Succinate Dehydrogenase Inhibitors as Triggers of Azole Resistance in Aspergillus fumigatus: The Role of sdh1 and Efflux Pathways

Succinate Dehydrogenase Inhibitors as Triggers of Azole Resistance in Aspergillus fumigatus: The Role of sdh1 and Efflux Pathways | 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 Succinate Dehydrogenase Inhibitors as Triggers of Azole Resistance in Aspergillus fumigatus: The Role of sdh1 and Efflux Pathways Heng Zhang, Zhangling Zhu, Xiao Gong, Wenxu Cheng, Weizu Liao, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5441096/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Aspergillus fumigatus is a major cause of invasive aspergillosis in immunocompromised patients. The rise in antifungal resistance is linked to the use of succinate dehydrogenase inhibitors (SDHIs). Objective This study investigates the mechanism of acquired azole resistance in A. fumigatus , triggered by SDHIs, which are widely used agricultural fungicides. Methods Conidia of A. fumigatus were co-cultured with four SDHIs (Boscalid, Thifluzamide, Fluopyram, Carboxin) to assess sensitivity to three azole drugs: voriconazole, itraconazole, and posaconazole. RT-qPCR identified genes related to resistance, focusing on sdh1 , a gene encoding a succinate dehydrogenase subunit. A sdh1 knockout strain was created to evaluate its impact on growth, azole sensitivity, ATP levels, superoxide dismutase (SOD) activity, and ergosterol biosynthesis. Results SDHI exposure increased resistance to azoles, with 4.12% of 2,496 strains showing higher minimum inhibitory concentration (MIC). Four strains had an eightfold MIC increase and reduced sdh1 expression. The sdh1 knockout strain showed impaired growth, increased azoles resistance, and lower reactive oxygen species (ROS), ATP production ( P < 0.001), and SOD activity ( P < 0.05). RNA sequencing indicated that sdh1 deletion upregulated efflux pump genes and enhanced ergosterol synthesis. Conclusion SDHIs may induce azole resistance in A. fumigatus by downregulating sdh1 . The findings highlight a potential new resistance mechanism, providing insights for managing A. fumigatus infections and azole resistance. Aspergillus fumigatus Succinate dehydrogenase sdh1 Efflux pumps Azole resistance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 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 ]. 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. 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[ 8 ]. 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[ 9 ]. 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[ 10 – 12 ]. 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 widespread agricultural use, SDHIs can enter water bodies through leaching, runoff, or atmospheric drift, contaminating soil and aquatic environments[ 13 ]. 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[ 14 ]. 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 Strains and plasmids A. fumigatus AF293 (Fungal Genetics Stock Center), used as the parent strain for all amplification in this investigation, includes the whole DNA sequence of A. fumigatus . A. fumigatus A1160 (Δ KU80, pyrG - ) (Fungal Genetics Stock Center) is an uracil (U)-deficient strain, defective in the pyrG gene, and unable to grow on U-deficient media. In the knockout process of this study, the pyrG gene was introduced and then allowed to grow in a U-free medium as the host strain. The screening marker pyrG was derived from the plasmid pBARGPE1-Pyrg-TagRFP (Wensheng Biotechnology, Hunan, China). The WT (Δ KU80, pyrG + ) strain was constructed using A1160. Plasmid pCT74 (Fenghui Biotechnology, Hunan, China) carries the hygromycin resistance ( hph ) gene. Transformants can be propagated in culture dishes containing hygromycin as a screening marker during the construction of the revertant. 12 A. fumigatus clinical isolates were identified by microscopic morphology and by molecular sequencing of the internal transcribed spacer (ITS) ribosomal DNA (rDNA)[15]. 2.2 Antifungals and SDHIs All tested agents, including ITC, 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. Fresh spores were harvested from cultures grown on SAB (Sabaurauds) solid medium for 3 days. 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 [16], 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 the standard M38-A2 broth microdilution method (described in section 2.4) was used to assess changes in susceptibility to three azole antifungal agents. 2.4 Minimum inhibitory concentration (MIC) assay The broth microdilution method of M38-A2 was conducted following the standards established by the Clinical Laboratory Standards Institute (CLSI)[17] 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 Detection of gene expression levels to related genes Fungal tissue cultured on SAB medium for three days was collected. Total RNA was extracted using TriEasy LS Total RNA Extraction Reagent (Yeasen Biotechnology, Shanghai, China) and reverse-transcripted into cDNA using a HI Fair II 1st strand cDNA Synthesis Kit (Yeasen Biotechnology, Shanghai, China). Antifungal susceptibility-related genes were selected for testing their expression (Table S1). The relative gene expression level (2 -ΔΔCT ) was calculated using the actin housekeeping gene control[18]. To minimize error, each repetition included three mutual control groups. 2.6 Construction of the sdh1 deletion strain and the complementation strain The targeted knockout of sdh1 was performed using a modified fusion PCR cassette and protoplast transformation technique[19], with the target gene being replaced by the pyrG gene in the pBARGPE1-Pyrg-TagRFP. Three distinct DNA pieces were amplified from AF293, each including a 1.2 kb region upstream and downstream of the sdh1 coding sequence and a pyrG cassette as the selectable marker (Table S2). Fusion PCR conditions were utilized as described [19]. A major band at 4.0 kb was shown after confirmation of the fusion PCR product by agarose gel electrophoresis (Fig.S1). The PEG-mediated protoplast method facilitated the transformation, and the construct was validated by sequencing at Sangon Biotech, confirming the successful generation of Δ sdh1 . For complementation, the plasmid pCT74, containing the hph gene, was employed. The sdh1 expression cassette from AF293 was amplified, ligated with the pCT74 plasmid using the Hieff Clone® Plus One Step Cloning Kit (Yeasen Biotechnology, Shanghai, China), and introduced into the Δ sdh1 strain through the same transformation procedure to generate the complementation strain Δ sdh1::sdh1 + (Fig.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. A 1μL aliquot of each suspension was inoculated onto solid CZA (Czapeck) and SAB media, and the colony diameter was recorded after 3 days. 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. The procedure was repeated three times on different days to minimize testing errors. 2.9 Mitochondrial membrane potential and reactive oxygen species (ROS) Assay DCFH‐DA (2′, 7′‐dichlorodihydrofluorescein diacetate), a cell‐permeable fluorescent probe, was employed to detect intracellular ROS[20]. The specific procedure involved collecting fresh spores and preparing a suspension with a concentration of 1×10⁶ CFU/mL. Pectinase was added, followed by incubation at 37°C and 2000 rpm for 6 hours to fully dissolve the cell wall. The suspension was washed twice with pre-cooled PBS (4°C, 2000 rpm, 5 minutes). DCFH-DA was then added to reach a final concentration of 10 mM. After a 30-minute incubation at 37°C, the spores were washed three times with PBS and resuspended. The flow cytometry data were generated on a Beckman Cytomics FC 500 BD FACSCanto II and analyzed with FlowJo v10 software. The excitation wavelength is 488 nm and the emission wavelength is 525 nm. 2.10 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. 2.11 RNA isolation and KEGG pathway enrichment analysis Spores (1x10⁶ CFU/mL) were added to SAB medium and incubated at 37°C for 3 days. Total RNA was extracted using the RNAprep Pure Plant Kit (Tiangen, Beijing, China). The RNA was used to construct cDNA libraries, which were sequenced on an Illumina NovaSeq platform to generate 150 bp paired-end reads. The raw reads were processed using the BMKCloud (www.biocloud.net) online platform. Gene expression levels were calculated based on fragmentsper kilobase of transcript per million fragments mapped (FPKM). Differential expression analysis of two conditions/groups was performed using the DESeq2（P-value < 0.01 & Fold Change≥2）. The KOBAS database and clusterProfiler software were used to analyze differentially expressed genes in KEGG pathways[21]. 2.12 Detection of ergosterol content Total intracellular sterols were extracted as reported by Branch with slight modifications[22]. Briefly, collect spores that have grown on SAB solid medium for three days and then place them in SAB liquid medium. The cultures were incubated for 16h with shaking at 35°C. The stationary-phase cells were harvested by centrifugation at 2,700rpm for 5min and washed once with sterile distilled water. The net wet weight of the cell pellet was determined. Three milliliters of 25% alcoholic potassium hydroxide solution (25g of KOH and 35mL of sterile distilled water, brought to 100mL with 100% ethanol), was added to each pellet and vortex mixed for 1min. Cell suspensions were transferred to 16- by 100-mm sterile borosilicate glass screw-cap tubes and were incubated in an 85°C water bath for 1h. Following incubation, tubes were allowed to cool to room temperature. Sterols were then extracted by addition of a mixture of 1mL of sterile distilled water and 3mL of n -heptane followed by vigorous vortex mixing for 3min. The heptane layer was transferred to a clean borosilicate glass screw-cap tube and stored at −20°C for as long as 24h. Prior to analysis, a 20-μL aliquot of sterol extract was diluted fivefold in 100% ethanol and scanned spectrophotometrically between 240 and 300nm with a Nano Drop One (Thermo Fisher Scientific, US). Ergosterol content was calculated as a percentage of the wet weight of the cell by the following equations: %ergosterol + %24(28)DHE = [( A 281.5/290) × F ]/pellet weight, %24(28)DHE = [( A 230/518) × F ]/pellet weight, and % ergosterol = [% ergosterol + % 24(28)DHE] − % 24(28)DHE, where F is the factor for dilution in ethanol and 290 and 518 are the E values (in percentages per centimeter) determined for crystalline ergosterol and 24(28)DHE, respectively. 2.13 Galleria mellonella in vivo toxicity assay G. mellonella lacks an adaptive immune system and specialized immune cells, making it a simple and cost-effective model, widely used in this study to evaluate fungal virulence[23-26]. G. mellonella larvae (200−250mg) were selected and injected with a spore suspension of A. fumigatus (2.0×10⁸ CFU/mL) for infection. After injection, the larvae were incubated at 37°C, with monitoring every 24 hours for a total of 5 days. Larval performance was assessed according to the G. mellonella Health Index Scoring System[27]. 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 Acquired Azole Resistance Culturing A. fumigatus in the presence of SDHIs altered resistance to three commonly used azole antifungal drugs: VOR, ITR, and POS. Of the 2,496 strains tested, 4 strains exhibited an eightfold increase in MIC, 5 strains showed a fourfold increase, 94 strains had a twofold increase, and 50 strains showed a 0.5-fold decrease in MIC. In total, 103 strains displayed reduced sensitivity to azole drugs, while 50 strains showed increased sensitivity. Notably, prolonged exposure to SDHIs did not lead to cumulative changes in azole susceptibility. Furthermore, the tested SDHIs did not exhibit a fungicidal effect on A. fumigatus , with MIC remaining above 32 µg/mL (Fig. 1 A). Subsequent analysis of SDH-related gene expression in the strains that demonstrated an eightfold increase in MIC revealed a significant reduction in sdh1 gene expression. In contrast, the expression levels of sdh2 , cybs , and b560 genes did not show consistent patterns of upregulation or downregulation. These findings suggest that decreased expression of the sdh1 may play a crucial role in the observed increase in MIC. Overall, the results indicate that SDHIs may contribute to azole resistance in A. fumigatus under complex environmental pressures by influencing sdh1 expression (Fig.B-E). 3.2 Biological information of Sdh1 in A. fumigatus Through a search of the NCBI database, the SDH subunit Sdh1 in A. fumigatus , numbered AFUA_3G07810, was found on chromosome 3. Sequence analysis revealed that it has 2247 bases, encodes 647 amino acids and contains 6 exons and 5 introns, resembles the flavoprotein subunit of SDH in Saccharomyces cerevisiae , and that it can pair oxidizing succinic acid to transfer electrons to ubiquinone. According to the amino acid sequence alignment, the flavoprotein subunit Sdh1 of SDH in S. cerevisiae had a 97% repeat rate and could match the protein's three-dimensional structure (root mean square deviation = 0.133) . 3.3 Disruption of sdh1 decreased A. fumigatus susceptibility to azoles We evaluated the effect of sdh1 on antifungal susceptibility using CLSI microdilution and E-test methods. Microdilution studies revealed no significant change in susceptibility between WT and Δ sdh1:: sdh1 + , with ITC, VOR, and POS MICs of 0.5 µg/mL, 0.25 µg/mL, and 0.25 µg/mL, respectively. However, the MICs of ITC, VOR, and POS against Δ sdh1 were 4 µg/mL, 2 µg/mL, and 2 µg/mL, respectively (Table 1 ). We also observed the same results in the E-test, with the three azoles not showing differences between WT and Δ sdh1:: sdh1 + but with a significantly reduced susceptibility in Δ sdh1 (Fig. 3 ). With the deletion of the sdh1 , the susceptibility of azoles was significantly decreased. The sdh1 gene is a negative regulator of triazole susceptibility in A. fumigatus . For CAS, we observed no significant susceptibility changes in WT, Δ sdh1 , and Δ sdh1:: sdh1 + . 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. 3.4 The effect of knockout of sdh1 on the activity of SDH, SOD and ROS, ATP content For continuous monitoring of SDH activity, the activity of SDH in Δ sdh1 slowly increases with increasing growth time, but the activity decreases at each time point (Fig. 4 A). When the three-day-old fungal tissue was tested on SAB solid medium, it was clearly observed that after the deletion of the s dh1 , the ATP (Fig. 4 B), ROS content (Fig. 4 C) and SOD activity (Fig. 4 D) decreased. It is noteworthy that SOD can catalyze the dismutation of superoxide anion to generate H 2 O 2 and O 2 , which is an important antioxidant enzyme in organisms. In organisms, SOD maintains the redox balance in cells by clearing excess ROS. Low SOD activity indicates that the balance of ROS in cells is also broken. 3.5 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. 5A-B). 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. 5C). These observations strongly indicate that the disruption of sdh1 adversely affects the reproductive capacity of A. fumigatus . Figure 5 s dh1 is necessary for the normal growth of A. fumigatus 3.6 sdh1 regulates genes related to ergosterol biosynthesis RNA-seq analysis of the transcriptome showed that, compared to WT, four transcription factors were significantly up-regulated among the 30 main transcription factors involved in ergosterol synthesis (Fig. 6 A-B). No significant down-regulated genes were observed. RT-qPCR analysis further confirmed that sdh1 was involved in the transcriptional regulation of mvd1 , erg1 , erg2 , and erg5 genes (Fig. 6 C). Therefore, the deletion of sdh1 leads to the overexpression of related genes in the ergosterol pathway. Diphosphomevalonate decarboxylase mvd1 involved in the biosynthesis of mevalonate, an essential precursor substrate for ergosterol synthesis, are up-regulated in sdh1 (Fig. 6 C). Consistent with our hypothesis, we also extracted the absolute ergosterol content of WT, Δ sdh1 , and Δ sdh1:: sdh1 + , and found that the ergosterol content in Δ sdh1 increased (Fig. 6 D). We observed that compared to WT and Δ sdh1:: sdh1 + , the ergosterol content of Δ sdh1 increased by 125% and 129% ( P 0.05). This result suggests that the deletion of the sdh1 activates the expression of genes related to the ergosterol pathway, ultimately increasing the production of ergosterol, which is a negative regulatory gene in the synthesis of the ergosterol pathway. 3.7 Deletion of s dh1 induces up-regulation of drug sensitivity related genes After the deletion of the sdh1 , the expression levels of the other three subunits did not show significant changes (Fig. 7 A). In order to investigate the reasons for the weakening of sdh1 sensitivity to drugs, we continued to analyze the genes related to drug sensitivity. Finally, we found in RNA-seq that KEGG gene pathway enrichment indicated that the deletion of sdh1 significantly affected the expression of 22 transporter genes involved in catalytic activity and transporter activity, of which 19 were up-regulated and 3 were down regulated (Table S3 ). It is worth noting that the expression level of the azole exporter cdr1B gene in strain Δ sdh1 was significantly increased (Fig. 7 B). In addition, the sensitivity of A. fumigatus to azole drugs is related to the expression of cyp51A/B , but we did not see high expression of cyp51A/B in Δ sdh1 . Therefore, we constructed the cdr1B gene-deficient strain Δ cdr1B by the same method and detected the changes of sdh1 in Δ cdr1B and WT strains, and finally found that the expression of sdh1 did not show significant changes in the two strains (Fig. 7 C). This indicates that sdh1 negatively regulates the expression of cdr1B , and sdh1 is a limiting factor for cdr1B expression. Overall, the absence of sdh1 promotes high expression of efflux pump genes, especially in cdr1B , which weakens sensitivity to azole drugs and is a negative regulatory factor for azole drug sensitivity. 3.8 RNA sequencing To decipher the role of sdh1 in the global regulation of gene expression in A. fumigatus , transcriptome analysis was performed (fold change > 2; P < 0.05), 2647 genes showed differential expression, 7436 genes showed no difference, of which 1568 genes were up-regulated and 1079 genes were deleted. Differential genes are significantly enriched in the "translation" pathway in the GO database. The top significant Genetic Information Processing in KEGG category is “Ribosome” and the top significant Cellular Processes in KEGG category is “Meiosis ”. The deletion of sdh1 will lead to cell proliferation and meiosis changes, and eventually lead to slow growth. Further differential gene detection of cell cycle and meiosis revealed that the expression of APSES protein related genes was changed, in which the expression of stua was increased[ 28 ]. The transcription factors of the APSES protein maybe affect the cellular processes of A. fumigatus , including growth, development and secondary metabolism, suggesting that sdh1 may also participate in the regulatory activities of the aspses family on A. fumigatus . To study the genes with subtle changes that are undetectable by GO and KEGG enrichments, GSEA was performed. The method uses priori defined gene sets to evaluate multiple genes involved in a single bio logical pathway, whether an individual gene is of statistical significance or not[ 29 ]. Consistent with the KEGG pathway enrichment results, Ribosome function-related biological pathways were altered. The bioprocesses that were not reflected by GO and KEGG, namely "Iron ion binding", were uncovered by GSEA, suggesting that the pathway of iron metabolism may be altered. This result may be associated with ferroptosis. 3.9 Disruption of sdh1 reduces the initial virulence of A. fumigatus The final survival rates of G. mellonella infected with Δ sdh1 , WT and Δ sdh1:: sdh1 + ranged from 35–45%, with no significant differences between the three strains ( P > 0.05) (Fig. 9 ). However, the survival rate of G. mellonella infected with Δ sdh1 was higher than that of WT and Δ sdh1:: sdh1 + during the first 3 days ( P < 0.05), indicating that the disruption of sdh1 reduced the virulence of A. fumigatus in the early stages but restored it in the later stages. 4 Discussion Resistance to azole drugs has always been an unavoidable challenge in the treatment of IA, and investigating the causes of resistance may be a breakthrough in preventing its occurrence. Although this study could not replicate the complex natural environment, we successfully induced strains with significantly reduced azole sensitivity, suggesting that SDHI induction may be a major contributing factor to azole resistance. Expression analysis pinpointed the key gene sdh1 . Subsequently, we constructed an Sdh1 subunit knockout strain and confirmed that the sdh1 is crucial for azole resistance, growth, and development in A. fumigatus . The knockout of the sdh1 resulted in a significant reduction in SDH activity, which subsequently impaired normal mitochondrial function. SDH integrity is linked to mitochondrial complex II function and the ability to produce ATP[ 30 ]. SDH activity also has a direct impact on critical biological processes such as fungal growth and mitosis[ 30 ]. The reduction in SDH activity disrupts this essential process, leading to decreased electron transport chain efficiency and, consequently, impairing cellular energy metabolism. With the electron transport chain compromised, the oxidative phosphorylation pathway is hindered, causing a marked reduction in ATP production. This energy deficit not only impairs normal cell growth and metabolism but also exacerbates overall mitochondrial dysfunction. Furthermore, the dysfunction in the electron transport chain leads to electron leakage, with electrons being erroneously transferred to oxygen, forming superoxide (a precursor to ROS)[ 31 , 32 ]. This increase in ROS production elevates oxidative stress within the cell, further damaging cellular structures and impairing function. Mitochondria are the primary source of ROS production and ATP production [ 33 – 36 ]. In recent years, studies suggest the associations between mitochondrial function, antifungal susceptibility, and virulence in fungi[ 37 , 38 ]. Although SOD, a key antioxidant enzyme, typically scavenges superoxide[ 39 , 40 ], its activity is significantly reduced following the sdh1 knockout. The combined effect of increased ROS production and decreased SOD activity results in excessive ROS accumulation, which further intensifies oxidative stress and disrupts cellular homeostasis. The cumulative impact of these changes ultimately hampers cell growth and function, underscoring the critical role of the sdh1 in maintaining mitochondrial integrity, oxidative balance, and energy metabolism. Studies on C. glabrata and S. cerevisiae have shown that mitochondrial dysfunction can result in azole resistance and susceptibility[ 37 ]. Several subunits of the respiratory complex located in the inner mitochondrial membrane (including the sdh1 ) are encoded on the mitochondrial genome, and the deletion of genes comprising the mitochondrial genome can act as key activators of the drug resistance pathway[ 37 ]. Impaired mitochondrial function leads to activation of the transcription factor pdr3 in S. cerevisiae and pdr1 in Candida glabrata , which in turn leads to altered activity of target genes encoding efflux pumps, such as cdr1 and cdr2 , ultimately leading to antifungal resistance [ 41 – 45 ]. Similarly, after the deletion of the sdh1 , mitochondrial changes activated efflux pump resistance pathways, including the cdr1B . RNA-seq analysis showed that the deletion of sdh1 led to the massive activation of ABC transporter genes, and 20 of the 22 genes with differences were up-regulated, including cdr1B . We continued to study the relationship between sdh1 and cdr1B , and found that the deletion of sdh1 would promote the expression of cdr1B , but the deletion of cdr1B could not change the expression of sdh1 , indicating that sdh1 regulates the expression of cdr1B unidirectionally. Ergosterol biosynthesis is a key process unique to fungi. Antifungal drugs such as azoles and polyenes directly target this biosynthetic pathway or ergosterol molecules. srbA was shown to regulate expression of multiple genes related to ergosterol biosynthesis pathway, including erg3 , erg24A , and erg25A . A mutant strain defective in srbA showed hypersensitivity to azoles[ 46 , 47 ]. Similar results were also found in this study. The deletion of sdh1 led to the high expression of mvd 1, erg 1, erg2 and erg5 in the ergosterol synthesis pathway, which ultimately led to the increase in the absolute content of ergosterol and the change in sensitivity to azole drugs. Ergosterol is involved in many biological functions such as membrane fluidity, regulation of overall membrane proteins, activity and distribution, and cell cycle control[ 48 ]. Studies in S. cerevisiae have highlighted that changes in the composition of the plasma membrane affect the function of transporters. For example, depletion in sterol levels caused by loss of erg4 or erg6 leads to a reduction in the activity of the multi-drug-resistance transporter Pdr5[ 49 ], and ergosterol is required to correctly localise the azole exporter Cdr1p in Candida albican s[ 50 ]. This leads us to speculate that even relatively small increases in ergosterol content in the cell membrane may lead to large increases in azole resistance via an indirect effect on azole transporter levels[ 51 ]. This conclusion also proves that sdh1 affects the function of plasma membrane. And from our in vivo study, the survival rate of G. mellonella infected with Δ sdh1 was higher than that of WT and Δ sdh1:: sdh1 + during the first 3 days ( P < 0.05), indicating that the disruption of sdh1 reduced the virulence of A. fumigatus in the early stages. However, Δ sdh1 was discovered to be resistant to triazoles and to exhibit no substantial alterations in susceptibility to CAS. sdh1 deletion may result in azole resistance by affecting the structure of the cell membrane rather than the cell wall. Our findings suggest that sdh1 potentially influences mitochondrial membrane potential and diminishes SDH activity by modifying plasma membrane permeability. This alteration subsequently results in reduced ATP production and ROS levels, while concurrently upregulating the expression of drug efflux pump genes like cdr1B and increasing ergosterol production. Ultimately, this cascade of events culminates in decreased sensitivity to azoles and impaired growth. Notably, the use of SDHIs may induce acquired azole resistance in A. fumigatus according Sdh1 suppression and further efflux upregulation, suggesting a potential new resistance mechanism. This is particularly concerning given the widespread agricultural use of SDHIs and their persistent environmental residues. Therefore, field sampling in regions where SDHIs are applied is crucial to isolate azole-resistant strains and conduct further investigations. This emerging resistance pattern underscores the importance of exploring resistance mechanisms linked to non-azole drugs. Additionally, SDH-related testing is recommended for clinical isolates with unclear resistance profiles. Declarations Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Availability of data and materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests: The authors declare that they have no competing interests. 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: Conceptualization, YS and HZ; methodology, ZLZ; software, XG and WXC; validation, ZLZ; formal analysis, WZL; investigation, TYM; resources, QWH; data curation, LYL; writing original draft preparation, HZ; writing review and editing, YS and HZ; visualization, ZD and LD; supervision, ZLZ; project administration, WXC; funding acquisition, YS and HZ. All authors have read and agreed to the published version of the manuscript. Acknowledgments: We thank everyone who contributed to the success of this research, including colleagues, institutions, and funding bodies. References Verweij PE, Snelders E, Kema GH, Mellado E, Melchers WJ. 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Brun S, Berges T, Poupard P, Vauzelle-Moreau C, Renier G, Chabasse D, Bouchara JP. Mechanisms of azole resistance in petite mutants of Candida glabrata . ANTIMICROB AGENTS CH. 2004;48(5):1788–96. Hallstrom TC, Moye-Rowley WS. Multiple signals from dysfunctional mitochondria activate the pleiotropic drug resistance pathway in Saccharomyces cerevisiae . J BIOL CHEM. 2000;275(48):37347–56. Fraczek MG, Bromley M, Buied A, Moore CB, Rajendran R, Rautemaa R, Ramage G, Denning DW, Bowyer P. The cdr1B efflux transporter is associated with non- cyp51a -mediated itraconazole resistance in Aspergillus fumigatus . J ANTIMICROB CHEMOTH. 2013;68(7):1486–96. Chung D, Barker BM, Carey CC, Merriman B, Werner ER, Lechner BE, Dhingra S, Cheng C, Xu W, Blosser SJ, et al. ChIP-seq and in vivo transcriptome analyses of the Aspergillus fumigatus SREBP SrbA reveals a new regulator of the fungal hypoxia response and virulence. PLOS PATHOG. 2014;10(11):e1004487. Hagiwara D, Miura D, Shimizu K, Paul S, Ohba A, Gonoi T, Watanabe A, Kamei K, Shintani T, Moye-Rowley WS, et al. A Novel Zn2-Cys6 Transcription Factor AtrR Plays a Key Role in an Azole Resistance Mechanism of Aspergillus fumigatus by Co-regulating cyp51A and cdr1B Expressions. PLOS PATHOG. 2017;13(1):e1006096. Alcazar-Fuoli L, Mellado E. Ergosterol biosynthesis in Aspergillus fumigatus : its relevance as an antifungal target and role in antifungal drug resistance. FRONT MICROBIOL. 2012;3:439. Kodedova M, Sychrova H. Changes in the Sterol Composition of the Plasma Membrane Affect Membrane Potential, Salt Tolerance and the Activity of Multidrug Resistance Pumps in Saccharomyces cerevisiae . PLoS ONE. 2015;10(9):e139306. Pasrija R, Panwar SL, Prasad R. Multidrug transporters CaCdr1p and CaMdr1p of Candida albicans display different lipid specificities: both ergosterol and sphingolipids are essential for targeting of CaCdr1p to membrane rafts. ANTIMICROB AGENTS CH. 2008;52(2):694–704. Furukawa T, van Rhijn N, Fraczek M, Gsaller F, Davies E, Carr P, Gago S, Fortune-Grant R, Rahman S, Gilsenan JM, et al. The negative cofactor 2 complex is a key regulator of drug resistance in Aspergillus fumigatus . NAT COMMUN. 2020;11(1):427. Additional Declarations No competing interests reported. Supplementary Files manuscriptsup.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5441096","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":383013859,"identity":"91fbfc9e-1bd6-40e8-9aaa-c4c0c0bd5866","order_by":0,"name":"Heng Zhang","email":"","orcid":"","institution":"Jingzhou Hospital Affiliated to Yangtze University","correspondingAuthor":false,"prefix":"","firstName":"Heng","middleName":"","lastName":"Zhang","suffix":""},{"id":383013860,"identity":"14a11636-687c-4e00-8c91-35a0a52dbcc5","order_by":1,"name":"Zhangling Zhu","email":"","orcid":"","institution":"Jingzhou Hospital Affiliated to Yangtze University","correspondingAuthor":false,"prefix":"","firstName":"Zhangling","middleName":"","lastName":"Zhu","suffix":""},{"id":383013861,"identity":"280253ea-fef5-4298-9ee9-2360da1a5ca3","order_by":2,"name":"Xiao Gong","email":"","orcid":"","institution":"Jingzhou Hospital Affiliated to Yangtze University","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Gong","suffix":""},{"id":383013862,"identity":"d99c0e99-2f56-4ac3-b3d9-b388cfb4510e","order_by":3,"name":"Wenxu Cheng","email":"","orcid":"","institution":"Jingzhou Hospital Affiliated to Yangtze University","correspondingAuthor":false,"prefix":"","firstName":"Wenxu","middleName":"","lastName":"Cheng","suffix":""},{"id":383013863,"identity":"a6129ddc-0308-4fa4-b6f5-94e745ba77d1","order_by":4,"name":"Weizu Liao","email":"","orcid":"","institution":"Yangtze University","correspondingAuthor":false,"prefix":"","firstName":"Weizu","middleName":"","lastName":"Liao","suffix":""},{"id":383013864,"identity":"409fd9e5-dabd-46a3-b480-7326516b1208","order_by":5,"name":"Tianyan Ma","email":"","orcid":"","institution":"Yangtze University","correspondingAuthor":false,"prefix":"","firstName":"Tianyan","middleName":"","lastName":"Ma","suffix":""},{"id":383013865,"identity":"3d6e3e2e-72d7-4471-86e9-0a8d70eeb06f","order_by":6,"name":"Qingwen Hu","email":"","orcid":"","institution":"Yangtze University","correspondingAuthor":false,"prefix":"","firstName":"Qingwen","middleName":"","lastName":"Hu","suffix":""},{"id":383013866,"identity":"b2164c27-264e-4dd7-868b-73a381740fea","order_by":7,"name":"Linyun Li","email":"","orcid":"","institution":"Jingzhou Hospital Affiliated to Yangtze University","correspondingAuthor":false,"prefix":"","firstName":"Linyun","middleName":"","lastName":"Li","suffix":""},{"id":383013867,"identity":"0fd032d8-d15a-49e3-92a4-5d76ed695e23","order_by":8,"name":"Zha-xi Dun-zhu","email":"","orcid":"","institution":"The Central Hospital of Jiacha County","correspondingAuthor":false,"prefix":"","firstName":"Zha-xi","middleName":"","lastName":"Dun-zhu","suffix":""},{"id":383013868,"identity":"5905dd69-0d12-4ea3-9084-164db4ef9be2","order_by":9,"name":"Lha-zom Drol-ga","email":"","orcid":"","institution":"The Central Hospital of Jiacha County","correspondingAuthor":false,"prefix":"","firstName":"Lha-zom","middleName":"","lastName":"Drol-ga","suffix":""},{"id":383013869,"identity":"efb88e87-4770-40af-bfda-720f6553f9e3","order_by":10,"name":"Yi Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYBACxgYog5+Z+fAD0rRItrOlGZBmncF5HgUJolQy9x9/Js3bVpe4+TAPgwFDjU00EQ47kGzM23Y4cdth3gMPGI6l5TYQ1NLYcPAxb9sBoBa+BAPGhsNEaGkGKgM7rJnHQII4LW3MjEBbmBM3MBOtpYeN2XDOucPGMw4DAzmBGL8YAkNM4k1ZnWx//+HDDz7U2BChBaiCiZeNwRGsMoGQchCQBznuxx8Ge2IUj4JRMApGwQgFACr4QDg9vz6MAAAAAElFTkSuQmCC","orcid":"","institution":"Jingzhou Hospital Affiliated to Yangtze University","correspondingAuthor":true,"prefix":"","firstName":"Yi","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2024-11-12 16:08:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5441096/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5441096/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71876049,"identity":"59129065-00da-4cb7-af02-bbdf3b364c22","added_by":"auto","created_at":"2024-12-19 10:54:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":228552,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in SDH-related genes in induced resistant strains\u003c/p\u003e\n\u003cp\u003eNote: The results of SDHI-induced resistance to azole drugs. \u003cstrong\u003eA\u003c/strong\u003e The x-axis indicates the detection of sensitivity to azole drugs after co-cultivation with SDHIs. It was ultimately found that 4 strains had their sensitivity to azole drugs increased to eight times the original level: The AF08 strain was cultured in 0.1 μg/mL Fluopyram for 96 hours, resulting in an eightfold increase in the MIC of ITR (from 0.5 to 4 μg/mL). The AF06 strain was cultured in 1mL boscalid for 96 hours, leading to an eightfold increase in the MIC of VOR (from 0.25 to 2 μg/mL). The AF05 strain was cultured in 1μg/mL boscalid for 72 hours, causing an eightfold increase in the MIC of POS (from 0.125 to 1μg/mL). The AF07 strain was cultured in 5 μg/mL fluopyram for 72 hours, resulting in an eightfold increase in the MIC of VOR (from 0.25 to 2 μg/mL). \u003cstrong\u003eB-E\u003c/strong\u003e The change in\u003cem\u003e sdh1\u003c/em\u003e expression levels with the greatest change in drug sensitivity is detected. Red bars represent post-induction measurements, while gray bars represent pre-induction measurements.\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5441096/v1/7cfd8aa257123836866d5431.png"},{"id":71876784,"identity":"10cc7093-4fab-4c1b-b89b-938216f32cb2","added_by":"auto","created_at":"2024-12-19 11:02:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1509896,"visible":true,"origin":"","legend":"\u003cp\u003eBiological information related to Sdh1\u003c/p\u003e\n\u003cp\u003eNote: \u003cstrong\u003eA-C\u003c/strong\u003e Protein structure model of protein Sdh1 in \u003cem\u003eS. cerevisiae, A. fumigatus and \u003c/em\u003eHomo sapiens; \u003cstrong\u003eD-E\u003c/strong\u003e Structural alignment results of Sdh1 with Sdh1p and SDH1. \u003cstrong\u003eF\u003c/strong\u003eStructural domain positions of Sdh1. \u003cstrong\u003eG \u003c/strong\u003eProtein sequence alignment results of Sdh1 with Sdh1p and SDH1.\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5441096/v1/7a9b9db0085879d28922bc84.png"},{"id":71876052,"identity":"6ff71a76-c52c-4b4d-8d70-8b50fd7a32e9","added_by":"auto","created_at":"2024-12-19 10:54:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6736186,"visible":true,"origin":"","legend":"\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. It can be visualized that the VOR, POS, and ITC zones of inhibition of Δ\u003cem\u003esdh1\u003c/em\u003e are significantly reduced and the sensitivity of Δ\u003cem\u003esdh1\u003c/em\u003e to VOR, POS, and ITC is also reduced.\u003c/p\u003e\n\u003cp\u003eComparison of the results of the E-test\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5441096/v1/c3b3aad8b55b59bac9768c07.png"},{"id":71875970,"identity":"e3bf3a25-269a-4d94-aae7-5ac150e6c280","added_by":"auto","created_at":"2024-12-19 10:54:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":425767,"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\u003e D.ROS 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":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5441096/v1/3fb76f89aaba16f84c07b7cb.png"},{"id":71876044,"identity":"a689aaba-dbcf-4bbf-84fa-f3c2afacfb83","added_by":"auto","created_at":"2024-12-19 10:54:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3454309,"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\u003c/strong\u003e and \u003cstrong\u003eB\u003c/strong\u003e show 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. Δ\u003cem\u003esdh1\u003c/em\u003e showed a marked slowdown in the growth at different concentrations or in different media, did not show a green color for the period when it had a significant proliferative capacity, and its ability to form conidia was greatly reduced. \u003cem\u003esdh1\u003c/em\u003e is indispensable for proliferation. \u003cstrong\u003eC\u003c/strong\u003e Deletion of \u003cem\u003esdh1\u003c/em\u003e significantly alters the morphology of the conidial head: Under conventional light microscopy or stained with lactophenol cotton blue, the terminal capsules of Δ\u003cem\u003esdh1\u003c/em\u003e conidial heads were clearly observed to be smooth and reduced in size, the peduncle and bottle peduncle were rare, the conidia were missing, and their ability to grow and proliferate was diminished. ( \u003cem\u003e****P \u003c/em\u003e\u0026lt;0.0001. )\u003c/p\u003e","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5441096/v1/5dcdad57c2e5cc8dd6b1ebfc.png"},{"id":71875971,"identity":"020c7ca2-2e43-4881-a198-297f90e056e6","added_by":"auto","created_at":"2024-12-19 10:54:06","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":841180,"visible":true,"origin":"","legend":"\u003cp\u003eThe Effect of\u003cem\u003e sdh1\u003c/em\u003e on the Synthesis of Ergosterol\u003c/p\u003e\n\u003cp\u003eNote:The deletion of the\u003cem\u003e sdh1 \u003c/em\u003egene significantly affects key genes in the ergosterol synthesis pathway, ultimately leading to an increase in ergosterol content. \u003cstrong\u003eA\u003c/strong\u003e Effects of\u0026nbsp;\u003cem\u003esdh1\u003c/em\u003e\u0026nbsp;deletion on the expression of the genes involved in ergosterol iosynthesis. Heatmap showing the RNA-seq expression levels of the genes involved in ergosterol biosynthesis. \u003cstrong\u003eB \u003c/strong\u003eThe putative ergosterol biosynthetic pathway in\u0026nbsp;\u003cem\u003eA. fumigatus\u003c/em\u003e.\u0026nbsp;\u0026nbsp;The genes highlighted in red are those whose expression levels were upregulated in the \u003cem\u003esdh1\u0026nbsp;\u003c/em\u003enull mutant compared with the WT. \u003cstrong\u003eC\u003c/strong\u003e Absolute quantitative detection results of ergosterol. \u003cstrong\u003eD\u003c/strong\u003e Relative expression levels of development genes, which were obtained by qRT-PCR and normalized against the \u003cem\u003eβ\u003c/em\u003e-tubulin gene. (\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":"OnlineFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5441096/v1/c81170e0c6cbb162327f1f27.png"},{"id":71876043,"identity":"7f9dd1ee-529f-4e8c-bb6e-db3797539645","added_by":"auto","created_at":"2024-12-19 10:54:08","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":274247,"visible":true,"origin":"","legend":"\u003cp\u003eThe Effect of\u003cem\u003e sdh1\u003c/em\u003e on the relative transcript level\u003c/p\u003e\n\u003cp\u003eNote:The Effect of\u003cem\u003e sdh1\u003c/em\u003e on the relative transcript level. \u003cstrong\u003eA\u003c/strong\u003e The expression levels of the other three subunits of SDH. \u003cstrong\u003eB\u003c/strong\u003eRelative expression levels of \u003cem\u003ecdr1B\u003c/em\u003e. \u003cstrong\u003eC\u003c/strong\u003e Relative expression levels of \u003cem\u003esdh1\u003c/em\u003e in WT and Δ\u003cem\u003ecdr1B. \u003c/em\u003e(\u003cem\u003e** P\u003c/em\u003e\u0026lt;0.01;\u003cem\u003e **** P\u003c/em\u003e\u0026lt; 0.0001; ns, not significant.)\u003c/p\u003e","description":"","filename":"OnlineFigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-5441096/v1/5e62eca08cfbb56c4aa97609.png"},{"id":71876055,"identity":"d4fc2e63-2c04-4ced-9b0a-b3ce9a587224","added_by":"auto","created_at":"2024-12-19 10:54:13","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":913059,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification differentially expressed genes and the data mining.\u003c/p\u003e\n\u003cp\u003eNote: Identification of \u003cem\u003esdh1\u003c/em\u003e-related differentially expressed genes (DEGs) and the data mining. \u003cstrong\u003eA\u003c/strong\u003e GO analysis on the \u003cem\u003esdh1\u003c/em\u003e-related DEGs. Top twenty pathways ranked by gene count as enriched by GO were presented. \u003cstrong\u003eB\u003c/strong\u003e Heatmap of top 20 up- and down-regulated DEGs triggered. \u003cstrong\u003eC\u003c/strong\u003e. KEGG enrichment of the \u003cem\u003esdh1\u003c/em\u003e-related DEGs. The \"ribosome\"-related biowriting pathway is significantly enriched. The \"Meiosis\"-related pathway is also significantly enriched. \u003cstrong\u003eD\u003c/strong\u003eVolcano plot of \u003cem\u003esdh1\u003c/em\u003e-related DEGs. The DEGs with fold change\u0026gt;2 and \u003cem\u003eP\u003c/em\u003e-value\u0026lt;0.05 were considered of significance. \u003cstrong\u003eE-F\u003c/strong\u003e GSEA analysis.The analysis was carried out by comparing the raw data between the \u003cem\u003esdh1\u003c/em\u003e group and the WT group . “Δ\u003cem\u003esdh1\u003c/em\u003e” was designated as the permutation type, and “Δ\u003cem\u003esdh1\u003c/em\u003eversus WT” was applied to display the result. Consistent with the KEGG enrichment results, the \"ribosome\"-related pathway was enriched, and the \"Iron ion binding\" pathway was also shown.\u003c/p\u003e","description":"","filename":"OnlineFigure8.png","url":"https://assets-eu.researchsquare.com/files/rs-5441096/v1/2dc6b17142c582e271276a6c.png"},{"id":71876045,"identity":"b242957b-aac2-41b7-827d-d9f9d55cc8e6","added_by":"auto","created_at":"2024-12-19 10:54:09","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":194377,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e test of Δ\u003cem\u003esdh1\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNote: The total number of \u003cem\u003eG. mellonella\u003c/em\u003e is 20. And the final survival of \u003cem\u003eG. mellonella\u003c/em\u003e did not change significantly on the sixth day after infection with the three \u003cem\u003eA. fumigatus\u003c/em\u003e spores, but the survival of infection with Δ\u003cem\u003esdh1\u003c/em\u003e was significantly higher in the first three days, probably due to the slow release of virulence under the premise of slow growth after the absence of \u003cem\u003esdh1\u003c/em\u003e. (Breslow: Log Rank test was used to determine the difference, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"OnlineFigure9.png","url":"https://assets-eu.researchsquare.com/files/rs-5441096/v1/eb76cd9125f9547394df5188.png"},{"id":77645956,"identity":"a0c083d5-a518-412a-9994-1e99207face1","added_by":"auto","created_at":"2025-03-03 23:47:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20738868,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5441096/v1/558aaca8-ee5a-4971-b71c-c4c5b88973fb.pdf"},{"id":71876048,"identity":"d11e3dcb-d568-4a64-a69b-7b74dd8896fe","added_by":"auto","created_at":"2024-12-19 10:54:10","extension":"docx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":560013,"visible":true,"origin":"","legend":"","description":"","filename":"manuscriptsup.docx","url":"https://assets-eu.researchsquare.com/files/rs-5441096/v1/34fd23a399a20ed858ee2781.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Succinate Dehydrogenase Inhibitors as Triggers of Azole Resistance in Aspergillus fumigatus: The Role of sdh1 and Efflux Pathways","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]. 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. 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 citationid=\"CR8\" class=\"CitationRef\"\u003e8\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=\"CR9\" class=\"CitationRef\"\u003e9\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=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\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 widespread agricultural use, SDHIs can enter water bodies through leaching, runoff, or atmospheric drift, contaminating soil and aquatic environments[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\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=\"CR14\" class=\"CitationRef\"\u003e14\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":"\u003cp\u003e\u003cstrong\u003e2.1 \u0026nbsp;Strains and plasmids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eA. fumigatus\u0026nbsp;\u003c/em\u003eAF293 (Fungal Genetics Stock Center), used as the parent strain for all amplification in this investigation, includes the whole DNA sequence of \u003cem\u003eA. fumigatus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eA. fumigatus\u003c/em\u003e A1160 (\u0026Delta;\u003cem\u003eKU80, pyrG\u003csup\u003e\u0026nbsp;-\u003c/sup\u003e\u003c/em\u003e) (Fungal Genetics Stock Center) is an uracil (U)-deficient strain, defective in the \u003cem\u003epyrG\u003c/em\u003e gene, and unable to grow on U-deficient media. In the knockout process of this study, the \u003cem\u003epyrG\u003c/em\u003e gene was introduced and then allowed to grow in a U-free medium as the host strain. The screening marker \u003cem\u003epyrG\u003c/em\u003e was derived from the plasmid pBARGPE1-Pyrg-TagRFP\u003cem\u003e\u0026nbsp;\u003c/em\u003e(Wensheng Biotechnology, Hunan, China). The WT (\u0026Delta;\u003cem\u003eKU80, pyrG\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e) strain was constructed using A1160. Plasmid\u003cem\u003e\u0026nbsp;\u003c/em\u003epCT74\u003cem\u003e\u0026nbsp;\u003c/em\u003e(Fenghui Biotechnology, Hunan, China)\u003cem\u003e\u0026nbsp;\u003c/em\u003ecarries the hygromycin resistance (\u003cem\u003ehph\u003c/em\u003e) gene. Transformants can be propagated in culture dishes containing hygromycin as a screening marker during the construction of the revertant.\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)[15].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Antifungals and SDHIs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll tested agents, including ITC, VOR, posaconazole (POS), caspofungin (CAS), Fluopyram, Boscalid, Thifluzamide, and\u0026nbsp;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\u0026thinsp;\u0026mu;g/mL).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Induction of Azole Resistance by SDHIs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e12 clinical isolates and WT were used as test strains. Fresh spores were harvested from cultures grown on SAB (Sabaurauds) solid medium for 3 days. The spores were suspended in RPMI 1640 liquid medium containing 0.1, 1, 2, 5, and 10 \u0026mu;g/mL SDHIs, based on the MIC of ipflufenoquin against\u0026nbsp;\u003cem\u003eA. fumigatus\u003c/em\u003e[16], 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 the standard M38-A2 broth microdilution method (described in section 2.4) was used to assess changes in susceptibility to three azole antifungal agents.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 \u0026nbsp;Minimum inhibitory concentration (MIC) assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe broth microdilution method of M38-A2 was conducted following the standards established by the Clinical Laboratory Standards Institute (CLSI)[17] to determine susceptibility to antifungal drugs. The antifungal agents were divided evenly into eight concentration gradients, ranging from 0.25 to 8 \u0026mu;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\u003e\u0026nbsp;Candida parapsilosis\u003c/em\u003e ATCC 22019 was selected as the quality control strain.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Detection of gene expression levels to related genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFungal tissue cultured on SAB medium for three days was collected. Total RNA was extracted using TriEasy LS Total RNA Extraction Reagent (Yeasen Biotechnology, Shanghai, China) and reverse-transcripted into cDNA using a HI Fair II 1st strand cDNA Synthesis Kit (Yeasen Biotechnology, Shanghai, China). Antifungal susceptibility-related genes were selected for testing their expression (Table S1). The relative gene expression level (2\u003csup\u003e-\u0026Delta;\u0026Delta;CT\u003c/sup\u003e) was calculated using the actin housekeeping gene control[18]. To minimize error, each repetition included three mutual control groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Construction of the sdh1 deletion strain and the complementation strain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe targeted knockout of \u003cem\u003esdh1\u003c/em\u003e was performed using a modified fusion PCR cassette and protoplast transformation technique[19], with the target gene being replaced by the \u003cem\u003epyrG\u003c/em\u003e gene in the pBARGPE1-Pyrg-TagRFP. Three distinct DNA pieces were amplified from AF293, each including a 1.2 kb region upstream and downstream of the \u003cem\u003esdh1\u003c/em\u003e coding sequence and a \u003cem\u003epyrG\u0026nbsp;\u003c/em\u003ecassette as the selectable marker (Table S2). Fusion PCR conditions were utilized as described [19]. A major band at 4.0 kb was shown after confirmation of the fusion PCR product by agarose gel electrophoresis (Fig.S1). The PEG-mediated protoplast method facilitated the transformation, and the construct was validated by sequencing at Sangon Biotech, confirming the successful generation of \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e. For complementation, the plasmid pCT74, containing the\u003cem\u003e\u0026nbsp;hph\u0026nbsp;\u003c/em\u003egene, was employed. The \u003cem\u003esdh1\u003c/em\u003e expression cassette from AF293 was amplified, ligated with the pCT74 plasmid using the Hieff Clone\u0026reg; Plus One Step Cloning Kit (Yeasen Biotechnology, Shanghai, China), and introduced into the \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e strain through the same transformation procedure to generate the complementation strain \u0026Delta;\u003cem\u003esdh1::sdh1\u003csup\u003e+\u003c/sup\u003e\u003c/em\u003e (Fig.S1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 E-test drug susceptibility testing\u003c/strong\u003e\u003c/p\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\u003cp\u003e\u003cstrong\u003e2.8 Growth rate determination and observation of mycelial morphology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFresh spores were collected and prepared into suspensions at concentrations of 10⁵, 10⁶, and 10⁷ CFU/mL. A 1\u0026mu;L aliquot of each suspension was inoculated onto solid CZA (Czapeck) and SAB media, and the colony diameter was recorded after 3 days. 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. The procedure was repeated three times on different days to minimize testing errors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9 Mitochondrial membrane potential and reactive oxygen species (ROS) Assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDCFH‐DA (2\u0026prime;, 7\u0026prime;‐dichlorodihydrofluorescein diacetate), a cell‐permeable fluorescent probe, was employed to detect intracellular ROS[20].\u0026nbsp;The specific procedure involved collecting fresh spores and preparing a suspension with a concentration of 1\u0026times;10⁶ CFU/mL. Pectinase was added, followed by incubation at 37\u0026deg;C and 2000 rpm for 6 hours to fully dissolve the cell wall. The suspension was washed twice with pre-cooled PBS (4\u0026deg;C, 2000 rpm, 5 minutes). DCFH-DA was then added to reach a final concentration of 10 mM. After a 30-minute incubation at 37\u0026deg;C, the spores were washed three times with PBS and resuspended. The flow cytometry data were generated on a Beckman Cytomics FC 500 BD FACSCanto II and analyzed with FlowJo v10 software. The excitation wavelength is 488\u0026nbsp;nm and the emission wavelength is 525\u0026nbsp;nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10 ATP Content, SOD, and SDH Activity Detection\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.11 RNA isolation and KEGG pathway enrichment analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpores (1x10⁶ CFU/mL) were added to SAB medium and incubated at 37\u0026deg;C for 3 days. Total RNA was extracted using the RNAprep Pure Plant Kit (Tiangen, Beijing, China). The RNA was used to construct cDNA libraries, which were sequenced on an Illumina NovaSeq platform to generate 150 bp paired-end reads. The raw reads were processed using the BMKCloud (www.biocloud.net) online platform. Gene expression levels were calculated based on fragmentsper kilobase of transcript per million fragments mapped (FPKM). Differential expression analysis of two conditions/groups was performed using the DESeq2（P-value \u0026lt; 0.01 \u0026amp; Fold Change\u0026ge;2）. The KOBAS database and clusterProfiler software were used to analyze differentially expressed genes in KEGG pathways[21].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.12 Detection of ergosterol content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal intracellular sterols were extracted as reported by Branch\u0026nbsp;with slight modifications[22]. Briefly, collect spores that have grown on SAB solid medium for three days and then place them in SAB liquid medium. The cultures were incubated for 16h with shaking at 35\u0026deg;C. The stationary-phase cells were harvested by centrifugation at 2,700rpm for 5min and washed once with sterile distilled water. The net wet weight of the cell pellet was determined. Three milliliters of 25% alcoholic potassium hydroxide solution (25g of KOH and 35mL of sterile distilled water, brought to 100mL with 100% ethanol), was added to each pellet and vortex mixed for 1min. Cell suspensions were transferred to 16- by 100-mm sterile borosilicate glass screw-cap tubes and were incubated in an 85\u0026deg;C water bath for 1h. Following incubation, tubes were allowed to cool to room temperature. Sterols were then extracted by addition of a mixture of 1mL of sterile distilled water and 3mL of\u0026nbsp;\u003cem\u003en\u003c/em\u003e-heptane followed by vigorous vortex mixing for 3min. The heptane layer was transferred to a clean borosilicate glass screw-cap tube and stored at \u0026minus;20\u0026deg;C for as long as 24h. Prior to analysis, a 20-\u0026mu;L aliquot of sterol extract was diluted fivefold in 100% ethanol and scanned spectrophotometrically between 240 and 300nm with a Nano Drop One (Thermo Fisher Scientific, US). Ergosterol content was calculated as a percentage of the wet weight of the cell by the following\u0026nbsp;equations: %ergosterol + %24(28)DHE = [(\u003cem\u003eA\u003c/em\u003e281.5/290) \u0026times;\u0026nbsp;\u003cem\u003eF\u003c/em\u003e]/pellet weight, %24(28)DHE = [(\u003cem\u003eA\u003c/em\u003e230/518) \u0026times;\u0026nbsp;\u003cem\u003eF\u003c/em\u003e]/pellet weight, and % ergosterol = [% ergosterol + % 24(28)DHE] \u0026minus; % 24(28)DHE, where\u0026nbsp;\u003cem\u003eF\u003c/em\u003e is the factor for dilution in ethanol and 290 and 518 are the E values (in percentages per centimeter) determined for crystalline ergosterol and 24(28)DHE, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.13 \u003cem\u003eGalleria mellonella\u003c/em\u003e \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003etoxicity assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eG. mellonella\u003c/em\u003e lacks an adaptive immune system and specialized immune cells, making it a simple and cost-effective model, widely used in this study to evaluate fungal virulence[23-26]. \u003cem\u003eG. mellonella\u003c/em\u003e larvae (200\u0026minus;250mg) were selected and injected with a spore suspension of\u003cem\u003e\u0026nbsp;A. fumigatus\u003c/em\u003e (2.0\u0026times;10⁸ CFU/mL) for infection. After injection, the larvae were incubated at 37\u0026deg;C, with monitoring every 24 hours for a total of 5 days. Larval performance was assessed according to the \u003cem\u003eG. mellonella\u0026nbsp;\u003c/em\u003eHealth Index Scoring System[27].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.14 Data processing software\u003c/strong\u003e\u003c/p\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 \u0026plusmn; 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\u0026nbsp;\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e"},{"header":"3 Result","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 SDHIs Induce Acquired Azole Resistance\u003c/h2\u003e\n \u003cp\u003eCulturing \u003cem\u003eA. fumigatus\u003c/em\u003e in the presence of SDHIs altered resistance to three commonly used azole antifungal drugs: VOR, ITR, and POS. Of the 2,496 strains tested, 4 strains exhibited an eightfold increase in MIC, 5 strains showed a fourfold increase, 94 strains had a twofold increase, and 50 strains showed a 0.5-fold decrease in MIC. In total, 103 strains displayed reduced sensitivity to azole drugs, while 50 strains showed increased sensitivity. Notably, prolonged exposure to SDHIs did not lead to cumulative changes in azole susceptibility. Furthermore, the tested SDHIs did not exhibit a fungicidal effect on \u003cem\u003eA. fumigatus\u003c/em\u003e, with MIC remaining above 32 \u0026micro;g/mL (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\n \u003cp\u003eSubsequent analysis of SDH-related gene expression in the strains that demonstrated an eightfold increase in MIC revealed a significant reduction in \u003cem\u003esdh1\u003c/em\u003e gene expression. In contrast, the expression levels of \u003cem\u003esdh2\u003c/em\u003e, \u003cem\u003ecybs\u003c/em\u003e, and \u003cem\u003eb560\u003c/em\u003e genes did not show consistent patterns of upregulation or downregulation. These findings suggest that decreased expression of the \u003cem\u003esdh1\u003c/em\u003e may play a crucial role in the observed increase in MIC. Overall, the results indicate that SDHIs may contribute to azole resistance in \u003cem\u003eA. fumigatus\u003c/em\u003e under complex environmental pressures by influencing \u003cem\u003esdh1\u003c/em\u003e expression (Fig.B-E).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Biological information of Sdh1 in \u003cem\u003eA. fumigatus\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eThrough a search of the NCBI database, the SDH subunit Sdh1 in \u003cem\u003eA. fumigatus\u003c/em\u003e, numbered AFUA_3G07810, was found on chromosome 3. Sequence analysis revealed that it has 2247 bases, encodes 647 amino acids and contains 6 exons and 5 introns, resembles the flavoprotein subunit of SDH in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, and that it can pair oxidizing succinic acid to transfer electrons to ubiquinone. According to the amino acid sequence alignment, the flavoprotein subunit Sdh1 of SDH in \u003cem\u003eS. cerevisiae\u003c/em\u003e had a 97% repeat rate and could match the protein\u0026apos;s three-dimensional structure (root mean square deviation\u0026thinsp;=\u0026thinsp;0.133) .\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Disruption of \u003cem\u003esdh1\u003c/em\u003e decreased \u003cem\u003eA. fumigatus\u003c/em\u003e susceptibility to azoles\u003c/h2\u003e\n \u003cp\u003eWe evaluated the effect of sdh1 on antifungal susceptibility using CLSI microdilution and E-test methods. Microdilution studies revealed no significant change in susceptibility between WT and \u0026Delta;\u003cem\u003esdh1:: sdh1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e, with ITC, VOR, and POS MICs of 0.5 \u0026micro;g/mL, 0.25 \u0026micro;g/mL, and 0.25 \u0026micro;g/mL, respectively. However, the MICs of ITC, VOR, and POS against \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e were 4 \u0026micro;g/mL, 2 \u0026micro;g/mL, and 2 \u0026micro;g/mL, respectively (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). We also observed the same results in the E-test, with the three azoles not showing differences between WT and \u0026Delta;\u003cem\u003esdh1:: sdh1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e but with a significantly reduced susceptibility in \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). With the deletion of the \u003cem\u003esdh1\u003c/em\u003e, the susceptibility of azoles was significantly decreased. The \u003cem\u003esdh1\u003c/em\u003e gene is a negative regulator of triazole susceptibility in \u003cem\u003eA. fumigatus\u003c/em\u003e. For CAS, we observed no significant susceptibility changes in WT, \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e, and \u0026Delta;\u003cem\u003esdh1:: sdh1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eResults of antifungal susceptibility tests\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003eAntifungal susceptibility\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eType\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eE-test MICs or MEC (\u0026micro;g/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eM38-A3 MICs or MEC (\u0026micro;g/mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStrain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026Delta;\u003cem\u003esdh1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026Delta;\u003cem\u003esdh1::sdh1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026Delta;\u003cem\u003esdh1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026Delta;\u003cem\u003esdh1::sdh1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePOS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVOR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eITC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCAS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003eNote: POS: posaconazole; VOR: voriconazole; ITC: itraconazole; CAS: caspofungin.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\u003cbr\u003e\n \u003cp\u003e\u003cstrong\u003e3.4 The effect of knockout of\u003c/strong\u003e \u003cstrong\u003esdh1\u003c/strong\u003e \u003cstrong\u003eon the activity of SDH, SOD and ROS, ATP content\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eFor continuous monitoring of SDH activity, the activity of SDH in \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e slowly increases with increasing growth time, but the activity decreases at each time point (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). When the three-day-old fungal tissue was tested on SAB solid medium, it was clearly observed that after the deletion of the s\u003cem\u003edh1\u003c/em\u003e, the ATP (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB), ROS content (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC) and SOD activity (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD) decreased. It is noteworthy that SOD can catalyze the dismutation of superoxide anion to generate H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e, which is an important antioxidant enzyme in organisms. In organisms, SOD maintains the redox balance in cells by clearing excess ROS. Low SOD activity indicates that the balance of ROS in cells is also broken.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 \u003cem\u003esdh1\u003c/em\u003e required for the normal growth of \u003cem\u003eA. fumigatus\u003c/em\u003e\u003c/h2\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\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\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. 5A-B). 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. 5C). 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\u003eFigure 5\u003c/strong\u003e s\u003cem\u003edh1\u003c/em\u003e is necessary for the normal growth of \u003cem\u003eA. fumigatus\u003c/em\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6 \u003cem\u003esdh1\u003c/em\u003e regulates genes related to ergosterol biosynthesis\u003c/h2\u003e\n \u003cp\u003eRNA-seq analysis of the transcriptome showed that, compared to WT, four transcription factors were significantly up-regulated among the 30 main transcription factors involved in ergosterol synthesis (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA-B). No significant down-regulated genes were observed. RT-qPCR analysis further confirmed that \u003cem\u003esdh1\u003c/em\u003e was involved in the transcriptional regulation of \u003cem\u003emvd1\u003c/em\u003e, \u003cem\u003eerg1\u003c/em\u003e, \u003cem\u003eerg2\u003c/em\u003e, and \u003cem\u003eerg5\u003c/em\u003e genes (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). Therefore, the deletion of \u003cem\u003esdh1\u003c/em\u003e leads to the overexpression of related genes in the ergosterol pathway. Diphosphomevalonate decarboxylase \u003cem\u003emvd1\u003c/em\u003e involved in the biosynthesis of mevalonate, an essential precursor substrate for ergosterol synthesis, are up-regulated in \u003cem\u003esdh1\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e\n \u003cp\u003eConsistent with our hypothesis, we also extracted the absolute ergosterol content of WT, \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e, and \u0026Delta;\u003cem\u003esdh1:: sdh1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e, and found that the ergosterol content in \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e increased (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD). We observed that compared to WT and \u0026Delta;\u003cem\u003esdh1:: sdh1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e, the ergosterol content of \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e increased by 125% and 129% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). WT and \u0026Delta;\u003cem\u003esdh1:: sdh1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e had similar ergosterol levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). This result suggests that the deletion of the \u003cem\u003esdh1\u003c/em\u003e activates the expression of genes related to the ergosterol pathway, ultimately increasing the production of ergosterol, which is a negative regulatory gene in the synthesis of the ergosterol pathway.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003e3.7 Deletion of s\u003cem\u003edh1\u003c/em\u003e induces up-regulation of drug sensitivity related genes\u003c/h2\u003e\n \u003cp\u003eAfter the deletion of the \u003cem\u003esdh1\u003c/em\u003e, the expression levels of the other three subunits did not show significant changes (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA). In order to investigate the reasons for the weakening of \u003cem\u003esdh1\u003c/em\u003e sensitivity to drugs, we continued to analyze the genes related to drug sensitivity. Finally, we found in RNA-seq that KEGG gene pathway enrichment indicated that the deletion of \u003cem\u003esdh1\u003c/em\u003e significantly affected the expression of 22 transporter genes involved in catalytic activity and transporter activity, of which 19 were up-regulated and 3 were down regulated (Table \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e). It is worth noting that the expression level of the azole exporter \u003cem\u003ecdr1B\u003c/em\u003e gene in strain \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e was significantly increased (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB). In addition, the sensitivity of \u003cem\u003eA. fumigatus\u003c/em\u003e to azole drugs is related to the expression of \u003cem\u003ecyp51A/B\u003c/em\u003e, but we did not see high expression of \u003cem\u003ecyp51A/B\u003c/em\u003e in \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e. Therefore, we constructed the \u003cem\u003ecdr1B\u003c/em\u003e gene-deficient strain \u0026Delta;\u003cem\u003ecdr1B\u003c/em\u003e by the same method and detected the changes of \u003cem\u003esdh1\u003c/em\u003e in \u0026Delta;\u003cem\u003ecdr1B\u003c/em\u003e and WT strains, and finally found that the expression of \u003cem\u003esdh1\u003c/em\u003e did not show significant changes in the two strains (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC). This indicates that \u003cem\u003esdh1\u003c/em\u003e negatively regulates the expression of \u003cem\u003ecdr1B\u003c/em\u003e, and \u003cem\u003esdh1\u003c/em\u003e is a limiting factor for \u003cem\u003ecdr1B\u003c/em\u003e expression. Overall, the absence of \u003cem\u003esdh1\u003c/em\u003e promotes high expression of efflux pump genes, especially in \u003cem\u003ecdr1B\u003c/em\u003e, which weakens sensitivity to azole drugs and is a negative regulatory factor for azole drug sensitivity.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\n \u003ch2\u003e3.8 RNA sequencing\u003c/h2\u003e\n \u003cp\u003eTo decipher the role of \u003cem\u003esdh1\u003c/em\u003e in the global regulation of gene expression in \u003cem\u003eA. fumigatus\u003c/em\u003e, transcriptome analysis was performed (fold change\u0026thinsp;\u0026gt;\u0026thinsp;2; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), 2647 genes showed differential expression, 7436 genes showed no difference, of which 1568 genes were up-regulated and 1079 genes were deleted. Differential genes are significantly enriched in the \u0026quot;translation\u0026quot; pathway in the GO database. The top significant Genetic Information Processing in KEGG category is \u0026ldquo;Ribosome\u0026rdquo; and the top significant Cellular Processes in KEGG category is \u0026ldquo;Meiosis \u0026rdquo;. The deletion of \u003cem\u003esdh1\u003c/em\u003e will lead to cell proliferation and meiosis changes, and eventually lead to slow growth. Further differential gene detection of cell cycle and meiosis revealed that the expression of APSES protein related genes was changed, in which the expression of \u003cem\u003estua\u003c/em\u003e was increased[\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. The transcription factors of the APSES protein maybe affect the cellular processes of \u003cem\u003eA. fumigatus\u003c/em\u003e, including growth, development and secondary metabolism, suggesting that \u003cem\u003esdh1\u003c/em\u003e may also participate in the regulatory activities of the aspses family on \u003cem\u003eA. fumigatus\u003c/em\u003e. To study the genes with subtle changes that are undetectable by GO and KEGG enrichments, GSEA was performed. The method uses priori defined gene sets to evaluate multiple genes involved in a single bio logical pathway, whether an individual gene is of statistical significance or not[\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. Consistent with the KEGG pathway enrichment results, Ribosome function-related biological pathways were altered. The bioprocesses that were not reflected by GO and KEGG, namely \u0026quot;Iron ion binding\u0026quot;, were uncovered by GSEA, suggesting that the pathway of iron metabolism may be altered. This result may be associated with ferroptosis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003e3.9 Disruption of \u003cem\u003esdh1\u003c/em\u003e reduces the initial virulence of \u003cem\u003eA. fumigatus\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eThe final survival rates of \u003cem\u003eG. mellonella\u003c/em\u003e infected with \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e, WT and \u0026Delta;\u003cem\u003esdh1:: sdh1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e ranged from 35\u0026ndash;45%, with no significant differences between the three strains (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e). However, the survival rate of \u003cem\u003eG. mellonella\u003c/em\u003e infected with \u0026Delta;\u003cem\u003esdh1\u003c/em\u003e was higher than that of WT and \u0026Delta;\u003cem\u003esdh1:: sdh1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e during the first 3 days (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating that the disruption of \u003cem\u003esdh1\u003c/em\u003e reduced the virulence of \u003cem\u003eA. fumigatus\u003c/em\u003e in the early stages but restored it in the later stages.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eResistance to azole drugs has always been an unavoidable challenge in the treatment of IA, and investigating the causes of resistance may be a breakthrough in preventing its occurrence. Although this study could not replicate the complex natural environment, we successfully induced strains with significantly reduced azole sensitivity, suggesting that SDHI induction may be a major contributing factor to azole resistance. Expression analysis pinpointed the key gene \u003cem\u003esdh1\u003c/em\u003e. Subsequently, we constructed an Sdh1 subunit knockout strain and confirmed that the \u003cem\u003esdh1\u003c/em\u003e is crucial for azole resistance, growth, and development in \u003cem\u003eA. fumigatus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe knockout of the \u003cem\u003esdh1\u003c/em\u003e resulted in a significant reduction in SDH activity, which subsequently impaired normal mitochondrial function. SDH integrity is linked to mitochondrial complex II function and the ability to produce ATP[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. SDH activity also has a direct impact on critical biological processes such as fungal growth and mitosis[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The reduction in SDH activity disrupts this essential process, leading to decreased electron transport chain efficiency and, consequently, impairing cellular energy metabolism. With the electron transport chain compromised, the oxidative phosphorylation pathway is hindered, causing a marked reduction in ATP production. This energy deficit not only impairs normal cell growth and metabolism but also exacerbates overall mitochondrial dysfunction. Furthermore, the dysfunction in the electron transport chain leads to electron leakage, with electrons being erroneously transferred to oxygen, forming superoxide (a precursor to ROS)[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. This increase in ROS production elevates oxidative stress within the cell, further damaging cellular structures and impairing function.\u003c/p\u003e \u003cp\u003eMitochondria are the primary source of ROS production and ATP production [\u003cspan additionalcitationids=\"CR34 CR35\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In recent years, studies suggest the associations between mitochondrial function, antifungal susceptibility, and virulence in fungi[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Although SOD, a key antioxidant enzyme, typically scavenges superoxide[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], its activity is significantly reduced following the \u003cem\u003esdh1\u003c/em\u003e knockout. The combined effect of increased ROS production and decreased SOD activity results in excessive ROS accumulation, which further intensifies oxidative stress and disrupts cellular homeostasis. The cumulative impact of these changes ultimately hampers cell growth and function, underscoring the critical role of the \u003cem\u003esdh1\u003c/em\u003e in maintaining mitochondrial integrity, oxidative balance, and energy metabolism. Studies on \u003cem\u003eC. glabrata\u003c/em\u003e and \u003cem\u003eS. cerevisiae\u003c/em\u003e have shown that mitochondrial dysfunction can result in azole resistance and susceptibility[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Several subunits of the respiratory complex located in the inner mitochondrial membrane (including the \u003cem\u003esdh1\u003c/em\u003e) are encoded on the mitochondrial genome, and the deletion of genes comprising the mitochondrial genome can act as key activators of the drug resistance pathway[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Impaired mitochondrial function leads to activation of the transcription factor \u003cem\u003epdr3\u003c/em\u003e in \u003cem\u003eS. cerevisiae\u003c/em\u003e and \u003cem\u003epdr1\u003c/em\u003e in \u003cem\u003eCandida glabrata\u003c/em\u003e, which in turn leads to altered activity of target genes encoding efflux pumps, such as \u003cem\u003ecdr1\u003c/em\u003e and \u003cem\u003ecdr2\u003c/em\u003e, ultimately leading to antifungal resistance [\u003cspan additionalcitationids=\"CR42 CR43 CR44\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Similarly, after the deletion of the \u003cem\u003esdh1\u003c/em\u003e, mitochondrial changes activated efflux pump resistance pathways, including the \u003cem\u003ecdr1B\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eRNA-seq analysis showed that the deletion of \u003cem\u003esdh1\u003c/em\u003e led to the massive activation of ABC transporter genes, and 20 of the 22 genes with differences were up-regulated, including \u003cem\u003ecdr1B\u003c/em\u003e. We continued to study the relationship between \u003cem\u003esdh1\u003c/em\u003e and \u003cem\u003ecdr1B\u003c/em\u003e, and found that the deletion of \u003cem\u003esdh1\u003c/em\u003e would promote the expression of \u003cem\u003ecdr1B\u003c/em\u003e, but the deletion of \u003cem\u003ecdr1B\u003c/em\u003e could not change the expression of \u003cem\u003esdh1\u003c/em\u003e, indicating that \u003cem\u003esdh1\u003c/em\u003e regulates the expression of \u003cem\u003ecdr1B\u003c/em\u003e unidirectionally. Ergosterol biosynthesis is a key process unique to fungi.\u003c/p\u003e \u003cp\u003eAntifungal drugs such as azoles and polyenes directly target this biosynthetic pathway or ergosterol molecules. \u003cem\u003esrbA\u003c/em\u003e was shown to regulate expression of multiple genes related to ergosterol biosynthesis pathway, including \u003cem\u003eerg3\u003c/em\u003e, \u003cem\u003eerg24A\u003c/em\u003e, and \u003cem\u003eerg25A\u003c/em\u003e. A mutant strain defective in \u003cem\u003esrbA\u003c/em\u003e showed hypersensitivity to azoles[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Similar results were also found in this study. The deletion of \u003cem\u003esdh1\u003c/em\u003e led to the high expression of \u003cem\u003emvd\u003c/em\u003e1, \u003cem\u003eerg\u003c/em\u003e1, \u003cem\u003eerg2\u003c/em\u003e and \u003cem\u003eerg5\u003c/em\u003e in the ergosterol synthesis pathway, which ultimately led to the increase in the absolute content of ergosterol and the change in sensitivity to azole drugs. Ergosterol is involved in many biological functions such as membrane fluidity, regulation of overall membrane proteins, activity and distribution, and cell cycle control[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Studies in \u003cem\u003eS. cerevisiae\u003c/em\u003e have highlighted that changes in the composition of the plasma membrane affect the function of transporters. For example, depletion in sterol levels caused by loss of \u003cem\u003eerg4\u003c/em\u003e or \u003cem\u003eerg6\u003c/em\u003e leads to a reduction in the activity of the multi-drug-resistance transporter Pdr5[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], and ergosterol is required to correctly localise the azole exporter Cdr1p in \u003cem\u003eCandida albican\u003c/em\u003es[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. This leads us to speculate that even relatively small increases in ergosterol content in the cell membrane may lead to large increases in azole resistance via an indirect effect on azole transporter levels[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. This conclusion also proves that \u003cem\u003esdh1\u003c/em\u003e affects the function of plasma membrane.\u003c/p\u003e \u003cp\u003eAnd from our \u003cem\u003ein vivo\u003c/em\u003e study, the survival rate of \u003cem\u003eG. mellonella\u003c/em\u003e infected with Δ\u003cem\u003esdh1\u003c/em\u003e was higher than that of WT and Δ\u003cem\u003esdh1:: sdh1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e during the first 3 days (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating that the disruption of \u003cem\u003esdh1\u003c/em\u003e reduced the virulence of \u003cem\u003eA. fumigatus\u003c/em\u003e in the early stages. However, Δ\u003cem\u003esdh1\u003c/em\u003e was discovered to be resistant to triazoles and to exhibit no substantial alterations in susceptibility to CAS. \u003cem\u003esdh1\u003c/em\u003e deletion may result in azole resistance by affecting the structure of the cell membrane rather than the cell wall.\u003c/p\u003e \u003cp\u003eOur findings suggest that \u003cem\u003esdh1\u003c/em\u003e potentially influences mitochondrial membrane potential and diminishes SDH activity by modifying plasma membrane permeability. This alteration subsequently results in reduced ATP production and ROS levels, while concurrently upregulating the expression of drug efflux pump genes like \u003cem\u003ecdr1B\u003c/em\u003e and increasing ergosterol production. Ultimately, this cascade of events culminates in decreased sensitivity to azoles and impaired growth.\u003c/p\u003e \u003cp\u003eNotably, the use of SDHIs may induce acquired azole resistance in \u003cem\u003eA. fumigatus\u003c/em\u003e according Sdh1 suppression and further efflux upregulation, suggesting a potential new resistance mechanism. This is particularly concerning given the widespread agricultural use of SDHIs and their persistent environmental residues. Therefore, field sampling in regions where SDHIs are applied is crucial to isolate azole-resistant strains and conduct further investigations. This emerging resistance pattern underscores the importance of exploring resistance mechanisms linked to non-azole drugs. Additionally, SDH-related testing is recommended for clinical isolates with unclear resistance profiles.\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:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The authors declare that they have no competing interests.\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 Conceptualization, YS and HZ; methodology, ZLZ; software, XG and WXC; validation, ZLZ; formal analysis, WZL; investigation, TYM; resources, QWH; data curation, LYL; writing original draft preparation, HZ; writing review and editing, YS and HZ; visualization, ZD and LD; supervision, ZLZ; project administration, WXC; funding acquisition, YS and HZ. All authors have read and agreed to the published version of the manuscript.\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\u003cli\u003e\u003cspan\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? LANCET INFECT DIS. 2009;9(12):789\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWiederhold NP, Verweij PE. \u003cem\u003eAspergillus fumigatus\u003c/em\u003e and pan-azole resistance: who should be concerned? CURR OPIN INFECT DIS. 2020;33(4):290\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeis JF, Chowdhary A, Rhodes JL, Fisher MC, Verweij PE. 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NAT COMMUN. 2020;11(1):427.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Aspergillus fumigatus, Succinate dehydrogenase, sdh1, Efflux pumps, Azole resistance","lastPublishedDoi":"10.21203/rs.3.rs-5441096/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5441096/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003eAspergillus fumigatus\u003c/em\u003e is a major cause of invasive aspergillosis in immunocompromised patients. The rise in antifungal resistance is linked to the use of succinate dehydrogenase inhibitors (SDHIs).\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eThis study investigates the mechanism of acquired azole resistance in \u003cem\u003eA. fumigatus\u003c/em\u003e, triggered by SDHIs, which are widely used agricultural fungicides.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eConidia of \u003cem\u003eA. fumigatus\u003c/em\u003e were co-cultured with four SDHIs (Boscalid, Thifluzamide, Fluopyram, Carboxin) to assess sensitivity to three azole drugs: voriconazole, itraconazole, and posaconazole. RT-qPCR identified genes related to resistance, focusing on \u003cem\u003esdh1\u003c/em\u003e, a gene encoding a succinate dehydrogenase subunit. A \u003cem\u003esdh1\u003c/em\u003e knockout strain was created to evaluate its impact on growth, azole sensitivity, ATP levels, superoxide dismutase (SOD) activity, and ergosterol biosynthesis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSDHI exposure increased resistance to azoles, with 4.12% of 2,496 strains showing higher minimum inhibitory concentration (MIC). Four strains had an eightfold MIC increase and reduced \u003cem\u003esdh1\u003c/em\u003e expression. The \u003cem\u003esdh1\u003c/em\u003e knockout strain showed impaired growth, increased azoles resistance, and lower reactive oxygen species (ROS), ATP production (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and SOD activity (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). RNA sequencing indicated that \u003cem\u003esdh1\u003c/em\u003e deletion upregulated efflux pump genes and enhanced ergosterol synthesis.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eSDHIs may induce azole resistance in \u003cem\u003eA. fumigatus\u003c/em\u003e by downregulating \u003cem\u003esdh1\u003c/em\u003e. The findings highlight a potential new resistance mechanism, providing insights for managing \u003cem\u003eA. fumigatus\u003c/em\u003e infections and azole resistance.\u003c/p\u003e","manuscriptTitle":"Succinate Dehydrogenase Inhibitors as Triggers of Azole Resistance in Aspergillus fumigatus: The Role of sdh1 and Efflux Pathways","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-19 10:53:26","doi":"10.21203/rs.3.rs-5441096/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"880208d4-5dea-4bb0-9876-598147df24c8","owner":[],"postedDate":"December 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-03T23:38:25+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-19 10:53:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5441096","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5441096","identity":"rs-5441096","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}