Dermatophytosis caused by Microsporum canis infection is a common cutaneous mycosis in small animals. As a zoonotic disease in humans^1,2, M. canis infection causes highly inflammatory tinea cruris or tinea corporis, which are treated with oral terbinafine (TBF) or itraconazole (ITCZ) and/ or topical antifungal agents. In recent years, dermatophyte resistance to antifungal drugs has emerged, impeding der- matophytosis treatment^3,4.

We have previously described the initial isolation of a TBF-resistant M. canis strain from a case of feline dermatophytosis in China in 2018⁵. The isolated strain exhibited a TBF minimum inhibitory concentration (MIC) of >32 μg/mL but remained susceptible to ITCZ (MIC: 0.023 μg/mL)^5,6. We later reported the first isolation of an ITCZ-tolerant M. canis strain from a case of human tinea corporis that was resistant to oral ITCZ therapy⁷. The isolate was ITCZ-tolerant (MIC: 1 μg/mL) and susceptible to TBF (MIC: <0.03 μg/mL), ravuconazole (RVCZ; MIC: 0.06 μg/mL), and luliconazole (LLCZ; MIC: <0.03 μg/ mL)⁷. As the patient had a pet cat diagnosed with dermato- phytosis, the infection was probably acquired from the cat⁷. Such cases have raised concerns that the incidence of pet-acquired infections with ITCZ-tolerant dermatophytes is in- creasing. However, the mechanism of ITCZ tolerance in zoophilic M. canis is not as clearly understood as in anthro- pophilic dermatophytes like Trichophyton interdigitale and T. rubrum⁸. In this study, as a One Health approach, we per- formed a transcriptome analysis to elucidate the tolerance mechanism of ITCZ-tolerant M. canis isolated from a case of human dermatophytosis.

1. Strain and antifungal susceptibility testing

Strain DTMU25-001 of M. canis was isolated from a patient with tinea faciei and corporis whose pet had a case of feline dermatophytosis in 2025⁷.

After determining the MICs for these agents^9,10, the cultures were resuspended, and a 10-μL aliquot of the suspension in each well was inoculated onto potato dextrose agar (Kanoto Chemical Co., Inc., Tokyo, Japan) plates using a pipette. After 7-10 days of incubation at 28℃, the minimum fungicidal concentration (MFC) was determined as the lowest con- centration of drug at which colony growth was observed¹¹.

2. Next-generation sequence analysis of whole-RNA samples

Strain DTMU25-001 was cultured in Sabouraud's dextrose broth (SDB: 1% peptone and 2% glucose) at 28℃ with shaking for 5 days. Culture medium containing mycelial cells was incubated with and without ITCZ at a concentration of 0.2 μg/mL for an additional 3 h at 28℃. Mycelia samples (approximately 100-200 mg) were flash-frozen in liquid nitrogen and ground to obtain a homogeneous powder. Total RNA was extracted using the RNeasy Total RNA kit (QIAGEN, Tokyo, Japan). Approximately 1 μg/μL of total RNA from ITCZ-treated and ITCZ-free cultures was stored at -80℃ until analysis.

The mRNA enrichment samples were sequenced using oligodT from whole RNA (300 ng of RNA) using a DNBSEQ-G400 system (MGI Tech Co., Ltd. Tokyo, Japan), and the resulting data were analyzed by Bioengineering Lab. Co., Ltd. (Kanagawa, Japan; https://gikenbio.com). Complementary DNA (cDNA) libraries were synthesized using an MGIEasy Fast RNA Library Prep Set (MGI Tech) and transformed into circular libraries using an MGIEasy Dual Barcode Circularization kit (MGI Tech) for sequencing analysis. For next-generation sequencing analysis of the cyclic cDNA libraries, a DNBSEQ-G400RS High-Throughput Sequencing kit (MGI Tech) and High-Throughput Paired-End Sequencing Primer kit (App-D; MGI Tech) were used to synthesize samples for DNA nano- balls. Whole-RNA sequencing (MGI DNBSEQ-G400RS) and data analysis of the two samples were performed by Bio- engineering Lab. Co., Ltd. (Kanagawa, Japan).

3. Real-time quantitative polymerase chain reaction analysis of heat shock protein and ABC transporter genes

The expression levels of heat shock protein and ABC trans- porter genes were analyzed using real-time quantitative polymerase chain reaction (RT-qPCR) as follows. First, cDNA samples synthesized from 0.5 μg of RNA were amplified using RT-qPCR (Thermal Cycler Dice; Takara) and analyzed as described in the previous report6.

The following primer pair was used to amplify fragments of the open reading frames of target genes: heat shock protein gene (293-bp fragment), forward primer 5'-GCCCT- TGGGCTAAACAATGCCGT-3' and reverse primer 5'-GTATAG- AGCCAACGAGAGTGAGGGCG-3', corresponding to nucleo- tides 56-78 and 339-358, respectively, of the M. canis heat shock protein gene (M. canis CBS 113480 heat shock pro- tein [MCYG_07264], partial mRNA, GenBank accession no. XM_002843435.1).

The following primer pair was used to analyze the ABC transporter gene (302-bp fragment): forward primer 5'-GGAACAACAGCCTTCAATCCACC-3' and reverse primer 5'-CACCGGGAATTTCTCCGTCG-3', corresponding to nucleo- tides 47-69 and 315-340, respectively, of the M. canis ABC transporter gene (M. canis CBS 113480 ABC transporter [MCYG_02928], partial mRNA, GenBank accession no. XM_002847376).

RT-qPCR assays and data analysis (ΔΔCT method) for relative quantitation were conducted using Thermal Cycler Dice Real-Time System software, version 5.11C (Takara). Basal expres- sion levels were estimated based on normalization to the level of actin-encoding transcripts in RNA from cultures under the assumption that an equivalent total RNA input and observed equal PCR efficiency would provide comparable threshold cycle (Ct) values. Gene expression levels were normalized to that of the actin-encoding gene, and values were compared between strains cultured in SDB with and without 0.2 μg/mL ITCZ. All RT-qPCR experiments were per- formed in triplicate. To identify any significant differences in the data, we performed a Student's nonpair t-test with Excel (Microsoft® Excel® 2021).

1. MFCs for M. canis strain DTMU25-001

The MFCs for strain DTMU25-001 were 0.25 μg/mL for TBF, >32 μg/mL for ITCZ, 2 μg/mL for RVCZ, and <0.03 μg/ mL for LLCZ.

2. Comparison of gene expression under ITCZ-treatment and ITCZ-free conditions

Approximately 7,500 Mb were sequenced for both samples, with approximately 24 million paired reads for each (Sup- plemental Fig. 1 and 2). After annotation, the data were mapped to M. canis CBS 113480 (assembly ASM15114v1), which is registered in GenBank, confirming approximately 98% identity between the RNA sequences of both samples.

Figure 1. Heatmap of 25-013 vs 25-013-ITZ

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Figure 2. (A) MA plot of 25-013 vs 25-013-ITZ, (B) Volcano of 25-013 vs 25-013-ITZ.

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A total of 8,849 expressed genes were detected in both sample RNAs, and the expression levels were compared between the ITCZ-treated and ITCZ-free samples. Among the compared genes, those for which the expression changed by a factor of >2 or ≤0.5 between ITCZ addition and no addition were plotted from highest to lowest expression level (Supplemental Tables 1 and 2). For 24 genes, the ex- pression increased >2-fold following treatment with ITCZ, with the heat shock protein gene (GenBank accession no. XM_002843435.1) and ABC transporter gene (GenBank accession no. XM_002847376.1) being the most highly ex- pressed, and thus presumed to be involved in tolerance (Supplemental Table 1). By comparison, the expression of 79 genes declined approximately ≤0.5-fold after the addition of ITCZ (Supplemental Table 2).

3. ITCZ-induced increase in gene expression

Transcript levels of the heat shock protein and ABC trans- porter genes in ITCZ-tolerant M. canis strain DTMU25-001 were compared between samples cultured in SDB containing 0.2 μg/mL ITCZ versus ITCZ-free conditions. The results of RT-qPCR analyses confirmed that ITCZ stimulation increased the expression of the ABC transporter gene approximately 2-fold and the heat shock protein gene 6-fold in M. canis cells compared with the unstimulated condition (Fig. 1).

Figure 1. Transcript levels of the genes encoding actin, the ABC transporter, and the heat shock protein in an ITCZ-tolerant M. canis strain (DTMU25-001). The strain was cultured in SDB con- taining 0.2 μg/mL ITCZ for 3 h at 28℃. Expression levels of all genes were nor- malized to that of the actin-encoding gene and are shown as relative expres- sion levels. RT-qPCR experiments were performed in triplicate. To determine any significant differences in the data, we performed a Student's nonpair t-test with Excel (Microsoft® Excel® 2021).

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4. Data summary

Updated sequences were submitted to GenBank under the following accession numbers: for data of the transcriptome analysis of ITCZ-induced/non-induced strain DTMU25-001, BioProject: PRJDB35662, and Run: DRR706743-DRR706744.

To the best of our knowledge, this is the first report of a transcriptome analysis conducted to clarify the ITCZ tolerance mechanism of an ITCZ-tolerant M. canis strain from human dermatophytosis. The MICs and MFCs for this strain suggest that it is particularly tolerant to ITCZ. Therefore, a trans- criptome analysis was performed to analyze gene expression in samples cultured in the presence and absence of ITCZ. To investigate the adaptive transcriptional responses in this experiment, RNA was extracted after culturing at an ITCZ concentration of 0.2 μg/mL, following Martins et al., who used a concentration of 0.14 μg/mL TBF to evaluate gene expression in four species of Trichophyton¹². Although TBF and ITCZ use different chemicals, this concentration was chosen to examine the response to the antifungal agent while avoiding cell death. The transcriptome analysis indicated that the expression levels of the heat shock protein gene (GenBank accession no. XM_002843435.1) and ABC transporter gene (GenBank accession no. XM_002847376.1) increased the most following culture in the presence of ITCZ (Supplemental Table 1). The results of RT-qPCR analyses confirmed that ITCZ stimulation increased the expression of the ABC transporter gene approximately 2-fold and that of the heat shock protein gene 6-fold in M. canis cells over the unstimulated condition (Fig. 1). The RT-qPCR analysis thus corroborated the results of the transcriptome analysis. However, because the RT-qPCR results only pertain to two gene variations per strain, they merely serve to confirm the findings of this transcriptome analysis.

The heat shock protein and ABC transporter are reportedly involved in the pathogenicity and antifungal drug resistance of anthropophilic dermatophytes^8,13. For example, different HSPs were detected during the infection and invasion of the host tissue, such as the overexpression of Hsp 30, HSP 60, HSP 70, HSP 78, and HSP 70 and of HSP 90, HSP-related gene hsf1, and HSPSSc1 in T. rubrum^8,13. Resistance to azoles has also been repeatedly reported and is associated with the overexpression of two genes (MDR2 and MDR3) encoding a multidrug transporter of the ABC transporter genes, giving rise to multidrug efflux outside the cell in T. rubrum8. In a recent study, we conducted a transcriptome analysis under the same conditions to clarify the multi-azole-resistance mechanism of a strain of M. canis isolated from a feline der- matophytosis¹⁴. The results of that experiment also showed increased expression of the same two genes. Moreover, we investigated whether sensitivity to azoles increased during cotreatment with milbemycin, which inhibits the ABC trans- porter in dermatophytes. The milbemycin counteracted the ITCZ and RVCZ tolerance of the M. canis isolate, suggesting that the strain acquired resistance through the high expression of the ABC transporter¹⁴. However, we have not investigated the heat shock protein for the azole-tolerant strains. Therefore, in future studies, we plan to generate heat shock protein and ABC transporter gene disruption in M. canis strains to investigate how these changes affect susceptibility to ITCZ. We would also like to investigate other genes that exhibited increased expression.

Additionally, 22 upregulated genes were identified; how- ever, based on the gene names registered in GenBank, it was unclear whether they were associated with ITCZ tolerance (Supplemental Table 1). Because it would be extremely difficult to create knockout strains for all of these genes, we plan to perform transcriptomic analysis on several ITCZ-tolerant strains and investigate the common upregulated genes.

In contrast, ITCZ stimulation decreased the expression of more genes than it increased. As reported by Carmo et al., Trichophyton interdigitale, which became resistant to ITCZ, ex- hibited a decrease in cellular metabolism¹⁵, which is thought to diminish the toxicity of ITCZ and is suspected to be related to a diversion of energy to sustain the high activity of ABC transporters. The results of our transcriptome analysis suggest that cellular metabolic activity was attenuated, which may contribute to the mechanism of ITCZ tolerance in a broad sense. Our study suggests that multiple intracellular functions are involved in the mechanism of tolerance to ITCZ in M. canis. However, further research is needed to fully elucidate the mechanism of resistance.