress (51). In contrast, fabG may function as a multifunctional oxidoreductase that not only regulates ROS homeostasis but also coordinates cell wall remodeling (such as β-glucan and chitin synthesis, Fig. 6) through modulation of the CWI pathway (Fig. 9). This results in enhanced tolerance to osmotic and cell wall stress—phenomena not previously reported in SOD or CAT mutants.
Expression levels of genes related to the CWI pathway in A. fumigatus. Relative expression levels of cell wall synthesis genes following caspofungin (2 µg/mL) and rapamycin (0.5 µg/mL) treatment for 5 hours. Gene expression was measured by RT-qPCR and normalized to the tubA gene, with normalization to WT. Expression was calculated using the 2−ΔΔCT method. Statistical analysis was performed using two-way ANOVA, and Tukey’s test was applied for multiple comparisons correction. A, P < 0.05; B, P < 0.01; C, P < 0.001; and D, P < 0.0001.
The fabG gene in A. fumigatus regulates its virulence and environmental adaptability through multiple mechanisms. A key function of oxidoreductases is the elimination of intracellular ROS (14), and fabG, as an oxidoreductase-related gene, similarly contributes to this process. Its deletion leads to reduced activity of oxidative enzymes such as SOD, resulting in ROS accumulation (52), suggesting that fabG may also play a role in counteracting ROS generation. As highly reactive molecules, ROS can induce oxidative stress (52), and their accumulation—along with redox imbalance—can further activate the cascade of the CWI signaling pathway (17). The biosynthesis of the A. fumigatus cell wall is regulated by the CWI pathway, which amplifies external signals through a signaling cascade and mediates downstream metabolic responses, especially during environmental stress, to regulate cell wall formation and remodeling (53). For example, the CWI pathway is the main signaling pathway controlling environmental stress response and cell wall component synthesis in Saccharomyces cerevisiae (17, 54). To counter persistent ROS, the fungal cell wall may undergo adaptive remodeling through compensatory thickening. In the absence of fabG, the thickened cell wall exhibited enhanced resistance to stressors such as oxidative and osmotic stress. This compensatory thickening, accompanied by abnormal increases in chitin and β-glucan, led to reduced sensitivity to cell wall-targeting antifungal drugs such as CAS. The CWI pathway is also critical for fungal virulence; for instance, the growth and virulence of Fusarium graminearum are highly dependent on it (55). Similarly, the thickened cell wall structure of the ΔfabG mutant results in slower growth, likely due to the need for reallocating energy and resources toward cell wall remodeling—a mechanism that may enhance fungal survival within the host, ultimately manifesting as reduced growth rate in the ΔfabG strain. Studies have shown that the fungal CW plays multiple roles in virulence, and A. fumigatus mutants with defective CWI show significantly attenuated virulence (54). Our study observed a similar phenomenon: the thickened cell wall exposed more immunogenic components such as β-1,3-glucan, making the fungus more recognizable and susceptible to immune cell attack. This may explain the reduced virulence caused by fabG deletion.
The cell wall of A. fumigatus plays a dual role: it not only maintains structural integrity but also serves as a critical immunological interface that mediates host–pathogen interactions (32, 56–58). Its composition directly influences fungal virulence and shapes the host immune response. During infection, pattern recognition receptors on host immune cells—such as Dectin-1—recognize β-1,3-glucan in the fungal cell wall, triggering downstream immune signaling cascades (59–62). Notably, increased surface exposure of β-1,3-glucan significantly enhances the phagocytosis of conidia by macrophages (63). Swollen conidia or early germ tubes, which expose large amounts of β-1,3-glucan, can strongly activate the Dectin-1 pathway and induce robust secretion of proinflammatory cytokines such as IL-1β and TNF-α (64). fabG may influence the structure and composition of the A. fumigatus CW by regulating the expression of genes involved in chitin and β-glucan biosynthesis. The elevated β-1,3-glucan content observed in the fabG mutant is associated with enhanced macrophage-mediated phagocytosis and fungal killing, suggesting that fabG may contribute to immune recognition and modulation during IA. This study found that the thickening of the A. fumigatus cell wall following fabG gene deletion is closely associated with the activation of the CWI pathway. Moreover, the increased resistance of the mutant to CAS may interfere with the recognition of pathogen-associated molecular patterns by host pattern recognition receptors. In summary, this study confirms that fabG likely plays a key role in the growth and virulence of A. fumigatus by regulating oxidoreductase activity. Additionally, fabG affects fungal sensitivity to cell wall-disrupting agents and cell wall thickness by modulating the expression of genes related to the CWI pathway, thereby coordinating fungal growth and virulence. However, current evidence is limited to molecular and basic phenotypic observations. The specific interactions between fabG and oxidoreductases such as SOD and CAT require further validation using methods such as yeast two-hybrid assays or co-immunoprecipitation. The connection between fabG and the CWI pathway also lacks definitive evidence—for example, confirmation via western blot using phospho-specific antibodies or phospho-proteomics would be necessary. It is also possible that the newly observed functions in the gene knockout strain arise from interactions between fabG deletion and KU80 deficiency. Overall, fabG exhibits novel functions and may play a role in the oxidoreductase system, representing a potential therapeutic target for combating A. fumigatus infections in the future.
Supplementary Material
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The reference list from the paper itself. Each links out to its DOI / PubMed record.
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