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Identification of the ADH gene family in Trichosporon asahii and the role of TaADH_like in pathogenicity and fluconazole resistance

BMC Genomics volume 26, Article number: 352 (2025) Cite this article

Abstract

Alcohol dehydrogenase has been studied in regulation of fungal growth and development, stress response and pathogenesis, but its function in T. asahii remains unexplored. In this study, we analyzed the ADH gene family in T. asahii for the first time, identifying six ADH genes and containing conserved ADH_N and ADH_Zinc_N domains. We constructed an overexpression strain of the most significantly differentially expressed gene TaADH_like and compared its phenotypes with those of the wild-type strain, focusing on colony morphology, biofilm biomass, stress response, drug resistance, and pathogenicity. The results showed that TaADH_like overexpression reduced sensitivity to hypoxic conditions, altered the hyphae-to-yeast transition, and led to slower growth, decreased colonization ability, reduced tissue damage, and lower lethality. Increased osmotic stress sensitivity and the involvement of the HOG MAPK pathway in the hyphae-to-yeast conversion contributed to the reduced colonization capacity of T. asahii. Furthermore, the overexpression of TaADH_like promoted biofilm formation and led to a slight enhancement in fluconazole resistance in T. asahii. This study is the first to elucidate the function of the alcohol dehydrogenase gene in T. asahii, providing a foundation for future genetic research on this pathogen.

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Introduction

Trichosporon asahii (T. asahii) belongs to the genus Trichosporon, which is widely found in nature and can colonize both animals and humans, invading the host through medical and surgical infections to cause disease [1]. It causes disseminated trichosporonosis, a complex fungal infection that can involve multiple organs, including superficial skin and hair infections, osteomyelitis, keratitis, meningitis, and endocarditis [2,3,4,5]. In recent years, an increasing number of clinical isolates have shown insensitivity to fluconazole in vitro drug sensitivity tests, leading to treatment failure in patients with invasive T. asahii infections [6, 7].

Alcohol dehydrogenase (ADH, EC 1.1.1.1), a zinc-containing enzyme that catalyzes the interconversion of aldehydes and alcohols, is ubiquitous in fungi, plants, and mammals. It plays critical roles in growth, morphogenesis, and pathogenesis under both aerobic and anaerobic condition across various fungal genera. In the dimorphic ascomycete Candida albicans (C. albicans), knockout of ADH1 significantly impairs hypha formation, resulting in the production of only conidia or short pseudohyphae in liquid media and reduced virulence [8]. Similarly, ADH1 deficiency in C. albicans inhibits hyphal development and mitochondrial oxidative phosphorylation, and lower energy production may lead to delayed trophic development [9]. In Botrytis cinerea, conidia from adh1 knockout mutant strains exhibit altered morphology, lower germination rates and reduced virulence [10]. T. asahii is a morphologically and physiologically complex, adaptable yeast-like fungus similar to C. albicans [11]. The yeast phase promotes rapid fungal growth and dissemination in circulation, while the hyphal phase facilitates attachment to and penetration of the infected host [12]. Moreover, T. asahii can adhere to and aggregate on implanted hypha devices to form biofilms, leading to persistent infections. Biofilm formation often results in therapeutic failure of antifungal drugs and evasion of the host immune response [13, 14], and it plays a crucial role in both virulence and drug resistance [15].

The alcohol dehydrogenase gene (Gene ID: evm.model.Chr08.320, Gene_symbol: TaADH_like) was identified as significantly down-regulated in previous experiments comparing wild-type and fluconazole-resistant strains of T. asahii through transcriptomic analyses (BioProject ID: PRJNA941075). In this study, we analyzed the ADH gene family and constructed TaADH_like overexpression strains. By comparing the colony morphology, biofilm biomass, stress response, drug resistance, and pathogenicity between the overexpression and wild-type strains, we initially explored the function of TaADH_like in T. asahii, thereby laying the foundation for further genetic studies of this fungus.

Materials and methods

Strains

The strains used in this study are listed in Table S1. The T. asahii YAN0802 (WT) was isolated from giant pandas and cultured in Sabouraud Dextrose Agar (SDA) medium at 28 °C for 3 d.

ADH gene family analysis

ADH gene family members were screened based on previous studies [16]. SMART (http://smart.embl.de/ (accessed on 18 June 2024)) was used to confirm the conserved domains. The phylogenetic trees of ADH were constructed using MEGA-X with maximum likelihood, the WAG + G amino acid model, and 1000 bootstrap replications, visualized by EvolView-v2 (https://evolgenius.info//evolview-v2/ (accessed on 18 June 2024)) [17, 18]. The MEME program (http://alternate.meme-suite.org/tools/meme (accessed on 18 June 2024)) was used to identify conserved motifs in the ADH sequence, with the number of motifs set to 10 and the minimum and maximum widths set to 10 and 50, respectively [19].ADH sequences were compared using MAFFT (https://mafft.cbrc.jp/ (accessed on 18 June 2024)), and TBTools v2.102 was used to visualize the results [20].

Construction of overexpression strains

Total RNA from the WT strain was extracted using the SteadyPure RNA Extraction Kit (Accurate Biotechnology (Hunan) Co., Ltd., Changsha, China) and a cDNA template was synthesized from the RNA via reverse transcription usingEvo M-MLV Plus cDNA Kit (Accurate Biotechnology (Hunan) Co., Ltd., Changsha, China). The DNA of the WT strain was extracted by Rapid Fungi Genomic DNA Isolation Kit (Sangon Biotech, Shanghai, China). The promoter (2000 bp) of the TDH3 gene with high transcriptome expression in the WT strain was selected as the promoter of the target gene TaADH_like (accession number PRJNA941075). The cDNA was used as the template to amplify the TaADH_like gene, and DNA was used as the template to amplify TDH3_Promoter. The fragments were ligated using the pclone007 Simple Vector Kit (TSINGKE, Beijing, China) and then transferred into E. coli Trelief™ 5α strains (TSINGKE, Beijing, China) for amplification [21].

Enzymes QuickCut™ ASC I (Yugong, Jiangsu, China) and Xho I (Takara, Beijing, China) sheared the pCAMBIA1300ura-GFP-Bar-MCS plasmid, and recombinant plasmids (P1300-HYG-TDH3_Promoter-TaADH_like) were obtained by multifragment recombination with linear plasmids, TDH3_Promoter, TaADH_like, and HYG according to ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, China). Recombinant plasmids were transferred into WT strains by A. tummefaciens AGL1 (WEIDI, Shanghai, China) mediated transformation method [21, 22]. Transformants were screened in SDA containing HYG (300 µg/mL) and cefotaxime sodium (200 µg/mL). PCR and fluorescence observation were used to verify the positive transformants, and RT-qPCR was used to determine the TaADH_like expression described previously [16], and the correct strain was named TaADH_likeOE. Primers are shown in Table S2.

Determination of the colony growth rate

Individual colonies in the medium were perforated using a 1 cm perforator and then transferred to fresh SDA medium, placed at 28 °C for incubation, and repeated 5 times. The colony diameter was measured daily for 25 consecutive days.

Morphological observation

Morphological differences in conidia and hyphae between the wild-type (WT) and TaADH_like overexpression (TaADH_likeOE) strains were examined using the copper ring culture method [23]. Conidia were eluted from the SDA medium with PBS buffer, and the concentration of conidia was 1 × 106 CFU/mL with hemocytometer plates. The 5 µL conidial suspension was inoculated into copper ring pores, cultured at 28 °C, and stained with lactophenol cotton blue (Biostime, Qingdao, China) at 4 h, 8 h, 12 h, 16 h, 20 h, 1 d, 3 d, 5 d and 7 d, and observed under a microscope (BX51, Olympus). The length and width of more than 100 conidia from WT and TaADH_likeOE strains were measured using ImageJ software.

Fluconazole sensitivity determination

The minimum inhibitory concentration (MIC) of fluconazole for the strains was determined according to the M27-A3 protocol recommended by the Clinical and Laboratory Standards Institute (CLSI) [24]. The MIC was assessed in 96-well plates using a 2-fold serial dilution of fluconazole, resulting in a final drug concentration range of 0.125–64 µg/mL. The inoculum concentration was adjusted to 5 × 10⁵ CFU/mL and 100 µL was added to each well. Plates were incubated for 48 h at 200 rpm/min at 28 °C. Each experiment was performed in triplicate.

Comparison of spot tests for susceptibility of WT and TaADH_likeOE strains to fluconazole inhibitory concentrations. SDA medium containing different concentrations of fluconazole (0, 2, 8, 16, 32, 64, 128, 256 µg/mL) was prepared. For each fluconazole concentration, 2 µL of conidial suspensions at 1 × 10⁷ CFU/mL, 1 × 10⁶ CFU/mL, and 1 × 10⁵ CFU/mL were spotted onto the respective SDA medium, incubated at 28℃ and observed after 48 h, with a 50% reduction of colonies as the observation threshold. Each experiment was performed in triplicate.

Stress experiment

SDA medium containing 0.2 mM CoCl2, SDS (0.004% W/V), CR (0.4 mg/mL and 1 mg/mL), 1.5 M NaCl, and 2 M sorbitol were prepared, respectively. Inoculate 2 µL of each concentration of conidial suspension 1 × 107 CFU/mL, 1 × 106 CFU/mL and 1 × 105 CFU/mL in medium and incubate at 28 ℃ for 3 d. The phenotypic differences between the strains were observed daily. The results were repeated three times.

Acetaldehyde content detection

The acetaldehyde content of the strains was determined using High Performance Liquid Chromatography (HPLC) (1260 infinityII, Agilent). Equal masses of WT and TaADH_likeOE strains were ground, and perchloric acid and sodium acetate were added in a volume twice that of the sample to adjust the pH to 4.0. The supernatant was transferred to a pre-cooled centrifuge tube, and an equal volume of DNPH (Aladdin, Shanghai, China) was added and shaken for 1 h. Subsequently, acetonitrile was added in a volume twice that of the sample and mixed by shaking for 30 min. The supernatant was spun dry in a rotary evaporator, and 2 mL of acetonitrile was dissolved for detection. The mobile phase was composed of 50% acetonitrile and 50% water, with a flow rate of 1 mL/min. The C18 solid phase extraction column was used, with a column temperature set at 30 °C and an injection volume of 20 µL. The results were repeated three times.

Determination of biofilm biomass

The quantification of biofilm biomass using the XTT reduction method was performed as previously described [25]. Each 96-well cell culture plate was added with 100 µL conidial suspension (1 × 106 CFU/mL), incubated at 28℃ for 2 h, lightly washed twice with PBS, and then re-added with 100 µL fresh SDB culture medium at 28℃. The culture medium was changed every 24 h. At 24, 48, and 72 h, the 96-well plates were removed, washed three times with PBS, and 200 µL of XTT-menadione solution was added to each well, and incubated at 28℃ for 4 h in the dark. Using 100 µL of liquid per well in a new 96-well plate, absorbance was measured at 492 nm (Varioskan Fish, Thermo). Each experiment was performed in triplicate.

ROS assay

The final concentration of 1 mg/mL Dihydroethidium (Beijing Solarbio Science & Technology Co., Ltd., China) 10 µL and 1 mL of conidial suspension (1 × 107 CFU/mL) were mixed thoroughly, incubated for 30 min at 37 ℃ in the dark, and then 200 µL of suspension was added into a black 96-well plate, and the fluorescence intensity (excitation wavelength of 518 nm, emission wavelength of 610 nm) was measured (Varioskan Fish, Thermo).The results were repeated three times. Meanwhile, 10 µL of suspension were aspirated to observe the fluorescence of the strain (BX51, Olympus).

Pathogenicity experiment

Animal

Eight-week-old female ICR mice (SPF(BEIJING)BIOTECHNOLOGY CO.,LTD., China) were immunosuppressed with 80 mg/kg cyclophosphamide and 25,000 units of penicillin sodium four days prior to the experiment. The mice were divided into three groups, A, B and C. Groups A and B were inoculated with 0.1 mL of 1 × 107 CFU/mL conidial suspensions of the WT and TaADH_likeOE strains, respectively, via the tail vein. Group C served as the control, inoculated with 0.1 mL of saline via the tail vein. The mice were fed and watered ad libitum, weighed at 1 d intervals, and recorded the survival rate.

Tissue fungal burden analysis

Mice in groups A and B were euthanized by cervical dislocation on 1, 3, 5 and 7 d (5 biological replicates), and heart, liver, spleen, lung, and kidney tissue fungal burden were determined by plate colony counting. Mouse hearts, livers, spleens, lungs, and kidneys were aseptically isolated, ground, and rinsed into sterile EP tubes with 2 mL of saline. Tissue solutions were diluted at 10− 1, 10− 2, and 10− 3 multiplicities, and 100 µL of each concentration was uniformly spread in SDA medium (3 replicates) and placed at 28 °C for 2–4 d. Colonies on the plates were counted, and the amount of fungal burden in the tissues was calculated. The fungal burden per gram of tissue was expressed as log (CFU/g), using the formula:

Fungal burden = log[X/(A/2 × 10− n×0.1)] [log(CFU/g)]

[X: number of colonies per plate count (CFU); A: mass of tissue organs (g); 10− n: dilution ratio; 2; volume of normal saline used in elution tissue (mL); 0.1: volume of suspended fluid used in inoculation plate (mL)]

Histopathological

Mice in groups A and B were euthanized by cervical dislocation on 1, 3, 5 and 7 d (5 biological replicates), and tissues of heart, liver, spleen, lungs and kidneys were removed and fixed in 4% paraformaldehyde for 24 h. They were routinely dehydrated and dipped in wax, embedded and sectioned, and then stained by Hematoxylin-Eosin staining (HE) and peroxodioic acid-Schiff staining (PAS), respectively.

RT-qPCR analysis

The strains were grown in Sabouraud Dextrose Broth (SDB) medium at 28 °C for 3 d at 200 rpm/min. RT-qPCR methods were as described previously [16]. WT and TaADH_likeOE strains total RNA was extracted and reverse transcribed to cDNA and 18sRNA was used as an internal reference for RT-qPCR assay. Primers are shown in Table S3-S5.

Data statistics

GraphPad Prism 9.5 was used to calculate the mean and standard deviation. Tissue fungal burden statistics were analyzed using a two-way ANOVA and all other tests were analyzed by a t-test using the Holm-Šídák method. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05.

Results

ADH gene family analysis

To understand the genetic evolutionary relationships of the alcohol dehydrogenase (ADH) gene family in T. asahii, comparative analysis of the ADH gene families in C. albicans and S. cerevisiae was conducted. Seventeen members of the ADH gene family containing both ADH_N and ADH_Zinc_N domains were identified, including six genes from T. asahii, six from S. cerevisiae, and five from C. albicans, which were labelled according to the genome annotations. The phylogenetic evolutionary tree divides 17 genes into three groups (Fig. 1-I). The predicted protein lengths encoded by the ADH genes ranged from 282 to 381 amino acids (aa). The molecular weights ranged from 30935.4 Da (CaADH3) to 40586.6 Da (CaFADH). The isoelectric point values of these TaADHs ranged from 5.58 to 8.65 and included 3 basic and 14 acidic proteins (Table 1).

The Zinc 1 binding motif, Zinc 2 binding motif and NADPH binding motif are important binding motifs for ADH. The Zinc 1 binding motif [GHE(x)2G(X)5G(X)2 V] contains catalytic zinc amino acid coordination residues. The Zinc 2 binding motif [GD(X) 9,10 C(X)2 C(X)2 C(X)7 C] contains structural zinc amino acid coordination residues (Fig. 1-III). The NADPH binding motif [GXG(X)2G] is highly conserved among ADH members (Fig. 1-III). The genes with higher homology have similar protein motif compositions. Motif1 and motif7 are unique to group B. Similarly, motif5 is a significant motif in groups A and B but absent in group C. All members of groups A and B, along with TaADH_like, contain motif3 and motif4, which may be the characteristic motifs of alcohol dehydrogenase (Fig. 1-IVA). The number of CDS regions varies from 1 to 6 across groups A-C. In C. albicans and S. cerevisiae, only CaFADH has two CDS regions, whereas the others have one CDS region, but all TaADH possess multiple CDS regions (Fig. 1-IVB).

Table 1 Detailed information of the ADH family members identified
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Fig. 1
figure 1

ADH gene family analysis. I, Phylogenetic analysis of the T. asahii, C. albicans and S. cerevisiae ADH gene family members. II, TaADH_like domain diagram. III, Partial results of amino acid sequence alignment of 17 ADH genes. IV, Phylogenetic tree, gene structure and conserved motifs of ADHs. A, Phylogenetic tree and motif analysis. B, Gene structure analysis of ADHs

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Transformant verification

The transformant was observed to green fluorescens in the dark field (Figure S1-I), and PCR amplification yielded a 4500 bp HYG-TDH3_Promoter-TaADH_like target band (Figure S1-II), with the mRNA level of TaADH_like about 59-fold higher than WT (P < 0.0001) (Figure S1-III). In conclusion, the TaADH_like overexpression strain was successfully constructed and named TaADH_likeOE strain.

Overexpression of TaADH_like decreased the germination and growth rate of T. asahii

TaADH_likeOE and WT were cultured on SDA medium for 25 days. WT exhibited significantly faster growth than TaADH_likeOE (P < 0.0001), with growth plateauing after 21 days (Fig. 2-I). Microscopic observations revealed differences in germination between TaADH_likeOE and WT (Fig. 2-II). Both TaADH_likeOE and WT conidia began to germinate at 4 h (Fig. 2-II A and a). At 8 h, WT conidia elongated and were significantly longer than TaADH_likeOE (Fig. 2-II B and b). At 12 h, TaADH_likeOE conidia were willow-like, and WT conidia developed septa and formed short hyphae (Fig. 2-II C and c). At 16 h, TaADH_likeOE willow-like conidia appeared septa, and a large number of conidia were still germinating. WT short hyphae with increased septa in the middle formed rectangular conidia (Fig. 2-II D and d). At 20 h, TaADH_likeOE was still germinating, WT conidia increased, and rectangular conidia gradually became round (Fig. 2-II E and e). In conclusion, TaADH_likeOE germinated slower than WT, consistent with the growth curve results. The length and width of over 100 conidia of both TaADH_likeOE and WT were measured, and the results showed that WT conidia tended to round while TaADH_likeOE conidia tended to oval (Fig. 2-III). The genes related to WT and TaADH_likeOE conidia were analyzed by RT-qPCR. The results indicated that genes associated with conidial development and maturation (apsA, umv1, vosA, DIT1, and fim1) were significantly down-regulated (Fig. 2-IV). These changes may have contributed to the altered conidial morphology and slower growth observed following TaADH_like overexpression.

Fig. 2
figure 2

Overexpression of TaADH_like decreased the germination and growth rate of T. asahii. I, Growth curve of TaADH_likeOE and WT. Results are expressed as mean ± SD. II, Microscopic observation on the growth difference between TaADH_likeOE and WT during germination. Scale, 10 μm. III, WT and TaADH_likeOE ratio of conidia length to width, Scale, 10 μm. IV, RT-qPCR analysis of regulating conidia-related genes. Positive and negative represent up- and down-regulation, and expression level is expressed as 2|−ΔΔCt|

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Overexpression of TaADH_like mediates T. asahii dimorphic transition

The morphology of TaADH_likeOE and WT showed obvious differences on SDA (Fig. 3-I). TaADH_likeOE colonies appeared small, creamy-white, and lustrous, with irregular growth and a moist surface characterized by prominent brain-like folds. In contrast, WT colonies are large, white and translucent, with neat radial outward growth at the edges, dry surface, and few downy folds.

The morphology differences were observed by microscope (Fig. 3-II). On Day 1, both TaADH_likeOE and WT strains showed a large number of conidia (Fig. 3-II A and a). On Day 3, TaADH_likeOE conidia had elongated, with the majority undergoing germination, while WT exhibited long chains of pseudohyphae and arthroconidia (Fig. 3-II B and b). On day 5, TaADH_likeOE conidia were mostly fusiform with large vacuoles in the middle, with many conidia linked together in clusters. WT displayed hyphae, accompanied by numerous pseudohyphae and arthroconidia, the conidia were mostly round (Fig. 3-II C and c). On Day 7, TaADH_likeOE had a large number of oval conidia and pseudohyphae, while WT showed elongated hyphae (Fig. 3-II D and d). These observations indicate that overexpression of TaADH_like caused T. asahii to transition from the hyphal phase to the yeast phase.

The genes related to WT and TaADH_likeOE hyphae were analyzed by RT-qPCR. BRG1, Ras1, Rac1, Rho1, CDC42 and cmkA genes related to hyphae growth, development and regulation were significantly down-regulated, and the NRG1 and RGF1 genes encoding hypha deterrent proteins were significantly up-regulated (Fig. 3-III). This coordinated regulation drove the transition of T. asahii from the hyphal phase to the yeast phase.

Fig. 3
figure 3

Overexpression of TaADH_like mediates T. asahii hyphae-to-yeast conversion. I, Colony morphology of WT and TaADH_likeOE. II, Morphology of conidia and hypha of WT and TaADH_likeOE. A-C and a-c scales are 10 μm, and D and d scales are 20 μm. III, RT-qPCR analysis of regulating hypha-related genes. Positive and negative represent up- and down-regulation, and an expression level is expressed as 2|−ΔΔCt|

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Slightly reduced fluconazole sensitivity

In the spot test, the concentration at which colony growth was reduced by 50% was 2 µg/mL for WT and 8 µg/mL for TaADH_likeOE (Fig. 4). In the MIC assay, the MIC values were approximately 4 µg/mL for WT and 8 µg/mL for TaADH_likeOE (Table 2). Due to the morphological characteristics of WT, some white floating colonies appeared in liquid culture, which caused discrepancies between the results and those of the spot test. Overall, TaADH_like overexpression led to a slight increase in resistance to T. asahii.

Fig. 4
figure 4

Fluconazole sensitivity determination of WT and TaADH_likeOE. The conidia concentration from left to right of the colony was 1 × 107, 1 × 106 and 1 × 105 CFU/mL

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Table 2 The minimum inhibitory concentration (MIC) was determined
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Stress experiment

CoCl2 is a chemical hypoxia mimetic in eukaryotic cells [26]. Compared to TaADH_likeOE, WT inoculated with 1 × 10⁶ and 1 × 10⁵ CFU/mL conidial suspensions on SDA exhibited significant inhibition (Fig. 5-I). This suggests that overexpression of TaADH_like reduces the sensitivity under hypoxic conditions. The integrity of the cell wall was assessed using cell wall disruptors sodium lauryl sulfate (SDS) and Congo red (CR) (Fig. 5-II). There was no significant difference in the growth of all tested strains, indicating that overexpression of TaADH_like did not affect the integrity of T. asahii cell wall. Additionally, it was found that WT growth was progressively inhibited with increasing CR concentration, primarily in the form of reduced radial hyphal growth. In contrast, TaADH_likeOE exhibited a yeast phase, and its growth was unaffected by CR concentration.

TaADH_likeOE exhibited significant suppression on SDA medium containing the osmotic stressors sorbitol and NaCl (Fig. 5-III). The expression of genes related to osmotic pressure regulation was analyzed by RT-qPCR (Fig. 5-IV). Following TaADH_like overexpression, genes associated with the osmolarity-related Sho1 and CnNIK1 (Sln1)-regulated HOG1 pathways were dysregulated. The Sho1 gene was most significantly downregulated, while the CnNIK1 gene was significantly upregulated. The Ste11 gene showed no differential expression, and the remaining genes in the regulatory pathway were downregulated. Overexpression of TaADH_like inhibited the Sho1 pathway, thereby enhancing the osmotic stress sensitivity of T. asahii, while the CnNIK1 pathway compensated for the Sho1 pathway.

Fig. 5
figure 5

Stress experiment. I, II and III were WT and TaADH_likeOE hypoxia, cell wall inhibitor and osmotic stress experiments, respectively. Control The conidia concentration from left to right of the colony was 1 × 107, 1 × 106 and 1 × 105 CFU/mL. IV, RT-qPCR analysis of regulating osmotic stress related genes. Positive and negative represent up- and down-regulation, and expression level is expressed as 2|−ΔΔCt|

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Acetaldehyde content and biofilm metabolic activity determination

Acetaldehyde, a toxic volatile organic compound with an irritating odor, was quantified by HPLC after being derivatized with 2,4-dinitrophenylhydrazine (DNPH) to form stable hydrazone compounds [27]. The regression equation of the standard curve was y=26005x + 4.047 (R2 = 0.9993, y = peak area, x = acetaldehyde-DNPH content). The acetaldehyde-DNPH content of the 20 µL sample volume of WT and TaADH_likeOE was 0.0081 µg and 0.0125 µg, respectively (Fig. 6-I). The overexpression of the TaADH_like increased acetaldehyde accumulation in T. asahii, potentially implicating its role in the interconversion between alcohols and aldehydes.

The biofilm metabolic activity of TaADH_likeOE was significantly higher than that of WT at 24, 48, and 72 h. The biofilm metabolic activity of TaADH_likeOE peaked at 48 h and maintained until 72 h (Fig. 6-II). Overexpression of TaADH_like enhanced biofilm metabolic activity in T. asahii.

Fig. 6
figure 6

Acetaldehyde content and biofilm metabolic activity determination. I, liquid chromatogram. A, B and C are chromatograms of acetaldehyde-DNPH standard, TaADH_likeOE and WT, respectively. II, WT and TaADH_likeOE biofilm metabolic activity assay

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ROS determination

Dihydroethidium (DHE) is a widely used cell-permeable fluorescent probe for detecting superoxide anion (O₂⁻) radicals [28]. When the level of superoxide anions inside the cell is high, more Ethidium is generated, resulting in stronger red fluorescence. At the same exposure time, the intensity of red fluorescence observed for TaADH_likeOE was significantly weaker than that of WT under the microscope (Fig. 7-I). Fluorescence intensity measurements revealed that TaADH_likeOE produced significantly lower fluorescence than WT (P < 0.001), consistent with the microscopic observations (Fig. 7-II).

Fig. 7
figure 7

WT and TaADH_likeOE ROS were detected. I, Microscopic observation of TaADH_likeOE and WT red fluorescence. Scale, 10 μm. II, Fluorescence intensity measurements for TaADH_likeOE and WT

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Pathogenicity

Mice inoculated with WT appeared to die on 1 d and experienced a significant mortality spike on 3 d, dropping the survival rate to less than 30%. In contrast, mice inoculated with TaADH_likeOE exhibited a slower rate of death and maintained a survival rate above 50% within 7 days (Fig. 8-I). Additionally, mice inoculated with WT and TaADH_likeOE showed a significant decrease in body weight compared to control mice (Fig. 8-II). Due to the high mortality of WT-inoculated mice within 3 days, the remaining number of mice was insufficient to continue the body weight change test, and thus only the body weight changes within the first 3 days were recorded. Overall, overexpression of TaADH_like reduced the lethality of T. asahii in mice.

The results of tissue fungal broaden analysis are shown in Fig. 8-III. The tissue fungal burden in mice inoculated with WT and TaADH_likeOE peaked at 3 d, which was significantly higher than that at 1 d (P < 0.0001). Subsequently, a decreasing trend was observed in the fungal burden of mice inoculated with TaADH_likeOE. On Day 1, the fungal burden in the heart, spleen, and kidney tissues of WT-inoculated mice was significantly higher (P < 0.0001) than that in TaADH_likeOE-inoculated mice. On Day 3, the fungal burden in the lung tissues of TaADH_likeOE-inoculated mice was significantly higher than that in WT-inoculated mice (P < 0.0001), while the remaining tissues exhibited lower burdens compared to WT-inoculated mice. Overall, WT colonised tissues stronger than the TaADH_likeOE strain.

Fig. 8
figure 8

Percent survival, weight and tissue fungal broaden analysis in mice inoculated with WT and TaADH_likeOE strains. I and II are WT and TaADH_likeOE survival and weight change, respectively. III, WT and TaADH_likeOE tissue fungal burden

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Histopathological

Pathological histology showed that WT-inoculated mice developed pathological damage earlier and exhibited more severe damage compared to TaADH_likeOE-inoculated mice. The TaADH_likeOE-inoculated mice showed infiltration of inflammatory cells between myocardial fibres on 3 d, and myocardial fibre lysis and rupture on 5 and 7 d, with an increase in inflammatory cells, which were similar to the results of the WT-inoculated mice on 3 d (Fig. 9-I). There were no obvious abnormalities in the liver tissues of TaADH_likeOE-inoculated mice on 1 d, and the cytoplasmal structure of some hepatocytes was damaged on 3 d, which was similar to WT-inoculated mice on 1 d. In both WT- and TaADH_likeOE-inoculated mice, the central vein and sinusoid space were enlarged and the lumen was filled with red blood cells on 3 and 5 d, respectively (Figure S2-I). The spleen tissues from WT-inoculated mice showed splenic pulp congestion at 1–3 d, with obvious connective tissue hyperplasia and thickened splenic trabeculae, leading to fibrosis (Figure S3-I). The kidney tissues from both WT- and TaADH_likeOE-inoculated mice exhibited adhesion of the renal capsule to the vascular balloon and occlusion of the balloon lumen on 1 and 3 d, respectively (Figure S4-I). However, the WT- and TaADH_likeOE-inoculated mice showed similar levels of damage in lung tissues (Figure S5-I).

All tissues exhibited the highest levels of conidial or hyphal infections on 3 d, which is consistent with the tissue fungal burden results (Fig. 9-II, S2-II, S3-II, S4-II, and S5-II). In the WT-inoculated mice, kidneys showed an elongated hyphal state on 3 d (Figure S4-II). No conidia were detected in the liver and spleen of the TaADH_likeOE-inoculated mice on 1 d (Figure S2-II and Figure S3-II). The higher tissue fungal burden in lung tissues of the TaADH_likeOE-inoculated mice on 3 d compared to the WT-inoculated mice. This may be due to the overexpression of TaADH_like, which caused the transition of T. asahii from the hyphal phase to the yeast phase. This shift may have altered the strain’s preference for tissue colonization, leading to a similar degree of pathological damage in lung tissues of WT- and TaADH_likeOE-inoculated mice after 3 d.

Fig. 9
figure 9

HE and PAS staining of heart tissue from mice infected with WT and TaADH_likeOE. I, HE staining of heart tissue. II, PAS staining of heart tissue. Representative HE and PAS stained sections of heart tissues 1–7 d and 1–3 d after infection of mice by control (A-D), TaADH_likeOE (E-H) and WT (I and J), respectively. Scale bar, 20 μm. Black arrows point to conidia or hyphae

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Discussion

Trichosporon asahii is an important pathogenic fungus for humans and animals, and the study of gene function is a crucial research tool for exploring its drug resistance and pathogenicity. In this study, we conducted a preliminary investigation into the function of the TaADH_like gene in T. asahii, which lays the foundation for in-depth research on the mechanism of drug resistance and pathogenicity of T. asahii.

ADH gene family analysis

S. cerevisiae and C. albicans serve as model fungi, with C. albicans being a common pathogenic fungus in humans and animals [29, 30]. These two model fungi were selected to investigate the genetic evolutionary relationships of ADH in T.asahii. S-(hydroxymethyl) glutathione dehydrogenase (FADH), considered the progenitor of the ADH gene family, together with alcohol dehydrogenase, constitutes the ADH gene family [31]. In this study, TaADH_like clusters with FADH in the phylogenetic tree. This may be because TaADH_like lacks motif5 of groups A and B, and is also deficient in group B - specific motif1 and motif7. Motif3 and motif 4 are present in both groups A and B, as well as in TaADH_like, and may be characteristic of alcohol dehydrogenase. However, sequence alignment results indicate significant differences between TaADH_like and other alcohol dehydrogenases. This may explain why TaADH_like is annotated as zinc-type alcohol dehydrogenase-like rather than alcohol dehydrogenase, and is not clustered with groups A and B. TaADH_like may be intermediate between FADH and alcohol dehydrogenase, but its correlation remains unclear. Most ADHs in C. albicans and S. cerevisiae possess a single CDS region, whereas T. asahii typically contains multiple CDS regions. The simpler gene structure of the saccharomycota (S. cerevisiae and C. albicans) has also been found in the Cyclophilin family [32]. C. albicans and S. cerevisiae exhibit higher homology than T. asahii. Although the basidiomycota (T. asahii) and the saccharomycota (S. cerevisiae and C. albicans) are genetically distinct in gene structure, the types and distribution of motifs are similar in the same group.

The overexpression of TaADH_like mediates the dimorphic transition in T. asahii

Dimorphic transitions are crucial for fungal growth. T. asahii, a morphologically and physiologically complex dimorphic fungus, can grow as yeast or exhibit filamentous growth, forming pseudohyphae and hyphae with abundant articulated conidia, similar to C. albicans [11]. In this study, overexpression of TaADH_like resulted in reduced hyphae formation, slowed conidia germination, and an arthroconidia formation of pseudohyphae, similar to the results after ADH1 gene knockdown in C. albicans [8], where TaADH_like exerted an opposing regulatory effect to the ADH1. In Aspergillus nidulans, vosA regulates conidial growth and development, promoting their maturation and germination [33]. Similarly, umv1 fulfills a comparable regulatory role in Ustilago maydis [34]. In Schizosaccharomyces pombe, fim1 is involved in sexual reproduction with conidia [35], whereas apsA is involved in asexual development in Aspergillus nidulans [36]. DIT1 encodes a spore wall maturation protein in yeast [37], and Arp2 is essential for the hydrophobin protein on the conidial surface in Aspergillus fumigatus [38]. Together, these genes regulate conidia growth and development, and TaADH_like overexpression reduced the expression of these genes, resulting in slower conidia germination in TaADH_likeOE.

Additionally, genes associated with hyphal growth, development, and regulation—BRG1, Ras1, Rac1, Rho1, CDC42, and cmkA—were significantly down-regulated in TaADH_likeOE, while the hyphal deterrent proteins NRG1 and RGF1 were significantly up-regulated. BRG1 encodes a DNA-binding protein that serves as a key regulator of fungal morphology. In C. albicans, mutants lacking BRG1 grew as yeast under all tested conditions, whereas overexpression of BRG1 promoted hyphal growth [39]. NRG1 encodes a hyphae deterrent protein that represses hyphae formation and hyphae-associated gene expression [40]. Nrg1p and Rfg1p are transcription factors that negatively regulate hyphal formation. Reducing Nrg1 transcript levels enhances hyphal growth and hyphae-specific gene expression, and overexpression of RGF1 drives pseudohyphae formation under yeast growth conditions [41, 42]. The Rho-related GTPase family is also plays a crucial role in fungal developmental and morphogenetic processes, including Rho, Cdc42, and Rac [43]. Under different culture conditions of T. asahii, the expression of CdC42 was found to significantly increased after the transition from the yeast phase to the hyphal phase, while the relative mRNA expression levels of Ras1, Rac1, and Rho1 were all significantly higher in the hyphal phase than in the yeast phase [44, 45]. The coordinated regulation of these genes induced the transition of T. asahii from the hyphal phase to the yeast phase.

The overexpression of TaADH_like slightly enhances fluconazole resistance in T. asahii

In C. albicans, a fluconazole MIC ≤ 8 µg/mL indicates susceptibility to fluconazole [46], but no such criteria have been established for T. asahii. In this study, fluconazole resistance in T. asahii increased from 2 to 4 µg/mL to 8 µg/mL following overexpression of TaADH_like. Biofilm activity was significantly higher in TaADH_likeOE than WT. Arthroconidia of T. asahii promote biofilm formation by enhancing cell adhesion [21, 47, 48]. TaADH_likeOE was transitioned from the hyphae phase to the yeast phase, and the increase in arthroconidia enhanced biofilm formation. Biofilm formation can hinder the entry of drugs into fungal cells [15]. In MIC experiment, the adhesion and growth of T. asahii on 96-well cell culture plates may contribute to the slight increase in fluconazole resistance observed in TaADH_likeOE. Although the overexpression of the TaADH_like only induces a slight increase in drug resistance, it does contribute to enhancing the drug resistance of T. asahii. Therefore, we speculate that TaADH_like is a potential gene associated with drug resistance in T. asahii.

The overexpression of TaADH_like maintains T. asahii growth under anaerobic conditions

In organisms, alcohol dehydrogenase (ADH) catalyzes the NAD⁺-mediated interconversion between ethanol and acetaldehyde [49]. This catalytic process is also accompanied by the production of the energy molecule ATP, which plays a crucial role in the resistance of organisms to adverse stress [50]. Moreover, ADH is regulated by oxygen. In Pichia pastoris, the transcription of ADH1 is regulated by oxygen, with higher expression under oxygen-limited conditions than under aerobic conditions. Its expression is inversely correlated with oxygen concentration, increasing as oxygen levels decrease [51]. Similarly, Adh1 is up-regulated under hypoxic conditions in Fusarium spinosum and Metarhizium [52, 53]. In this study, we found that overexpression of TaADH_like increased the tolerance of strains under hypoxic conditions, consistent with the results of previous studies.

HOG-MAPK pathway May reduce pathogenicity by influencing its colonisation

In S. cerevisiae, the HOG1 pathway is activated by two distinct upstream branches, Sln1 and Sho1 [54]. The Sln1 branch consists of the transmembrane histidine kinase receptor Sln1, the phosphotransporter protein Ypd1, the response regulator Ssk1, and the MAPKKK Ssk2/Ssk22, and the Sho1 branch consists of the transmembrane osmolality receptor Sho1, the small G protein Cdc42, the PAK kinase Ste20, and the MAPKKK Ste11 [55]. PBS2 is a scaffolding protein that can be activated by Ssk2/Ssk22 and Ste11 to further activate HOG1 in response to hyperosmotic stress [56]. In C. albicans, Ssk2 and Ssk22 merge into a single gene, Ssk2, and Ste11 is not involved in the regulation of the Hog1 signaling pathway [57]. In C. albicans, Hog1 is regulated only by Ssk2 and is not affected by Ste11 [58]. This is a marked difference from the HOG1 signaling pathway in S. cerevisiae. In this study, SSK2 was not annotated in T. asahii, and only SSK22 was present. The Sln1 pathway consists of CnNIK1 (Sln1), CnHHK2 (Ypd1), SSK1, and SSK22.The Sho1 branch consists of Sho1, Cdc42, Ste20, and Ste11. However, Ste11 was not differentially expressed under high osmotic conditions, and it is presumed that Ste11 is also not involved in the regulation of the HOG1 signaling pathway in T. asahii. Except for Ste11, Sho1 of HOG1 pathway was significantly down-regulated and CnNIK1 gene appeared significantly up-regulated in T. asahii, presumably the Sho1 pathway was suppressed by overexpression of TaADH_like, and CnNIK1 acted as the main pathway in response to osmotic stress. There are no unannotated PBS2-associated genes in T. asahii, which may be similar to C. albicans, where HOG1 is directly regulated by genes such as SSK22.

In C. albicans, the Hog MAPK pathway regulates the formation of hypha, which affects the virulence of the strain, and Hog1 inhibits the yeast-to-hyphae transition through downstream components [59]. Hog1 can act as a repressor of the yeast-to-hypha transition and an inducer of chlamydospore formation in C. albicans [60]. Additionally, Hog1 has been found to be involved in the colonization of the mouse intestine by C. albicans [61]. The virulence of hog1 defective mutant of T. asahii to silkworm was weakened [62]. In this study, HOG1 was significantly down-regulated, and it is speculated that the Hog MAPK pathway was involved in the hyphae-to-yeast transition of T. asahii and reduced the colonisation ability of TaADH_likeOE.

The overexpression of TaADH_like reduces the pathogenicity of T. asahii

The ability to switch between yeast and hyphal is necessary for virulence [63, 64]. Both forms play crucial roles during infection. The yeast form may disseminate via the bloodstream, spreading the organism to various host ecological niches, while the hyphal form is invasive, enabling the organism to evade phagocytosis [65]. In dimorphic ascomyces C. albicans, the reduction of hyphal formation ability reduced the virulence of C. albicans in mice, Caenorhabditis Elegans and Galleria mellonella [8]. The transformation of T. asahii clinical isolates from the hyphal phase to the yeast phase reduced the lethality of infection in mice, with the organism predominantly in the pseudohyphal form within host tissues [66]. In this study, overexpression of TaADH_like led to a shift of T. asahii from the hyphal phase to the yeast phase, resulting in reduced lethality in mice. WT exhibited stronger colonization of organs compared to TaADH_likeOE. Pathological histology revealed that the WT-inoculated mice developed pathological damage earlier and exhibited more severe damage than the TaADH_likeOE-inoculated mice. In conclusion, overexpression of TaADH_like caused T. asahii to shift from the hypha phase to the yeast phase, thereby reducing its pathogenicity.

Alcohol dehydrogenase is widely present in organisms, but the genome-wide information of many fungi has not been precisely annotated. The genome-wide information of T. asahii was annotated to the chromosome level for the first time in this laboratory. Although the article was explored by model strains C. albicans and S. cerevisiae, there is a lack of fungi from the same family and genus to jointly explore the evolutionary level of T. asahii. It is hoped that this will be addressed in future studies.

Conclusion

Alcohol dehydrogenase is involved in pathogenicity regulation of T. asahii. The T. asahii ADH gene family contains six members. Overexpression of TaADH_like reduced sensitivity to hypoxic environments, conversion of T. asahii from hyphae to yeast phase, slower growth, diminishes colonisation ability, reduced damage to organ tissues and lowers lethality. Increased osmotic stress sensitivity and the involvement of the HOG MAPK pathway in the hyphae-to-yeast transition reduce the colonization ability of T. asahii. Furthermore, the overexpression of TaADH_like promoted biofilm formation and led to a slight enhancement in fluconazole resistance in T. asahii.

Data availability

The T. asahii datasets generated during the current study are available in the Genome Sequence Archive at the National Genomics Data Center, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences repository, GSA: CRA014197. The S. cerevisiae datasets generated during the current study are available in the Saccharomyces Genome Database repository, GCA_000146045. The C. albicans datasets generated during the current study are available in the International Nucleotide Sequence Database Collaboration repository, GCA_000182965. The data of GCA_000146045 and GCA_000182965 are publicly available on Ensembl Fungi (http://fungi.ensembl.org/index.html). The genome assemblies are R64-1-1 and GCA000182965v3 respectively.

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Acknowledgements

Not applicable.

Funding

This work was supported by the Sichuan Beef Cattle Innovation team of the National Modern Agricultural Industry Technology System (grant number SCCXTD_2023-13).

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Author notes

  1. Zhen Liu, Xiaoping Ma and Xiangwen Zeng contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Animal Disease and Human Health of Sichuan Province, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, China

    Zhen Liu, Xiaoping Ma, Xiangwen Zeng, Zhiguo Li, Ruiguo Liu, Rongyan Luo, Weichen Wang & Muhammad Salman Tahir

  2. China Conservation and Research Center for the Giant Panda, Chengdu, 611800, China

    Chengdong Wang

  3. College of Life Sciences, Sichuan Agricultural University, Chengdu, 611130, China

    Yu Gu

Authors
  1. Zhen Liu
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  2. Xiaoping Ma
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  4. Zhiguo Li
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Contributions

ZL (Zhen Liu), XM and XZ analyse the data and Write-Original draft preparation. ZL (Zhiguo Li) prepares the sample. RL (Rongyan Luo) and RL (Ruiguo Liu) check the data. WW and MS (Muhammad Salman Tahir) have been substantially revised. CW and YG conceived and designed the analysis. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Xiaoping Ma or Yu Gu.

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All animal experiments were approved by the Institutional Animal Care and Use Committee of Sichuan Agricultural University (permit number: DYY-S20231020).

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Liu, Z., Ma, X., Zeng, X. et al. Identification of the ADH gene family in Trichosporon asahii and the role of TaADH_like in pathogenicity and fluconazole resistance. BMC Genomics 26, 352 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12864-025-11546-5

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12864-025-11546-5

Keywords

BMC Genomics

ISSN: 1471-2164

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