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Article

Genome-Wide Identification and Characterization of the Medium-Chain Dehydrogenase/Reductase Superfamily of Trichosporon asahii and Its Involvement in the Regulation of Fluconazole Resistance

by
Xiaoping Ma
1,*,†,
Zhen Liu
1,†,
Xiangwen Zeng
1,
Zhiguo Li
1,
Rongyan Luo
1,
Ruiguo Liu
1,
Chengdong Wang
2 and
Yu Gu
3,*
1
Key Laboratory of Animal Disease and Human Health of Sichuan Province, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China
2
China Conservation and Research Center for the Giant Panda, Chengdu 611800, China
3
College of Life Sciences, Sichuan Agricultural University, Chengdu 611130, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
J. Fungi 2024, 10(2), 123; https://doi.org/10.3390/jof10020123
Submission received: 4 January 2024 / Revised: 27 January 2024 / Accepted: 30 January 2024 / Published: 1 February 2024
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Abstract

:
The medium-chain dehydrogenase/reductase (MDR) superfamily contains many members that are widely present in organisms and play important roles in growth, metabolism, and stress resistance but have not been studied in Trichosporon asahii. In this study, bioinformatics and RNA sequencing methods were used to analyze the MDR superfamily of T. asahii and its regulatory effect on fluconazole resistance. A phylogenetic tree was constructed using Saccharomyces cerevisiae, Candida albicans, Cryptococcus neoformans, and T. asahii, and 73 MDRs were identified, all of which contained NADPH-binding motifs. T. asahii contained 20 MDRs that were unevenly distributed across six chromosomes. T. asahii MDRs (TaMDRs) had similar 3D structures but varied greatly in their genetic evolution at different phylum levels. RNA-seq and gene expression analyses revealed that the fluconazole-resistant T. asahii strain upregulates xylitol dehydrogenase, and downregulated alcohol dehydrogenase and sorbitol dehydrogenase concluded that the fluconazole-resistant T. asahii strain was less selective toward carbon sources and had higher adaptability to the environment. Overall, our study contributes to our understanding of TaMDRs, providing a basis for further analysis of the genes associated with drug resistance in T. asahii.

1. Introduction

Trichosporon asahii (T. asahii) is a fungus belonging to the genus Trichosporon and is the most common and important conditionally pathogenic fungus among all Trichosporon species, causing disease in both humans and animals. It is the main causative agent of invasive, systemically disseminated trichosporonosis [1,2]. Trichosporonosis is a complex fungal infection that can involve multiple organs, is difficult to treat using antifungal drugs, is prone to recurrence, and is associated with a poor prognosis [3,4,5]. Triazole antifungals, such as fluconazole, have become the most commonly used antifungal drugs in clinics due to their high safety and efficacy [6]. However, in recent years, there have been increasing reports of azole resistance in clinical isolates of T. asahii and therapeutic failure after azole application. A number of clinical isolates have been found to be insensitive to fluconazole in in vitro drug sensitivity tests, resulting in a poor response to antifungal therapy in patients with invasive trichosporonosis [7,8].
The medium-chain dehydrogenase/reductase (MDR) superfamily is composed of alcohol dehydrogenases (ADH), cinnamyl alcohol dehydrogenases (CAD), sorbitol dehydrogenases (SDH), quinone oxidoreductases (QOR), and many other enzymes [9]. MDR proteins have two conserved structural domains: a GroES-like structural domain with the catalytic structural domain of the enzyme (ADH_N) and a typical Rossmann folded zinc-binding (ADH_zinc_N) coenzyme-binding domain [10,11]. ADHs are widely distributed in all types of organisms, are involved in organismal virulence, growth, metabolism, and resistance, and are the most frequently reported family in the MDR superfamily [12,13,14,15]. Recently, ADH has been found to be involved in fungal drug resistance. ADH1 is differentially expressed in fluconazole-resistant and -sensitive strains of Candida albicans [16,17]. A clinical comparison of 20 clinical C. albicans strains isolated from patients with vulvovaginal candidiasis (VVC) and C. albicans-susceptible and -resistant strains revealed that ADH1 expression was 10.63 to 17.61 times higher in the resistant strains than in the susceptible strains [18]. ADH1p is involved in fluconazole resistance in C. albicans and plays an important role in energy metabolism [19]. In addition to ADH proteins, SDH and xylitol dehydrogenases (XDH) are involved in energy metabolism. SDH play a fundamental role in polyol metabolism, and the sorbitol metabolic pathway is a key bypass for glycolysis during glucose metabolism [20]. Some microorganisms use sorbitol as an alternative carbon and energy source [21]. XDH is involved in xylose metabolism, and its metabolites enter the pentose phosphate pathway for translocation to produce energy [22].
Many MDR superfamily members have been identified and functionally analyzed in bacteria and plants; however, no such studies have been performed in fungi. We identified MDR superfamily members in T. asahii for the first time using bioinformatics and analyzed the gene structure, chromosomal location, conserved motifs, interspecies phylogenetic evolution, and three-dimensional (3D) structure of the encoded proteins, as well as the transcriptional expression of these T. asahii MDRs (TaMDRs). The aim of this study was to gain a comprehensive understanding of TaMDRs and preliminarily investigate their expression patterns in drug-resistant T. asahii strains.

2. Materials and Methods

2.1. Strain Material

Wild-type T. asahii strains (WT) and fluconazole-resistant strains (PB) were obtained from the Clinical Veterinary Medicine Laboratory of the College of Veterinary Medicine, Sichuan Agricultural University, China. WT and PB were used for RNA sequencing, with three replicates per group.

2.2. Acquisition of Genomic Information

The complete dataset of T. asahii was obtained from the China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA014197). Cryptococcus neoformans, C. albicans, and Saccharomyces cerevisiae genome and protein sequences were downloaded from the Ensembl fungi genome database (https://fungi.ensembl.org/index.html (accessed on 27 August 2023)). The cds, genome, gff, and pep files were selected for subsequent analysis.

2.3. Identification of MDR Superfamily Members

The hidden Markov models (HMM) of the zinc-binding dehydrogenase domain (ADH_Zinc_N, PF00107) and the alcohol dehydrogenase GroES-like domain (ADH_N, PF08240) were obtained from the Pfam database (http://pfam.xfam.org/ (accessed on 13 January 2023)). Sequences containing both the ADH_Zinc_N and ADH_N domains were screened and identified using HMMSEARCH (threshold e-value < 0.001). The Conserved Domain Database (CDD, https://www.ncbi.nlm.nih.gov/cdd (accessed on 1 September 2023)) was used to confirm the conserved domains of the collected sequences, and sequences containing ADH_Zinc_N and ADH_N domains were considered to belong to the MDR superfamily.

2.4. Analysis of the Protein Properties of TaMDRs

ExPASy (http://web.expasy.org/protparam/ (accessed on 13 September 2023)) was used to predict the basic properties of MDR gene-encoded proteins, including their length, isoelectric point (pI), and molecular weight (MW) [23]. Subcellular localization was predicted using WoLF PSORT (https://wolfpsort.hgc.jp/ (accessed on 13 September 2023)) [24].

2.5. Phylogenetic Analysis

To explore the evolutionary relationships of TaMDRs, C. neoformans (CnMDR), C. albicans (CaMDR), and S. cerevisiae (ScMDR) maximum likelihood phylogenetic evolutionary trees were constructed using Molecular Evolutionary Genetics Analyses (MEGA 7.0), and tree topology support was assessed by bootstrap analyses with 1000 replicates [25]. ClustalW 2.0.10 software was used for multiple sequence comparisons to assess the evolutionary relationships of MDR members among different species using default parameters. The WAG + G + I amino acid substitution model was used. Furthermore, the phylogenetic trees of TaMDRs were constructed using MEGA-X with maximum likelihood, the WAG + G amino acid model, and 1000 bootstrap replications. The constructed phylogenetic tree was annotated and visualized using EvolView-v2 (https://www.evolgenius.info/evolview/ (accessed on 27 September 2023)) [26].

2.6. Gene Structure and Conserved Motif Analysis

To analyze the TaMDR, CnMDR, CaMDR, and ScMDR gene structures and conserved motifs, the exon introns of MDR proteins were analyzed and visualized using the online Gene Structure Display Server tools (GSDS, http://gsds.cbi.pku.edu.cn/ (accessed on 2 October 2023)) [27]. The MEME program (http://alternate.meme-suite.org/tools/meme (accessed on 4 October 2023)) was used to identify conserved motifs in the MDR sequence, with the number of motifs set to 15 and the minimum and maximum widths set to 10 and 50, respectively [28].

2.7. Comparison of Multiple Sequences and Protein Structure Prediction of TaMDRs

TaMDR sequences were compared using ClustalW in MEGA software (version 7.0), and ESPript 3.0 (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi (accessed on 18 October 2023)) was used to visualize the results [29]. Three-dimensional protein models were constructed using the SWISS-MODEL website (http://swis model.expasy.org (accessed on 12 November 2023)) [30]. The generated models were evaluated using SAVES v6.0 software (https://saves.mbi.ucla.edu/ (accessed on 15 November 2023)), which provides six methods evaluations simultaneously, three of which display a pass, indicating that the model is available. The protein structure was visualized using the SWISS-PDB viewer 4.10.

2.8. Collinearity Analysis

Genome-wide covariance analyses of T. asahii, C. neoformans, C. albicans, and S. cerevisiae were performed using the one-step MCScanX module of TBtools v2.012 software to obtain genome-wide replication events [31].

2.9. Analysis of the Localization of the TaMDR Gene on Chromosomes

To understand the chromosomal distribution of the MDR members in the T. asahii genome, the chromosomal location information of the MDR members was obtained from the Gene Structure Annotation Information file. Visualization was performed using TBtools software v 2.027 [31].

2.10. Analysis of TaMDR Expression Pattern in Fluconazole Resistance

Transcriptome sequencing was performed to investigate the expression patterns of MDR members in clinical isolates (WT) and fluconazole-resistant strains (PB) of T. asahii. The complete dataset has been submitted to the NCBI SRA database (accession number PRJNA941075). Gene expression levels were estimated as FPKM (fragments per kilobase of transcript per million mapped reads) values. Heatmaps of the transcript expression levels of differentially expressed TaMDRs were plotted using a heatmapper (http://www.heatmapper.ca/ (accessed on 30 November 2023)) [32].

2.11. Gene Expression Analysis

Total RNA from the WT and PB strains was extracted using the SteadyPure RNA Extraction Kit (Accurate Biotechnology (Hunan) Co., Ltd., Changsha, China) according to the manufacturer’s instructions. A cDNA template was synthesized from the RNA via reverse transcription using an Evo M-MLV RT Kit with clean gDNA (Accurate Biotechnology (Hunan) Co., Ltd., Changsha, China). Primer Premier 5 software was used to design gene-specific primers; 18s rRNA was used as the internal reference gene [33]. Each qPCR contained 10 uL SYBR Green ProTaq HS Premix (Accurate Biotechnology (Hunan) Co., Ltd., Changsha, China), 1 uL cDNA, and 0.4 µM of each gene-specific primer (Supplementary Table S1) at a final volume of 20 mL and was performed on a CFX96 real-time PCR system (BioRad, Hercules, CA, USA). The following reaction procedure was used: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, and 60 °C for 30 s. qRT-PCR data were analyzed using the 2−∆∆Ct method to calculate the relative expression levels. All assays were performed in triplicate [34]. All statistical analyses were performed using GraphPad Prism 8.01 (GraphPad Software Inc., La Jolla, CA, USA).

3. Results

3.1. Identification and Basic Properties of TaMDRs

A HMM was used to identify MDR superfamily members in T. asahii, and 20 genes containing both the ADH_Zinc_N and ADH_N domains were identified and named TaMDR_1–TaMDR_20 (Table 1). The predicted lengths of the proteins encoded by the TaMDRs ranged from 308 to 416 amino acids (aa). The molecular weights ranged from 32,819.7 Da (TaMDR_6) to 44,464.9 Da (TaMDR_1). The isoelectric point values of these TaMDRs ranged from 5.74 to 8.08 and included 5 basic and 15 acidic proteins. Subcellular localization predictions suggested that 16 TaMDR proteins were localized in the cytoplasm, and 4 proteins were localized in the mitochondria.

3.2. Phylogenetic Analysis

To understand the evolutionary relationships between the proteins encoded by TaMDRs, we selected three model fungal species, S. cerevisiae, C. albicans, and C. neoformans, to construct a phylogenetic tree with the TaMDRs (Figure 1, Supplementary Table S2). A total of 73 MDRs were identified in the four fungal strains, including 20 TaMDRs from T. asahii, 27 CnMDRs from C. neoformans, 16 CaMDRs from C. albicans, and 10 ScMDRs from S. cerevisiae. Phylogenetic analysis was used to categorize the predicted MDRs into five major branches (Group A–E), revealing high homology between C. neoformans and T. asahii and between C. albicans and S. cerevisiae.
In addition, a phylogenetic tree of TaMDRs was constructed, which was divided into five branches, as shown in Figure 1, and the relevant annotation information for each gene was marked (Figure 2). Among the 20 TaMDRs identified, only TaMDR_11 encoding S-(hydroxymethyl) glutathione dehydrogenase was present in Group A. Group B encoded a GroES-like protein, which is a marker protein encoded by the ADH_N domain. Group C contained three xylitol dehydrogenases, two sorbitol dehydrogenases, and one L-arabinitol 4-dehydrogenase. Group D included two quinone oxidoreductases, two zinc-bound dehydrogenases, and one zinc-type alcohol dehydrogenase-like protein. A total of four alcohol dehydrogenases were identified.

3.3. Multiple Sequence Comparisons of TaMDRs

Multiple sequence comparisons were performed for the 20 proteins of the TaMDRs (Figure 3). Zn 1-binding motifs, Zn 2-binding motifs, and NADPH-binding motifs are highly important binding motifs for MDRs. The Zn1-binding motif [GHE(x)2G(X)5G(X)2V] contains a catalytic zinc amino acid coordination residue, and the Zn-binding motif [GD(X)9,10C(X)2C(X)2C(X)7C] contains a structural zinc amino acid coordination residue. Groups A and E have a conserved Zn 1-binding motif and Zn 2-binding motif. Group D lacks both Zn-1-binding motifs and Zn-2-binding motifs. The NADPH-binding domain [GXG(X)2G] was highly conserved in all TaMDRs.

3.4. Gene Structure and Protein Motif Analysis of the MDRs

To study the structural diversity of MDRs, their exon–intron structures and motif distributions were analyzed (Figure 4B). The coding sequences (CDSs) of Groups A-E ranged from 1 to 11. A total of 26 genes were identified in C. albicans and S. cerevisiae; only CaMDR_15 had two CDSs, whereas the remaining 25 genes had one CDS. Approximately 26 genes had no untranslated regions (UTRs) at the C-terminus or N-terminus. A total of 47 genes were identified in T. asahii and C. neoformans, of which three genes had only one CDS region (TaMDR_15, TaMDR_18, and CnMDR_10). Genes containing three or seven CDS regions were the most abundant, followed by genes containing four CDS regions.
Next, we analyzed the conserved motifs present in the MDRs and found a total of 15 conserved motifs in genes with higher homology and similar protein motif compositions (Figure 4A and Figure 5, and Supplementary Figure S1). Motifs 1, 2, 3, 5, and 12 were detected in most of the MDR gene sequences. Motif 1 is a Zn 1-binding motif that was detected in 63 MDRs. Motif 3 (an NADPH-binding motif) was detected in all MDRs. Together, motifs 4 and 5 form the Zn 2-binding motif, with motif 4 containing four cysteine coordination residues of the zinc structure. Motif 5 was not detected in Sc_MDR7, and 51 genes contained motif 4. Motif 11 was the characteristic motif of Groups A and E. Motifs 7 and 15 were the characteristic motifs of Group C. Motifs 4 and 7 were absent in Group D, similar to the results of TaMDRs multiple sequence comparisons.

3.5. D Structure Prediction of TaMDRs

TaMDR_11, TaMDR_9, TaMDR_12, TaMDR_19, and TaMDR_17 genes were selected as representatives to analyze the related 3D structures (Figure 6). The Zn 1-binding motifs of TaMDR_11, TaMDR_9 and TaMDR_12 belonged mainly to the β-sheet (Figure 6(A1,B1,C1)), and those of TaMDR_19 and TaMDR_17 belonged to the β-sheet and the random coil (Figure 6(D1,E1)). The Zn 2-binding motifs in all five genes were located in a β-sheet, an α-helix, and an irregular coil (Figure 6(A2,B2,C2,D2,E2)). The NADPH-binding motif was located in the middle of the ADH_N and ADH_Zinc_N domains and was immediately adjacent to the ADH_Zinc_N domain (Figure 6A–E). Motif 11 is a characteristic motif in both Groups A and E, but in the 3D structure, it exhibited a large disparity; motif 11 in TaMDR_11 consisted of two β-sheets and one α-helix, while in TaMDR_17, it consisted of one β-sheet and two α-helixes (Figure 6(A3,E3)). Motif 15 and motif 7 were the characteristic sequences in Group C, and both motifs consisted of one β-sheet and one α-helix (Figure 6(C3,C4)).

3.6. Chromosomal Localization of TaMDRs

According to the whole-genome sequencing results, T. asahii has eight chromosomes, and TaMDRs are unevenly distributed among six chromosomes (Figure 7). Six genes were distributed on chromosome 4, followed by four on chromosomes 2 and 3. The least number of genes was distributed on chromosome 6, with only one gene.

3.7. Collinearity Analysis

We did not observe intraspecific collinearity in T. asahii, and further comparative collinearity maps were constructed at the genomic level for four fungi: Basidiomycota (T. asahii and C. neoformans) and Saccharomycotina (S. cerevisiae and C. albicans) (Figure 8 and Supplementary Figure S2). Two TaMDRs on chromosome 1 (TaMDR_1) and chromosome 2 (TaMDR_4) were co-linked with the MDRs of C. neoformans (CnMDR_7, CnMDR_12, CnMDR_18, CnMDR_19, and CnMDR_20). Only one TaMDR on chromosome 4 (TaMDR_13) exhibited a collinear relationship with S. cerevisiae (ScMDR_6), whereas there were no collinear genes with C. albicans. All collinear genes were present in Groups C and D (Figure 1). In addition, with the exception of three MDRs, S. cerevisiae and C. albicans had very few genes that were collinear with T. asahii, whereas C. neoformans had many collinearities with T. asahii.

3.8. Transcription and Expression of TaMDRs in Drug-Resistant Strains

Ribonucleic acid sequencing (RNA-seq) was performed on WT and PB strains, and the |Log2 fold change (Log2FC)| > 0.5 and false discovery rate < 0.05 were used to identify differentially expressed genes (DEGs). Eleven DEGs were differentially altered in the resistant strain, and seven were downregulated in the resistant strain (Figure 9). TaMDR_19 and TaMDR_20 exhibited the most significant differences in expression among the resistant strains and were downregulated by 5.8- and 5-fold, respectively. TaMDR_3 was the most significantly upregulated gene, with a 2.25-fold upregulation.

3.9. QRT-PCR Analysis of TaMDRs in Fluconazole-Resistant Strains

To further investigate the differences in TaMDR expression in fluconazole-resistant T. asahii strains, qRT-PCR was performed to verify the expression of seven genes with significantly different expression levels (Figure 10). The relative expression levels of TaMDR_2 and TaMDR_3 were upregulated in the resistant strain compared to those in the wild-type strain, with TaMDR_3 exhibiting the highest expression level (2.58-fold higher than that in the wild-type strain). In contrast, TaMDR_12, TaMDR_14, TaMDR_18, TaMDR_19, and TaMDR_20 were significantly downregulated in the resistant strains, especially TaMDR_19, which exhibited a 6.3-fold decrease in expression compared to that in the wild-type strain. Overall, the qRT-PCR results were consistent with the transcriptomic data.

4. Discussion

Trichosporon asahii is the causative agent of invasive trichosporonosis, and fluconazole-resistant T. asahii strains make its clinical treatment a major challenge [35]. In this study, we explored, for the first time, the composition of the MDR superfamily members of T. asahii and their expression patterns in drug-resistant strains, providing new insights into the role of MDRs in the drug resistance of T. asahii.
S. cerevisiae, C. albicans, and C. neoformans are model fungal species, of which C. neoformans and C. albicans are common pathogenic fungi in humans and animals [36,37,38]. C. neoformans and T. asahii belong to Basidiomycota, whereas C. albicans and S. cerevisiae belong to Saccharomycota; therefore, these three fungi were selected to jointly explore the genetic evolutionary relationships of TaMDRs. In this study, Basidiomycota (T. asahii and C. neoformans) were shown to have 20 and 27 MDRs, respectively, whereas Saccharomycotina (C. albicans and S. cerevisiae) had 16 and 10 MDRs, respectively. Basidiomycota can thus be inferred to possess a higher number of MDRs than Saccharomycotina. The motif distribution and gene structure of fungal MDRs associated with phylogenetic trees are important tools for sequence characterization in genetic studies. Fungi in the same phylum exhibited high homology, and genes within the same group had similar protein motifs. MDRs in Basidiomycota had a more complex gene structure than those in Saccharomycotina, with most MDRs in C. albicans and S. cerevisiae having only a single CDS region, whereas T. asahii and C. neoformans were more inclined to have multiple CDS regions. A simpler gene structure for Saccharomycotina has also been reported in studies of the Cyclophilin family [39]. In addition, T. asahii exhibited a greater collinearity with C. neoformans and very little collinearity with C. albicans and S. cerevisiae. Our results suggest that fungi have evolved with significant evolutionary differences at the phylum level.
We constructed a phylogenetic tree for TaMDRs, and the S-(hydroxymethyl) glutathione dehydrogenase (FADH) in Group A was considered as the origin of the alcohol dehydrogenase family, which, together with alcohol dehydrogenase, constitutes the ADH family [40]. Motif 11 is a characteristic motif of Groups A and E and is also present in the PRK09422 family within the MDR superfamily (alcohol-active dehydrogenase/acetaldehyde-active reductase). It is detected in Group B’s CaMDR_1 and Group C (TaMDR_19, CnMDR_1, and CaMDR_5), but only TaMDR_19 and CnMDR_1 are annotated as alcohol dehydrogenase. Therefore, MDRs containing motif 11 are inferred to possess aldehyde reductase activity. Group C was composed of xylitol dehydrogenase, sorbitol dehydrogenase, L-arabinitol 4-dehydrogenase, and zinc-binding dehydrogenase, which belong to the PDH (polyol dehydrogenase) family of the MDR superfamily [11]. Characteristic motifs 7 and 15 in Group C have both been identified in zinc-dependent alcohol dehydrogenase-like family proteins. Therefore, Group C MDRs may possess the ability to catalyze NAD(P)(H)-dependent reversible conversion of alcohols to their corresponding aldehydes. In Group D, TaMDR_5, TaMDR_18, and TaMDR_19 contained QOR-specific sites. However, TaMDR_19 was annotated as a zinc-type alcohol dehydrogenase-like protein instead of a quinone oxidoreductase, leading to the inference that TaMDR_19 is an atypical alcohol dehydrogenase. In the multiple sequence comparisons, these genes did not have complete catalytic or structural zinc-binding sites, and we speculated that TaMDR_19 clustered in Group D, which is consistent with the characteristic of Group D lacking the Zn 1- (motif 1) and Zn 2-binding motifs (motifs 4 and 5). Here, we believed that although the Basidiomycota (T. asahii and C. neoformans) and the Saccharomycotina (C. albicans and S. cerevisiae) exhibit genetic structural differences, the types and distributions of motifs are similar within the same group.
MDR proteins consist of two domains (ADH_N and ADH_Zinc_N), where the C-terminal domain is a typical Rossmann fold, which consists of six parallel β-sheets with two α-helices on each side [41]. The N-terminal domain consists of antiparallel β-sheets and surface-positioned α-helices, with long-range homology to the GroES structure [42]. This is consistent with our predicted 3D structure of some MDRs. Zinc-dependent alcohol dehydrogenases usually form dimers (higher plants and mammals) or tetramers (yeast and bacteria), each of which has two tightly bound zinc atoms per subunit, a catalytic zinc at the active site, and a structural zinc in the lobe of the catalytic domain in the Zn 1 (motif 1)- and Zn 2 (motif 4 and motif 5)-binding motifs, respectively [43]. In S. cerevisiae, the catalytic zinc is coordinated in two ways: a “classical” coordination of Cys-43, His-66, and Cys-153 and an “alternative” coordination of Cys-43, His-66, Glu-67, and Cys-153. Catalytic zinc coordination is relatively flexible, leading to the replacement of zinc-bound water with alcohols or aldehydes, thus contributing to the catalytic process of alcohols and aldehydes [44,45]. With the exception of Groups C and D, the catalytic zinc shown in the multiple sequence comparisons of TaMDRs is the binding mode of “classical” coordination. In plants, the amino acid coordination residues of structural zinc are four cysteine residues located in the Zn 2-binding motif [46]. TaMDR proteins had similar structural zinc coordination residues. The side chain of the residues of the structural zinc interacts with the residues of the coenzyme-binding structural domain of another monomer, thus linking multiple monomers into a dimeric or tetrameric structure, while the NADPH motif site is a characteristic sequence for coenzyme binding, and the typical Rossmann fold acts as a coenzyme-binding domain, where the coenzyme binds at the carboxy-terminal end [44].
MDRs are also involved in the regulation of fungal carbon sources. Both aerobically and anaerobically, S. cerevisiae Adh1p can use glucose as a carbon source to produce ethanol and NAD, whereas ADH2 in the cytoplasm can utilize ethanol as a carbon source to catalyze the conversion of ethanol to acetaldehyde under anoxic conditions [47]. Candida maltosa is able to ferment xylose, with ADH1 and ADH2 promoting xylose metabolism [48]. Under oxygen-limited conditions, ADH1 is absent, and the growth of Pichia stipitis in the xylose medium is inhibited [49]. Cytoplasmic TaMDR_2 and mitochondrial TaMDR_3 were identified as xylitol dehydrogenases and were significantly upregulated in drug-resistant T. asahii strains. XDH is involved in xylose metabolism; xylulose produced by oxidation is phosphorylated and transported via the pentose phosphate pathway [50]. The pentose phosphate pathway produces NADPH in the aerobic phase, enters the glycolytic pathway in the anaerobic phase, and provides energy to the body in anaerobic environments. The significant upregulation of TaMDR_2 and TaMDR_3 in T. asahii provided more energy and enhanced the resistance of the resistant strain to anaerobic environments.
MDRs are involved in a variety of mechanisms, including growth and energy metabolism, under aerobic and anaerobic conditions [51]. Alcohol dehydrogenase catalyzes the interconversion of alcohols and aldehydes, which play an important role in the exposure of organisms to adverse stresses [15,52]. Adh3p is located in the mitochondria and participates in redox shuttling in S. cerevisiae, where it is formed during biosynthetic reactions and transported to the cytoplasm [53]. TaMDR_19 and TaMDR_20 were identified as ethanol dehydrogenases that were significantly downregulated in T. asahii. In addition, sorbitol dehydrogenase is involved in the reversible NAD conversion of D-sorbitol to D-fructose and forms a part of the sorbitol pathway, which is a key bypass for glycolysis during glucose metabolism [20]. TaMDR_12 was identified as a sorbitol dehydrogenase that was significantly downregulated in the resistant strain. In fungal drug resistance mechanisms, the expulsion of drugs through efflux pumps, alterations in cell membrane permeability, and maintenance of cell wall integrity necessitate energy consumption [54,55]. In drug-resistant strains of C. albicans, the expression of ADH1 is positively correlated with the expression of efflux pump genes CDR1 and CDR2 [18]. However, energy metabolism decreases after acquiring the resistant strain compared to the wild-type strain. Therefore, the wild-type strain is inferred to require more energy for the maintenance of normal metabolism than the resistant strain, and the energy requirement is relatively low; therefore, the resistant strain is more environmentally adaptive. Due to the relatively few studies on T. asahii and the limited transcriptome data, the broader exploration of TaMDRs is limited. Therefore, more transcriptome data related to carbon source changes or energy metabolism will contribute to a better understanding of the regulatory role of the MDR superfamily of the fluconazole-resistance T. asahii. This will hopefully be realized in future experiments.

5. Conclusions

Twenty MDRs were identified in the T. asahii genome, which were unevenly distributed on six chromosomes. The genetic evolution of TaMDRs varied greatly at the phylum level. TaMDRs had similar 3D structures, and all had NADPH-binding motifs. Fluconazole-resistant T. asahii strains upregulated xylitol dehydrogenase and participated in xylitol energy metabolism. However, the expression of both alcohol dehydrogenase and sorbitol dehydrogenase was downregulated compared to that in the wild-type strain, indicating that fluconazole-resistant T. asahii strains had lower carbon source selectivity, lower energy requirements for self-maintenance, and higher adaptability to the environment. In conclusion, our study fills a gap in the MDR superfamily in T. asahii and provides a basis for further analysis of genes associated with drug resistance in T. asahii.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/jof10020123/s1, Figure S1: Basic composition of motifs for MDRs; Figure S2: Comparative linear relationship of MDRs in C. albicans, T. asahii, and S. cerevisiae; Table S1: Fluorescent quantitative primers for TaMDRs; Table S2: Detailed information of alcohol dehydrogenase proteins (MDRs) identified in C. neoformans, C. albicans, and S. cerevisiae.

Author Contributions

Conceptualization, X.M. and Y.G.; methodology, X.M. and Z.L. (Zhen Liu); software, X.Z. and Z.L. (Zhiguo Li); visualization, R.L. (Rongyan Luo) and R.L. (Ruiguo Liu); writing—original draft preparation, X.M., Z.L. (Zhen Liu) and X.Z.; writing—review and editing, C.W. and Y.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Sichuan Beef Cattle Innovation team of the National Modern Agricultural Industry Technology System, grant number SCCXTD_2023-13.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw sequence data of T. asahii reported in this paper have been deposited in the Genome Sequence Archive at the National Genomics Data Center, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA014197), and are publicly accessible at https://ngdc.cncb.ac.cn/gsa (accessed on 29 December 2023).

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Phylogenetic analysis of the Trichosporon asahii, Cryptococcus neoformans, Candida albicans, and Saccharomyces cerevisiae medium-chain dehydrogenase/reductase (MDR) superfamily members. Arcs of different colors indicate different groups. Circles and triangles represent MDRs of the Basidiomycota (T. asahii and C. neoformans) and Saccharomycotina (C. albicans and S. cerevisiae), respectively.
Figure 1. Phylogenetic analysis of the Trichosporon asahii, Cryptococcus neoformans, Candida albicans, and Saccharomyces cerevisiae medium-chain dehydrogenase/reductase (MDR) superfamily members. Arcs of different colors indicate different groups. Circles and triangles represent MDRs of the Basidiomycota (T. asahii and C. neoformans) and Saccharomycotina (C. albicans and S. cerevisiae), respectively.
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Figure 2. Phylogenetic analysis of the T. asahii MDRs (TaMDRs). Arcs of different colors indicate different groups.
Figure 2. Phylogenetic analysis of the T. asahii MDRs (TaMDRs). Arcs of different colors indicate different groups.
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Figure 3. Protein sequence alignment of all identified TaMDRs. The red background means the amino acids are identical. The red frames represent the binding motifs of Zn1, Zn2, and NADPH, respectively. White triangles represent catalytic zinc ligand residues, and black triangles represent structural zinc ligand residues.
Figure 3. Protein sequence alignment of all identified TaMDRs. The red background means the amino acids are identical. The red frames represent the binding motifs of Zn1, Zn2, and NADPH, respectively. White triangles represent catalytic zinc ligand residues, and black triangles represent structural zinc ligand residues.
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Figure 4. Phylogenetic tree, gene structure, and conserved motifs of MDRs. (A) Phylogenetic tree and motif analysis; the length of the solid line represents the length of the protein sequence. Colored boxes represent different motifs. (B) Gene structure analysis of the MDRs. Yellow boxes indicate untranslated regions (UTR) regions; green boxes indicate CDS regions; introns are indicated by black lines. The scale at the bottom is in base pairs (bp).
Figure 4. Phylogenetic tree, gene structure, and conserved motifs of MDRs. (A) Phylogenetic tree and motif analysis; the length of the solid line represents the length of the protein sequence. Colored boxes represent different motifs. (B) Gene structure analysis of the MDRs. Yellow boxes indicate untranslated regions (UTR) regions; green boxes indicate CDS regions; introns are indicated by black lines. The scale at the bottom is in base pairs (bp).
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Figure 5. Basic composition of MDR motifs.
Figure 5. Basic composition of MDR motifs.
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Figure 6. The protein 3D structures in TaMDRs. (A) TaMDR_11; (B) TaMDR_9; (C) TaMDR_12; (D) TaMDR_19; (E) TaMDR_17. The red α-helix and the yellow β-sheet form the ADH_Zinc_N domain. The green α-helix and the blue β-sheet form the ADH_N domain. The blue-green region represents the NADPH-binding motifs. A1–E1 and A2–E2 represent the Zn1- and Zn2-binding motifs, respectively. A3 and E3 represent motif 11, C3 represents motif 7, and C4 represents motif 15.
Figure 6. The protein 3D structures in TaMDRs. (A) TaMDR_11; (B) TaMDR_9; (C) TaMDR_12; (D) TaMDR_19; (E) TaMDR_17. The red α-helix and the yellow β-sheet form the ADH_Zinc_N domain. The green α-helix and the blue β-sheet form the ADH_N domain. The blue-green region represents the NADPH-binding motifs. A1–E1 and A2–E2 represent the Zn1- and Zn2-binding motifs, respectively. A3 and E3 represent motif 11, C3 represents motif 7, and C4 represents motif 15.
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Figure 7. Distribution of TaMDR genes on the chromosomes of T. asahii. The chromosomes are represented by green barred boxes, and different horizontal lines on the chromosomes represent genes at different locations. The scale on the left is in megabases (Mb).
Figure 7. Distribution of TaMDR genes on the chromosomes of T. asahii. The chromosomes are represented by green barred boxes, and different horizontal lines on the chromosomes represent genes at different locations. The scale on the left is in megabases (Mb).
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Figure 8. Comparative linear relationship of MDRs in C. neoformans, T. asahii, and S. cerevisiae. 1–14 represents the chromosome number of C. neoformans, and I–XVI represents the chromosome number of S. cerevisiae.
Figure 8. Comparative linear relationship of MDRs in C. neoformans, T. asahii, and S. cerevisiae. 1–14 represents the chromosome number of C. neoformans, and I–XVI represents the chromosome number of S. cerevisiae.
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Figure 9. Heatmap of MDR transcript expression levels in wild-type (WT) and resistant (PB) T. asahii strains, expressed as Log2FC values. Colored blocks indicate upregulated (red) or downregulated (blue) levels. White blocks indicate genes that were not differentially expressed.
Figure 9. Heatmap of MDR transcript expression levels in wild-type (WT) and resistant (PB) T. asahii strains, expressed as Log2FC values. Colored blocks indicate upregulated (red) or downregulated (blue) levels. White blocks indicate genes that were not differentially expressed.
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Figure 10. Gene expression of TaMDRs in WT and PB strains. The x-axis indicates the codes of the seven differentially expressed genes (DEGs) in T. asahii, and the y-axis indicates the gene expression levels. The values are expressed as mean ± SDs. Green bars represent transcriptome results, and red bars represent qRT-PCR results.
Figure 10. Gene expression of TaMDRs in WT and PB strains. The x-axis indicates the codes of the seven differentially expressed genes (DEGs) in T. asahii, and the y-axis indicates the gene expression levels. The values are expressed as mean ± SDs. Green bars represent transcriptome results, and red bars represent qRT-PCR results.
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Table 1. Detailed information of the MDR superfamily members identified in T. asahii.
Table 1. Detailed information of the MDR superfamily members identified in T. asahii.
GeneGene Accession NoLengthMW (Da)PISubcellular Localization
TaMDR_1evm.model.Chr01.12141644,464.96.78cytoplasm
TaMDR_2evm.model.Chr01.19440943,551.66.64cytoplasm
TaMDR_3evm.model.Chr02.24538640,935.16.96cytoplasm
TaMDR_4evm.model.Chr02.48338040,665.75.65cytoplasm
TaMDR_5evm.model.Chr02.69434137,556.97.90cytoplasm
TaMDR_6evm.model.Chr02.95530832,819.77.39cytoplasm
TaMDR_7evm.model.Chr03.111936939,170.87.19cytoplasm
TaMDR_8evm.model.Chr03.3136639,926.45.89mitochondrial
TaMDR_9evm.model.Chr03.65337340,057.96.24cytoplasm
TaMDR_10evm.model.Chr03.89932635,447.75.98cytoplasm
TaMDR_11evm.model.Chr04.18837139,685.26.50cytoplasm
TaMDR_12evm.model.Chr04.28737339,975.86.83mitochondrial
TaMDR_13evm.model.Chr04.38740543,332.57.84mitochondrial
TaMDR_14evm.model.Chr04.7334036,507.96.13cytoplasm
TaMDR_15evm.model.Chr04.7632234,674.55.69mitochondrial
TaMDR_16evm.model.Chr04.93838741,339.58.08cytoplasm
TaMDR_17evm.model.Chr06.24735938,170.55.74cytoplasm
TaMDR_18evm.model.Chr08.30734136,122.16.25cytoplasm
TaMDR_19evm.model.Chr08.32034436,829.06.96cytoplasm
TaMDR_20evm.model.Chr08.39136638,786.36.18cytoplasm
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Ma, X.; Liu, Z.; Zeng, X.; Li, Z.; Luo, R.; Liu, R.; Wang, C.; Gu, Y. Genome-Wide Identification and Characterization of the Medium-Chain Dehydrogenase/Reductase Superfamily of Trichosporon asahii and Its Involvement in the Regulation of Fluconazole Resistance. J. Fungi 2024, 10, 123. https://doi.org/10.3390/jof10020123

AMA Style

Ma X, Liu Z, Zeng X, Li Z, Luo R, Liu R, Wang C, Gu Y. Genome-Wide Identification and Characterization of the Medium-Chain Dehydrogenase/Reductase Superfamily of Trichosporon asahii and Its Involvement in the Regulation of Fluconazole Resistance. Journal of Fungi. 2024; 10(2):123. https://doi.org/10.3390/jof10020123

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Ma, Xiaoping, Zhen Liu, Xiangwen Zeng, Zhiguo Li, Rongyan Luo, Ruiguo Liu, Chengdong Wang, and Yu Gu. 2024. "Genome-Wide Identification and Characterization of the Medium-Chain Dehydrogenase/Reductase Superfamily of Trichosporon asahii and Its Involvement in the Regulation of Fluconazole Resistance" Journal of Fungi 10, no. 2: 123. https://doi.org/10.3390/jof10020123

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