Agricultural SDHIs Induce Azole Resistance in Aspergillus fumigatus via Mitochondrial Sdh1 Suppression

Agricultural SDHIs Induce Azole Resistance in Aspergillus fumigatus via Mitochondrial Sdh1 Suppression | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Agricultural SDHIs Induce Azole Resistance in Aspergillus fumigatus via Mitochondrial Sdh1 Suppression Heng Zhang, Zhangling Zhu, Mengqi Peng, Sijie Liu, Xiao Gong, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6487242/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Sep, 2025 Read the published version in Mycopathologia → Version 1 posted 6 You are reading this latest preprint version Abstract Introduction: Aspergillus fumigatus poses significant clinical challenges due to its increasing azole resistance. This study investigates the sdh1 gene's role in regulating azole susceptibility, mitochondrial function, and virulence. Materials and Methods: Fungal strains were co-cultured with varying concentrations of succinate dehydrogenase inhibitors (SDHIs). Post-treatment azole minimum inhibitory concentrations (MICs) were determined using broth microdilution method, while succinate dehydrogenase subunit (SDH) expression changes were analyzed via RT-qPCR. Using A. fumigatus MFIG001 as the parental strain, sdh1 knockout mutant (Δ sdh1 ) and complemented strain (Δ sdh1::sdh1 + ) were constructed through homologous recombination. Detect the hyphal growth rate of Δ sdh1 , MICs and the changes in virulence within the Galleria mellonella infection model. Mitochondrial function was evaluated by measuring SDH activity, ATP content, and reactive oxygen species (ROS) levels. Transcriptomic changes were analyzed using RNA-seq and RT-qPCR, with efflux pump activity validated through Rhodamine 6G accumulation assays. Results: Exposure to subinhibitory concentrations of SDHIs induced azole resistance in A. fumigatus , with 4.12% of strains exhibiting reduced susceptibility to voriconazole, itraconazole , and posaconazole. RT-qPCR analysis revealed significant downregulation of sdh1 in resistant strains, implicating its role in resistance development. Deletion of sdh1 resulted in an 8- to 16-fold increase in triazole MICs, confirming its role as a negative regulator of azole susceptibility. Phenotypically, the Δ sdh1 strain exhibited impaired growth, reduced sporulation, and diminished efficacy of azole treatment in the G. mellonella infection model. Furthermore, Δ sdh1 exhibited severe mitochondrial dysfunction, including reduced SDH activity, decreased ATP levels, elevated ROS, and impaired antioxidant defenses. RNA-seq analysis revealed that the deletion of sdh1 upregulated the expression of efflux pump genes (e.g., cdr1B , abcB , mdr4 ), while Rhodamine 6G efflux assays demonstrated significantly enhanced efflux activity. Discussion: These results identify sdh1 as a critical determinant of azole susceptibility through dual mechanisms: mitochondrial function maintenance and efflux pump regulation. The observed SDHI-induced cross-resistance suggests agricultural fungicides may drive environmental selection of azole-resistant strains. While sdh1 deletion increased drug tolerance through efflux activation, the concurrent mitochondrial damage reduced pathogenic fitness, revealing compensatory evolutionary constraints. This work highlights the need to monitor non-target effects of agricultural SDHIs on clinical antifungal resistance. Aspergillus fumigatus Succinate dehydrogenase sdh1 Efflux pumps Azole resistance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction Aspergillus fumigatus is the leading cause of human invasive aspergillosis (IA), particularly affecting immunocompromised individuals, with a high mortality rate of up to 90%[ 1 – 3 ]. Inhalation of A. fumigatus conidia, which germinate into hyphae, can invade the tissues and blood vessels of immunocompromised patients, leading to the gradual development of IA[ 4 – 6 ]. The emergence of antifungal-resistant strains, particularly those resistant to multiple azole drugs, poses a significant challenge to treatment. The most common azole resistance mechanisms in A. fumigatus involve tandem-repeat insertions in the promoter region along with mutations in the cyp51A gene (such as TR34/L98H, TR46/Y121F/T289A, and TR53), associated with resistance to itraconazole (ITC) and voriconazole (VOR) in both clinical and environmental settings[ 7 ].These combinations of promoter tandem repeats and amino acid exchanges are thought to have arisen through the use of agricultural fungicides which are structurally similar to clinically used azoles[ 8 – 10 ]. The frontline antifungals used to combat IA are members of the triazoles, an antifungal class of compounds that inhibit 14α-sterol-demethylase activity to deplete cellular sterols[ 11 ]. Multiple transporters, including cdr1B , abcB , and mdr4 , contribute to the emergence of drug resistance[ 12 – 14 ]. However, the mechanisms underlying some resistance cases remain poorly understood[ 15 ]. Researches link theses resistance to extensive agricultural use of demethylation inhibitor (DMI) azoles, such as difenoconazole, epoxiconazole, propiconazole, and tebuconazole, which are structurally similar to medical triazoles like isavuconazole and VOR[ 16 ]. These fungicides persist in the environment for extended periods—47 days for tebuconazole and up to 120 days for epoxiconazole—potentially promoting resistance in opportunistic fungi. Reports of azole-resistant A. fumigatus in the environment and among patients with no prior antifungal treatment history suggest an eco-evolutionary link between environmental and clinical resistance, especially in 'hotspot' areas like composters, urban spaces, and greenhouses, where sub-MIC azole concentrations foster adaptation[ 17 – 19 ]. In contrast to DMIs, non-azole agricultural fungicides, such as succinate dehydrogenase inhibitors (SDHIs), may avoid triggering similar clinical resistance. SDHIs, including thifluzamide, boscalid, and isoflucypram, are highly effective agricultural fungicides and classified as Group C2 by the Fungicide Resistance Action Committee[ 16 ]. SDHIs act by binding to the ubiquinone-binding site in mitochondrial complex II (succinate dehydrogenase, SDH), thereby disrupting electron transport, hindering energy production, and ultimately suppressing fungal growth[ 20 – 22 ]. A. fumigatus encodes four SDH subunits ( sdh1 , sdh2 , cybs , b560 ), with structural similarities to SDH subunits in other fungi and humans. However, in our preliminary study, most SDHIs lack fungicidal effects on A. fumigatus , and their synergy with azoles. Four commonly used SDHIs displayed no fungicidal activity against A. fumigatus in vitro , although co-cultivation revealed altered azole sensitivity and potential resistance to azoles. Due to their high efficacy, SDHIs have secured a position among the top three fungicides in the global market. According to Phillips McDougall, SDHI fungicide sales are projected to exceed USD 3.00 billion by 2024[ 23 , 24 ]. This success raises critical concerns about their evolutionary impact on A. fumigatus —specifically, the widespread use of SDHIs may directly or indirectly drive azole resistance in this human pathogen through environmental selection pressure. Due to widespread agricultural use, SDHIs can enter water bodies through leaching, runoff, or atmospheric drift, contaminating soil and aquatic environments[ 25 ]. For instance, thifluzamide residues have been found in paddy soils in China and river channels in Korea, with a half-life of 28.64 days in paddy soil in Jiangsu Province, China[ 26 ]. The environmental persistence of SDHIs underpins their potential role in acquired azole resistance of A. fumigatus , which might threaten human healthy. This study investigates the mechanisms of SDHIs inducting acquired azole resistance, offering insights into managing A. fumigatus infections and combating resistance. 2 Materials and methods 2.1 A. fumigatus strains, media, and growth conditions In this study, the A. fumigatus MFIG001 strain (a uracil auxotroph strain lacking pyrG and akuB genes) was used as the wild-type genetic background to construct the sdh1 gene deletion strain (Δ sdh1 ) and its complemented strain (Δ sdh1::sdh1 + ). The media used in this study included SAB(Sabaurauds), CZA(Czapek), LB(Luria-Bertani), and RPMI(Roswell Park Memorial Institute) 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. 12 A. fumigatus clinical isolates were identified by microscopic morphology and by molecular sequencing of the internal transcribed spacer (ITS) ribosomal DNA (rDNA)[ 27 ]. All the sequences of the analyzed strains were uploaded to GenBank (PP069948-PP070390). 2.2 Antifungals and SDHIs All tested agents, including itraconazole (ITC), voriconazole (VOR), posaconazole (POS), caspofungin (CAS), Fluopyram, Boscalid, Thifluzamide, and Carboxin, were purchased in powder form from Aladdin Biochemical Technology Co., Ltd., Shanghai, China, and diluted in dimethyl sulfoxide or water as stock solutions (6400 µg/mL). 2.3 Induction of Azole Resistance by SDHIs 12 clinical isolates and WT were used as test strains. The spores were suspended in RPMI 1640 liquid medium containing 0.1, 1, 2, 5, and 10 µg/mL SDHIs, based on the MIC of ipflufenoquin against A. fumigatus [ 28 ], and the spore concentration was adjusted to a final concentration of 1×10⁵ CFU/mL. The suspension was incubated at 37°C with shaking at 130 rpm. Samples were collected every 24 hours, and spores were washed 3× with PBS to remove residual SDHIs, and MICs for three azoles (ITC, VOR, POS) were determined using Clinical Laboratory Standards Institute (CLSI) M38-A3. 2.4 Minimum inhibitory concentration (MIC) assay The broth microdilution method of M38-A3 was conducted following the standards established by the CLSI[ 29 ] to determine susceptibility to antifungal drugs. The antifungal agents were divided evenly into eight concentration gradients, ranging from 0.25 to 8 µg/mL. The final inoculum concentration was 2×10⁴ CFU/mL. MIC and minimum effective concentration (MEC) were recorded after 48 hours of incubation at 35°C. Meanwhile, Candida parapsilosis ATCC 22019 was selected as the quality control strain. 2.5 Mutant strains generation A. fumigatus ku80 was used as the parental isolate to generate knockout mutants. Knockout cassettes were generated using the protocol from Zhao et al(Fig. S1 )[ 30 ]. 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 min. In total, 200 µL of PEG was added and the mixture was then incubated at room temperature for 10 min. Transformation mixture was plated on CZA agar. Transformants were twice purified on CZA agar and PCR validated(Fig. S1 ). All primers used in this study are listed in Supplementary Table S1 . 2.6 Construction of the Δ sdh1 complemented strain The sdh1 gene was amplified from the genomic DNA (gDNA) of A. fumigatus using primers Aim-F and Aim-R in a PCR experiment. The amplified sdh1 gene was subsequently subcloned into the Nael and KpnI sites of the plasmid PCT74(Fenghui,China), resulting in the recombinant plasmid. The constructed plasmid was transformed into the Δ sdh1 deletion strain to generate the Δ sdh1 complemented strain (Δ sdh1::sdh1 + )(Fig. S1 ). All primers used in this experiment are listed in Supplementary Table S1 . 2.7 E-test drug susceptibility testing A fresh spore suspension containing 1×10⁶ CFU/mL was evenly spread onto RPMI 1640 agar plates using sterile cotton swabs. POS, ITC, VOR, and CAS test strips (YiMan Biotechnology, Guangzhou, China) were gently placed in the center of a shallow dish, sealed with parafilm, and incubated at 35°C for 48 hours to read the results. The experiment was repeated three times. 2.8 Growth rate determination and observation of mycelial morphology Fresh spores were collected and prepared into suspensions at concentrations of 10⁵, 10⁶, and 10⁷ CFU/mL. On CZA, SAB agar plates, 1 µL of each spore suspension was centrally inoculated. The plates were incubated at 37°C for 48 hours, after which the colony diameter was measured. Additionally, three hyphal samples were collected from SAB agar, stained with lactophenol cotton blue, and the morphology of the mycelia and conidial heads was observed under a standard 40× optical microscope. 2.9 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 48h. 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 (NGS) based on the Illumina sequencing platform. A fold change of ≥ 2 and a P value of < 0.05 were set as the threshold values for differentially expressed genes. The detailed procedures were performed by Beijing Biomaker Biotechnology Co., Ltd. (China). For RT-qPCR analysis, total RNA was extracted with theTRIeasy™ 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[ 31 ]. All the RT-qPCR primers are given in Table S2. 2.10 Detection of ROS content A. fumigatus conidia were collected after 48 hours of incubation on SAB solid medium at 35°C and resuspended in 1640 liquid medium to a final concentration of 5×10⁴ cfu/mL. All samples were incubated at 37°C in a shaking incubator at 130 rpm for 16 hours. At the end of the incubation, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) was added to each sample and incubated at 37°C for 1 hour to detect intracellular ROS levels. Flow cytometry data acquisition was performed using a Beckman Cytomics FC 500 BD FACSCanto II, and data analysis was conducted using FlowJo v10 software. The excitation wavelength was set at 488 nm, and the emission wavelength at 525 nm. 2.11 ATP Content, SOD, and SDH Activity Detection Fresh mycelial tissue was collected after three days of incubation on SAB solid medium, ground into powder using a high-speed shaker with an appropriate amount of glass beads. ATP content (Boxbio Biotechnology, Beijing, China), SOD activity (Boxbio Biotechnology, Beijing, China), and SDH activity (Solarbio, Beijing, China) were measured using commercial assay kits. This experiment was repeated three times on different days. 2.12 Rhodamine 6G efflux by A. fumigatus cells The efflux of rhodamine 6G (R6G) from intact A. fumigatus cells was determined by adapting the method described by Kolaczkowski et al[ 32 ]. Conidia cells from SAB cultures in the exponential growth phase (OD600, 0.5) were collected by centrifugation (3,000g, 5 min, 20℃) and washed three times with water. The cells were resuspended at a concentration of 0.5×10 6 to 1.0× 10 7 CFU/mL in HEPES-NaOH (50 mM; pH 7.0) containing 5 mM 2-deoxyglucose and 10 µM R6G. Cell suspensions were incubated at 30°C with shaking (200 rpm) for 90 min to allow rhodamine accumulation under glucose starvation conditions. The starved cells were washed twice in HEPES-NaOH, and portions (400 µL) were incubated at 30°C for 5 min before the addition of glucose (2 mM) to initiate rhodamine efflux. At specified intervals after the addition of glucose, the cells were removed by centrifugation, and triplicate 100µL volumes of the cell supernatants were transferred to the wells of 96-well flat-bottom microtiter plates (BKMAM Biotechnology, Hunan, China). The rhodamine fluorescence of the sample was measured by microplate reader (ALLSHENG, Wuhan, China). The microplate reader was read at the excitation wavelength of 529 nm. 2.13 Galleria mellonella virulence assay The G. mellonella virulence assay was performed using a method described previously[ 33 – 35 ]. Groups of 20 larvae was maintained in wood shavings in the dark at room temperature before use. Suspensions of tested strains that had been grown on SDA for 72h at 37°C were harvested by gentle scraping of colony surfaces with sterile plastic loops, washed twice, and adjusted to 1 × 10 8 CFU/mL in sterile saline. Conidia suspension and therapeutic and control solutions were injected into the larvae via the last right proleg using a Hamilton syringe (25 gauge, 50 µl). To determine the in vivo effects of azoles against pathogenic fungi, injection treatments were performed using ITC, VOR, and POS. The larvae were infected with the conidia suspension and injected with the tested agents (0.5 µg per agent) 2 hours post-infection. The death of larvae was monitored by visual inspection of the color (brown-dark/brown) every 24 h for a duration of 5 days. The experiments were repeated triplicate using larvae from different batches. The G. mellonella survival curves were analyzed by the Kaplan–Meier method. Differences between groups were considered significant at P < 0.05. 2.14 Data processing software GraphPad Prism 9 software and Origin 2018 were used for mapping, SPSS 26.0 software was used for statistical analysis, and mean ± s was used for data representation. Single factor analysis of variance (ANOVA) was used. The mean between the two groups was compared by the t -test, and the difference was statistically significant ( P < 0.05). 3 Result 3.1 SDHIs Induce Azole Resistance in A. fumigatus via Mitochondrial sdh1 Downregulation To investigate whether SDHIs promote azole resistance in A. fumigatus , conidia were exposed to subinhibitory concentrations of four SDHIs (Boscalid, Thifluzamide, Fluopyram, Carboxin) and subsequently tested for sensitivity to three azole drugs: VOR, ITR, and POS. Among 2,496 strains analyzed, 4.12% (103/2,496) exhibited reduced azole susceptibility, with 4 strains showing an eightfold increase, 5 strains a fourfold increase, and 94 strains a twofold increase in MIC(Table S3-19). Conversely, 50 strains (2.0%) displayed increased azole sensitivity (MIC reduced by half), suggesting a bidirectional but non-cumulative response to SDHI exposure (Fig.1A). Critically, prolonged SDHI treatment did not amplify these MIC shifts, indicating a threshold effect rather than progressive adaptation. To explore the molecular basis of resistance, we focused on strains with the most pronounced MIC increases (eightfold). RT-qPCR analysis revealed significant downregulation of sdh1 , a gene encoding the flavoprotein subunit of SDH, in all four resistant strains (Fig.1B-E). In contrast, expression of other SDH complex genes (sdh2 , cybs , b560 ) showed no consistent trends, ruling out compensatory upregulation of paralogs. These results implicate sdh1 suppression as a key driver of SDHI-induced azole resistance. Notably, the tested SDHIs themselves lacked direct fungicidal activity against A. fumigatus , confirming that resistance emerged indirectly via metabolic adaptation rather than selective pressure from SDHI toxicity. Collectively, these findings suggest that agricultural SDHIs may prime azole resistance in environmental A. fumigatus populations by dysregulating mitochondrial metabolism via Sdh1 suppression. 3.2 sdh1 Deletion Reduces Azole Susceptibility in A. fumigatus Sdh1 is similar in Homo sapiens and Saccharomyces cerevisiae (Fig.2). To determine the role of sdh1 in antifungal susceptibility, we performed CLSI microdilution and E-test assays. Both methods consistently demonstrated that Δ sdh1 exhibited significantly reduced susceptibility to triazoles compared to WT and the Δ sdh1::sdh1 + . For WT and Δ sdh1::sdh1 + , MIC values remained identical: ITC = 0.5 μg/mL, VOR= 0.25 μg/mL, and POS = 0.25 μg/mL. In contrast, MICs for Δ sdh1 increased 8- to 16-fold (ITC = 4 μg/mL, VOR = 2 μg/mL, POS = 2 μg/mL; Table 1). E-test results corroborated these findings, with Δ sdh1 showing pronounced resistance halos, whereas WT and Δ sdh1::sdh1 + displayed equivalent susceptibility profiles (Fig.2). Notably, caspofungin (CAS) susceptibility remained unchanged across all strains (MIC = 0.25 μg/mL; Table 1), confirming that sdh1 specifically modulates triazole tolerance rather than broadly affecting antifungal responses. These results establish sdh1 as a negative regulator of triazole susceptibility in A. fumigatus . Table 1 Results of antifungal susceptibility tests Antifungal susceptibility Type E-test MICs or MEC (μg/mL) M38-A3 MICs or MEC (μg/mL) Strain WT Δ sdh1 Δ sdh1::sdh1 + WT Δ sdh1 Δ sdh1::sdh1 + POS 0.38 1.5 0.25 0.25 2 0.25 VOR 0.25 2 0.38 0.25 2 0.25 ITC 1 4 1 0.5 4 0.5 CAS 0.5 0.25 0.38 0.5 0.25 0.5 Note: POS: posaconazole; VOR: voriconazole; ITC: itraconazole; CAS: caspofungin. 3.3 sdh1 Deficiency Disrupts Mitochondrial Function To assess the impact of sdh1 deletion on mitochondrial function, we measured SDH activity and metabolic parameters in the Δ sdh1 strain. SDH activity in Δ sdh1 exhibited a delayed recovery over time, reaching 58.3% of WT levels at 48 hours and 72.5% at 72 hours, but remained significantly lower than WT at all time points (Fig.3A). In 3-day-old colonies cultured on SAB agar, Δ sdh1 displayed severe mitochondrial dysfunction: ATP levels plummeted to 34.7% of WT (Fig.3B), ROS content surged to 162% of WT (Figure 3C), and SOD activity dropped to 45% of WT (Fig.3D). These results confirm that sdh1 deletion disrupts mitochondrial energy metabolism, exacerbates oxidative stress, and impairs antioxidant defenses. 3.4 sdh1 required for the normal growth of A. fumigatus The colony of Δ sdh1 exhibited markedly sluggish growth and displayed a white, shrunken appearance. In comparison to the WT and Δ sdh1::sdh1 + , there was a notable reduction in the growth diameters of Δ sdh1 when cultivated on SAB or CZA agar (Fig.4A-D). Furthermore, Δ sdh1 exhibited a strikingly diminished production of conidial heads, with a complete absence of bottle pedicles or conidial heads bearing pedicel bases (Fig.4E). These observations strongly indicate that the disruption of sdh1 adversely affects the reproductive capacity of A. fumigatus . 3.5 sdh1 Regulates Efflux Pump Activity To elucidate the molecular mechanisms underlying azole resistance in the Δ sdh1 strain, transcriptomic and functional analyses revealed alterations in efflux pump activity. A total of 2667 genes showed differential expression, with 1587 upregulated and 1080 downregulated(Fig.5A). KEGG pathway enrichment analysis showed that the differentially expressed genes (DEGs) were significantly enriched in metabolic pathways and prominently enriched in the ribosome function category, indicating significant differences. We specifically focused on the enrichment of differentially expressed genes related to drug sensitivity. Within the category of Environmental Information Processing, genes associated with ABC transporters were significantly enriched(Fig.5B). This indicates that deletion of the sdh1 gene leads to changes in efflux pump-related genes. Specifically, among these efflux pump genes, 48 genes showed more than a 4-fold change in expression, including 33 significantly upregulated and 15 downregulated genes(Fig.5C). Notably, the highest fold change among the downregulated genes was a 10.96-fold decrease, whereas among the upregulated genes, 15 exhibited expression levels exceeding 10.96-fold, with the highest reaching 472.5-fold. Notably, abcB increased by 113.5-fold, and mfsB by 34.1-fold. RT-qPCR further confirmed elevated expression of critical genes, including mdr4 , cdr1B , abcB , abcF , mfsA , and mfsB (Fig.5D)—all previously characterized to modulate azole susceptibility[12-14]. This suggests that sdh1 deletion enhances transporter efficiency. Consistent with this hypothesis, efflux pump activity assays conducted across three strains demonstrated a marked increase in drug efflux in Δ sdh1 . Specifically, significant efflux differences emerged within 15 minutes and persisted throughout the assay (Fig.5E). In contrast, expression levels of the ergosterol biosynthesis-specific transcription factors srbA and atrF , as well as the azole target genes cyp51A and cyp51B , remained unchanged[36-38] (Fig.5D). These findings indicate that sdh1 deletion primarily drives reduced azole susceptibility through efflux pump hyperactivation rather than ergosterol pathway modulation. 3.6 Disruption of sdh1 reduces the initial virulence of A. fumigatus The final survival rate of G. mellonella infected with Δ sdh1 was higher than that of WT ( P >0.05) (Fig.6). However, after treatment with POS, VOR, and ITC, Δ sdh1 did not reduce the mortality of G. mellonella , while a decrease in mortality was observed in WT. This also indicates that the virulence of Δ sdh1 has decreased,but it maintains low sensitivity to azole drug. 4 Discussion The emergence of azole resistance has posed a significant challenge in treating IA[ 39 ], and elucidating its mechanisms may provide critical insights for preventing resistance. In this study, exposure to sub-inhibitory concentrations of SDHIs successfully induced a marked reduction in azole susceptibility in A. fumigatus , suggesting SDHIs as potential drivers of azole resistance. Through transcriptional profiling, we identified and validated the pivotal role of the sdh1 gene in mediating resistance. SDHIs exert fungicidal effects by targeting fungal mitochondrial SDH, disrupting energy metabolism, and inducing oxidative damage, thereby impairing mitochondrial function[ 40 ]. SDH, a key enzyme in the mitochondrial electron transport chain and TCA cycle, catalyzes the oxidation of succinate to fumarate while transferring electrons to ubiquinone (CoQ)[ 41 – 43 ]. The catalytic core of SDH is highly conserved across species[ 41 ]. The widespread use and environmental persistence of SDHIs inevitably affect A. fumigatus SDH activity. In this study, knockout of sdh1 significantly reduced SDH activity, thereby disrupting Complex II function. Diminished SDH activity not only compromised electron transport efficiency but also reduced ATP production and elevated ROS levels. Excessive ROS accumulation exacerbated oxidative stress, further destabilizing cellular homeostasis. Additionally, reduced SOD activity weakened antioxidant defenses. Mitochondrial function is intricately linked to antifungal drug susceptibility and fungal virulence[ 44 , 45 ]. Mitochondrial mutations can confer drug sensitivity or resistance, with most non- cyp51A mutant isolates exhibiting mitochondrial dysfunction, overexpression of efflux pump genes, and multidrug resistance[ 44 , 46 – 49 ]. Both Candida glabrata and S. cerevisiae can live without mitochondrial DNA (mtDNA), and the drug-resistant mutants most commonly lost their mitochondrial genome[ 44 ]. In fact, mtDNA loss appears to be a key activating mutation for the drug resistance pathway. Loss of mtDNA and associated drug resistance is relatively common for in vitro cultures of both S. cerevisiae (about 2% of cells) and C. glabrata (frequencies of 2×10 − 4 to 4×10 − 4 ) [ 50 , 51 ]. Impaired mitochondrial function activates transcription factors such as Pdr3 in S. cerevisiae and Pdr1 in C. glabrata , altering the activity of efflux pump-encoding genes (e.g., cdr1 and cdr2 ), ultimately driving antifungal resistance[ 44 ]. In filamentous fungi, the role of ABC and MFS transporters in effluxing natural and synthetic toxins is well established. In A. fumigatus , efflux pump activity is a key contributor to azole resistance, mediated by MDR transporters from the ABC and MFS families[ 52 – 54 ]. These pumps reduce intracellular drug concentrations, diminishing drug efficacy. Genomic analysis reveals 278 MFS transporters and 49 ABC transporters in A. fumigatus [ 53 ]. Studies have demonstrated the role of A. fumigatus MDR pumps, such as cdr1B , abcB , and mdr4 , in enhancing ITC resistance[ 55 – 57 ]. In our study, upregulated expression of these efflux pump genes correlated with increased efflux activity, likely directly reducing susceptibility to ITR, POS, and VOR. Mitochondria are primary sources of ATP and ROS, requiring a balance between energy production and ROS generation for optimal enzyme activity[ 58 – 61 ]. Reduced ATP levels likely contributed to growth defects in our strains. While ROS induction may promote antifungal drug tolerance or resistance[ 62 , 63 ], the precise mechanisms remain unclear[ 46 ]. SDHI can enhance the activity of efflux pumps that mediate the development of resistance. Reports have shown that in field-resistant strains of Botrytis cinerea , overexpression of the ABC transporter gene bcatrB is significantly associated with resistance to SDHI fungicides, such as boscalid[ 64 – 66 ]. Similarly, in A. fumigatus , increased efflux pump activity reduces intracellular SDHI and azole concentrations, lowering drug sensitivity. In SDHI-resistant strains, the expression of bcatrB is significantly upregulated (> 10-fold), which enhances the efflux activity against SDHI. In filamentous fungi, the role of ABC and MFS transporters in the efflux of natural and synthetic toxins is well-known[ 64 , 67 ]. Similarly, in A. fumigatus , increased efflux pump activity reduces intracellular SDHI concentration, and the enhanced activity also decreases the concentration of azole drugs, ultimately leading to a significant reduction in sensitivity. Notably, SDHI-induced sdh1 inhibition and efflux pump upregulation suggest a novel mechanism of acquired azole resistance in A. fumigatus . However, sdh1 inhibition may concurrently slow growth, potentially limiting the environmental proliferation of SDHI-resistant strains. Given the widespread agricultural use and environmental persistence of both SDHIs and azoles, this dual induction of resistance warrants urgent attention. Field sampling in SDHI-treated areas to isolate azole-resistant strains and further mechanistic studies are imperative. This emerging resistance pattern underscores the need to explore non-azole-related resistance mechanisms. Additionally, clinical isolates with unexplained resistance should undergo SDH-related profiling to guide therapeutic strategies. Abbreviations Succinate dehydrogenase--SDH; succinate dehydrogenase inhibitors--SDHI; superoxide dismutase--SOD; minimum inhibitory concentration--MIC; invasive aspergillosis--IA; itraconazole--ITC; voriconazole--VOR; demethylation inhibitor--DMI; posaconazole--POS; caspofungin--CAS; minimum effective concentration--MEC; 2′, 7′‐dichlorodihydrofluorescein diacetate--DCFH‐DA; reactive oxygen species--ROS Rhodamine 6G--R6g. Declarations Ethics approval and consent to participate: Not applicable. Consent for publication : Not applicable. Availability of data and materials: All RNA-seq data files are available at the NCBI Sequence Read Archive (SRA) database under the accession number PRJNA1192589 for WT, Δ sdh1 and Δ sdh1:: sdh1 + of A.fumigatus conidia RNA-seq. Competing interests: The authors have no relevant financial or non-financial interests to disclose. Funding: This work was supported by the Jingzhou Science and Technology Plan Project (2024HD34); the Yangtze University Science and Technology Aid to Tibet Medical Talent Training Program Project (2023YZ06); and the Key Research and Development program of Hubei Province (2024BCB043). Author Contribution: All authors contributed to the study conception and design. Conceptualization, Yi Sun and Heng Zhang; methodology, Heng Zhang; software, Xiao Gong; validation, Mengqi Peng; formal analysis, Mengqi Peng; investigation, Tian Chen; resources, Qingwen Hu; data curation, Linyun Li; writing original draft preparation, Heng Zhang; writing review and editing, Sijie Liu and Heng Zhang; visualization, Zha-xi Dun-zhu and Lha-zom Drol-ga; supervision, Zhangling Zhu; project administration, Mengqi Peng; funding acquisition, Yi Sun and Heng Zhang. Acknowledgments: We thank everyone who contributed to the success of this research, including colleagues, institutions, and funding bodies. 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Hospital","correspondingAuthor":false,"prefix":"","firstName":"Linyun","middleName":"","lastName":"Li","suffix":""},{"id":449614587,"identity":"be8d9968-7c73-4bb2-8496-d090cde374c9","order_by":8,"name":"Zha-xi Dun-zhu","email":"","orcid":"","institution":"Tibet University Medical College","correspondingAuthor":false,"prefix":"","firstName":"Zha-xi","middleName":"","lastName":"Dun-zhu","suffix":""},{"id":449614588,"identity":"712019ad-4d31-42b9-b466-3f022b9ef14b","order_by":9,"name":"Lha-zom Drol-ga","email":"","orcid":"","institution":"Tibet University Medical College","correspondingAuthor":false,"prefix":"","firstName":"Lha-zom","middleName":"","lastName":"Drol-ga","suffix":""},{"id":449614589,"identity":"037f3c00-a3f0-49a6-8995-25228f5fee75","order_by":10,"name":"Yi Sun","email":"","orcid":"https://orcid.org/0000-0002-4489-3803","institution":"Jingzhou Central Hospital: Yangtze University Jingzhou Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2025-04-20 03:42:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6487242/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6487242/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11046-025-00992-0","type":"published","date":"2025-09-15T15:57:03+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81939713,"identity":"4fe31e9e-4730-424f-8dac-0948d4f01779","added_by":"auto","created_at":"2025-05-05 06:46:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4202616,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in SDH-related genes in SDHI-induced azole-resistant strains\u003c/p\u003e\n\u003cp\u003eNote: (A) Three-dimensional analysis of SDHI-induced azole resistance. The X-axis represents strain types, the Y-axis indicates sensitivity to azole drugs after co-culture with SDHIs, and the Z-axis shows the fold change in sensitivity. Colors are coded to SDHI concentrations, and shapes denote co-culture durations. (B–E) Expression levels of \u003cem\u003esdh1\u003c/em\u003e in strains with the most significant alterations in drug sensitivity. Red bars: post-induction; gray bars: pre-induction\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6487242/v1/1795ed3d72477c41a5300071.png"},{"id":81938477,"identity":"e8a4b3d7-1806-4832-ba06-4b820ae54ec8","added_by":"auto","created_at":"2025-05-05 06:38:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6820246,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the results of the E-test\u003c/p\u003e\n\u003cp\u003eNote: \u003cem\u003esdh1\u003c/em\u003e is able to significantly alter the sensitivity to triazoles. The first row represents the E-test inhibition circle plot of Δ\u003cem\u003esdh1\u003c/em\u003e against VOR, POS, ITC, and CAS\u003cem\u003e. \u003c/em\u003eThe second three rows denote WT and Δ\u003cem\u003esdh1::sdh1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6487242/v1/0558e5462d059a647a839ae1.png"},{"id":81939714,"identity":"125007d3-bceb-4a9c-95ee-317a52ffb180","added_by":"auto","created_at":"2025-05-05 06:46:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2063369,"visible":true,"origin":"","legend":"\u003cp\u003eSDH, SOD activity and ROS, ATP content\u003c/p\u003e\n\u003cp\u003eNote: \u003cstrong\u003eA\u003c/strong\u003e After the deletion of the\u003cem\u003e sdh1\u003c/em\u003e, the activity of SDH slowly increases with increasing growth time, but overall, the activity of SDH significantly decreases. \u003cstrong\u003eB\u003c/strong\u003e ATP content. \u003cstrong\u003eC \u003c/strong\u003eROS level significantly increased in Δ\u003cem\u003esdh1\u003c/em\u003e. \u003cstrong\u003eD\u003c/strong\u003e SOD activity significantly decreased in Δ\u003cem\u003esdh1\u003c/em\u003e. (*\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05；***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001；****\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.0001.)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6487242/v1/7d7236def82703e145e7b357.png"},{"id":81940392,"identity":"ad18e71d-1d8f-47eb-81f8-b73c1d4de8d2","added_by":"auto","created_at":"2025-05-05 06:54:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6319586,"visible":true,"origin":"","legend":"\u003cp\u003es\u003cem\u003edh1\u003c/em\u003e is necessary for the normal growth of \u003cem\u003eA. fumigatus\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNote: \u003cem\u003esdh1\u003c/em\u003e is necessary for the normal growth of\u003cem\u003e A. fumigatus\u003c/em\u003e. \u003cstrong\u003eA-D \u003c/strong\u003eshow the growth of Δ\u003cem\u003esdh1\u003c/em\u003e, WT, and Δ\u003cem\u003esdh1::sdh1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e at various concentrations on CZA and SAB, rows 1-3 indicate strains Δ\u003cem\u003esdh1\u003c/em\u003e, WT, and Δ\u003cem\u003esdh1::sdh1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e, respectively, and columns 1-3 indicate concentrations of 10\u003csup\u003e5\u003c/sup\u003e, 10\u003csup\u003e6\u003c/sup\u003e, and 10\u003csup\u003e7\u003c/sup\u003e CFU/mL, respectively. \u003cstrong\u003eE \u003c/strong\u003eConidiophore morphology under light microscopy (original) and after lactophenol cotton blue staining (color). Scale bars: 100 μm. ( \u003cem\u003e****P \u003c/em\u003e\u0026lt;0.0001. )\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6487242/v1/1949cd5438e25b013501e425.png"},{"id":81939715,"identity":"7dacfc04-bb2b-486d-8525-ed36bfa11667","added_by":"auto","created_at":"2025-05-05 06:46:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1656875,"visible":true,"origin":"","legend":"\u003cp\u003eSdh1 regulates efflux pump activity.\u003c/p\u003e\n\u003cp\u003eNote: \u003cstrong\u003eA\u003c/strong\u003e Volcano plot of differentially expressed genes (DEGs). \u003cstrong\u003eB\u003c/strong\u003e Pathway enrichment of DEGs. \u003cstrong\u003eC\u003c/strong\u003e Membrane transporters associated with multidrug efflux, hypothesized to mediate drug efflux. \u003cstrong\u003eD\u003c/strong\u003e Relative expression levels of azole susceptibility-associated genes, determined by RT-qPCR and normalized to the β-tubulin reference gene. \u003cstrong\u003eE\u003c/strong\u003e Efflux pump activity measured via Rhodamine 6G (R6G) efflux at different time points. (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05; \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt;0.01;\u003cem\u003e****P \u0026lt; \u003c/em\u003e0.0001.)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6487242/v1/99a5ecf8395177f2f0894539.png"},{"id":81938474,"identity":"a4952f7b-0702-4de3-8d2a-ab5db8f85187","added_by":"auto","created_at":"2025-05-05 06:38:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1180525,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival curve of\u003cem\u003e\u0026nbsp; Galleria mellonella\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNote: POS, posaconazole; VOR: voriconazole; ITC: itraconazole. The\u003cem\u003e\u0026nbsp;G. mellonella\u0026nbsp;\u003c/em\u003esurvival curves were analyzed by the Kaplan–Meier method. (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05; \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt;0.01.)\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6487242/v1/d4348c05bf2e5caf12004eb5.png"},{"id":91889774,"identity":"c200aeb1-a2fb-4937-86f2-ba8fc4257b19","added_by":"auto","created_at":"2025-09-22 16:01:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":23010204,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6487242/v1/7abe6c9c-5463-4203-88df-ba170b32e0ee.pdf"},{"id":81938490,"identity":"53d96de9-964e-4a2d-9873-f6b7a1f1cfa0","added_by":"auto","created_at":"2025-05-05 06:38:13","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":1482714,"visible":true,"origin":"","legend":"","description":"","filename":"KDASHsup.docx","url":"https://assets-eu.researchsquare.com/files/rs-6487242/v1/dd3a65fdcb26e2102cb38ca2.docx"},{"id":81938483,"identity":"bdb22e19-2db1-479b-993b-0250b8ce4c36","added_by":"auto","created_at":"2025-05-05 06:38:13","extension":"xlsx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":30100,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3S19.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6487242/v1/b8ffb9e354500cdc2001bbbe.xlsx"}],"financialInterests":"","formattedTitle":"Agricultural SDHIs Induce Azole Resistance in Aspergillus fumigatus via Mitochondrial Sdh1 Suppression","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003e \u003cem\u003eAspergillus fumigatus\u003c/em\u003e is the leading cause of human invasive aspergillosis (IA), particularly affecting immunocompromised individuals, with a high mortality rate of up to 90%[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Inhalation of \u003cem\u003eA. fumigatus\u003c/em\u003e conidia, which germinate into hyphae, can invade the tissues and blood vessels of immunocompromised patients, leading to the gradual development of IA[\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe emergence of antifungal-resistant strains, particularly those resistant to multiple azole drugs, poses a significant challenge to treatment. The most common azole resistance mechanisms in \u003cem\u003eA. fumigatus\u003c/em\u003e involve tandem-repeat insertions in the promoter region along with mutations in the \u003cem\u003ecyp51A\u003c/em\u003e gene (such as TR34/L98H, TR46/Y121F/T289A, and TR53), associated with resistance to itraconazole (ITC) and voriconazole (VOR) in both clinical and environmental settings[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].These combinations of promoter tandem repeats and amino acid exchanges are thought to have arisen through the use of agricultural fungicides which are structurally similar to clinically used azoles[\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The frontline antifungals used to combat IA are members of the triazoles, an antifungal class of compounds that inhibit 14α-sterol-demethylase activity to deplete cellular sterols[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Multiple transporters, including \u003cem\u003ecdr1B\u003c/em\u003e, \u003cem\u003eabcB\u003c/em\u003e, and \u003cem\u003emdr4\u003c/em\u003e, contribute to the emergence of drug resistance[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, the mechanisms underlying some resistance cases remain poorly understood[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eResearches link theses resistance to extensive agricultural use of demethylation inhibitor (DMI) azoles, such as difenoconazole, epoxiconazole, propiconazole, and tebuconazole, which are structurally similar to medical triazoles like isavuconazole and VOR[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. These fungicides persist in the environment for extended periods\u0026mdash;47 days for tebuconazole and up to 120 days for epoxiconazole\u0026mdash;potentially promoting resistance in opportunistic fungi. Reports of azole-resistant \u003cem\u003eA. fumigatus\u003c/em\u003e in the environment and among patients with no prior antifungal treatment history suggest an eco-evolutionary link between environmental and clinical resistance, especially in 'hotspot' areas like composters, urban spaces, and greenhouses, where sub-MIC azole concentrations foster adaptation[\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn contrast to DMIs, non-azole agricultural fungicides, such as succinate dehydrogenase inhibitors (SDHIs), may avoid triggering similar clinical resistance. SDHIs, including thifluzamide, boscalid, and isoflucypram, are highly effective agricultural fungicides and classified as Group C2 by the Fungicide Resistance Action Committee[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. SDHIs act by binding to the ubiquinone-binding site in mitochondrial complex II (succinate dehydrogenase, SDH), thereby disrupting electron transport, hindering energy production, and ultimately suppressing fungal growth[\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. \u003cem\u003eA. fumigatus\u003c/em\u003e encodes four SDH subunits (\u003cem\u003esdh1\u003c/em\u003e, \u003cem\u003esdh2\u003c/em\u003e, \u003cem\u003ecybs\u003c/em\u003e, \u003cem\u003eb560\u003c/em\u003e), with structural similarities to SDH subunits in other fungi and humans. However, in our preliminary study, most SDHIs lack fungicidal effects on \u003cem\u003eA. fumigatus\u003c/em\u003e, and their synergy with azoles. Four commonly used SDHIs displayed no fungicidal activity against \u003cem\u003eA. fumigatus in vitro\u003c/em\u003e, although co-cultivation revealed altered azole sensitivity and potential resistance to azoles.\u003c/p\u003e \u003cp\u003eDue to their high efficacy, SDHIs have secured a position among the top three fungicides in the global market. According to Phillips McDougall, SDHI fungicide sales are projected to exceed USD 3.00\u0026nbsp;billion by 2024[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This success raises critical concerns about their evolutionary impact on \u003cem\u003eA. fumigatus\u003c/em\u003e\u0026mdash;specifically, the widespread use of SDHIs may directly or indirectly drive azole resistance in this human pathogen through environmental selection pressure. Due to widespread agricultural use, SDHIs can enter water bodies through leaching, runoff, or atmospheric drift, contaminating soil and aquatic environments[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. For instance, thifluzamide residues have been found in paddy soils in China and river channels in Korea, with a half-life of 28.64 days in paddy soil in Jiangsu Province, China[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The environmental persistence of SDHIs underpins their potential role in acquired azole resistance of \u003cem\u003eA. fumigatus\u003c/em\u003e, which might threaten human healthy.\u003c/p\u003e \u003cp\u003eThis study investigates the mechanisms of SDHIs inducting acquired azole resistance, offering insights into managing \u003cem\u003eA. fumigatus\u003c/em\u003e infections and combating resistance.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 \u003cem\u003eA. fumigatus\u003c/em\u003e strains, media, and growth conditions\u003c/h2\u003e\n \u003cp\u003eIn this study, the \u003cem\u003eA. fumigatus\u003c/em\u003e MFIG001 strain (a uracil auxotroph strain lacking \u003cem\u003epyrG\u003c/em\u003e and \u003cem\u003eakuB\u003c/em\u003e genes) was used as the wild-type genetic background to construct the \u003cem\u003esdh1\u003c/em\u003e gene deletion strain (\u0026Delta;\u003cem\u003esdh1\u003c/em\u003e) and its complemented strain (\u0026Delta;\u003cem\u003esdh1::sdh1\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e).\u003c/p\u003e\n \u003cp\u003eThe media used in this study included SAB(Sabaurauds), CZA(Czapek), LB(Luria-Bertani), and RPMI(Roswell Park Memorial Institute) 1640. All liquid media were prepared from the corresponding solid media without agar. Fresh \u003cem\u003eA. fumigatus\u003c/em\u003e 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\u0026deg;C.\u003c/p\u003e\n \u003cp\u003e12 \u003cem\u003eA. fumigatus\u003c/em\u003e clinical isolates were identified by microscopic morphology and by molecular sequencing of the internal transcribed spacer (ITS) ribosomal DNA (rDNA)[\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. All the sequences of the analyzed strains were uploaded to GenBank (PP069948-PP070390).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Antifungals and SDHIs\u003c/h2\u003e\n \u003cp\u003eAll tested agents, including itraconazole (ITC), voriconazole (VOR), posaconazole (POS), caspofungin (CAS), Fluopyram, Boscalid, Thifluzamide, and Carboxin, were purchased in powder form from Aladdin Biochemical Technology Co., Ltd., Shanghai, China, and diluted in dimethyl sulfoxide or water as stock solutions (6400 \u0026micro;g/mL).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Induction of Azole Resistance by SDHIs\u003c/h2\u003e\n \u003cp\u003e12 clinical isolates and WT were used as test strains. The spores were suspended in RPMI 1640 liquid medium containing 0.1, 1, 2, 5, and 10 \u0026micro;g/mL SDHIs, based on the MIC of ipflufenoquin against \u003cem\u003eA. fumigatus\u003c/em\u003e[\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e], and the spore concentration was adjusted to a final concentration of 1\u0026times;10⁵ CFU/mL. The suspension was incubated at 37\u0026deg;C with shaking at 130 rpm. Samples were collected every 24 hours, and spores were washed 3\u0026times; with PBS to remove residual SDHIs, and MICs for three azoles (ITC, VOR, POS) were determined using Clinical Laboratory Standards Institute (CLSI) M38-A3.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Minimum inhibitory concentration (MIC) assay\u003c/h2\u003e\n \u003cp\u003eThe broth microdilution method of M38-A3 was conducted following the standards established by the CLSI[\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e] to determine susceptibility to antifungal drugs. The antifungal agents were divided evenly into eight concentration gradients, ranging from 0.25 to 8 \u0026micro;g/mL. The final inoculum concentration was 2\u0026times;10⁴ CFU/mL. MIC and minimum effective concentration (MEC) were recorded after 48 hours of incubation at 35\u0026deg;C. Meanwhile, \u003cem\u003eCandida parapsilosis\u003c/em\u003e ATCC 22019 was selected as the quality control strain.\u003c/p\u003e\u003cspan\u003e\n \u003cp\u003e\u003cstrong\u003e2.5 Mutant strains generation\u003c/strong\u003e\u003c/p\u003e\n \u003c/span\u003e\n \u003cp\u003e\u003cem\u003eA. fumigatus\u003c/em\u003e ku80 was used as the parental isolate to generate knockout mutants. Knockout cassettes were generated using the protocol from Zhao et al(Fig.\u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e)[\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. Briefly, 1.2 kb flanking regions of the gene of interest were PCR amplified and fused to a \u003cem\u003epyrG\u003c/em\u003e cassette via additional fusion PCR. The \u003cem\u003eA. fumigatus\u003c/em\u003e ku80 strain was cultured overnight at 37\u0026deg;C and 120 rpm in CZA liquid medium supplemented with uracil, followed by a 5-hour protoplasting treatment in Sabouraud agar\u0026thinsp;+\u0026thinsp;protoplasting solution (pectinase\u0026thinsp;+\u0026thinsp;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\u0026thinsp;+\u0026thinsp;200 mM CaCl\u003csub\u003e2\u003c/sub\u003e. Fusion PCR product was added to 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e protoplasts, followed by addition of PEG. This was incubated on ice for 30 min. In total, 200 \u0026micro;L of PEG was added and the mixture was then incubated at room temperature for 10 min. Transformation mixture was plated on CZA agar. Transformants were twice purified on CZA agar and PCR validated(Fig.\u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). All primers used in this study are listed in Supplementary Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6 Construction of the \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e complemented strain\u003c/h2\u003e\n \u003cp\u003eThe \u003cem\u003esdh1\u003c/em\u003e gene was amplified from the genomic DNA (gDNA) of \u003cem\u003eA. fumigatus\u003c/em\u003e using primers Aim-F and Aim-R in a PCR experiment. The amplified \u003cem\u003esdh1\u003c/em\u003e gene was subsequently subcloned into the Nael and KpnI sites of the plasmid PCT74(Fenghui,China), resulting in the recombinant plasmid. The constructed plasmid was transformed into the \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e deletion strain to generate the \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e complemented strain (\u0026Delta;\u003cem\u003esdh1::sdh1\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e)(Fig.\u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). All primers used in this experiment are listed in Supplementary Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7 E-test drug susceptibility testing\u003c/h2\u003e\n \u003cp\u003eA fresh spore suspension containing 1\u0026times;10⁶ CFU/mL was evenly spread onto RPMI 1640 agar plates using sterile cotton swabs. POS, ITC, VOR, and CAS test strips (YiMan Biotechnology, Guangzhou, China) were gently placed in the center of a shallow dish, sealed with parafilm, and incubated at 35\u0026deg;C for 48 hours to read the results. The experiment was repeated three times.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8 Growth rate determination and observation of mycelial morphology\u003c/h2\u003e\n \u003cp\u003eFresh spores were collected and prepared into suspensions at concentrations of 10⁵, 10⁶, and 10⁷ CFU/mL. On CZA, SAB agar plates, 1 \u0026micro;L of each spore suspension was centrally inoculated. The plates were incubated at 37\u0026deg;C for 48 hours, after which the colony diameter was measured. Additionally, three hyphal samples were collected from SAB agar, stained with lactophenol cotton blue, and the morphology of the mycelia and conidial heads was observed under a standard 40\u0026times; optical microscope.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9 RNA-seq analysis and Real time Quantitative PCR\u003c/h2\u003e\n \u003cp\u003eFresh \u003cem\u003eA. fumigatus\u003c/em\u003e conidia were grown in liquid SAB in a rotary shaker at 220 rpm at 37\u0026deg;C for 48h. 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 (NGS) based on the Illumina sequencing platform. A fold change of \u0026ge;\u0026thinsp;2 and a \u003cem\u003eP\u003c/em\u003e value of \u0026lt;\u0026thinsp;0.05 were set as the threshold values for differentially expressed genes. The detailed procedures were performed by Beijing Biomaker Biotechnology Co., Ltd. (China).\u003c/p\u003e\n \u003cp\u003eFor RT-qPCR analysis, total RNA was extracted with theTRIeasy\u0026trade; Total RNA Extraction Reagent (Yeasen, China) according to the manufacturer\u0026rsquo;s directions. The Hifair\u0026reg; 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 (\u0026Delta;CT) and normalized against the mRNA expression of \u003cem\u003etubA\u003c/em\u003e in \u003cem\u003eA. fumigatus\u003c/em\u003e. The 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;CT\u003c/sup\u003e method was used to determine the changes in mRNA expression[\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. All the RT-qPCR primers are given in Table\u0026ensp;S2.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.10 Detection of ROS content\u003c/h2\u003e\n \u003cp\u003e\u003cem\u003eA. fumigatus\u003c/em\u003e conidia were collected after 48 hours of incubation on SAB solid medium at 35\u0026deg;C and resuspended in 1640 liquid medium to a final concentration of 5\u0026times;10⁴ cfu/mL. All samples were incubated at 37\u0026deg;C in a shaking incubator at 130 rpm for 16 hours. At the end of the incubation, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) was added to each sample and incubated at 37\u0026deg;C for 1 hour to detect intracellular ROS levels. Flow cytometry data acquisition was performed using a Beckman Cytomics FC 500 BD FACSCanto II, and data analysis was conducted using FlowJo v10 software. The excitation wavelength was set at 488 nm, and the emission wavelength at 525 nm.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e2.11 ATP Content, SOD, and SDH Activity Detection\u003c/h2\u003e\n \u003cp\u003eFresh mycelial tissue was collected after three days of incubation on SAB solid medium, ground into powder using a high-speed shaker with an appropriate amount of glass beads. ATP content (Boxbio Biotechnology, Beijing, China), SOD activity (Boxbio Biotechnology, Beijing, China), and SDH activity (Solarbio, Beijing, China) were measured using commercial assay kits. This experiment was repeated three times on different days.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e2.12 Rhodamine 6G efflux by \u003cem\u003eA. fumigatus\u003c/em\u003e cells\u003c/h2\u003e\n \u003cp\u003eThe efflux of rhodamine 6G (R6G) from intact \u003cem\u003eA. fumigatus\u003c/em\u003e cells was determined by adapting the method described by Kolaczkowski et al[\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]. Conidia cells from SAB cultures in the exponential growth phase (OD600, 0.5) were collected by centrifugation (3,000g, 5 min, 20℃) and washed three times with water. The cells were resuspended at a concentration of 0.5\u0026times;10\u003csup\u003e6\u003c/sup\u003e to 1.0\u0026times; 10\u003csup\u003e7\u003c/sup\u003e CFU/mL in HEPES-NaOH (50 mM; pH 7.0) containing 5 mM 2-deoxyglucose and 10 \u0026micro;M R6G. Cell suspensions were incubated at 30\u0026deg;C with shaking (200 rpm) for 90 min to allow rhodamine accumulation under glucose starvation conditions. The starved cells were washed twice in HEPES-NaOH, and portions (400 \u0026micro;L) were incubated at 30\u0026deg;C for 5 min before the addition of glucose (2 mM) to initiate rhodamine efflux. At specified intervals after the addition of glucose, the cells were removed by centrifugation, and triplicate 100\u0026micro;L volumes of the cell supernatants were transferred to the wells of 96-well flat-bottom microtiter plates (BKMAM Biotechnology, Hunan, China). The rhodamine fluorescence of the sample was measured by microplate reader (ALLSHENG, Wuhan, China). The microplate reader was read at the excitation wavelength of 529 nm.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e2.13 \u003cem\u003eGalleria mellonella\u003c/em\u003e virulence assay\u003c/h2\u003e\n \u003cp\u003eThe \u003cem\u003eG. mellonella\u003c/em\u003e virulence assay was performed using a method described previously[\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]. Groups of 20 larvae was maintained in wood shavings in the dark at room temperature before use. Suspensions of tested strains that had been grown on SDA for 72h at 37\u0026deg;C were harvested by gentle scraping of colony surfaces with sterile plastic loops, washed twice, and adjusted to 1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e CFU/mL in sterile saline. Conidia suspension and therapeutic and control solutions were injected into the larvae via the last right proleg using a Hamilton syringe (25 gauge, 50 \u0026micro;l). To determine the \u003cem\u003ein vivo\u003c/em\u003e effects of azoles against pathogenic fungi, injection treatments were performed using ITC, VOR, and POS. The larvae were infected with the conidia suspension and injected with the tested agents (0.5 \u0026micro;g per agent) 2 hours post-infection. The death of larvae was monitored by visual inspection of the color (brown-dark/brown) every 24 h for a duration of 5 days. The experiments were repeated triplicate using larvae from different batches. The \u003cem\u003eG. mellonella\u003c/em\u003e survival curves were analyzed by the Kaplan\u0026ndash;Meier method. Differences between groups were considered significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e2.14 Data processing software\u003c/h2\u003e\n \u003cp\u003eGraphPad Prism 9 software and Origin 2018 were used for mapping, SPSS 26.0 software was used for statistical analysis, and mean\u0026thinsp;\u0026plusmn;\u0026thinsp;s was used for data representation. Single factor analysis of variance (ANOVA) was used. The mean between the two groups was compared by the \u003cem\u003et\u003c/em\u003e-test, and the difference was statistically significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3 Result","content":"\u003cp\u003e\u003cstrong\u003e3.1 SDHIs Induce Azole Resistance in \u003cem\u003eA. fumigatus\u003c/em\u003e via Mitochondrial \u003cem\u003esdh1\u0026nbsp;\u003c/em\u003eDownregulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate whether SDHIs promote azole resistance in\u003cem\u003e\u0026nbsp;A. fumigatus\u003c/em\u003e, conidia were exposed to subinhibitory concentrations of four SDHIs (Boscalid, Thifluzamide, Fluopyram, Carboxin) and subsequently tested for sensitivity to three azole drugs: VOR, ITR, and POS. Among 2,496 strains analyzed, 4.12% (103/2,496) exhibited reduced azole susceptibility, with 4 strains showing an eightfold increase, 5 strains a fourfold increase, and 94 strains a twofold increase in MIC(Table S3-19). Conversely, 50 strains (2.0%) displayed increased azole sensitivity (MIC reduced by half), suggesting a bidirectional but non-cumulative response to SDHI exposure (Fig.1A). Critically, prolonged SDHI treatment did not amplify these MIC shifts, indicating a threshold effect rather than progressive adaptation.\u003c/p\u003e\n\u003cp\u003eTo explore the molecular basis of resistance, we focused on strains with the most pronounced MIC increases (eightfold). RT-qPCR analysis revealed significant downregulation of \u003cem\u003esdh1\u003c/em\u003e, a gene encoding the flavoprotein subunit of SDH, in all four resistant strains (Fig.1B-E). In contrast, expression of other SDH complex genes \u003cem\u003e(sdh2\u003c/em\u003e, \u003cem\u003ecybs\u003c/em\u003e, \u003cem\u003eb560\u003c/em\u003e) showed no consistent trends, ruling out compensatory upregulation of paralogs. These results implicate \u003cem\u003esdh1\u003c/em\u003e suppression as a key driver of SDHI-induced azole resistance.\u003c/p\u003e\n\u003cp\u003eNotably, the tested SDHIs themselves lacked direct fungicidal activity against \u003cem\u003eA. fumigatus\u003c/em\u003e, confirming that resistance emerged indirectly via metabolic adaptation rather than selective pressure from SDHI toxicity. Collectively, these findings suggest that agricultural SDHIs may prime azole resistance in environmental \u003cem\u003eA. fumigatus\u003c/em\u003e populations by dysregulating mitochondrial metabolism via Sdh1 suppression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 \u003cem\u003esdh1\u003c/em\u003e Deletion Reduces Azole Susceptibility in \u003cem\u003eA. fumigatus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSdh1 is similar in Homo sapiens and \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e(Fig.2).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eTo determine the role of \u003cem\u003esdh1\u003c/em\u003e in antifungal susceptibility, we performed CLSI microdilution and E-test assays. Both methods consistently demonstrated that \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e exhibited significantly reduced susceptibility to triazoles compared to WT and the \u0026Delta;\u003cem\u003esdh1::sdh1\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e. For WT and \u0026Delta;\u003cem\u003esdh1::sdh1\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e, MIC values remained identical: ITC = 0.5 \u0026mu;g/mL, VOR= 0.25 \u0026mu;g/mL, and POS = 0.25 \u0026mu;g/mL. In contrast, MICs for \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e increased 8- to 16-fold (ITC = 4 \u0026mu;g/mL, VOR = 2 \u0026mu;g/mL, POS = 2 \u0026mu;g/mL; Table 1). E-test results corroborated these findings, with \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e showing pronounced resistance halos, whereas WT and \u0026Delta;\u003cem\u003esdh1::sdh1\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e displayed equivalent susceptibility profiles (Fig.2). Notably, caspofungin (CAS) susceptibility remained unchanged across all strains (MIC = 0.25 \u0026mu;g/mL; Table 1), confirming that \u003cem\u003esdh1\u003c/em\u003e specifically modulates triazole tolerance rather than broadly affecting antifungal responses. These results establish\u003cem\u003e\u0026nbsp;sdh1\u003c/em\u003e as a negative regulator of triazole susceptibility in \u003cem\u003eA. fumigatus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u0026nbsp;\u003c/strong\u003eResults of antifungal susceptibility tests\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" style=\"width: 568px;\"\u003e\n \u003cp\u003eAntifungal susceptibility\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003eType\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 247px;\"\u003e\n \u003cp\u003eE-test MICs or MEC (\u0026mu;g/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 247px;\"\u003e\n \u003cp\u003eM38-A3 MICs or MEC (\u0026mu;g/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003eStrain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003eWT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e\u0026Delta;\u003cem\u003esdh1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e\u0026Delta;\u003cem\u003esdh1::sdh1\u003csup\u003e+\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003eWT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e\u0026Delta;\u003cem\u003esdh1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e\u0026Delta;\u003cem\u003esdh1::sdh1\u003csup\u003e+\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003ePOS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003eVOR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003eITC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003eCAS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote: POS: posaconazole; VOR: voriconazole; ITC: itraconazole; CAS: caspofungin.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3\u003c/strong\u003e \u003cem\u003esdh1\u003c/em\u003e Deficiency Disrupts Mitochondrial Function\u003c/p\u003e\n\u003cp\u003eTo assess the impact of \u003cem\u003esdh1\u003c/em\u003e deletion on mitochondrial function, we measured SDH activity and metabolic parameters in the \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e strain. SDH activity in \u0026Delta;\u003cem\u003esdh1\u0026nbsp;\u003c/em\u003eexhibited a delayed recovery over time, reaching 58.3% of WT levels at 48 hours and 72.5% at 72 hours, but remained significantly lower than WT at all time points (Fig.3A). In 3-day-old colonies cultured on SAB agar, \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e displayed severe mitochondrial dysfunction: ATP levels plummeted to 34.7% of WT (Fig.3B), ROS content surged to 162% of WT (Figure 3C), and SOD activity dropped to 45% of WT (Fig.3D). These results confirm that \u003cem\u003esdh1\u003c/em\u003e deletion disrupts mitochondrial energy metabolism, exacerbates oxidative stress, and impairs antioxidant defenses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 \u003cem\u003esdh1\u003c/em\u003e required for the normal growth of \u003cem\u003eA. fumigatus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe colony of \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e exhibited markedly sluggish growth and displayed a white, shrunken appearance. In comparison to the WT and \u0026Delta;\u003cem\u003esdh1::sdh1\u003csup\u003e+\u003c/sup\u003e\u003c/em\u003e, there was a notable reduction in the growth diameters of \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e when cultivated on SAB or CZA agar (Fig.4A-D). Furthermore, \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e exhibited a strikingly diminished production of conidial heads, with a complete absence of bottle pedicles or conidial heads bearing pedicel bases (Fig.4E). These observations strongly indicate that the disruption of \u003cem\u003esdh1\u003c/em\u003e adversely affects the reproductive capacity of \u003cem\u003eA. fumigatus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5\u003cem\u003e\u0026nbsp;sdh1\u003c/em\u003e Regulates Efflux Pump Activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the molecular mechanisms underlying azole resistance in the \u0026Delta;\u003cem\u003esdh1\u0026nbsp;\u003c/em\u003estrain, transcriptomic and functional analyses revealed alterations in efflux pump activity. A total of 2667 genes showed differential expression, with 1587 upregulated and 1080 downregulated(Fig.5A). KEGG pathway enrichment analysis showed that the differentially expressed genes (DEGs) were significantly enriched in metabolic pathways and prominently enriched in the ribosome function category, indicating significant differences. We specifically focused on the enrichment of differentially expressed genes related to drug sensitivity. Within the category of Environmental Information Processing, genes associated with ABC transporters were significantly enriched(Fig.5B). This indicates that deletion of the \u003cem\u003esdh1\u003c/em\u003e gene leads to changes in efflux pump-related genes. Specifically, among these efflux pump genes, 48 genes showed more than a 4-fold change in expression, including 33 significantly upregulated and 15 downregulated genes(Fig.5C). Notably, the highest fold change among the downregulated genes was a 10.96-fold decrease, whereas among the upregulated genes, 15 exhibited expression levels exceeding 10.96-fold, with the highest reaching 472.5-fold. Notably, \u003cem\u003eabcB\u003c/em\u003e increased by 113.5-fold, and \u003cem\u003emfsB\u003c/em\u003e by 34.1-fold. RT-qPCR further confirmed elevated expression of critical genes, including \u003cem\u003emdr4\u003c/em\u003e,\u003cem\u003e\u0026nbsp;cdr1B\u003c/em\u003e, \u003cem\u003eabcB\u003c/em\u003e, \u003cem\u003eabcF\u003c/em\u003e, \u003cem\u003emfsA\u003c/em\u003e, and \u003cem\u003emfsB\u003c/em\u003e (Fig.5D)\u0026mdash;all previously characterized to modulate azole susceptibility[12-14]. This suggests that \u003cem\u003esdh1\u003c/em\u003e deletion enhances transporter efficiency.\u003c/p\u003e\n\u003cp\u003eConsistent with this hypothesis, efflux pump activity assays conducted across three strains demonstrated a marked increase in drug efflux in \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e. Specifically, significant efflux differences emerged within 15 minutes and persisted throughout the assay (Fig.5E). In contrast, expression levels of the ergosterol biosynthesis-specific transcription factors \u003cem\u003esrbA\u003c/em\u003e and \u003cem\u003eatrF\u003c/em\u003e, as well as the azole target genes \u003cem\u003ecyp51A\u003c/em\u003e and \u003cem\u003ecyp51B\u003c/em\u003e, remained unchanged[36-38] (Fig.5D). These findings indicate that \u003cem\u003esdh1\u003c/em\u003e deletion primarily drives reduced azole susceptibility through efflux pump hyperactivation rather than ergosterol pathway modulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 Disruption of\u003cem\u003e\u0026nbsp;sdh1\u003c/em\u003e reduces the initial virulence of \u003cem\u003eA. fumigatus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe final survival rate of \u003cem\u003eG. mellonella\u003c/em\u003e infected with \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e was higher than that of WT (\u003cem\u003eP\u003c/em\u003e\u0026gt;0.05) (Fig.6). However, after treatment with POS, VOR, and ITC, \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e did not reduce the mortality of\u003cem\u003e\u0026nbsp;G. mellonella\u003c/em\u003e, while a decrease in mortality was observed in WT. This also indicates that the virulence of \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e has decreased,but it maintains low sensitivity to azole drug.\u003c/p\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThe emergence of azole resistance has posed a significant challenge in treating IA[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], and elucidating its mechanisms may provide critical insights for preventing resistance. In this study, exposure to sub-inhibitory concentrations of SDHIs successfully induced a marked reduction in azole susceptibility in \u003cem\u003eA. fumigatus\u003c/em\u003e, suggesting SDHIs as potential drivers of azole resistance. Through transcriptional profiling, we identified and validated the pivotal role of the \u003cem\u003esdh1\u003c/em\u003e gene in mediating resistance.\u003c/p\u003e \u003cp\u003eSDHIs exert fungicidal effects by targeting fungal mitochondrial SDH, disrupting energy metabolism, and inducing oxidative damage, thereby impairing mitochondrial function[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. SDH, a key enzyme in the mitochondrial electron transport chain and TCA cycle, catalyzes the oxidation of succinate to fumarate while transferring electrons to ubiquinone (CoQ)[\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The catalytic core of SDH is highly conserved across species[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The widespread use and environmental persistence of SDHIs inevitably affect \u003cem\u003eA. fumigatus\u003c/em\u003e SDH activity. In this study, knockout of \u003cem\u003esdh1\u003c/em\u003e significantly reduced SDH activity, thereby disrupting Complex II function. Diminished SDH activity not only compromised electron transport efficiency but also reduced ATP production and elevated ROS levels. Excessive ROS accumulation exacerbated oxidative stress, further destabilizing cellular homeostasis. Additionally, reduced SOD activity weakened antioxidant defenses.\u003c/p\u003e \u003cp\u003eMitochondrial function is intricately linked to antifungal drug susceptibility and fungal virulence[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Mitochondrial mutations can confer drug sensitivity or resistance, with most non-\u003cem\u003ecyp51A\u003c/em\u003e mutant isolates exhibiting mitochondrial dysfunction, overexpression of efflux pump genes, and multidrug resistance[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan additionalcitationids=\"CR47 CR48\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Both \u003cem\u003eCandida glabrata\u003c/em\u003e and \u003cem\u003eS. cerevisiae\u003c/em\u003e can live without mitochondrial DNA (mtDNA), and the drug-resistant mutants most commonly lost their mitochondrial genome[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In fact, mtDNA loss appears to be a key activating mutation for the drug resistance pathway. Loss of mtDNA and associated drug resistance is relatively common for in vitro cultures of both \u003cem\u003eS. cerevisiae\u003c/em\u003e (about 2% of cells) and \u003cem\u003eC. glabrata\u003c/em\u003e (frequencies of 2\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e to 4\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e) [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Impaired mitochondrial function activates transcription factors such as Pdr3 in \u003cem\u003eS. cerevisiae\u003c/em\u003e and Pdr1 in \u003cem\u003eC. glabrata\u003c/em\u003e, altering the activity of efflux pump-encoding genes (e.g., \u003cem\u003ecdr1\u003c/em\u003e and \u003cem\u003ecdr2\u003c/em\u003e), ultimately driving antifungal resistance[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn filamentous fungi, the role of ABC and MFS transporters in effluxing natural and synthetic toxins is well established. In \u003cem\u003eA. fumigatus\u003c/em\u003e, efflux pump activity is a key contributor to azole resistance, mediated by MDR transporters from the ABC and MFS families[\u003cspan additionalcitationids=\"CR53\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. These pumps reduce intracellular drug concentrations, diminishing drug efficacy. Genomic analysis reveals 278 MFS transporters and 49 ABC transporters in \u003cem\u003eA. fumigatus\u003c/em\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Studies have demonstrated the role of \u003cem\u003eA. fumigatus\u003c/em\u003e MDR pumps, such as \u003cem\u003ecdr1B\u003c/em\u003e, \u003cem\u003eabcB\u003c/em\u003e, and \u003cem\u003emdr4\u003c/em\u003e, in enhancing ITC resistance[\u003cspan additionalcitationids=\"CR56\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. In our study, upregulated expression of these efflux pump genes correlated with increased efflux activity, likely directly reducing susceptibility to ITR, POS, and VOR.\u003c/p\u003e \u003cp\u003eMitochondria are primary sources of ATP and ROS, requiring a balance between energy production and ROS generation for optimal enzyme activity[\u003cspan additionalcitationids=\"CR59 CR60\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Reduced ATP levels likely contributed to growth defects in our strains. While ROS induction may promote antifungal drug tolerance or resistance[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], the precise mechanisms remain unclear[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSDHI can enhance the activity of efflux pumps that mediate the development of resistance. Reports have shown that in field-resistant strains of \u003cem\u003eBotrytis cinerea\u003c/em\u003e, overexpression of the ABC transporter gene \u003cem\u003ebcatrB\u003c/em\u003e is significantly associated with resistance to SDHI fungicides, such as boscalid[\u003cspan additionalcitationids=\"CR65\" citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Similarly, in \u003cem\u003eA. fumigatus\u003c/em\u003e, increased efflux pump activity reduces intracellular SDHI and azole concentrations, lowering drug sensitivity. In SDHI-resistant strains, the expression of \u003cem\u003ebcatrB\u003c/em\u003e is significantly upregulated (\u0026gt;\u0026thinsp;10-fold), which enhances the efflux activity against SDHI. In filamentous fungi, the role of ABC and MFS transporters in the efflux of natural and synthetic toxins is well-known[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Similarly, in \u003cem\u003eA. fumigatus\u003c/em\u003e, increased efflux pump activity reduces intracellular SDHI concentration, and the enhanced activity also decreases the concentration of azole drugs, ultimately leading to a significant reduction in sensitivity.\u003c/p\u003e \u003cp\u003eNotably, SDHI-induced \u003cem\u003esdh1\u003c/em\u003e inhibition and efflux pump upregulation suggest a novel mechanism of acquired azole resistance in \u003cem\u003eA. fumigatus\u003c/em\u003e. However, \u003cem\u003esdh1\u003c/em\u003e inhibition may concurrently slow growth, potentially limiting the environmental proliferation of SDHI-resistant strains. Given the widespread agricultural use and environmental persistence of both SDHIs and azoles, this dual induction of resistance warrants urgent attention. Field sampling in SDHI-treated areas to isolate azole-resistant strains and further mechanistic studies are imperative. This emerging resistance pattern underscores the need to explore non-azole-related resistance mechanisms. Additionally, clinical isolates with unexplained resistance should undergo SDH-related profiling to guide therapeutic strategies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eSuccinate dehydrogenase--SDH; succinate dehydrogenase inhibitors--SDHI; superoxide dismutase--SOD; minimum inhibitory concentration--MIC; invasive aspergillosis--IA; itraconazole--ITC; voriconazole--VOR; demethylation inhibitor--DMI; posaconazole--POS; caspofungin--CAS; minimum effective concentration--MEC; 2\u0026prime;, 7\u0026prime;‐dichlorodihydrofluorescein diacetate--DCFH‐DA; reactive oxygen species--ROS Rhodamine 6G--R6g.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e All RNA-seq data files are available at the NCBI Sequence Read Archive (SRA) database under the accession number PRJNA1192589 for WT, \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e and \u0026Delta;\u003cem\u003esdh1:: sdh1\u003csup\u003e+\u003c/sup\u003e\u0026nbsp;\u003c/em\u003eof \u003cem\u003eA.fumigatus\u003c/em\u003e conidia RNA-seq.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by the Jingzhou Science and Technology Plan Project (2024HD34); the Yangtze University Science and Technology Aid to Tibet Medical Talent Training Program Project (2023YZ06); and the Key Research and Development program of Hubei Province (2024BCB043).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution:\u003c/strong\u003e All authors contributed to the study conception and design. Conceptualization, Yi Sun and Heng Zhang; methodology, Heng Zhang; software, Xiao Gong; validation, Mengqi Peng; formal analysis, Mengqi Peng; investigation, Tian Chen; resources, Qingwen Hu; data curation, Linyun Li; writing original draft preparation, Heng Zhang; writing review and editing, Sijie Liu and Heng Zhang; visualization, Zha-xi Dun-zhu and Lha-zom Drol-ga; supervision, Zhangling Zhu; project administration, Mengqi Peng; funding acquisition, Yi Sun and Heng Zhang.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We thank everyone who contributed to the success of this research, including colleagues, institutions, and funding bodies.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eVerweij PE, Snelders E, Kema GH, Mellado E, Melchers WJ. Azole resistance in \u003cem\u003eAspergillus fumigatus\u003c/em\u003e: a side-effect of environmental fungicide use? 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Illumina identification of RsrA, a conserved C2H2 transcription factor coordinating the NapA mediated oxidative stress signaling pathway in \u003cem\u003eAspergillus\u003c/em\u003e. Bmc Genomics. 2014;15:1011. https://doi.org/10.1186/1471-2164-15-1011.\u003c/li\u003e\n \u003cli\u003eLambeth JD. NOX enzymes and the biology of reactive oxygen. Nat Rev Immunol. 2004;4:181-9. https://doi.org/10.1038/nri1312.\u003c/li\u003e\n \u003cli\u003eKowaltowski AJ, de Souza-Pinto NC, Castilho RF, Vercesi AE. Mitochondria and reactive oxygen species. Free Radic Biol Med. 2009;47:333-43. https://doi.org/10.1016/j.freeradbiomed.2009.05.004.\u003c/li\u003e\n \u003cli\u003eLiu Z, Raj S, van Rhijn N, Fraczek M, Michel JP, Sismeiro O, Legendre R, Varet H, Fontaine T, Bromley M, Latge JP. Functional Genomic and Biochemical Analysis Reveals Pleiotropic Effect of Congo Red on \u003cem\u003eAspergillus fumigatus\u003c/em\u003e. Mbio. 2021;12. https://doi.org/10.1128/mBio.00863-21.\u003c/li\u003e\n \u003cli\u003eDickwella WM, Gautam I, Sarkar D, Mentink-Vigier F, Vermaas JV, Ding SY, Lipton AS, Fontaine T, Latge JP, Wang P, Wang T. Adaptative survival of \u003cem\u003eAspergillus fumigatus\u003c/em\u003e to echinocandins arises from cell wall remodeling beyond beta-1,3-glucan synthesis inhibition. Nat Commun. 2024;15:6382. https://doi.org/10.1038/s41467-024-50799-8.\u003c/li\u003e\n \u003cli\u003eKretschmer M, Leroch M, Mosbach A, Walker AS, Fillinger S, Mernke D, Schoonbeek HJ, Pradier JM, Leroux P, De Waard MA, Hahn M. Fungicide-driven evolution and molecular basis of multidrug resistance in field populations of the grey mould fungus Botrytis cinerea. Plos Pathog. 2009;5:e1000696. https://doi.org/10.1371/journal.ppat.1000696.\u003c/li\u003e\n \u003cli\u003eLiu M, Peng J, Wang X, Zhang W, Zhou Y, Wang H, Li X, Yan J, Duan L. Transcriptomic Analysis of Resistant and Wild-Type Botrytis cinerea Isolates Revealed Fludioxonil-Resistance Mechanisms. Int J Mol Sci. 2023;24. https://doi.org/10.3390/ijms24020988.\u003c/li\u003e\n \u003cli\u003eFernandez-Ortuno D, Tores JA, Chamorro M, Perez-Garcia A, de Vicente A. Characterization of Resistance to Six Chemical Classes of Site-Specific Fungicides Registered for Gray Mold Control on Strawberry in Spain. Plant Dis. 2016;100:2234-9. https://doi.org/10.1094/PDIS-03-16-0280-RE.\u003c/li\u003e\n \u003cli\u003ede Waard MA, Andrade AC, Hayashi K, Schoonbeek HJ, Stergiopoulos I, Zwiers LH. Impact of fungal drug transporters on fungicide sensitivity, multidrug resistance and virulence. Pest Manag Sci. 2006;62:195-207. https://doi.org/10.1002/ps.1150.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"mycopathologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"myco","sideBox":"Learn more about [Mycopathologia](https://www.springer.com/journal/11046)","snPcode":"11046","submissionUrl":"https://submission.nature.com/new-submission/11046/3","title":"Mycopathologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Aspergillus fumigatus, Succinate dehydrogenase, sdh1, Efflux pumps, Azole resistance","lastPublishedDoi":"10.21203/rs.3.rs-6487242/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6487242/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIntroduction:\u0026nbsp;\u003cem\u003eAspergillus fumigatus\u003c/em\u003e poses significant clinical challenges due to its increasing azole resistance. This study investigates the \u003cem\u003esdh1\u003c/em\u003e gene's role in regulating azole susceptibility, mitochondrial function, and virulence.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMaterials and Methods: Fungal strains were co-cultured with varying concentrations of succinate dehydrogenase inhibitors (SDHIs). Post-treatment azole minimum inhibitory concentrations (MICs) were determined using broth microdilution method, while succinate dehydrogenase subunit (SDH) expression changes were analyzed via RT-qPCR. Using\u0026nbsp;\u003cem\u003eA. fumigatus\u003c/em\u003e\u0026nbsp;MFIG001 as the parental strain, \u003cem\u003esdh1\u003c/em\u003e knockout mutant (Δ\u003cem\u003esdh1\u003c/em\u003e) and complemented strain (Δ\u003cem\u003esdh1::sdh1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e) were constructed through homologous recombination. Detect the hyphal growth rate of Δ\u003cem\u003esdh1\u003c/em\u003e, MICs and the changes in virulence within the \u003cem\u003eGalleria mellonella \u003c/em\u003einfection model. Mitochondrial function was evaluated by measuring SDH activity, ATP content, and reactive oxygen species (ROS) levels. Transcriptomic changes were analyzed using RNA-seq and RT-qPCR, with efflux pump activity validated through Rhodamine 6G accumulation assays.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eResults: Exposure to subinhibitory concentrations of SDHIs induced azole resistance in\u003cem\u003e A. fumigatus\u003c/em\u003e, with 4.12% of strains exhibiting reduced susceptibility to voriconazole, itraconazole , and posaconazole. RT-qPCR analysis revealed significant downregulation of\u003cem\u003e sdh1 \u003c/em\u003ein resistant strains, implicating its role in resistance development. Deletion of\u003cem\u003e sdh1\u003c/em\u003e resulted in an 8- to 16-fold increase in triazole MICs, confirming its role as a negative regulator of azole susceptibility. Phenotypically, the Δ\u003cem\u003esdh1\u003c/em\u003e strain exhibited impaired growth, reduced sporulation, and diminished efficacy of azole treatment in the \u003cem\u003eG. mellonella\u003c/em\u003e infection model. Furthermore, Δ\u003cem\u003esdh1\u003c/em\u003e exhibited severe mitochondrial dysfunction, including reduced SDH activity, decreased ATP levels, elevated ROS, and impaired antioxidant defenses. RNA-seq analysis revealed that the deletion of \u003cem\u003esdh1\u003c/em\u003e upregulated the expression of efflux pump genes (e.g., \u003cem\u003ecdr1B\u003c/em\u003e, \u003cem\u003eabcB\u003c/em\u003e, \u003cem\u003emdr4\u003c/em\u003e), while Rhodamine 6G efflux assays demonstrated significantly enhanced efflux activity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDiscussion: These results identify \u003cem\u003esdh1\u003c/em\u003e as a critical determinant of azole susceptibility through dual mechanisms: mitochondrial function maintenance and efflux pump regulation. The observed SDHI-induced cross-resistance suggests agricultural fungicides may drive environmental selection of azole-resistant strains. While \u003cem\u003esdh1\u003c/em\u003e deletion increased drug tolerance through efflux activation, the concurrent mitochondrial damage reduced pathogenic fitness, revealing compensatory evolutionary constraints. This work highlights the need to monitor non-target effects of agricultural SDHIs on clinical antifungal resistance.\u003c/p\u003e","manuscriptTitle":"Agricultural SDHIs Induce Azole Resistance in Aspergillus fumigatus via Mitochondrial Sdh1 Suppression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-05 06:38:08","doi":"10.21203/rs.3.rs-6487242/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revisions","date":"2025-06-22T20:49:15+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-05-02T09:03:37+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-29T09:57:36+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Mycopathologia","date":"2025-04-28T19:56:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-26T15:15:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"Mycopathologia","date":"2025-04-23T22:13:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"mycopathologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"myco","sideBox":"Learn more about [Mycopathologia](https://www.springer.com/journal/11046)","snPcode":"11046","submissionUrl":"https://submission.nature.com/new-submission/11046/3","title":"Mycopathologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"880208d4-5dea-4bb0-9876-598147df24c8","owner":[],"postedDate":"May 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-22T15:58:31+00:00","versionOfRecord":{"articleIdentity":"rs-6487242","link":"https://doi.org/10.1007/s11046-025-00992-0","journal":{"identity":"mycopathologia","isVorOnly":false,"title":"Mycopathologia"},"publishedOn":"2025-09-15 15:57:03","publishedOnDateReadable":"September 15th, 2025"},"versionCreatedAt":"2025-05-05 06:38:08","video":"","vorDoi":"10.1007/s11046-025-00992-0","vorDoiUrl":"https://doi.org/10.1007/s11046-025-00992-0","workflowStages":[]},"version":"v1","identity":"rs-6487242","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6487242","identity":"rs-6487242","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}