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10.1186/s13568-024-01755-8
Original Article
An efficient gene targeting system using Δku80 and functional analysis of Cyp51A in Trichophyton rubrum
http://orcid.org/0000-0003-0687-3147
Ishii Masaki m_ishii@musashino-u.ac.jp

1
http://orcid.org/0000-0002-1394-5455
Yamada Tsuyoshi 23
http://orcid.org/0000-0003-4333-4433
Ohata Shinya shiohata@musashino-u.ac.jp

1
1 https://ror.org/04bcbax71 grid.411867.d 0000 0001 0356 8417 Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, Tokyo, 202-8585 Japan
2 https://ror.org/01gaw2478 grid.264706.1 0000 0000 9239 9995 Teikyo University Institute of Medical Mycology, Teikyo University, Hachioji, Tokyo 192-0395 Japan
3 https://ror.org/01gaw2478 grid.264706.1 0000 0000 9239 9995 Asia International Institute of Infectious Disease Control, Teikyo University, Tokyo, Japan
31 8 2024
31 8 2024
2024
14 9625 7 2024
14 8 2024
© The Author(s) 2024
2024
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Trichophyton rubrum is one of the most frequently isolated fungi in patients with dermatophytosis. Despite its clinical significance, the molecular mechanisms of drug resistance and pathogenicity of T. rubrum remain to be elucidated because of the lack of genetic tools, such as efficient gene targeting systems. In this study, we generated a T. rubrum strain that lacks the nonhomologous end-joining-related gene ku80 (Δku80) and then developed a highly efficient genetic recombination system with gene targeting efficiency that was 46 times higher than that using the wild-type strain. Cyp51A and Cyp51B are 14-α-lanosterol demethylase isozymes in T. rubrum that promote ergosterol biosynthesis and are the targets of azole antifungal drugs. The expression of cyp51A mRNA was induced by the addition of the azole antifungal drug efinaconazole, whereas no such induction was detected for cyp51B, suggesting that Cyp51A functions as an azole-responsive Cyp51 isozyme. To explore the contribution of Cyp51A to susceptibility to azole drugs, the neomycin phosphotransferase (nptII) gene cassette was inserted into the cyp51A 3′-untranslated region of Δku80 to destabilize the mRNA of cyp51A. In this mutant, the induction of cyp51A mRNA expression by efinaconazole was diminished. The minimum inhibitory concentration for several azole drugs of this strain was reduced, suggesting that dermatophyte Cyp51A contributes to the tolerance for azole drugs. These findings suggest that an efficient gene targeting system using Δku80 in T. rubrum is applicable for analyzing genes encoding drug targets.

Key Points

A novel gene targeting system using Δku80 strain was established in T. rubrum

Cyp51A in T. rubrum responds to the azole antifungal drug efinaconazole

Cyp51A contributes to azole drug tolerance in T. rubrum

Keywords

Dermatophyte
Trichophyton rubrum
Ku80
Cyp51A
http://dx.doi.org/10.13039/501100001691 Japan Society for the Promotion of Science 21K15438 23K06533 Ishii Masaki Nihon Nohyaku Co., Ltd.issue-copyright-statement© Springer-Verlag GmbH Germany, part of Springer Nature 2024
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pmcIntroduction

Dermatophytosis is a superficial fungal infection with symptoms such as itching, redness, and nail abnormalities. Tinea pedis (athlete’s foot), a type of dermatophytosis, affects approximately 10% of the world’s population (Havlickova et al. 2008). Trichophyton rubrum, the most common dermatophyte (Zhan and Liu 2017), is a clinically important organism that reduces the quality of life and has a unique life cycle as an anthropophilic dermatophyte that specifically inhabits human surface tissues. A limited class of antifungals, such as azole antifungals, are used in dermatophytosis treatment. Although drug resistance issues in T. rubrum have resulted in a need to elucidate the detailed molecular mechanisms of its drug resistance and to identify and analyze drug targets (Yamada et al. 2017; Monod et al. 2019), these issues have not been completely clarified because of the underdevelopment of genetic methods in T. rubrum.

Homologous recombination (HR), a repair mechanism for DNA double-strand, is one of the most commonly used genetic engineering methods (Smithies et al. 1985). This technique allows not only the precise insertion of any DNA fragment into the desired genomic region, but also the introduction of mutations, deletions, and replacements based on sequence homology. Nevertheless, eukaryotes also possess a nonhomologous end-joining (NHEJ) repair mechanism for double-strand breaks, which competes with HR-mediated insertion of DNA into target regions (Krappmann 2007). To efficiently promote targeted integration via HR, several fungal species have been engineered by disrupting either of the Ku70/Ku80 complexes involved in NHEJ (Yamada et al. 2009; Matsumoto et al. 2021). These strains have demonstrated the effectiveness of improving HR efficiency in various fungi (Yamada et al. 2009; Matsumoto et al. 2021).

Azole antifungal drugs used for treating dermatophytosis target the lanosterol demethylase Cyp51, which functions in the ergosterol synthesis pathway. XP_003235929 and XP_003236980 in T. rubrum have been identified as Cyp51A and Cyp51B homologs, respectively (Celia-Sanchez et al. 2022). It has been reported that the addition of azole antifungal drugs induces fungal Cyp51 expression (Henry et al. 2000; Roundtree et al. 2020). This result suggests that Cyp51A functions as a responsible Cyp51 isozyme when ergosterol biosynthesis is hindered, such as during treatment with azole antifungals. Because the cyp51 homolog erg11 is an essential gene in budding yeast (Kalb et al. 1987), a deficiency of dermatophyte cyp51A could cause strong growth defects. In budding yeast, disruption of the natural 3′-untranslated region (UTR) by the insertion of an antibiotic-resistant marker was found to destabilize the corresponding mRNAs, and this strategy has been used to analyze essential genes (Schuldiner et al. 2005; Breslow et al. 2008).

In this study, we established a highly efficient HR system using a ku80-deficient strain of T. rubrum CBS118892 (Martinez et al. 2012), a clinically isolated strain from a patient’s nail. This strain has been used for whole genome analysis (Martinez et al. 2012) and several transcriptome analyses (Persinoti et al. 2014; Mendes et al. 2018; Martins et al. 2019; Cao et al. 2022), as well as to produce genetically modified strains (Lang et al. 2020; Ishii et al. 2023, 2024a,b). Therefore, we used this strain as a parent strain of ku80 deletion strain. Using this established system, we developed a mutant in which the neomycin phosphotransferase (nptII) gene was inserted into the 3′-UTR of cyp51A, which encodes a target for azole antifungals. When the azole antifungal drug efinaconazole was added, the magnitude of increase in cyp51A expression decreased in this mutant, which also exhibited sensitivity to ravuconazole and efinaconazole. This study would accelerate the production of genetically engineered strains to investigate the pathogenicity and drug resistance of T. rubrum and provide novel insights into antifungal targets.

Materials and methods

Fungal and bacterial strains and culture conditions

Trichophyton rubrum CBS118892 was cultured on Sabouraud dextrose agar (SDA; 1% Bacto peptone, 4% glucose, 1.5% agar, pH unadjusted) at 28 °C. The conidia of T. rubrum were prepared as described previously (Uchida et al. 2003). We confirmed the sequence of cyp51A and cyp51B as well as their promoters and terminators.

Plasmid construction

To construct a ku80-targeting vector, pAg1-Δku80-flp, approximately 2.1 and 1.5 kb of the 5′- and 3′-UTR fragments, respectively, of the ku80 open reading frame (ORF) were amplified from T. rubrum genomic DNA by polymerase chain reaction (PCR). The PCR products of the 5′- and 3′-UTR fragments were cleaved by SpeI/ApaI and BglII/KpnI, respectively. The plasmid backbone of pAg1 (Zhang et al. 2003) and the FLP/FRT module (Reuß et al. 2004) of pMRV-TmKu80/T2 were cleaved by SpeI/KpnI and ApaI/BamHI, respectively (Yamada et al. 2014). These fragments were joined using Ligation high version 2 (TOYOBO, Osaka, Japan). To construct a cyp51A 3′-UTR-targeting vector, pAg1-cyp51A-3′-UTR, 1.6 kbp of the cyp51A ORF and 1.5 kbp of the 3′-UTR fragment of cyp51 ORF were amplified from T. rubrum genomic DNA by PCR. The neomycin phosphotransferase gene cassette, which consists of E. coli neomycin phosphotransferase gene (nptII), Aspergillus nidulans trpC promoter (PtrpC), and Aspergillus fumigatus cgrA terminator (TcgrA), was cleaved from pMRV-TmKu80/T2 using ApaI and ClaI. These fragments were joined using an In-Fusion HD Cloning Kit (TaKaRa Bio, Shiga, Japan). The primers used in this study are shown in Table 1.Table 1 Primers used in this study

Primer name	Sequences	
ku80-5′-F-SpeI	5′-CGC ACT AGT CCA CTG GAG ATC CCC AAC AG-3′	
ku80-5′-R-ApaI	5′-CGC GGG CCC TCG GGT CAA ACA GCC ACA AT-3′	
ku80-3′-F-BglII	5′-CGC AGA TCT GCT GCT GGT GGG TAT GTA GG-3′	
ku80-3′-R-KpnI	5′-CGC GGT ACC TTC GTT TGA GCC GAG AGA CC-3′	
cyp51A-F-SpeI	5′-ACT AGT ATG GCC GTG CTC ACA GTG-3′	
cyp51A-R-ApaI	5′-GGG CCC TAA CGT GAA TTA GAA CGT CGT TC-3′	
cyp51A-3′-F-ClaI	5′-CGA TCG ATA CTC ACA GTT ATT GAA CAG TTT CTG TA-3′	
cyp51A-3′-R-KpnI	5′-GCG GGT ACC AGC TCG GAA ATG CCT TGA CA-3′	
Primer 1	5′-TGA GGA AGG CCA GGG GAA CTT AT-3′	
Primer 2	5′-CCT TCC TGC TCT TTG CTT TCC CT-3′	
Primer 3	5′-AGC TGG TCT CGG AAA GTT GG-3′	
Primer 4	5′-AAG CCA CCA AAG CTC TCT CC-3′	
Primer 5	5′-AGC TCC TTC AAT TGA CCC GG-3′	
Primer 6	5′-AGA TGA TTC ATG ACG TAT ATT CAC CG-3′	
Primer 7	5′-GAT GGA TTG CAC GCA GGT TC-3′	
Primer 8	5′-CAC TGT TTT CTG GAC CTA TGA AAC C-3′	
Primer 9	5′-GCG AAT ACA GCA GAG AGA AAA TTG A-3′	
chs1-RT-F	5′-GGC CAC AAC GAA GCC TAT GA-3′	
chs1-RT-R	5′-GCT GGG AGG TAC TGT TTG ATC AA-3′	
cyp51A-RT-F	5′-CAA TCG GCC TGG GAG ATG-3′	
cyp51A-RT-R	5′-TTG GAC TTA GCT CCT TCG CG-3′	
cyp51B-RT-F	5′-GAA CAA CGT TGG TGT CAC CG-3′	
cyp51B-RT-R	5′-ACA TCT GTG TCT GCC TGA GC-3′	

Transformation of T. rubrum

Trichophyton rubrum was transformed using the polyethylene glycol (PEG) method as described previously (Yamada et al. 2008). The desired transformants and purified genomic DNA were analyzed by PCR. The ku80 ORF was replaced with a cassette with the nptII and the flippase gene (flp) flanked by flippase recognition sequences (Fig. 1a). As flp was inserted downstream of the copper ion-responsive promoter Pctr4, nptII and flp were removed from the ku80-deficient genome by adding the copper ion chelator bathocuproinedisulfonic acid to induce FLP recombinase expression (Fig. 1a). Total DNA was extracted using the Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research, Irvine, USA). Fungal cells were disrupted by μT-01 (TAITEC, Saitama, Japan) using 5-mm stainless beads.Fig. 1 ku80 locus targeting and nptII marker excision. a Schematic representation of the ku80 locus before and after excision of the copper ion-responsive promoter Pctr4, nptII and flp in T. rubrum. Site-specific recombination between the flanking FRT sequences (black box) was performed by the conditional expression of flp. b–e PCR analysis of total DNA samples from transformants. WT was used as a control. b Fragments were amplified with primer pairs (Primers 1 and 2). c Fragments were amplified with primer pairs (Primers 3 and 4). d Internal fragments of the ku80 ORF were amplified with primer pairs (Primers 5 and 4). e Internal fragments of nptII were amplified with primer pairs (Primers 6 and 7). The nptII-harboring strain (Δcla4) was used as a positive control. f Southern blot analysis of genome DNA samples from wild-type and Δku80 strains. g Mycelial growth of WT and Δku80 strains on SDA at 28 °C for 16 days

Antifungal susceptibility assay

Conidia (2 × 103) were incubated with two-fold serial dilutions of antifungal agents in 200 μl 3-Morpholinopropanesulfonic acid (MOPS)-buffered Roswell Park Memorial Institute (RPMI)1640 medium (pH 7.0) at 28 °C for 7 days, and the minimum inhibitory concentration MIC100 (minimal concentration required to inhibit growth by 100%) was determined. Efinaconazole was purchased from BLD Pharmatech Ltd, Shanghai, China, and ravuconazole was purchased from Merck, Darmstadt, Germany.

Quantitative reverse transcription-PCR (qRT-PCR)

Total RNAs were purified using NucleoSpin RNA (Macherey–Nagel, Düren, Germany) and reverse-transcribed into cDNAs using ReverTra Ace (Toyobo, Osaka, Japan) according to the manufacturers’ instructions. qRT-PCR was performed using TB Green Premix Ex Taq II (TaKaRa Bio, Shiga, Japan) on a StepOne Real-time PCR (Thermo Fisher Scientific, Waltham, USA). The relative mRNA expression level was determined using the 2−∆∆Ctcsh1) as an endogenous control to normalize the samples (Jacob et al. 2012). The primers used in this study are listed in Table 1.

Statistical analysis

Mean values of three or more groups with two variables were compared using two-way ANOVA with Šidák correction and Tukey’s post hoc test, according to the recommendation of Prism 10 (GraphPad, Boston, USA). The difference in the efficiency of HR in wild-type (WT) and Δku80 strains was analyzed by two-sided Fisher’s exact test using Prism 10 (GraphPad, Boston, USA). Differences were considered significant at P < 0.05.

Results

To increase gene targeting efficiency, we attempted to delete the gene encoding Ku80. WT strain was transformed using the disruption cassette and 14 of the 261 transformants obtained (5.4%) were found to be deficient in ku80 gene. The cloned fungi were cultivated under conditions in which bathocuproinedisulfonic acid was incorporated into the medium to induce FLP recombinase and facilitate the removal of the nptII gene cassette. Subsequent cloning was performed to obtain the deficient strain candidates. To confirm that the ku80-deficient strain (Δku80) was generated as designed, PCR was performed using genomic DNA purified from WT and Δku80 strains (Fig. 1a, top and bottom, respectively) as templates. PCR performed using WT genomic DNA and primers designed for the 5′- and 3′-UTR of ku80 (Primers 1 and 2 in Fig. 1a, respectively) amplified the PCR products with the expected size (6.6 kbp; Fig. 1b, left lane). The size of PCR products in Δku80 was reduced as expected (3.8 kbp; Fig. 1b, right lane). In contrast, PCR performed using primers designed against sequences in the 5′-UTR (Primer 3 in Fig. 1a) and the ORF of ku80 (Primers 4 and 5 in Fig. 1a) yielded PCR products of the expected size for WT (Fig. 1c, d, left lanes) but not for Δku80 (Fig. 1c, d, right lanes) strain. The deletion of nptII from the genome of Δku80 + nptII strain (Fig. 1a, middle) was confirmed by PCR using primers designed against the sequences in the promoter and terminator of nptII (Primers 6 and 7 in Fig. 1a, respectively, Fig. 1e). The deletion of Δku80 was also confirmed by Southern blot analysis of genomic DNA from WT and Δku80 strains (Fig. 1f). These data indicated that the Δku80 strain was successfully generated with no reduction in the number of available drug markers. To ascertain the extent of the impact of Ku80 protein on growth, we compared mycelial growth between WT and Δku80 strains, which revealed comparable mycelial growth (Fig. 1g).

The mRNA expression of cyp51A in T. rubrum was upregulated by the addition of the azole antifungal drug efinaconazole, but that of cyp51B was not upregulated (Fig. 2a). We attempted to insert the nptII cassette into the downstream of cyp51A ORF of T. rubrum, as demonstrated in budding yeast studies (Schuldiner et al. 2005; Breslow et al. 2008). Using the obtained Δku80 strain, we inserted the nptII cassette into cyp51A 3′-UTR (hereinafter termed the insertional mutant; Fig. 2b, c). Homologous recombinant strains were obtained in 12 of 26 strains (46.2%; Table 2) in which the insertion of the drug resistance gene within target region was confirmed by PCR using primers designed within the ORF and 3′-UTR of cyp51A (Primers 8 and 9, respectively; Fig. 2b, c). The HR efficiency of the Δku80 strain was 46 times higher than that of the WT strain (1/98; 1.0%; Table 2). These data demonstrated that a highly efficient HR method had been established in T. rubrum.Fig. 2 Production and characterization of the cyp51A 3′-UTR insertional mutants of T. rubrum. a The mRNA expression of cyp51A and cyp51B with or without 1 ng/ml efinaconazole in WT. Data are expressed as mean ± SD. The dots on the graph represent biological replicates (n = 4). n.s., not significant. ****, P < 0.0001. Two variables were compared using two-way ANOVA with Šidák correction. b Schematic representation of the cyp51A locus of WT and insertional mutant. c PCR analysis of total DNA samples from the independently isolated insertional mutant #1 and #2. The fragments were amplified with primer pairs (Primer 8 and 9). Δku80 was used as a control. d The mRNA expression of cyp51A in Δku80 and insertional mutant #1 and #2 with or without 1 ng/ml efinaconazole. The bars represent the standard deviation of the data obtained from three independent experiments. Data are expressed as mean ± SD. The dots on the graph represent biological replicates (n = 4–10). n.s., not significant. ****, P < 0.0001. Two variables were compared using two-way ANOVA with Tukey’s post hoc test. (e) Mycelial growth of Δku80 and insertional mutant #1 and #2 on SDA at 28℃ for 13 days

Table 2 Gene targeting efficiency of WT and Δku80 strains

Strain	Total
transformants	Homologous replacement	Efficiency (%)	
WT	98	1	1.0	
Δku80	26	12	46.2	
In homologous replacement cells, the drug resistance gene was inserted between the ORF and 3′-UTR of cyp51A. Two-sided Fisher’s exact test, P < 0.0001

Under efinaconazole-free conditions, the mRNA level of cyp51A in the two independently isolated insertional mutants, which were derived from the Δku80 strain, were comparable to that in the parent strain Δku80 (Fig. 2d). Nevertheless, efinaconazole-induced elevation of cyp51A mRNA level decreased in the insertional mutants (Fig. 2d). These findings suggest that the insertion of the nptII gene cassette into the 3′-UTR of cyp51A causes mRNA perturbation at least under the condition of cyp51A induction in T. rubrum. The insertional mutants exhibited similar mycelial growth as that of the parent strain Δku80 (Fig. 2e), but it showed increased sensitivity to the azole antifungals efinaconazole and ravuconazole (Table 3). However, the MICs of itraconazole and luliconazole remained unchanged in the insertional mutants. These findings suggest that Cyp51A functions as a factor for azole antifungal tolerance in T. rubrum.Table 3 MIC values (μg/ml) of efinaconazole and ravuconazole

Azole drugs	WT	Δku80
(Parent strain)	Insertional mutant #1	Insertional
mutant #2	
Efinaconazole	0.02	0.02	0.005	0.005	
Ravuconazole	0.08	0.08	0.04	0.04	
Itraconazole	1	1	1	1	
Luliconazole	0.00063	0.00063	0.00063	0.00063	
Two biological repeats were performed

Discussion

Trichophyton rubrum is an anthropophilic dermatophyte specialized for human parasitism, whereas several other dermatophytes are zoophilic or geophilic (Reiss Errol et al. 2011). The nature of this fungus is of great interest from not only a medical but also biological point of view. In recent years, transcriptomic, proteomic, and immunological studies of this fungus have been conducted extensively (Xu et al. 2018, 2022; Burstein et al. 2020; Peres et al. 2022; Galvão-Rocha et al. 2023). Nevertheless, molecular and cellular biological studies of T. rubrum have been limited partially due to a lack of genetic tools for this organism. In this study, we generated a ku80-deficient strain of this fungus and demonstrated that this strain can be applied in efficient HR methods, similar to a system established in a zoophilic dermatophyte, Trichophyton mentagrophytes (formerly Arthroderma vanbreuseghemii) (Yamada et al. 2009). The method established in this study might serve as a fundamental technique to promote research that will advance the findings of previous comprehensive analyses and immunological analyses observed on the host side.

The insertional mutant, in which the expression induction of cyp51A by efinaconazole was attenuated, exhibited increased sensitivity to efinaconazole and ravuconazole. Considering that cyp51A expression was upregulated in response to efinaconazole addition, we speculated that T. rubrum Cyp51A is an inducible Cyp51 isozyme crucial for tolerance to azole antifungals. Indeed, it has been reported that itraconazole treatment also induces an increase in cyp51A expression (Diao et al. 2009). In A. fumigatus, loss or suppression of cyp51A expression enhances sensitivity to the azole antifungal fluconazole (Hu et al. 2007). Conversely, cyp51B deficiency does not significantly alter fluconazole sensitivity (Hu et al. 2007). This difference may be partially explained by the lower binding affinity of Cyp51A for fluconazole than for Cyp51B (Andrew et al. 2010). Nevertheless, a difference in the induction of the expression of each cyp51 gene in response to azoles may also contribute to this disparity in sensitivity. Regarding T. rubrum, no studies have investigated the contribution of Cyp51A and Cyp51B isozymes to the resistance to azole antifungal drugs. It has been reported that strains of T. mentagrophytes with a deficiency in Cyp51B exhibit a 2–3-fold reduction in MICs and enhanced susceptibility to itraconazole and voriconazole (Yamada et al. 2022). This observation highlights the necessity for further investigation into the susceptibility of the Cyp51 isozymes in dermatophytes to azole drugs. In the future, it is important to generate T. rubrum strains that are deficient in cyp51A and cyp51B, followed by analyzing their involvement in growth and resistance to azole antifungal drugs.

The induction of Cyp51 expression has been extensively studied in Aspergillus species. It is known that in response to ergosterol depletion by azole treatment, the membrane-bound transcription factor SrbA is cleaved by proteases and activated (Dhingra et al. 2016; Bat-Ochir et al. 2016), translocating into the nucleus and upregulating the expression of enzymes involved in the ergosterol synthesis pathway, including Cyp51A (Zhang et al. 2021). A deficiency in SrbA in A. fumigatus has been demonstrated to markedly enhance the azole susceptibility of the azole-sensitive and -resistant fungi (Willger et al. 2008; Hagiwara et al. 2016). Moreover, the SrbA-binding region located upstream of the cyp51A gene has been shown to contribute to drug resistance by forming tandem repeats (Gsaller et al. 2016; Kühbacher et al. 2022). As the regulatory mechanism of cyp51 expression in T. rubrum is anticipated to represent a novel drug target for azole drug susceptibility and a cornerstone of research for elucidating the mechanism of azole resistance. Consequently, it is of great interest to elucidate the detailed molecular mechanism of the regulation of cyp51A expression by utilizing genetic tools, such as Δku80, which can be employed to elucidate the molecular mechanisms underlying the regulation of cyp51A expression in T. rubrum.

Abbreviations

HR Homologous recombination

MOPS 3-Morpholinopropanesulfonic acid

NHEJ Nonhomologous end-joining

ORF Open reading frame

PEG Polyethylene glycol

PCR Polymerase chain reaction

RPMI Roswell Park Memorial Institute

SDA Sabouraud dextrose agar

UTR Untranslated region

Acknowledgements

The authors thank H. Uga, N. Hori, H. Hamanaka for their technical help. This work was supported by the Japan Society for the Promotion of Science.

Author contributions

MI conceived and designed research. MI conducted experiments. MI, TY and SO analyzed data. MI, TY and SO wrote the manuscript. All authors read and approved the manuscript.

Funding

This work was supported by the Japan Society for the Promotion of Science (Grant numbers 23K06533 and 21K15438) and funding from Nihon Nohyaku Co., Ltd..

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

Authors have no competing interests.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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References

Andrew SWG Melo N Martel MC Parker EJ Nes DW Kelly LS Kelly ED Expression, purification, and characterization of Aspergillus fumigatus sterol 14-α demethylase (CYP51) isoenzymes A and B Antimicrob Agents Chemother 2010 54 4225 4234 10.1128/aac.00316-10 20660663
Andrew SWG, Melo N, Martel MC, Parker EJ, Nes DW, Kelly LS, Kelly ED (2010) Expression, purification, and characterization of Aspergillus fumigatus sterol 14-α demethylase (CYP51) isoenzymes A and B. Antimicrob Agents Chemother 54:4225–4234. 10.1128/aac.00316-1020660663 10.1128/aac.00316-10
Bat-Ochir C Kwak J-Y Koh S-K Jeon M-H Chung D Lee Y-W Chae S-K The signal peptide peptidase SppA is involved in sterol regulatory element-binding protein cleavage and hypoxia adaptation in Aspergillus nidulans Mol Microbiol 2016 100 635 655 10.1111/mmi.13341 26822492
Bat-Ochir C, Kwak J-Y, Koh S-K, Jeon M-H, Chung D, Lee Y-W, Chae S-K (2016) The signal peptide peptidase SppA is involved in sterol regulatory element-binding protein cleavage and hypoxia adaptation in Aspergillus nidulans. Mol Microbiol 100:635–655. 10.1111/mmi.1334126822492 10.1111/mmi.13341
Breslow DK Cameron DM Collins SR Schuldiner M Stewart-Ornstein J Newman HW Braun S Madhani HD Krogan NJ Weissman JS A comprehensive strategy enabling high-resolution functional analysis of the yeast genome Nat Methods 2008 5 711 718 10.1038/nmeth.1234 18622397
Breslow DK, Cameron DM, Collins SR, Schuldiner M, Stewart-Ornstein J, Newman HW, Braun S, Madhani HD, Krogan NJ, Weissman JS (2008) A comprehensive strategy enabling high-resolution functional analysis of the yeast genome. Nat Methods 5:711–718. 10.1038/nmeth.123418622397 10.1038/nmeth.1234
Burstein VL Beccacece I Guasconi L Mena CJ Cervi L Chiapello LS Skin immunity to dermatophytes: from experimental infection models to human disease Front Immunol 2020 11 605644 10.3389/fimmu.2020.605644 33343578
Burstein VL, Beccacece I, Guasconi L, Mena CJ, Cervi L, Chiapello LS (2020) Skin immunity to dermatophytes: from experimental infection models to human disease. Front Immunol 11:605644. 10.3389/fimmu.2020.60564433343578 10.3389/fimmu.2020.605644
Cao X Xu X Dong J Xue Y Sun L Zhu Y Liu T Jin Q Genome-wide identification and functional analysis of circRNAs in Trichophyton rubrum conidial and mycelial stages BMC Genomics 2022 23 21 10.1186/s12864-021-08184-y 34983376
Cao X, Xu X, Dong J, Xue Y, Sun L, Zhu Y, Liu T, Jin Q (2022) Genome-wide identification and functional analysis of circRNAs in Trichophyton rubrum conidial and mycelial stages. BMC Genomics 23:21. 10.1186/s12864-021-08184-y34983376 10.1186/s12864-021-08184-y
Celia-Sanchez BN Mangum B Brewer M Momany M Analysis of Cyp51 protein sequences shows 4 major Cyp51 gene family groups across fungi G3 Genes Genomes Genetics 2022 12 249 10.1093/g3journal/jkac249
Celia-Sanchez BN, Mangum B, Brewer M, Momany M (2022) Analysis of Cyp51 protein sequences shows 4 major Cyp51 gene family groups across fungi. G3 Genes Genomes Genetics 12:249. 10.1093/g3journal/jkac24910.1093/g3journal/jkac249
Dhingra Sourabh Kowalski CH Thammahong A Beattie SR Bultman KM Cramer RA RbdB, a rhomboid protease critical for SREBP activation and virulence in Aspergillus fumigatus mSphere 2016 10.1128/msphere.00035-16 27303716
Dhingra S, Kowalski CH, Thammahong A, Beattie SR, Bultman KM, Cramer RA (2016) RbdB, a rhomboid protease critical for SREBP activation and virulence in Aspergillus fumigatus. mSphere. 10.1128/msphere.00035-1627303716 10.1128/msphere.00035-16
Diao Y Zhao R Deng X Leng W Peng J Jin Q Transcriptional profiles of Trichophyton rubrum in response to itraconazole Med Mycol 2009 47 237 247 10.1080/13693780802227308 18663659
Diao Y, Zhao R, Deng X, Leng W, Peng J, Jin Q (2009) Transcriptional profiles of Trichophyton rubrum in response to itraconazole. Med Mycol 47:237–247. 10.1080/1369378080222730818663659 10.1080/13693780802227308
Galvão-Rocha FM Rocha CHL Martins MP Sanches PR Bitencourt TA Sachs MS Martinez-Rossi NM Rossi A The antidepressant sertraline affects cell signaling and metabolism in Trichophyton rubrum J Fungi 2023 10.3390/jof9020275
Galvão-Rocha FM, Rocha CHL, Martins MP, Sanches PR, Bitencourt TA, Sachs MS, Martinez-Rossi NM, Rossi A (2023) The antidepressant sertraline affects cell signaling and metabolism in Trichophyton rubrum. J Fungi. 10.3390/jof902027510.3390/jof9020275
Gsaller F Hortschansky P Furukawa T Carr PD Rash B Capilla J Müller C Bracher F Bowyer P Haas H Brakhage AA Bromley MJ Sterol biosynthesis and azole tolerance Is governed by the opposing actions of SrbA and the CCAAT binding complex PLoS Pathog 2016 12 e1005775 10.1371/journal.ppat.1005775 27438727
Gsaller F, Hortschansky P, Furukawa T, Carr PD, Rash B, Capilla J, Müller C, Bracher F, Bowyer P, Haas H, Brakhage AA, Bromley MJ (2016) Sterol biosynthesis and azole tolerance Is governed by the opposing actions of SrbA and the CCAAT binding complex. PLoS Pathog 12:e100577527438727 10.1371/journal.ppat.1005775
Hagiwara D Watanabe A Kamei K Sensitisation of an azole-resistant Aspergillus fumigatus strain containing the Cyp51A-related mutation by deleting the SrbA gene Sci Rep 2016 6 38833 10.1038/srep38833 27934927
Hagiwara D, Watanabe A, Kamei K (2016) Sensitisation of an azole-resistant Aspergillus fumigatus strain containing the Cyp51A-related mutation by deleting the SrbA gene. Sci Rep 6:38833. 10.1038/srep3883327934927 10.1038/srep38833
Havlickova B Czaika VA Friedrich M Epidemiological trends in skin mycoses worldwide Mycoses 2008 51 2 15 10.1111/j.1439-0507.2008.01606.x 18783559
Havlickova B, Czaika VA, Friedrich M (2008) Epidemiological trends in skin mycoses worldwide. Mycoses 51:2–15. 10.1111/j.1439-0507.2008.01606.x18783559 10.1111/j.1439-0507.2008.01606.x
Henry WK Nickels TJ Edlind DT Upregulation of ERG genes in Candida species by azoles and other sterol biosynthesis Inhibitors Antimicrob Agents Chemother 2000 44 2693 2700 10.1128/aac.44.10.2693-2700.2000 10991846
Henry WK, Nickels TJ, Edlind DT (2000) Upregulation of ERG genes in Candida species by azoles and other sterol biosynthesis Inhibitors. Antimicrob Agents Chemother 44:2693–2700. 10.1128/aac.44.10.2693-2700.200010991846 10.1128/aac.44.10.2693-2700.2000
Hu W Sillaots S Lemieux S Davison J Kauffman S Breton A Linteau A Xin C Bowman J Becker J Jiang B Roemer T Essential gene identification and drug target prioritization in Aspergillus fumigatus PLoS Pathog 2007 3 e24 10.1371/journal.ppat.0030024 17352532
Hu W, Sillaots S, Lemieux S, Davison J, Kauffman S, Breton A, Linteau A, Xin C, Bowman J, Becker J, Jiang B, Roemer T (2007) Essential gene identification and drug target prioritization in Aspergillus fumigatus. PLoS Pathog 3:e2417352532 10.1371/journal.ppat.0030024
Ishii M Matsumoto Y Yamada T Uga H Katada T Ohata S TrCla4 promotes actin polymerization at the hyphal tip and mycelial growth in Trichophyton rubrum Microbiol Spectr 2023 10.1128/spectrum.02923-23 37905917
Ishii M, Matsumoto Y, Yamada T, Uga H, Katada T, Ohata S (2023) TrCla4 promotes actin polymerization at the hyphal tip and mycelial growth in Trichophyton rubrum. Microbiol Spectr. 10.1128/spectrum.02923-2337905917 10.1128/spectrum.02923-23
Ishii M Matsumoto Y Yamada T Uga H Katada T Ohata S Targeting dermatophyte Cdc42 and Rac GTPase signaling to hinder hyphal elongation and virulence iScience 2024 27 110139 10.1016/j.isci.2024.110139 38952678
Ishii M, Matsumoto Y, Yamada T, Uga H, Katada T, Ohata S (2024a) Targeting dermatophyte Cdc42 and Rac GTPase signaling to hinder hyphal elongation and virulence. iScience 27:110139. 10.1016/j.isci.2024.11013938952678 10.1016/j.isci.2024.110139
Ishii M Yamada T Ishikawa K Ichinose K Monod M Ohata S The Ptk2-Pma1 pathway enhances tolerance to terbinafine in Trichophyton rubrum Antimicrob Agents Chemother 2024 68 e01609 e1623 10.1128/aac.01609-23 38567956
Ishii M, Yamada T, Ishikawa K, Ichinose K, Monod M, Ohata S (2024b) The Ptk2-Pma1 pathway enhances tolerance to terbinafine in Trichophyton rubrum. Antimicrob Agents Chemother 68:e01609-e1623. 10.1128/aac.01609-2338567956 10.1128/aac.01609-23
Jacob TR Peres NTA Persinoti GF Silva LG Mazucato M Rossi A Martinez-Rossi NM rpb2 is a reliable reference gene for quantitative gene expression analysis in the dermatophyte Trichophyton rubrum Med Mycol 2012 50 368 377 10.3109/13693786.2011.616230 21958376
Jacob TR, Peres NTA, Persinoti GF, Silva LG, Mazucato M, Rossi A, Martinez-Rossi NM (2012) rpb2 is a reliable reference gene for quantitative gene expression analysis in the dermatophyte Trichophyton rubrum. Med Mycol 50:368–377. 10.3109/13693786.2011.61623021958376 10.3109/13693786.2011.616230
Kalb VF Woods CW Turi TG Dey CR Sutter TR Loper JC Primary structure of the P450 lanosterol demethylase gene from Saccharomyces cerevisiae DNA 1987 6 529 537 10.1089/dna.1987.6.529 3322742
Kalb VF, Woods CW, Turi TG, Dey CR, Sutter TR, Loper JC (1987) Primary structure of the P450 lanosterol demethylase gene from Saccharomyces cerevisiae. DNA 6:529–537. 10.1089/dna.1987.6.5293322742 10.1089/dna.1987.6.529
Krappmann S Gene targeting in filamentous fungi: the benefits of impaired repair Fungal Biol Rev 2007 21 25 29 10.1016/j.fbr.2007.02.004
Krappmann S (2007) Gene targeting in filamentous fungi: the benefits of impaired repair. Fungal Biol Rev 21:25–29. 10.1016/j.fbr.2007.02.00410.1016/j.fbr.2007.02.004
Kühbacher A Peiffer M Hortschansky P Merschak P Bromley MJ Haas H Brakhage AA Gsaller F Azole resistance-associated regulatory motifs within the promoter of cyp51A in Aspergillus fumigatus Microbiol Spectr 2022 10 e01209 e1222 10.1128/spectrum.01209-22 35575535
Kühbacher A, Peiffer M, Hortschansky P, Merschak P, Bromley MJ, Haas H, Brakhage AA, Gsaller F (2022) Azole resistance-associated regulatory motifs within the promoter of cyp51A in Aspergillus fumigatus. Microbiol Spectr 10:e01209-e1222. 10.1128/spectrum.01209-2235575535 10.1128/spectrum.01209-22
Lang EAS Bitencourt TA Peres NTA Lopes L Silva LG Cazzaniga RA Rossi A Martinez-Rossi NM The stuA gene controls development, adaptation, stress tolerance, and virulence of the dermatophyte Trichophyton rubrum Microbiol Res 2020 241 126592 10.1016/j.micres.2020.126592 33002720
Lang EAS, Bitencourt TA, Peres NTA, Lopes L, Silva LG, Cazzaniga RA, Rossi A, Martinez-Rossi NM (2020) The stuA gene controls development, adaptation, stress tolerance, and virulence of the dermatophyte Trichophyton rubrum. Microbiol Res 241:126592. 10.1016/j.micres.2020.12659233002720 10.1016/j.micres.2020.126592
Martinez DA Oliver BG Gräser Y Goldberg JM Li W Martinez-Rossi NM Monod M Shelest E Barton RC Birch E Brakhage AA Chen Z Gurr SJ Heiman D Heitman J Kosti I Rossi A Saif S Samalova M Saunders CW Shea T Summerbell RC Xu J Young S Zeng Q Birren BW Cuomo CA White TC Comparative genome analysis of Trichophyton rubrum and related dermatophytes reveals candidate genes involved in infection Mbio 2012 3 e00259 e312 10.1128/mBio.00259-12 22951933
Martinez DA, Oliver BG, Gräser Y, Goldberg JM, Li W, Martinez-Rossi NM, Monod M, Shelest E, Barton RC, Birch E, Brakhage AA, Chen Z, Gurr SJ, Heiman D, Heitman J, Kosti I, Rossi A, Saif S, Samalova M, Saunders CW, Shea T, Summerbell RC, Xu J, Young S, Zeng Q, Birren BW, Cuomo CA, White TC (2012) Comparative genome analysis of Trichophyton rubrum and related dermatophytes reveals candidate genes involved in infection. Mbio 3:e00259-e312. 10.1128/mBio.00259-1222951933 10.1128/mBio.00259-12
Martins MP Silva LG Rossi A Sanches PR Souza LDR Martinez-Rossi NM Global analysis of cell wall genes revealed putative virulence factors in the dermatophyte Trichophyton rubrum Front Microbiol 2019 10 2168 10.3389/fmicb.2019.02168 31608026
Martins MP, Silva LG, Rossi A, Sanches PR, Souza LDR, Martinez-Rossi NM (2019) Global analysis of cell wall genes revealed putative virulence factors in the dermatophyte Trichophyton rubrum. Front Microbiol 10:216831608026 10.3389/fmicb.2019.02168
Matsumoto Y Nagamachi T Yoshikawa A Yamazaki H Yamasaki Y Yamada T Sugita T Development of an efficient gene-targeting system for elucidating infection mechanisms of the fungal pathogen Trichosporon asahii Sci Rep 2021 11 18270 10.1038/s41598-021-97287-3 34521867
Matsumoto Y, Nagamachi T, Yoshikawa A, Yamazaki H, Yamasaki Y, Yamada T, Sugita T (2021) Development of an efficient gene-targeting system for elucidating infection mechanisms of the fungal pathogen Trichosporon asahii. Sci Rep 11:18270. 10.1038/s41598-021-97287-334521867 10.1038/s41598-021-97287-3
Mendes NS Bitencourt TA Sanches PR Silva-Rocha R Martinez-Rossi NM Rossi A Transcriptome-wide survey of gene expression changes and alternative splicing in Trichophyton rubrum in response to undecanoic acid Sci Rep 2018 8 2520 10.1038/s41598-018-20738-x 29410524
Mendes NS, Bitencourt TA, Sanches PR, Silva-Rocha R, Martinez-Rossi NM, Rossi A (2018) Transcriptome-wide survey of gene expression changes and alternative splicing in Trichophyton rubrum in response to undecanoic acid. Sci Rep 8:2520. 10.1038/s41598-018-20738-x29410524 10.1038/s41598-018-20738-x
Monod M Feuermann M Salamin K Fratti M Makino M Alshahni MM Makimura K Yamada T Trichophyton rubrum azole resistance mediated by a new ABC transporter, TruMDR3 Antimicrob Agents Chemother 2019 63 e00863 e919 10.1128/AAC 31501141
Monod M, Feuermann M, Salamin K, Fratti M, Makino M, Alshahni MM, Makimura K, Yamada T (2019) Trichophyton rubrum azole resistance mediated by a new ABC transporter, TruMDR3. Antimicrob Agents Chemother 63:e00863-e919. 10.1128/AAC31501141 10.1128/AAC
Peres NTA Lang EAS Bitencourt TA Oliveira VM Fachin AL Rossi A Martinez-Rossi NM The bZIP Ap1 transcription factor is a negative regulator of virulence attributes of the anthropophilic dermatophyte Trichophyton rubrum Curr Res Microb Sci 2022 3 100132 10.1016/j.crmicr.2022.100132 35909615
Peres NTA, Lang EAS, Bitencourt TA, Oliveira VM, Fachin AL, Rossi A, Martinez-Rossi NM (2022) The bZIP Ap1 transcription factor is a negative regulator of virulence attributes of the anthropophilic dermatophyte Trichophyton rubrum. Curr Res Microb Sci 3:100132. 10.1016/j.crmicr.2022.10013235909615 10.1016/j.crmicr.2022.100132
Persinoti GF de Aguiar Peres NT Jacob TR Rossi A Vêncio RZ Martinez-Rossi NM RNA-sequencing analysis of Trichophyton rubrum transcriptome in response to sublethal doses of acriflavine BMC Genomics 2014 15 S1 10.1186/1471-2164-15-S7-S1 25573029
Persinoti GF, de Aguiar Peres NT, Jacob TR, Rossi A, Vêncio RZ, Martinez-Rossi NM (2014) RNA-sequencing analysis of Trichophyton rubrum transcriptome in response to sublethal doses of acriflavine. BMC Genomics 15:S1. 10.1186/1471-2164-15-S7-S125573029 10.1186/1471-2164-15-S7-S1
Reiss E Shadomy HJ Lyon GM Chambers KE Dermaatophytosis Fundamental Medical Mycology 2011 Hoboken Wiley-Blackwell 527 565
Reiss E, Shadomy HJ, Lyon GM (2011) Dermaatophytosis. In: Chambers KE (ed) Fundamental Medical Mycology. Wiley-Blackwell, Hoboken, pp 527–565
Reuß O Vik Å Kolter R Morschhäuser J The SAT1 flipper, an optimized tool for gene disruption in Candida albicans Gene 2004 341 119 127 10.1016/j.gene.2004.06.021 15474295
Reuß O, Vik Å, Kolter R, Morschhäuser J (2004) The SAT1 flipper, an optimized tool for gene disruption in Candida albicans. Gene 341:119–127. 10.1016/j.gene.2004.06.02115474295 10.1016/j.gene.2004.06.021
Roundtree TM Juvvadi RP Shwab KE Cole CD Steinbach JW Aspergillus fumigatus Cyp51A and Cyp51B proteins are compensatory in function and localize differentially in response to antifungals and cell wall Inhibitors Antimicrob Agents Chemother 2020 10.1128/aac.00735-20.10.1128/aac.00735-20 32660997
Roundtree TM, Juvvadi RP, Shwab KE, Cole CD, Steinbach JW (2020) Aspergillus fumigatus Cyp51A and Cyp51B proteins are compensatory in function and localize differentially in response to antifungals and cell wall Inhibitors. Antimicrob Agents Chemother. 10.1128/aac.00735-20.10.1128/aac.00735-2032660997 10.1128/aac.00735-20.10.1128/aac.00735-20
Schuldiner M Collins SR Thompson NJ Denic V Bhamidipati A Punna T Ihmels J Andrews B Boone C Greenblatt JF Weissman JS Krogan NJ Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile Cell 2005 123 507 519 10.1016/j.cell.2005.08.031 16269340
Schuldiner M, Collins SR, Thompson NJ, Denic V, Bhamidipati A, Punna T, Ihmels J, Andrews B, Boone C, Greenblatt JF, Weissman JS, Krogan NJ (2005) Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile. Cell 123:507–519. 10.1016/j.cell.2005.08.03116269340 10.1016/j.cell.2005.08.031
Smithies O Gregg RG Boggs SS Koralewski MA Kucherlapati RS Insertion of DNA sequences into the human chromosomal β-globin locus by homologous recombination Nature 1985 317 230 234 10.1038/317230a0 2995814
Smithies O, Gregg RG, Boggs SS, Koralewski MA, Kucherlapati RS (1985) Insertion of DNA sequences into the human chromosomal β-globin locus by homologous recombination. Nature 317:230–234. 10.1038/317230a02995814 10.1038/317230a0
Uchida K Tanaka T Yamaguchi H Achievement of complete mycological cure by topical antifungal agent NND-502 in guinea pig model of tinea pedis Microbiol Immunol 2003 47 143 146 10.1111/j.1348-0421.2003.tb02797.x 12680717
Uchida K, Tanaka T, Yamaguchi H (2003) Achievement of complete mycological cure by topical antifungal agent NND-502 in guinea pig model of tinea pedis. Microbiol Immunol 47:143–146. 10.1111/j.1348-0421.2003.tb02797.x12680717 10.1111/j.1348-0421.2003.tb02797.x
Willger SD Puttikamonkul S Kim K-H Burritt JB Grahl N Metzler LJ Barbuch R Bard M Lawrence CB Cramer RA Jr A sterol-regulatory element binding protein is required for cell polarity, hypoxia adaptation, azole drug resistance, and virulence in Aspergillus fumigatus PLoS Pathog 2008 4 e1000200 10.1371/journal.ppat.1000200 18989462
Willger SD, Puttikamonkul S, Kim K-H, Burritt JB, Grahl N, Metzler LJ, Barbuch R, Bard M, Lawrence CB, Cramer RA Jr (2008) A sterol-regulatory element binding protein is required for cell polarity, hypoxia adaptation, azole drug resistance, and virulence in Aspergillus fumigatus. PLoS Pathog 4:e100020018989462 10.1371/journal.ppat.1000200
Xu X Liu T Yang J Chen L Liu B Wang L Jin Q The first whole-cell proteome- and lysine-acetylome-based comparison between Trichophyton rubrum conidial and mycelial stages J Proteome Res 2018 17 1436 1451 10.1021/acs.jproteome.7b00793 29564889
Xu X, Liu T, Yang J, Chen L, Liu B, Wang L, Jin Q (2018) The first whole-cell proteome- and lysine-acetylome-based comparison between Trichophyton rubrum conidial and mycelial stages. J Proteome Res 17:1436–1451. 10.1021/acs.jproteome.7b0079329564889 10.1021/acs.jproteome.7b00793
Xu X Hu X Dong J Xue Y Liu T Jin Q Proteome-wide identification and functional analysis of lysine crotonylation in Trichophyton rubrum conidial and mycelial stages Front Genet 2022 10.3389/fgene.2022.832668 36755874
Xu X, Hu X, Dong J, Xue Y, Liu T, Jin Q (2022) Proteome-wide identification and functional analysis of lysine crotonylation in Trichophyton rubrum conidial and mycelial stages. Front Genet. 10.3389/fgene.2022.83266836755874 10.3389/fgene.2022.832668
Yamada Tsuyoshi Koichi Makimura Tatsuya Hisajima Maki Ito Yoshiko Umeda Shigeru Abe Genetic transformation of the dermatophyte, Trichophyton mentagrophytes, based on the use of G418 resistance as a dominant selectable marker Journal of Dermatological Science 2008 49 1 53 61 10.1016/j.jdermsci.2007.08.009 18055182
Yamada T, Makimura K, Hisajima T, Ito M, Umeda Y, Abe S (2008) Genetic transformation of the dermatophyte, Trichophyton mentagrophytes, based on the use of G418 resistance as a dominant selectable marker. J Dermatol Sci, 49:53–61. 10.1016/j.jdermsci.2007.08.00918055182 10.1016/j.jdermsci.2007.08.009
Yamada T Makimura K Hisajima T Ishihara Y Umeda Y Abe S Enhanced gene replacements in Ku80 disruption mutants of the dermatophyte, Trichophyton mentagrophytes FEMS Microbiol Lett 2009 298 208 217 10.1111/j.1574-6968.2009.01714.x 19659498
Yamada T, Makimura K, Hisajima T, Ishihara Y, Umeda Y, Abe S (2009) Enhanced gene replacements in Ku80 disruption mutants of the dermatophyte, Trichophyton mentagrophytes. FEMS Microbiol Lett 298:208–217. 10.1111/j.1574-6968.2009.01714.x19659498 10.1111/j.1574-6968.2009.01714.x
Yamada Y Maeda M Alshahni MM Monod M Staib P Yamada T Flippase (FLP) recombinase-mediated marker recycling in the dermatophyte Arthroderma vanbreuseghemii Microbiology (n y) 2014 160 2122 2135 10.1099/mic.0.076562-0
Yamada Y, Maeda M, Alshahni MM, Monod M, Staib P, Yamada T (2014) Flippase (FLP) recombinase-mediated marker recycling in the dermatophyte Arthroderma vanbreuseghemii. Microbiology (n y) 160:2122–2135. 10.1099/mic.0.076562-010.1099/mic.0.076562-0
Yamada T Maeda M Alshahni MM Tanaka R Yaguchi T Bontems O Salamin K Fratti M Monod M Terbinafine resistance of Trichophyton clinical isolates caused by specific point mutations in the squalene epoxidase gene Antimicrob Agents Chemother 2017 61 e00115-17 10.1128/AAC.00115-17 28416557
Yamada T, Maeda M, Alshahni MM, Tanaka R, Yaguchi T, Bontems O, Salamin K, Fratti M, Monod M (2017) Terbinafine resistance of Trichophyton clinical isolates caused by specific point mutations in the squalene epoxidase gene. Antimicrob Agents Chemother 61:e00115-17. 10.1128/AAC.00115-1728416557 10.1128/AAC.00115-17
Yamada T Yaguchi T Maeda M Alshahni MM Salamin K Guenova E Feuermann M Monod M Gene amplification of CYP51B: a new mechanism of resistance to azole compounds in Trichophyton indotineae Antimicrob Agents Chemother 2022 66 e00059-22 10.1128/aac.00059-22 35546111
Yamada T, Yaguchi T, Maeda M, Alshahni MM, Salamin K, Guenova E, Feuermann M, Monod M (2022) Gene amplification of CYP51B: a new mechanism of resistance to azole compounds in Trichophyton indotineae. Antimicrob Agents Chemother 66:e00059-22. 10.1128/aac.00059-2235546111 10.1128/aac.00059-22
Zhan P Liu W The changing face of dermatophytic infections worldwide Mycopathologia 2017 182 77 86 10.1007/s11046-016-0082-8 27783316
Zhan P, Liu W (2017) The changing face of dermatophytic infections worldwide. Mycopathologia 182:77–86. 10.1007/s11046-016-0082-827783316 10.1007/s11046-016-0082-8
Zhang A Lu P Dahl-Roshak AM Paress PS Kennedy S Tkacz JS An Z Efficient disruption of a polyketide synthase gene (pks1) required for melanin synthesis through Agrobacterium-mediated transformation of Glarea lozoyensis Mol Genet Genomics 2003 268 645 655 10.1007/s00438-002-0780-4 12589439
Zhang A, Lu P, Dahl-Roshak AM, Paress PS, Kennedy S, Tkacz JS, An Z (2003) Efficient disruption of a polyketide synthase gene (pks1) required for melanin synthesis through Agrobacterium-mediated transformation of Glarea lozoyensis. Mol Genet Genomics 268:645–655. 10.1007/s00438-002-0780-412589439 10.1007/s00438-002-0780-4
Zhang C Gao L Ren Y Gu H Zhang Y Lu L The CCAAT-binding complex mediates azole susceptibility of Aspergillus fumigatus by suppressing SrbA expression and cleavage Microbiologyopen 2021 10 e1249 10.1002/mbo3.1249 34964293
Zhang C, Gao L, Ren Y, Gu H, Zhang Y, Lu L (2021) The CCAAT-binding complex mediates azole susceptibility of Aspergillus fumigatus by suppressing SrbA expression and cleavage. Microbiologyopen 10:e1249. 10.1002/mbo3.124934964293 10.1002/mbo3.1249
