Nootkatone antifungal activity and calcium signaling-mediated resistance in Aspergillus fumigatus

Abstract

Abstract

Tobacco is rich in natural bioactive compounds, yet the antifungal potential of its essential oil remains largely unexplored. In this study, we demonstrate for the first time that tobacco essential oil exhibits significant antifungal activity against the opportunistic human pathogen Aspergillus fumigatus. At high concentrations, the oil effectively inhibits hyphal growth and suppresses conidial production. Chemical profiling identified nootkatone as the principal active compound. Nootkatone exerts a concentration-dependent dual antifungal effect, simultaneously inhibiting hyphal elongation and spore formation. Mechanistic analyses revealed that the calcium signaling pathway, particularly the crzA and cchA genes, plays a central regulatory role in mediating fungal resistance to nootkatone. Deletion of either gene (ΔcrzA or ΔcchA) led to significantly increased sensitivity to nootkatone, highlighting their role in nootkatone stress response. Furthermore, under nootkatone stress, CrzA was found to regulate the expression of multiple efflux pump genes (abcC, abcE, atrA, mdr1, and atrB), uncovering a previously unrecognized link between calcium signaling and nootkatone efflux. In addition to targeting cellular signaling, nootkatone also disrupted fungal cell wall integrity and triggered the release of host immune mediators, suggesting a synergistic antifungal mechanism that combines structural disruption with immune activation. In the Galleria mellonella infection model, nootkatone significantly enhanced host survival and reduced fungal burden. Notably, treatment of ΔcrzA-infected larvae resulted in a 70% survival rate, markedly higher than that observed in wild-type (WT) infections. Moreover, nootkatone retained strong inhibitory activity against itraconazole-resistant isolates, underscoring its potential as a novel, broad-spectrum antifungal agent.

Key points

• Nootkatone inhibits fungal growth—suppresses Aspergillus fumigatus hyphae and spores via dual concentration-dependent action

• Targets calcium signaling—key genes (crzA/cchA) regulate resistance; deletion enhances susceptibility and links to efflux pump activation.

• Dual antifungal mechanisms and drug resistance overcoming—disrupts cell wall integrity, activates host immunity, and is effective against azole-resistant strains, boosting survival in infection models.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00253-026-13753-w.

Introduction

Fungal infections have emerged as a major global public health challenge. With the widespread use of immunosuppressive therapies and the continuous rise of drug-resistant fungal strains, the incidence of invasive fungal infections has increased significantly. Among these, aspergillosis caused by Aspergillus fumigatus is particularly concerning due to its high mortality rate, prolonged treatment duration, and high recurrence rate, posing a significant challenge in clinical therapy (Cadena et al. 2021; Latgé and Chamilos 2019). Although first-line antifungal agents such as itraconazole (ITZ) and voriconazole (VRC) are widely used, their long-term use often leads to resistance mutations, severely limiting clinical efficacy (Lockhart et al. 2023; Wei et al. 2017). Therefore, there is an urgent need to explore new antifungal strategies, particularly those based on natural products that are safe, effective, and less likely to induce resistance. As alternatives to conventional drugs, plant-derived natural products—such as essential oils—have demonstrated promising antifungal potential with fewer side effects (Adaszyńska-Skwirzyńska et al. 2023; Cai et al. 2022; D'Agostino et al. 2019; Kumari et al. 2022).

Plant-derived essential oils (EOs) have gained increasing attention as potent antifungal agents due to their complex chemical composition and broad-spectrum bioactivities (Kokoska et al. 2019; Paudel et al. 2022; Zapata-Zapata et al. 2022). Extracted from various plant parts—such as flowers, leaves, roots, and bark—these oils typically consist of terpenes, aromatic hydrocarbons, aldehydes, and alcohols. These compounds are not only essential for plant development and defense but also contribute to the biological activities of the oils (Chen et al. 2025; Jirovetz et al. 2003; Padalia et al. 2010). Recent research has demonstrated that many essential oils exhibit significant antifungal properties, with mechanisms of action including disruption of fungal cell membranes, impairment of cell wall integrity, and interference with intracellular metabolic processes (Abd El-Hack et al. 2023; da Silva et al. 2021; Li et al. 2022; Ruiz-Duran et al. 2023). Compared to synthetic antifungal drugs, plant essential oils offer several advantages, including natural origin, relatively low toxicity, and efficacy against drug-resistant fungal strains (Aziz et al. 2018; Muntean et al. 2019). Consequently, essential oils are increasingly considered as promising complementary agents or alternatives to conventional antifungal therapies.

Tobacco (Nicotiana tabacum), a member of the Solanaceae family, is a globally cultivated crop of economic significance (Jassbi et al. 2017; Xie et al. 2021). Although traditionally valued for its use in the tobacco industry, tobacco leaves have also been employed in ethnomedicine, particularly for alleviating anxiety and depression (Berlowitz et al. 2020). As a rich source of aromatic compounds, tobacco harbors a diverse array of secondary metabolites. To date, over 3000 chemical constituents have been identified across different tobacco species, including terpenoids, carotenoid derivatives, and volatile aromatic compounds (Popova et al. 2019a; Xie et al. 2021). Some of these constituents are unique to tobacco, endowing it with distinct phytochemical characteristics.

While extensive studies have been conducted on the aroma profiles and sensory quality of tobacco (Popova et al. 2019a), research into the biological activities of tobacco essential oil (TEO) remains relatively limited. Recent in vitro and in vivo studies have revealed that TEO and its major components exhibit various pharmacological properties, such as antibacterial (Popova et al. 2019b; Xie et al. 2021; Yuan et al. 2019), anti-inflammatory (Yan et al. 2019), antioxidant, antitumor (Yuan et al. 2019), and neuroprotective effects (Yan et al. 2019). However, its potential antifungal activity—particularly against pathogenic fungi—has not been thoroughly investigated. This represents a critical knowledge gap and an opportunity for future research. A comprehensive exploration of the antifungal properties of TEO could provide a scientific basis for its application in antifungal drug development. Moreover, such studies may facilitate the broader medicinal use of tobacco in modern healthcare, moving beyond its traditional industrial roles and harnessing its therapeutic potential as a source of novel, natural antifungal agents.

The calcium signaling pathway plays a pivotal role in regulating fungal growth, differentiation, stress response, and, notably, antifungal drug resistance (Bowyer et al. 2020; Giuraniuc et al. 2023; Li et al. 2019; Song et al. 2016a; Wang et al. 2016). As a widespread secondary messenger, calcium modulates the activity of several downstream effectors, including calmodulin (CaM), calcium-dependent protein kinases (CDPKs), and specific calcium channels such as CchA and MidA (de Castro et al. 2014b). CaM is a highly conserved calcium-binding protein containing EF-hand motifs, which are helix–loop–helix structural domains responsible for binding Ca²⁺ ions and translating calcium signals into conformational changes that activate downstream targets (Ohya and Anraku 1992; Park et al. 2019; Zuo et al. 2025). These molecules collectively enable fungal cells to sense intracellular calcium fluctuations and adapt to external antifungal stress, thereby modifying their drug susceptibility profiles. A key regulatory module within this pathway is the calcineurin-CrzA axis (Park et al. 2019). Calcineurin is a Ca²⁺/calmodulin-dependent serine/threonine phosphatase composed of a catalytic subunit (CnaA) and a regulatory subunit (CnaB). The cnaA gene encodes the catalytic subunit of calcineurin and is essential for calcineurin phosphatase activity, playing a central role in calcium signal transduction, cell wall integrity maintenance, stress adaptation, and fungal virulence (Park et al. 2019). Upon calcium influx, Ca²⁺-bound calmodulin interacts with CnaA, activating calcineurin phosphatase activity. Activated calcineurin then dephosphorylates the transcription factor CrzA (calcineurin-responsive zinc finger transcription factor), enabling its translocation into the nucleus (Shwab et al. 2019; Zhang et al. 2021). Once in the nucleus, CrzA functions as a transcriptional regulator by binding to promoter regions of target genes involved in vital biological processes, such as cell wall biosynthesis and remodeling (e.g., chitin synthases), ion homeostasis, oxidative stress responses, and xenobiotic efflux (de Castro et al. 2014a; Ries et al. 2017; Shwab et al. 2019). Perturbation of this regulatory axis has been shown to significantly influence fungal tolerance to triazole-class antifungals, including itraconazole (Li et al. 2020; Liu et al. 2015). Building on these insights, the present study aims to elucidate the molecular mechanisms underlying the tolerance of the pathogenic fungus A. fumigatus to bioactive compounds in tobacco essential oil (TEO), with a specific focus on the role of the calcium–CrzA signaling pathway. Additionally, the study employs the Galleria mellonella infection model to assess the in vivo antifungal efficacy of TEO-derived components against A. fumigatus, including strains known to exhibit resistance to conventional antifungal agents. The outcomes of this investigation are expected to provide both theoretical and experimental foundations for the development of novel antifungal therapies or combination strategies leveraging natural products. Specifically, characterizing the physiological and biochemical responses of A. fumigatus to TEO and identifying the associated tolerance mechanisms may open new avenues for integrating plant-derived compounds into the antifungal pharmacopeia. Ultimately, this research holds substantial implications for addressing the growing challenge of antifungal drug resistance and advancing the application of natural products in infectious disease management.

Materials and methods

Strains and cultivation conditions

A. fumigatusA1160 (ΔKU80 pyrG1), obtained from the Fungal Genetics Stock Center (FGSC) (Dai et al. 2023; Jiang et al. 2014), is a strain originally isolated from a clinical sample. This strain was used to construct the ΔcrzA, ΔcchA, and ΔcnaA null mutants. The A1160 strain was routinely cultured on YUU medium (0.5% yeast extract, 2% dextrose, 1 mL/L 1000 × trace elements, 1.2% uracil, 1.1% uridine, and 2% agar) at 37 °C. The media used in this study included YAG, minimal medium (MM), and YUU. Solid media contained 2% agar, whereas liquid YAG, MM, and YUU lacked agar. The compositions were as follows: YAG: 2% glucose, 0.5% yeast extract, and 1 mL/L 1000 × trace elements; MM: 1% glucose, 1 mL/L 1,000 × trace elements, and 50 mL/L of 20 × salts; 20 × salts (per liter): 120 g NaNO₃, 10.4 g KCl, 30.4 g KH₂PO₄, 10.4 g MgSO₄·7H₂O; 100 mL 1000 × trace elements: 2.20 g ZnSO₄·7H₂O, 1.10 g H₃BO₃, 0.50 g MnCl₂·4H₂O, 0.16 g FeSO₄·7H₂O, 0.16 g CoCl₂·5H₂O, 0.16 g CuSO₄·5H₂O, 0.11 g (NH₄)₆Mo₇O₂₄·4H₂O, and 5.00 g Na₄EDTA. Conidia were harvested from plates after 48 h of incubation at 37 °C. Spores were collected by gently washing the colony surface with sterile solution, filtered to remove mycelial debris, diluted as required, and counted using a Neubauer hemocytometer. The components of the culture media used in our experiments were sourced from Shanghai Shenggong Biotechnology Co., Ltd. located in Shanghai, China.

Tobacco essential oil extraction and GC-MS analysis

The extraction of tobacco essential oil was performed with minor modifications based on previously published methods (Xie et al. 2021). Briefly, 60 g of tobacco leaves (Yunyan 87) was placed in a 1000-mL three-necked flask containing 800 mL of distilled water supplemented with 5% NaCl and soaked for 1.5 h. The mixture was then heated to a gentle boil, and hydrodistillation was initiated once reflux was observed. Distillation was continued for 8 h, after which the system was allowed to cool to room temperature. The distillate was washed three times with a small volume of ether, and the organic phase was collected, dried if necessary, and transferred into a glass vial for storage. The remaining ether was removed using a rotary evaporator, yielding the tobacco essential oil. The oil was analyzed qualitatively and quantitatively using gas chromatography-mass spectrometry (GC-MS) (Agilent Technologies, Santa Clara, CA, USA) (Xie et al. 2021). The chromatographic conditions included a DB-5 fused silica capillary column (30 m × 250 µm × 0.25 µm) (Agilent Technologies, Santa Clara, CA, USA), with helium as the carrier gas at a flow rate of 1 mL/min. The injector temperature was set to 250 °C, and the flame ionization detector (FID) temperature was set to 280 °C. The initial column temperature was 60 °C, held for 1 min, then increased at a rate of 15 °C/min to 250 °C, where it was held for 10 min. The injection volume was 0.4 μL. Mass spectrometry was performed using electron ionization (EI) (Agilent Technologies, Santa Clara, CA, USA) with an ion source temperature of 250 °C and a quadrupole temperature of 150 °C. The electron energy was set to 70 eV, with a scanning range of 33–450 m/z and an electron multiplier voltage of 1347 V.

Transcriptional profile analyses using RNA-seq

The aim of this study was to investigate transcriptomic changes in A. fumigatus upon stimulation with nootkatone (Sigma-Aldrich, St. Louis, MO, USA) using RNA sequencing (RNA-seq). A. fumigatus spores were inoculated into liquid minimal medium (MM) and cultured under shaking conditions for 15 h to allow the development of mature hyphae. Nootkatone was then added to the cultures at a final concentration of 0.3 mg/mL to induce cellular stress. Samples were collected at 10, 30, and 60 min post-treatment to capture time-resolved transcriptional responses, while an untreated group served as the control. The harvested hyphae were immediately flash-frozen in liquid nitrogen to preserve RNA integrity. Total RNA was extracted using a commercial RNA extraction kit (Vazyme, Nanjing, China) following the manufacturer’s instructions. Polyadenylated mRNA was isolated from total RNA using oligo (dT)-based magnetic bead purification to remove ribosomal and non-coding RNAs. The purified mRNA was subsequently reverse-transcribed to generate complementary DNA (cDNA), which was then used for RNA-seq library construction. High-throughput sequencing was performed to obtain comprehensive transcriptome profiles for all samples. All procedures, including RNA isolation, mRNA purification, cDNA synthesis, and sequencing, were conducted by Wuhan Benagen Tech Co., Ltd., located in Wuhan, China. The raw sequencing reads were aligned to the reference genome of A. fumigatus, and differential gene expression analysis was performed to identify transcripts with significant expression changes in response to nootkatone treatment. These results provide important insights into the molecular mechanisms underlying stress responses and transcriptional regulation in A. fumigatus.

RNA extraction and qRT-PCR

Spores of A. fumigatus were inoculated into liquid minimal medium (MM) and incubated at 37 °C with shaking at 200–250 rpm for 16 h to promote spore germination. After 16 h, nootkatone was added to the experimental group at a final concentration of 0.3 mg/mL for drug treatment, while the control group remained untreated with nootkatone. Both groups were incubated at 37 °C with shaking for an additional 30 min. Following the treatment, mycelia were immediately collected and rapidly frozen in liquid nitrogen to preserve RNA integrity under RNase-free conditions. Total RNA was extracted using TRIzol reagent (Vazyme, Nanjing, China) according to the manufacturer’s instructions. The extracted RNA was assessed for quality, and RNA of satisfactory quality was reverse transcribed into cDNA using a PrimeScript RT Reagent Kit (Vazyme, Nanjing, China). Quantitative real-time PCR (RT-qPCR) was then performed using SYBR Green dye (Vazyme, Nanjing, China), with actin as the internal reference gene. Each reaction included at least three technical replicates. Data analysis was carried out using the 2^−ΔΔCT method to compare gene expression levels between the experimental and control groups, thereby assessing the impact of nootkatone on target gene expression.

Construction of crzA, cchA, and cnaA gene deletions

Fusion PCR was used to construct the crzA knockout cassette, following the previously described methodology (Dai et al. 2023). In brief, approximately 1-kb sections of DNA flanking the crzA gene were amplified using the primers crzA P1/P3 and crzA P4/P6. The pyr4 selection marker, derived from the plasmid pAL5 (Dai et al. 2023), was amplified using the primers Pyr4 F/R. These three PCR products (the two flanking regions and the selection marker) were then used as templates to generate the crzA deletion cassette via fusion PCR, using primers crzA P2/P5. The resultant knockout cassette was subsequently transformed into the parental A. fumigatus strain A1160 using the previously described methodology (Song et al. 2016b). The transformants were screened and verified by diagnostic PCR, employing the primers crzA P8/P9, crzA P1/P7, and crzA P6/P10 to confirm the successful integration of the knockout cassette.

Following the same approach, the cchA and cnaA genes were also knocked out. For each of these genes, approximately 1-kb regions flanking the cchA and cnaA genes were amplified using the primers cchA P1/P3/cchA P4/P6 and cnaA P1/P3/cnaA P4/P6, respectively. The pyr4 marker was amplified from pAL5 with the same primers, Pyr4 F/R. Fusion PCR was then performed with these products to generate the cchA and cnaA deletion cassettes using the respective primers cchA P2/P5 and cnaA P2/P5. These deletion cassettes were transformed into the A. fumigatus strain A1160, and transformants were verified by diagnostic PCR using the primers cchA P8/P9, cchA P1/P7, and cchA P6/P10, and cnaA P8/P9, cnaA P1/P7, and cnaA P6/P10.

To generate the crzA-complemented strain crzA^C, the crzA-complemented fragments, which include 1.0 kb of the upstream flanking regions and the crzA open reading frame (ORF), were amplified with the primer pair Com-crzA-F/Com-crzA-R. Subsequently, it was cloned into plasmid pAN7-1 (Dai et al. 2024) at the HindIII cleavage site using the ClonExpress MultiS Single-Step Cloning Kit (Vazyme, Nanjing, China). Then, the complemented plasmid was transformed into ΔcrzA to generate the crzA^C strain. The cchA-complemented strain (cchA^C) was constructed using the same strategy. The sequences of all primers used in this study are provided in Supplemental Table S1.

Construction of the CaM point mutation strains

A CaM mutant carrying a single amino acid substitution (E32A) in the first EF-hand loop of the CaM protein was constructed as follows. The selectable marker pyr4 was amplified from plasmid pAL5 (Dai et al. 2023) using the primer pair Pyr4-F/Pyr4-R. The 5′ flanking region (1727 bp) and the 3′ flanking region (1653 bp) were amplified from wild-type (WT) A. fumigatus genomic DNA using the primer pairs CaM-mutant-P1/CaM-mutant-P3 and CaM-mutant-P4/CaM-mutant-P6, respectively. The WT strain refers to A. fumigatus carrying the unmodified CaM allele, encoding the wild-type CaM protein. The three PCR products were fused to generate a 5502 bp DNA fragment with the primers CaM-mutant-P2/CaM-mutant-P5, which was then cloned into the vector Blunt-Zero (Beijing Solabao Technology Co., Ltd., Beijing, China) to generate the plasmid Pfr01. The 5′ flanking sequence (970 bp) and 3′ flanking sequence (4542 bp) were amplified from plasmid Pfr01 using the primer pairs CaM-mutant-P2/CaM(1)-DOWN and CaM(1)-UP/CaM-mutant-P5, respectively, and the complementary regions contained the desired mutation (glutamate32–alanine32). Using these two fragments as templates, the final 5502 bp fragment was generated by fusion PCR using primers CaM-mutant-P2 and CaM-mutant-P5, which was further cloned into the vector Blunt-Zero to generate plasmid Pfr02. We generated CaM^mut(1) by transforming Pfr02 into the WT strain. A similar strategy was used to construct other CaM point mutation strains.

In vivo antifungal efficacy of nootkatone against A. fumigatus

Virulence assays were performed using G. mellonella larvae with minor modifications to previously described methods (Fallon et al. 2011); G. mellonella is a widely used invertebrate infection model for assessing the virulence of A. fumigatus and for evaluating the in vivo efficacy of antifungal agents. Healthy larvae of similar size and weight were randomly assigned to experimental groups. Infection was established by injecting larvae via the hind prolegs with 10 µL of phosphate-buffered saline (PBS) containing 5 × 10⁵ conidia of the respective A. fumigatus strain per larva. Control larvae received an equal volume (10 µL) of PBS alone. To evaluate the in vivo antifungal activity of nootkatone, larvae were treated with nootkatone by injection into the hind prolegs at a defined time point following infection. Specifically, nootkatone was administered as a single dose of 0.1 mg per larva, dissolved in 10 µL of PBS, 1 h post-infection. Infected but untreated larvae, as well as solvent-treated larvae when applicable, were included as control groups. Following infection and treatment, larvae were incubated at 37 °C in the dark and monitored daily for survival over a 7-day period. Larvae were considered dead when they failed to respond to physical stimulation. Survival curves were generated using the Kaplan–Meier method, and statistical significance between groups was determined using the log-rank (Mantel–Cox) test with GraphPad Prism software (GraphPad Software, San Diego, CA, USA).

Immune cell cytokine detection

To investigate whether nootkatone-induced alterations in A. fumigatus spore properties affect host immune responses, an immune cell cytokine detection assay was performed to evaluate macrophage cytokine expression following exposure to A. fumigatus spores. A. fumigatus spores were cultured on YAG agar plates for 5 days, then suspended in PBS or sterile distilled water to prepare a spore suspension. The suspension was filtered to remove mycelia, and the spore concentration was counted. The spore concentration was adjusted to 1 × 10⁷ spores/mL. Macrophages were cultured in RPMI 1640 medium (Sangon Biotech, Shanghai, China) supplemented with 10% fetal bovine serum (FBS) (Sangon Biotech, Shanghai, China) and 1% penicillin–streptomycin (P/S) (Sangon Biotech, Shanghai, China) at 37 °C and 5% CO₂. Once the cell density reached an appropriate level, the prepared spore suspension was added for infection. The infection times were 2 h and 4 h, with an uninfected group serving as the control. After the infection period, total RNA was extracted from the infected cells using an RNA extraction kit (Vazyme, Nanjing, China). RNA concentration and purity were measured using a spectrophotometer to ensure RNA quality for reverse transcription. cDNA was synthesized using a reverse transcription kit. Real-time quantitative PCR (RT-qPCR) was performed using SYBR Green dye (Vazyme, Nanjing, China), with β-actin as the internal reference gene. The mRNA expression levels of target cytokines, including IL-1β, IL-6, and TNF-α, were analyzed. Each reaction was set up with at least three technical replicates and at least three biological replicates per group. Data analysis was performed using the 2^−ΔΔCT method, with statistical analysis conducted using GraphPad Prism software (GraphPad Software, San Diego, CA, USA). Significance was determined by ANOVA, with a p-value < 0.01 considered statistically significant.

Statistical analysis

All experiments were performed at least three times independently. Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). Comparisons between two groups were conducted using Student’s t-test, and multiple-group comparisons were analyzed by one-way ANOVA followed by Tukey’s post hoc test. p-value < 0.05 was considered statistically significant.

Results

Antifungal activity of tobacco essential oil against A. fumigatus and its synergistic effect with ITZ in inhibiting drug resistance

In this study, we investigated the antifungal activity of tobacco essential oil (TEO), extracted from tobacco leaves by distillation, and its effect on the growth of A. fumigatus. Our results demonstrated that TEO inhibited the growth of A. fumigatus in a concentration-dependent manner, with progressively stronger inhibition observed as the concentration increased (Fig. 1A). In the control group, in which the MM medium contained no TEO, A. fumigatus colonies exhibited normal growth, sporulation, and colony morphology, indicating that the strain could grow and reproduce in the absence of TEO (Fig. 1A). In contrast, increasing concentrations of TEO resulted in a gradual reduction in colony growth and spore formation (Fig. 1A).

Fig. 1.

Antifungal activity and resistance suppression by tobacco essential oil against A. fumigatus. A Dose-dependent inhibition of A. fumigatus growth by tobacco essential oil. Conidia (1 × 10⁷ per plate) were spotted on MM plates supplemented with increasing concentrations (0, 0.1, 0.2, and 0.3 µg/mL) of tobacco essential oil. Fungal colony size decreased with increasing essential oil concentrations, indicating strong antifungal activity. B Synergistic effect of tobacco essential oil with ITZ in preventing the emergence of resistant colonies. Plates containing 3 µg/mL ITZ alone showed abundant colony growth, indicative of resistant mutants. In contrast, plates with 3 µg/mL ITZ combined with 1 µg/mL tobacco essential oil showed complete inhibition of resistant colony formation. C Quantification of mutation frequency under antifungal pressure. The mutation rate of A. fumigatus in the presence of ITZ alone was significantly higher compared to the combination treatment with ITZ and tobacco essential oil, which effectively reduced the emergence of resistant mutants to near-zero levels. Data are expressed as mean ± SD from independent experiments

Specifically, at a concentration of 0.1 µg/mL, colony growth was slowed, colonies appeared paler, and sporulation was reduced. At 0.2 µg/mL, colony size was markedly decreased, with little to no spore formation, indicating strong growth inhibition at this concentration. At 0.3 µg/mL, colony growth was almost completely suppressed, with no visible colonies observed, demonstrating that this concentration effectively inhibited the growth of A. fumigatus (Fig. 1A). These results indicate that TEO significantly inhibits both the growth and sporulation of A. fumigatus in a dose-dependent manner.

Given the clinical challenges associated with the widespread use of azole antifungals and the resulting emergence of drug-resistant fungal strains, we further evaluated the combined effects of TEO and ITZ against resistant A. fumigatus strains. When the medium contained 3 µg/mL ITZ alone, resistant colonies appeared after 6 days of incubation (Fig. 1B), likely due to the presence of pre-existing resistant mutants. In contrast, no visible colonies were detected after 1 week of incubation when TEO was combined with ITZ, indicating that the combined treatment either significantly reduced the survival of resistant strains or suppressed the emergence of resistance mutations (Fig. 1B).

To further assess the effect of this combination on resistance development, the mutation frequency of ITZ resistance was analyzed. When ITZ was used alone, a high mutation frequency was observed, with approximately 30 resistant mutants per 10⁷ spores (Fig. 1C), suggesting that prolonged ITZ exposure promotes the emergence of resistance. However, when TEO was combined with ITZ, the mutation frequency was significantly reduced compared with ITZ treatment alone (Fig. 1C). These findings indicate that TEO can effectively suppress or reduce the development of ITZ-induced resistance mutations, thereby potentially enhancing the long-term efficacy of antifungal therapy.

In conclusion, tobacco essential oil exhibits potent antifungal activity against A. fumigatus, particularly at higher concentrations, effectively inhibiting fungal growth and sporulation. Moreover, the combination of TEO with ITZ not only reduces the survival of drug-resistant strains but also significantly limits the emergence of resistance mutations. Together, these results support the potential application of tobacco essential oil in combination with ITZ as a promising strategy for the treatment of azole-resistant fungal infections.

Identification of active compounds in tobacco essential oil

To identify the compounds responsible for the antifungal activity of tobacco essential oil (TEO), we extracted TEO from tobacco leaves by steam distillation and analyzed its chemical composition using GC–MS. GC–MS profiling revealed that TEO is a chemically diverse natural mixture containing multiple classes of compounds (Table 1), including ketones, terpenes, alcohols, aldehydes, and other aromatic constituents. Among the ketones, nootkatone, ionone, and megastigmatrienone were detected, all of which have been reported to possess notable biological activities. The major terpenes and hydrocarbons identified included neophytadiene and 1-nonadecene, whereas alcohols such as linalool and trans-geranylgeraniol were also present. In addition, several aldehydes—including 3-furaldehyde, pentadecanal, and 2-hexyl-1-decanol—were detected, along with phenolic and ester-derived aromatic compounds. Collectively, these results highlight the chemical complexity of TEO and suggest its potential biological relevance. To evaluate the antifungal activity of the identified constituents, several commercially available monomeric compounds were purchased and screened in vitro. Among the tested compounds, nootkatone displayed the strongest inhibitory activity against A. fumigatus and thus was selected for further investigation (Supplemental Fig. S1A). In subsequent experiments, we aimed to elucidate the molecular mechanisms underlying A. fumigatus resistance to nootkatone and to assess its potential as a therapeutic candidate for aspergillosis. These findings provide a foundation for further exploration of nootkatone as a novel antifungal agent and may contribute to the development of alternative strategies for combating A. fumigatus infections, particularly those resistant to conventional antifungal therapies.

Table 1.

Main chemical components identified in tobacco essential oil

No	Tentatively identified compounds	Retention time (min)	CAS no	Relative content (%)
1	Nootkatone	27.715	4674-50-4	0.249
2	Linalool	12.235	78-70-6	0.096
3	beta-Damascenone	20.284	23726-93-4	3.492
4	Ionone	20.684	8013-90-9	0.152
5	1-(2,6,6-Trimethyl-1-cyclohexen-1-yl)−2-buten-1-one	20.975	35044-68-9	0.601
6	(5E)−6,10-Dimethylundeca-5,9-dien-2-one	21.888	3796-70-1	0.685
7	Isobornyl acetate	22.821	125-12-2	1.224
8	Thymol	23.850	89-83-8	0.193
9	trans-Geranylgeraniol	24.007	24034-73-9	0.182
10	Megastigmatrienone	24.996	38818-55-2	0.355
11	Pentadecanal	27.938	2765-11-9	0.365
12	1-Nonadecene	28.281	18435-45-5	0.481
13	3-Furaldehyde	31.997	498-60-2	27.591
14	1,3-Benzenediol, 5-hexyl-	31.934	5465-20-3	3.359
15	n-Butyric acid tetrahydrofurfuryl ester	31.975	637-65-0	2.061
16	Neophytadiene	32.263	504-96-1	0.160
17	Hexadecanoic acid, methyl ester	34.099	112-39-0	0.254
18	2-Hexyl-1-decanol	33.996	2425-77-6	0.690
19	1,3,6,10-Cyclotetradecatetraene	36.862	1898-13-1	0.088
20	2-Hexadecenal	34.281	22644-96-8	0.437

Dose-dependent inhibition of A. fumigatus growth and sporulation by nootkatone

To systematically evaluate the antifungal activity of nootkatone against A. fumigatus, dose–response experiments were conducted using increasing concentrations of the compound. The results showed that nootkatone exerted strong, concentration-dependent antifungal effects, markedly suppressing both colony expansion and conidial production (Fig. 2A). For colony growth assessment, colony diameters were measured on MM plates. In the untreated control group (MM without nootkatone), colonies grew vigorously and reached an average diameter of approximately 3 cm (Fig. 2B). In contrast, treatment with 0.1 mg/mL nootkatone significantly reduced colony size. Further increases in nootkatone concentration (0.2, 0.3, and 0.4 mg/mL) resulted in progressively greater growth inhibition (Fig. 2B). Statistical analysis confirmed that all treatment groups exhibited highly significant reductions in colony diameter compared with the control (p < 0.001), indicating that even low concentrations of nootkatone effectively impair hyphal development. Nootkatone also strongly inhibited spore formation. The highest conidial yield, approximately 2 × 10⁸ spores, was observed in the untreated control group. At 0.1 mg/mL nootkatone, spore production declined sharply to approximately 0.3 × 10⁸ spores, suggesting early disruption of the conidiation process (Fig. 2C). At higher concentrations (0.2–0.4 mg/mL), conidial production was almost completely abolished. All treated groups showed statistically significant reductions in spore counts compared with the control (p < 0.001), further confirming the potent inhibitory effect of nootkatone on fungal reproduction. To determine whether these effects were strain-dependent, we additionally tested another A. fumigatus strain, Af293. Nootkatone treatment similarly inhibited mycelial growth and reduced conidiation in Af293, consistent with the results observed in strain A1160 (Supplemental Fig. S1B). These findings indicate that the antifungal activity of nootkatone is not strain-specific. In summary, nootkatone exhibits dual antifungal effects on A. fumigatus, significantly inhibiting both hyphal growth and conidial production in a dose-dependent manner. As a naturally derived bioactive compound, nootkatone may represent a promising candidate for antifungal applications.

Fig. 2.

Nootkatone inhibits the growth and sporulation of A. fumigatus in a dose-dependent manner. A Representative images showing colony morphology of A. fumigatus cultured on MM plates supplemented with increasing concentrations of nootkatone (0, 0.1, 0.2, 0.3, and 0.4 mg/mL). Higher nootkatone concentrations resulted in progressive suppression of fungal colony expansion. B Quantitative analysis of colony diameter after incubation with different nootkatone concentrations. Data show a significant and concentration-dependent reduction in mycelial growth compared to the MM control. C Quantification of spore yield (× 10⁸) under varying concentrations of nootkatone. Spore production was markedly inhibited in a dose-dependent manner. Error bars represent mean ± SD from three independent experiments. Statistical significance: ***p < 0.001 versus MM control

Critical role of calcium signaling pathways in the tolerance mechanism of A. fumigatus under nootkatone-induced stress

To elucidate the molecular basis of A. fumigatus tolerance under nootkatone-induced stress, we adopted an integrated strategy combining transcriptome sequencing (RNA-seq), RT-qPCR validation, and physiological modulation assays. Collectively, these results indicate that calcium homeostasis and calcium-dependent signaling pathways play a central role in the adaptive response of A. fumigatus to nootkatone exposure. Transcriptomic profiling revealed that nootkatone treatment markedly perturbed the Ca²⁺ homeostasis regulatory network in A. fumigatus. Several genes essential for intracellular calcium regulation were significantly downregulated, including the genes for endoplasmic reticulum Ca²⁺-ATPase (AFUA_6G06740), the plasma membrane calcium channel Cch1 (AFUA_1G11110), the calmodulin-like protein Sha1 (AFUA_1G04920), and calcium transporters such as Agc1 and Vcx1 (Fig. 3A). The coordinated suppression of these genes suggests that nootkatone interferes with key processes governing calcium influx, intracellular storage, and efflux, thereby destabilizing intracellular Ca²⁺ balance. Given that calcium signaling regulates multiple stress-response pathways, such disruption is likely to impair essential physiological processes, including cell wall maintenance, stress adaptation, and survival under antifungal pressure. Notably, although most calcium regulatory genes were suppressed, a subset of stress-associated genes—including the calcium homeostasis protein regucalcin and annexin Anxc4—displayed modest upregulation (Fig. 3A). This expression pattern suggests the activation of a partial compensatory response aimed at counteracting Ca²⁺ dysregulation. However, the limited magnitude of induction implies that this feedback response is insufficient to restore calcium homeostasis under nootkatone-induced stress.

Fig. 3.

Nootkatone disrupts calcium signaling in A. fumigatus and its antifungal effect is influenced by calcium homeostasis. A Heatmap showing differential expression of calcium-related genes in A. fumigatus treated with nootkatone compared to the untreated wild-type strain. RNA-Seq and RT-PCR results both confirm downregulation (blue) and upregulation (red) of several calcium signaling and transport-related genes. Notably, genes encoding calcium channels (e.g., Cch1 and Sha1), ATPases, and calcium-binding proteins are significantly affected. B Colony morphology of A. fumigatus grown on MM plates for 48 h under different conditions: untreated (MM), with EGTA (10 mM), with CaCl₂ (50 mM), and with 0.1 mg/mL nootkatone alone or in combination with EGTA or CaCl₂. Calcium supplementation (CaCl₂) failed to reverse growth inhibition caused by nootkatone, while calcium chelation by EGTA exacerbated the antifungal effect. C Quantitative analysis of colony diameter under each treatment condition. Nootkatone significantly reduced fungal growth, and this inhibition was further enhanced by EGTA. In contrast, CaCl₂ mitigated the inhibitory effect of nootkatone. Data represent mean ± SD from three independent experiments. Statistical significance: p < 0.01; ns, not significant

To further assess the functional importance of calcium signaling in nootkatone tolerance, we performed calcium modulation assays. Co-treatment with the extracellular calcium chelator ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) significantly enhanced the antifungal effect of nootkatone (Fig. 3B), resulting in stronger inhibition of colony growth than nootkatone treatment alone (p < 0.01) (Fig. 3C). This finding indicates that depletion of extracellular Ca²⁺ exacerbates calcium signaling disruption, thereby increasing physiological stress and reducing fungal tolerance. In contrast, supplementation with exogenous CaCl₂ did not restore normal growth, suggesting that simply increasing extracellular calcium availability cannot compensate for the impaired calcium regulatory machinery or signaling cascade (Fig. 3C). In summary, these results provide strong evidence that calcium homeostasis and calcium-dependent signaling are critical components of the adaptive tolerance response of A. fumigatus under nootkatone-induced stress. Disruption of this regulatory network compromises fungal stress adaptation and significantly potentiates the antifungal efficacy of nootkatone.

Functional analysis of key calcium signaling components in A. fumigatus reveals crucial roles for CrzA and CchA in nootkatone stress tolerance

Building on our previous observation that calcium signaling is central to the adaptive response of A. fumigatus under nootkatone-induced stress, we further dissected the roles of key pathway components using targeted gene deletion, genetic complementation, and site-directed mutagenesis. Knockout mutants were generated for three major calcium signaling genes—crzA, cchA, and cnaA—to assess their respective contributions to nootkatone tolerance. Phenotypic characterization showed that both ΔcrzA and ΔcchA mutants exhibited colony growth comparable to WT strain on MM plates, indicating that deletion of either gene does not substantially affect basal growth under non-stress conditions. However, when exposed to 0.1 mg/mL or 0.2 mg/mL nootkatone, both mutants displayed significantly reduced colony diameters compared with WT (p < 0.001), demonstrating markedly increased susceptibility to the compound (Fig. 4A, B). Importantly, genetic complementation of crzA and cchA fully restored WT phenotype, confirming that the hypersensitive phenotypes observed in the deletion mutants were specifically attributable to loss of crzA or cchA (Supplemental Fig. S1C). Collectively, these findings identify CrzA and CchA as key regulators of calcium-dependent stress responses that promote fungal resilience under nootkatone exposure. In contrast, the ΔcnaA mutant exhibited pronounced growth impairment even in the absence of nootkatone, indicating that CnaA is essential for normal fungal growth and basal physiology. Notably, nootkatone treatment did not significantly exacerbate the growth defect of ΔcnaA beyond its baseline impairment, suggesting that although CnaA participates in calcium signaling, its apparent contribution to nootkatone-specific tolerance may be limited, or alternatively, masked by its fundamental role in cellular viability.

Fig. 4.

Disruption of calcium signaling enhances A. fumigatus susceptibility to nootkatone. A Representative images of colony morphology for WT and calcium signaling-related mutant strains (ΔcrzA, ΔcchA, and ΔcnaA) of A. fumigatus grown on MM plates supplemented with nootkatone (0.1 mg/mL and 0.2 mg/mL). Compared to WT, all mutants displayed increased sensitivity to nootkatone, as evidenced by reduced colony size. B Quantitative analysis of colony diameter under each treatment condition. In the absence of nootkatone (MM), all strains grew comparably, except for ΔcnaA, which showed a slight growth defect. Upon treatment with nootkatone, ΔcrzA, ΔcchA, and ΔcnaA exhibited significantly greater growth inhibition than WT, particularly at 0.2 mg/mL. Data represent mean ± SD from three independent experiments. Statistical significance was calculated using one-way ANOVA with Tukey’s multiple comparisons test. ns, not significant; ***p < 0.001

To further evaluate the role of the calcium signaling core factor calmodulin (CaM), we constructed a series of site-directed mutants harboring single, double, triple, or quadruple substitutions at its Ca²⁺-binding sites (Supplemental Fig. S2A and 2B). The single-point mutants (CaM^mut(1), CaM^mut(2), CaM^mut(3), CaM^mut(4)) and the double-point mutant CaM^mut(1,2) showed WT-like growth and exhibited no significant change in sensitivity to nootkatone (Supplemental Fig. S2C), suggesting that partial disruption of Ca²⁺ binding does not markedly influence tolerance. By contrast, triple-point mutants (CaM^mut(1,2,3), CaM^mut(1,2,4), CaM^mut(1,3,4), and CaM^mut(2,3,4)) displayed severe growth inhibition even on MM, indicating that extensive disruption of CaM Ca²⁺-binding capacity profoundly compromises basal fungal physiology (Supplemental Fig. S2C). Consistent with this interpretation, nootkatone exposure did not further intensify the already severe growth defects of these mutants. These results suggest that while CaM is indispensable for fungal viability, its direct involvement in nootkatone-specific tolerance is likely limited and difficult to resolve due to the dominant baseline growth defect caused by CaM dysfunction.

In summary, our functional analyses highlight CrzA and CchA as critical calcium signaling regulators mediating A. fumigatus tolerance to nootkatone-induced stress. Deletion of either gene significantly increased fungal sensitivity to nootkatone, whereas CnaA and CaM appear to be primarily essential for basal growth and survival, with only a limited or indirect contribution to nootkatone-specific tolerance.

CrzA-dependent regulation of efflux pumps enhances A. fumigatus tolerance to nootkatone

Our previous findings identified the transcription factor CrzA as a central regulator of the adaptive response of A. fumigatus to nootkatone-induced stress. To further elucidate the molecular basis of CrzA-mediated tolerance, we examined whether CrzA regulates efflux pump expression and thereby contributes to nootkatone detoxification. Transcript analysis revealed that exposure to nootkatone (MM + nootkatone) significantly induced the expression of multiple efflux pump–associated genes in the WT strain, including abcC, abcE, atrA, mdr1, and atrB (Fig. 5A–E). This coordinated induction suggests that these transporters may facilitate nootkatone export, thereby limiting intracellular accumulation. In contrast, transcriptional activation of these efflux-related genes was markedly attenuated in the ΔcrzA mutant under nootkatone treatment, with expression levels significantly lower than those observed in WT. These results indicate that CrzA acts as a key transcriptional activator of efflux pump genes during nootkatone stress and that CrzA-dependent transporter induction likely contributes to enhanced fungal tolerance.

Fig. 5.

CrzA regulates the expression of efflux pump genes and influences intracellular nootkatone accumulation in A. fumigatus. A–E Relative mRNA expression levels of efflux pump-related genes (abcC, abcE, atrA, mdr1, and atrB) in WT and ΔcrzA strains cultured in MM with or without 0.2 mg/mL nootkatone. Upon nootkatone treatment, transcription of all five genes was significantly upregulated in the WT strain, whereas this induction was markedly reduced or abolished in the ΔcrzA mutant. F Intracellular nootkatone accumulation in WT, ΔcrzA, and crzA^C (complemented) strains. The ΔcrzA mutant exhibited significantly higher intracellular nootkatone levels compared to WT, while complementation restored drug accumulation to WT levels. These results indicate that CrzA regulates nootkatone efflux, likely through control of drug transporter gene expression. Data represent mean ± SD from three biological replicates. Statistical significance was determined using Student’s t-test or one-way ANOVA (**p < 0.01)

To directly evaluate whether the loss of CrzA affects intracellular nootkatone retention, intracellular nootkatone levels were quantified using gas chromatography-mass spectrometry (GC-MS). Under nootkatone exposure, the ΔcrzA strain accumulated significantly higher intracellular nootkatone concentrations than the WT strain (Fig. 5F), providing strong evidence that CrzA promotes nootkatone tolerance by enhancing efflux activity and maintaining intracellular nootkatone levels below toxic thresholds. In summary, CrzA is a critical regulator of efflux-mediated defense in A. fumigatus under nootkatone-induced stress. Loss of CrzA compromises efflux pump gene induction, resulting in increased intracellular accumulation of nootkatone and consequently reducing fungal tolerance to the compound.

Nootkatone disrupts fungal cell wall integrity and modulates host immune response in A. fumigatus

Previous studies have established that nootkatone exerts strong inhibitory activity against A. fumigatus. To determine whether this antifungal effect involves disruption of fungal cell wall integrity, we systematically assessed the impact of nootkatone on both the structural stability and biosynthetic capacity of the cell wall. To evaluate cell wall robustness, the cell wall–perturbing agents Congo Red (CR) and Calcofluor White (CFW) were incorporated into MM to induce cell wall stress. As expected, CR or CFW alone significantly impaired A. fumigatus growth, confirming their disruptive effects on cell wall integrity. Notably, co-treatment with nootkatone and either CR or CFW resulted in markedly stronger inhibition of colony expansion than any single treatment (Fig. 6A). This enhanced growth inhibition is consistent with the possibility that nootkatone compromises cell wall integrity, thereby increasing fungal susceptibility to cell wall stress. To explore the underlying molecular basis, we performed transcriptome profiling (RNA-seq) followed by RT-qPCR validation to examine the expression of genes involved in cell wall biosynthesis (Fig. 6B). Nootkatone treatment significantly downregulated multiple key genes, including the genes for the conidial hydrophobin RodB (AFUA_1G17250), a cell wall proline-rich protein (AFUA_5G02770), 1,3-β-glucanosyltransferase (AFUA_3G13200), mannosyltransferase PIG-M (AFUA_7G01300), β−1,6-glucan synthase Kre6 (AFUA_2G11870), and chitin synthase B (ChsB) (AFUA_4G04180). Importantly, the strong agreement between RNA-seq and RT-qPCR results supports the conclusion that nootkatone suppresses core pathways responsible for cell wall polysaccharide matrix assembly, thereby weakening structural integrity and potentially impairing cell wall functionality. Given that the fungal cell wall is a major determinant of host–pathogen interactions—particularly in immune recognition—we next assessed whether nootkatone-treated conidia alter host immune responses. Conidia pre-exposed to nootkatone were co-incubated with host immune cells, and transcription of pro-inflammatory cytokines was quantified at 2 and 4 h post-infection. Compared with untreated conidia, nootkatone-treated conidia induced significantly higher expression of IL-6, TNF-α, and IL-1β (p < 0.01) (Fig. 6C). These findings suggest that nootkatone may remodel or destabilize cell wall architecture, potentially increasing exposure of immunostimulatory components such as β-glucans and chitin, thereby enhancing immune recognition and activation.

Fig. 6.

Nootkatone disrupts fungal cell wall integrity and activates host immune responses. A Colony morphology of A. fumigatus cultured on MM plates with or without nootkatone (0.2 mg/mL) in the presence or absence of cell wall stress agents Congo Red (CR, 200 µg/mL) or Calcofluor White (CFW, 100 µg/mL). Nootkatone-treated colonies exhibited growth defects that were exacerbated under additional cell wall stress (MM + nootkatone + CR or + CFW), indicating compromised cell wall integrity. B Heatmap showing differential expression (log₂ fold change) of cell wall biosynthesis and remodeling genes in A. fumigatus following nootkatone treatment. Data from RNA-Seq and RT-PCR consistently show downregulation of genes involved in glucan and chitin biosynthesis (e.g., rodB, kre6, bgt1, and chsB), and upregulation of some cell wall remodeling genes (e.g., chiA and phiA), suggesting broad disruption of cell wall homeostasis. Quantitative RT-PCR analysis of proinflammatory cytokine expression (IL-6, TNF-α, and IL-1β) in macrophages treated with or without nootkatone-treated A. fumigatus conidia for 0, 2, or 4 h. Nootkatone-treated A. fumigatus conidia significantly induced the transcription of all three cytokines in a time-dependent manner, suggesting activation of the innate immune response. Data are presented as mean ± SD from three biological replicates. Statistical significance: p < 0.01, compared to untreated controls

In summary, nootkatone appears to exert antifungal activity through a dual mechanism involving both direct impairment of fungal cell wall biosynthesis and indirect modulation of host immune responses. Specifically, nootkatone downregulates key cell wall biosynthetic genes, weakening the structural and functional integrity of the cell wall, while also promoting pro-inflammatory cytokine induction by host immune cells. This combined pathogen-targeted and host-directed activity not only strengthens antifungal efficacy but also highlights nootkatone as a promising candidate for therapeutic development. Collectively, these results provide mechanistic insight into nootkatone-mediated antifungal action and support future efforts to develop antifungal strategies that integrate both microbial inhibition and immune enhancement.

In vivo antifungal efficacy of nootkatone against A. fumigatus: CrzA-dependent activity and potent action against drug-resistant strains

Previous studies have demonstrated that nootkatone exhibits strong in vitro inhibitory activity against A. fumigatus. To further evaluate its therapeutic potential in vivo, we employed the G. mellonella infection model to assess the antifungal efficacy of nootkatone in both wild-type and drug-resistant infection settings. Kaplan–Meier survival analysis showed that nootkatone treatment significantly improved host survival following infection. In the untreated control group, mortality reached 100% by day 7 post-infection, consistent with the high virulence of A. fumigatus. In contrast, nootkatone-treated larvae exhibited approximately 50% survival at day 7, demonstrating potent in vivo antifungal efficacy (Fig. 7A). To corroborate these results, fungal burden was quantified by measuring A. fumigatus DNA levels in infected larvae. Nootkatone treatment significantly reduced fungal DNA content relative to untreated controls. Notably, fungal proliferation was already strongly suppressed by day 3 post-infection, and DNA levels remained consistently low through day 7, indicating a sustained antifungal effect (Fig. 7B). We next investigated whether the calcium-responsive transcription factor CrzA contributes to nootkatone tolerance in vivo. In the ΔcrzA infection model, nootkatone treatment resulted in survival rates exceeding 60% at day 7, which was significantly higher than that observed in nootkatone-treated wild-type infections (Fig. 7A). Consistently, fungal DNA levels in the ΔcrzA + nootkatone group were reduced to near the detection limit (Fig. 7B). These findings indicate that CrzA contributes to fungal tolerance during nootkatone exposure and that loss of CrzA markedly enhances susceptibility in vivo. Although the precise mechanisms require further investigation, the absence of CrzA likely compromises calcium-dependent stress adaptation pathways, thereby potentiating nootkatone-mediated killing. Together, these data identify CrzA-associated signaling as a promising target for combination antifungal strategies aimed at enhancing nootkatone efficacy.

Fig. 7.

In vivo antifungal efficacy of nootkatone against A. fumigatus, including itraconazole-resistant strains. A Kaplan-Meier survival curves of G. mellonella larvae infected with WT or ΔcrzA strains of A. fumigatus and treated with nootkatone or PBS. Nootkatone significantly prolonged host survival, with the ΔcrzA mutant showing the highest sensitivity. B Fungal burden in G. mellonella larvae post-infection. Quantification of fungal DNA at 3 and 7 days post-infection shows that nootkatone treatment significantly reduced fungal load compared to WT control. Data represent mean ± SD of three independent experiments. p < 0.01 compared to WT. C Growth of wild-type and itraconazole-resistant A. fumigatus strains (sjx02, sjx03, sjx04, sjx05, sjx06) on MM, MM + ITZ, or MM + nootkatone. Nootkatone effectively inhibited the growth of ITZ-resistant strains, while ITZ treatment alone allowed resistant colony formation. D Kaplan-Meier survival curves of G. mellonella larvae infected with WT or sjx06 (ITZ-resistant) strains and treated with ITZ, nootkatone, or PBS. Nootkatone treatment markedly improved survival even in infections caused by the ITZ-resistant sjx06 strain, highlighting its distinct antifungal mechanism

To further assess activity against azole-resistant strains, five clinical A. fumigatus isolates with confirmed ITZ resistance were evaluated. All isolates displayed robust growth on medium containing 2 μg/mL ITZ, confirming high-level azole resistance (Fig. 7C). Remarkably, nootkatone supplementation strongly inhibited the growth of all resistant isolates, with inhibition levels comparable to those observed in susceptible strains (Fig. 7C). These results suggest that nootkatone acts through a mechanism distinct from azoles and is largely unaffected by common azole resistance determinants. To validate these findings in vivo, we established a G. mellonella infection model using a representative ITZ-resistant isolate (sjx06). As expected, ITZ treatment failed to improve survival, with complete mortality by day 6 post-infection (Fig. 7D). In contrast, nootkatone treatment maintained larval survival at approximately 65% throughout the 7-day observation period, slightly exceeding its protective effect in wild-type infections (Fig. 7D). These findings confirm that sjx06 remains highly susceptible to nootkatone and further indicate that azole resistance does not confer cross-resistance to this compound. In conclusion, our results establish nootkatone as a promising antifungal candidate with robust in vivo efficacy against A. fumigatus. Nootkatone displayed enhanced therapeutic activity in CrzA-deficient infections and retained potent efficacy against ITZ-resistant clinical isolates, highlighting its potential utility against drug-resistant aspergillosis. By bypassing common azole resistance mechanisms, nootkatone may provide an effective alternative therapeutic option, either as a standalone agent or in combination with targeted pathway inhibitors to further improve antifungal outcomes.

Discussion

Fungal infections, particularly those caused by A. fumigatus, represent a growing global health concern due to increasing immunosuppression and the rapid emergence of antifungal resistance (Cadena et al. 2021; Latgé and Chamilos 2019). Although azole antifungals such as itraconazole and voriconazole remain first-line therapies, their long-term efficacy is increasingly compromised by resistance development. These limitations highlight the urgent need for antifungal agents with novel mechanisms of action and reduced susceptibility to resistance. In this study, we identified nootkatone, a natural sesquiterpenoid derived from tobacco essential oil, as a potent antifungal compound against A. fumigatus. Previous studies have reported antifungal activity of nootkatone against several fungal species; however, its molecular targets and tolerance mechanisms in A. fumigatus have remained largely unexplored (Carneiro et al. 2025; Yang et al. 2025). Our findings therefore extend current knowledge by providing mechanistic insights into how nootkatone inhibits fungal growth and how A. fumigatus responds to this stress.

Unlike azole antifungals, which primarily target ergosterol biosynthesis, nootkatone exerts multifaceted antifungal effects. We demonstrated that nootkatone significantly suppresses both hyphal growth and conidiospore formation (Fig. 2), indicating interference with fundamental processes of fungal development. Inhibition of sporulation is particularly relevant, as conidia are essential for fungal dissemination and pathogenicity. This effect suggests that nootkatone may limit the spread of A. fumigatus by weakening its reproductive capacity, further supporting its potential as a lead antifungal compound.

A key finding of this study is the involvement of the calcium signaling pathway in A. fumigatus tolerance to nootkatone. Calcium signaling is a highly conserved regulatory system that enables fungi to adapt to environmental stresses, including antifungal exposure (Liu et al. 2015). Transcriptomic analysis revealed that nootkatone disrupts calcium homeostasis (Fig. 3A), and pharmacological chelation of extracellular calcium further enhanced its antifungal activity (Fig. 3B). These observations indicate that nootkatone-induced stress is closely linked to calcium-dependent adaptive responses rather than simple calcium deprivation. Genetic analyses further clarified the roles of specific calcium signaling components. While the transcription factor CrzA was essential for tolerance to nootkatone (Fig. 4), deletion of upstream regulators such as CnaA or calmodulin did not result in comparable sensitivity (Fig. 4). This finding contrasts with the classical model in which the calcineurin–calmodulin complex is considered central to CrzA activation. Our results suggest the presence of compensatory or alternative calcium-dependent pathways that can partially maintain CrzA activity under nootkatone-induced stress. Importantly, we demonstrate that CrzA-mediated regulation of efflux pump genes is a critical determinant of nootkatone resistance (Fig. 5). Efflux pumps are well-established contributors to multidrug resistance in fungi, particularly in response to azole antifungals (Li et al. 2020). Our data show that nootkatone induces efflux pump gene expression in a CrzA-dependent manner and that loss of CrzA leads to increased intracellular accumulation of nootkatone (Fig. 5).

In addition to its direct antifungal effects, nootkatone also appears to influence fungal–host interactions. We observed that nootkatone treatment enhances host immune responses, likely through altered exposure of fungal cell wall components such as β-glucans and chitin (Fig. 6). This dual mechanism—direct inhibition of fungal growth combined with immune potentiation—may reduce the likelihood of resistance development and improve therapeutic outcomes. Finally, in vivo experiments using the G. mellonella infection model demonstrated that nootkatone is effective against both azole-sensitive and azole-resistant A. fumigatus strains. The retained efficacy against resistant isolates highlights its potential clinical value and supports the hypothesis that nootkatone operates through mechanisms distinct from those of current azole drugs (Fig. 7). Although further validation in mammalian models is required, these findings position nootkatone as a promising candidate for antifungal drug development.

In summary, this study reveals that nootkatone exerts antifungal activity through a combination of calcium signaling disruption, CrzA-dependent efflux regulation, cell wall interference, and immune activation. By integrating fungal stress adaptation with host responses, our work provides new insights into antifungal tolerance mechanisms and identifies CrzA as a potential therapeutic target. These findings broaden the current understanding of antifungal resistance and support the development of natural compounds with novel and multifaceted modes of action.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contribution

JS and JT conceived and designed research. JX, LS, YC, JT, XL, and HD conducted experiments and analyzed data. JX, LS,and JS wrote the manuscript. All authors read and approved the manuscript.

Funding

This work was supported by Chongqing China Tobacco Industry Co., Ltd. Research Project (HX20230203).

Data availability

We have now deposited all RNA-seq raw data in the NCBI. The accession number is PRJNA1372267.

Declarations

Ethics approval and consent to participate

This article does not contain any studies with human participants or animals performed by any of the authors.

Competing interests

The authors declare no competing interests.