The Ptk2-Pma1 pathway enhances tolerance to terbinafine in Trichophyton rubrum

ABSTRACT The increasing prevalence of dermatophyte resistance to terbinafine, a key drug in the treatment of dermatophytosis, represents a significant obstacle to treatment. Trichophyton rubrum is the most commonly isolated fungus in dermatophytosis. In T. rubrum, we identified TERG_07844, a gene encoding a previously uncharacterized putative protein kinase, as an ortholog of budding yeast Saccharomyces cerevisiae polyamine transport kinase 2 (Ptk2), and found that T. rubrum Ptk2 (TrPtk2) is involved in terbinafine tolerance. In both T. rubrum and S. cerevisiae, Ptk2 knockout strains were more sensitive to terbinafine compared with the wild types, suggesting that promotion of terbinafine tolerance is a conserved function of fungal Ptk2. Pma1 is activated through phosphorylation by Ptk2 in S. cerevisiae. Overexpression of T. rubrum Pma1 (TrPma1) in T. rubrum Ptk2 knockout strain (ΔTrPtk2) suppressed terbinafine sensitivity, suggesting that the induction of terbinafine tolerance by TrPtk2 is mediated by TrPma1. Furthermore, omeprazole, an inhibitor of plasma membrane proton pump Pma1, increased the terbinafine sensitivity of clinically isolated terbinafine-resistant strains. These findings suggest that, in dermatophytes, the TrPtk2-TrPma1 pathway plays a key role in promoting intrinsic terbinafine tolerance and may serve as a potential target for combinational antifungal therapy against terbinafine-resistant dermatophytes.

to terbinafine.Our findings suggest that the TERG_07844 gene product has a homolo gous function to the protein kinase Ptk2 of budding yeast and that the proton pump Pma1 functions downstream of the TERG_07844 gene product in terbinafine tolerance.We also found that omeprazole, a proton pump inhibitor approved for clinical use, potentiated the antifungal effect of terbinafine on terbinafine-resistant isolates.These results suggest that the Ptk2-Pma1 pathway enhances resistance to terbinafine in Trichophyton rubrum and could be a potential target for antifungal treatment.

TERG_07844 is involved in terbinafine tolerance
Protein kinases are involved in a wide range of physiological activities, including the regulation of intracellular ion concentrations and responses to external stresses such as antifungal drugs (11).Whole-genome analyses of dermatophytes have revealed a large number of genes encoding kinases of unknown function (12).Among these genes, we focused on several genes that are conserved among dermatophytes and are highly expressed in T. rubrum (13)(14)(15).In efforts to characterize the deletion mutants of these genes, we found that deletion of TERG_07844 leads to terbinafine sensitivity.
A TERG_07844 knockout stain was generated from the terbinafine-susceptible T. rubrum strain CBS118892 by replacing the TERG_07844 open reading frame (ORF) with the neomycin resistance gene (nptII) cassette (ΔTERG_07844; Fig. 1A).We also generated a revertant strain (eYFP-TERG_07844C) by random integration of the eYFP-TERG_07844 gene, which expresses the TERG_07844 gene product (XP_047604827) tagged with enhanced yellow fluorescent protein (eYFP) at the N-terminus (eYFP-XP_047604827) to obtain an information of the TrPtk2 subcellular localization, in the genome of ΔTERG_07844.
To confirm the loss of the TERG_07844 ORF, PCR was performed using primer pairs designed within the TERG_07844 ORF (primers 1 and 2 in Fig. 1A) and the neomycin resistance gene nptII cassette (primers 3 and 4 in Fig. 1A).PCR using the former primer pair amplified the PCR products in the parental strain CBS118892 and eYFP-TERG_07844C, but not in ΔTERG_07844 (Fig. 1B).Conversely, PCR using the latter primer pair did not amplify the PCR products in CBS118892 but did in ΔTERG_07844 and eYFP-TERG_07844C (Fig. 1C).In eYFP-TERG_07844C, two bands were amplified because of two selection markers, nptII and hph genes (Fig. 1C).Intracellular eYFP signals in eYFP-TERG_07844C were confirmed by confocal microscopy (Fig. 1D).
To analyze the terbinafine susceptibility of T. rubrum CBS118892, ΔTERG_07844, and eYFP-TERG_07844C, we cultured these strains on agar plates in the presence and absence of low concentrations of terbinafine (Fig. 1E and G) and measured the diameter of the colonies (Fig. 1F and H).The mycelial growth of ΔTERG_07844 was comparable to that of CBS118892 and eYFP-TERG_07844C on the agar medium without terbinafine (Fig. 1E and F).However, on agar medium containing terbinafine, the mycelial growth of ΔTERG_07844 was significantly reduced (Fig. 1G and H).These results suggest that TERG_07844 is involved in terbinafine tolerance in T. rubrum.

XP_047604827 encoded by TERG_07844 in T. rubrum is phylogenetically and functionally similar to S. cerevisiae Ptk2
To gain insight into TERG_07844, we performed a phylogenetic tree analysis to deter mine which kinases in S. cerevisiae are similar to XP_047604827 encoded by TERG_07844 (Fig. 2A).The phylogenetic tree revealed that XP_047604827 is grouped with the halotolerance kinases Sat4 (accession number NP_009934) and Hal5 (accession number NP_012370) from S. cerevisiae.Deficiencies in these kinases result in the decrease of high salt tolerance in S. cerevisiae (16).The polyamine transport kinase, Ptk2 (accession number NP_012593), was also found in proximity to XP_047604827.In contrast to Sat4 and Hal5, the absence of Ptk2 has been reported to cause high salt tolerance in S. cerevisiae (17,18).Near XP_047604827, the protein XP_964224, identified as a Ptk2 ortholog in the filamentous fungus Neurospora crassa (19), was also found (Fig. 2A).To determine whether XP_047604827 is functionally related to either Sat4/Hal5 or Ptk2, we examined the response of T. rubrum ΔTERG_07844 in a medium containing high salt concentrations.Compared with the terbinafine-sensitive strain CBS118892, ΔTERG_07844 exhibited enhanced mycelial growth in the presence of 0.5 M NaCl and displayed high salt tolerance, like the Ptk2-deficient S. cerevisiae strain ΔScPtk2 (17,18).Moreover, the sensitivity of ΔTERG_07844 to compounds to which ΔScPtk2 is resistant was investigated (18).The results showed that ΔTERG_07844 is resistant to spermine and lithium chloride (Fig. 2B and C).These salt tolerances were significantly reduced in eYFP-TERG_07844C (Fig. 2B and C).These results suggest that XP_047604827 encoded by TERG_07844 has phylogenetic and functional similarities to the Ptk2 protein of budding yeast.Consequently, we refer to T. rubrum XP_047604827 encoded by TERG_07844 as TrPtk2 in this study.
To investigate the general impact of fungal Ptk2 on terbinafine resistance, we assessed the sensitivity of the Ptk2-deficient S. cerevisiae strain ΔScPtk2 (Table 1) to terbinafine.As previously reported, ΔScPtk2 was resistant to spermine (Fig. 2D, left and center panels).Interestingly, ΔScPtk2 was sensitive to terbinafine (Fig. 2D, right panel), similar to ΔTrPtk2 (Fig. 1H).These observations suggest that the contribution of fungal Ptk2 to terbinafine tolerance is evolutionarily conserved in fungi.

Overexpression of TrPma1 suppresses the terbinafine sensitivity of ΔTrPtk2
In S. cerevisiae, the proton pump Pma1 is an essential protein for fungal growth (20).Pma1 is the most established substrate of Ptk2 and is activated by this kinase through phosphorylation in S. cerevisiae (21,22).To investigate if TrPma1 functions downstream of TrPtk2, we overexpressed TrPma1 tagged with eYFP at its C-terminus (TrPma1-eYFP) in ΔTrPtk2 and examined whether TrPma1 could complement the terbinafine sensitivity of ΔTrPtk2.On terbinafine-free agar medium (control), T. rubrum CBS118892 (parent), ΔTrPtk2, eYFP-TrPtk2C (revertant), and TrPma1OE-eYFP (ΔTrPtk2 overexpressing TrPma1-eYFP) (Table 1) showed similar growth rates (Fig. 3A).Mycelial growth was inhibited in T. rubrum ΔTrPtk2 not only on agar media containing terbinafine but also on agar media containing other squalene epoxidase inhibitors, namely, liranaftate and butenafine.Conversely, the mycelial growth in the presence of squalene epoxidase inhibitors was restored in the revertant strain eYFP-TrPtk2C and TrPma1OE-eYFP (Fig. 3A).These results suggest that TrPma1 acts downstream of TrPtk2 in the promotion of squalene epoxidase inhibitor resistance.
Since Pma1 functions as a proton pump on the plasma membrane in S. cerevisiae (23), TrPtk2 could potentially enhance resistance to terbinafine by regulating the subcellular localization of TrPma1.We overexpressed TrPma1-eYFP in CBS118892 and ΔTrPtk2, then cultivated these strains with or without terbinafine, and examined the subcellular localization of TrPma1-eYFP (Fig. 3B).In CBS118892, TrPma1-eYFP localized to the fungal cell surface, as reported for other fungal Pma1 (Fig. 3B).The membrane localization of TrPma1-eYFP was not affected in this strain cultured on terbinafine-containing agar medium, indicating that terbinafine does not disrupt the subcellular localization of TrPma1-eYFP.The localization of TrPma1-eYFP on the fungal cell surface was not disrupted in ΔTrPtk2 in the presence or absence of terbinafine.These results suggest that TrPtk2 is not involved in the regulation of TrPma1 subcellular localization and that TrPtk2

Omeprazole enhances the antifungal activity of terbinafine in both terbina fine-susceptible and resistant strains
TrPtk2 inhibitors may be effective compounds for combination therapy for dermatophy tosis, as ΔTrPtk2 displayed greater sensitivity to terbinafine compared with the terbina fine-susceptible strain CBS118892 (Fig. 1F and G).However, no fungal Ptk2 inhibitors have been identified to date.We hypothesized that pharmacological inhibition of Pma1 might improve dermatophyte sensitivity to terbinafine, since TrPma1 functions downstream of TrPtk2 (Fig. 3C).We assessed the growth characteristics of CBS118892 on agar medium containing terbinafine and omeprazole, an inhibitor of Pma1 in the yeast (24).Terbinafine alone had a significant inhibitory effect on the mycelial growth of CBS118892 dermatophytes (Fig. 4A and B).Furthermore, the combination of omeprazole and terbinafine resulted in greater inhibition of mycelial growth than either omeprazole or terbinafine treatment alone (Fig. 4A and B).These results suggest that omeprazole increases the terbinafine sensitivity of terbinafine-susceptible dermatophyte strains.
The resistance of T. rubrum to terbinafine is mainly due to specific mutations in the squalene epoxidase.The mutations L393F and F397L showed the highest minimum inhibitory concentrations (7).We investigated if omeprazole could enhance terbinafine sensitivity in resistant strains.For this purpose, we used clinical isolates of terbinafineresistant strains that had specific mutations in the squalene epoxidase gene.These included strain TIMM20092 with the F397L mutation and strains TIMM20093 and TIMM20094, both with the L393F mutation (6).In these terbinafine-resistant strains, both omeprazole and terbinafine exhibited inhibition of mycelial growth individually, except for terbinafine-treated TIMM20094, whose mycelial diameter was comparable with that of the vehicle control (Fig. 4C and D).Interestingly, co-administration of terbinafine and omeprazole resulted in more pronounced inhibitory effects than either medicine alone and ΔScPtk2 were grown in synthetic defined medium, and serial dilutions were dropped on synthetic defined agar plates with 2 mM spermine or 50 µg/mL terbinafine.Growth was measured after 3 days.

Species and strains Description Reference
Trichophyton rubrum

CBS118892
A terbinafine-sensitive clinical isolate from a patient nail sample.
ΔTERG_07844 (ΔTrPtk2) The TERG_07844 ORF was replaced with the neomycin resistance gene (nptII) in the strain.This strain was derived from CBS118892.

DISCUSSION
The present study suggests that the fungal Ptk2-Pma1 pathway promotes tolerance to squalene epoxidase inhibitors, including terbinafine (Fig. 5).Although terbinafine has potent antifungal activity on its own, the finding in this study that inhibition of the TrPtk2-TrPma1 pathway enhances the efficacy of terbinafine is clinically important in terms of overcoming terbinafine-resistant strains.Inhibition of the ATPases, including kinases and proton pumps, has recently emerged as a novel therapeutic approach against drug-resistant dermatophytes (25).Our finding that the antifungal efficacy of terbinafine against terbinafine-resistant dermatophytes is enhanced by omeprazole underscores the importance of this strategy.The veterinary antiparasitic milbemycin has been reported to promote the activity of the antifungal drugs itraconazole and voriconazole via inhibition of the dermatophyte efflux pump MDR3 (26).Omeprazole, which was found to enhance the antifungal effect of terbinafine in this study, has an advantage over milbemycin in terms of clinical applicability as it is a drug approved for human use.This study also suggests that compounds that potentiate the activity of antifungals can be found by repurposing non-antifungal drugs.
Based on the studies in S. cerevisiae (21,22), we propose that TrPma1 functions downstream of TrPtk2 in the promotion of tolerance to terbinafine.Consistent with this idea, we observed that the terbinafine susceptibility of T. rubrum was increased when TrPma1 was inhibited by omeprazole and that the terbinafine sensitivity of the Trptk2 mutant was ameliorated upon TrPma1 overexpression.Nevertheless, it remains inconclusive whether and how TrPtk2 functions upstream of TrPma1 in T. rubrum, as these observations could have a different basis.In future studies, it will be important to demonstrate the TrPtk2-dependent phosphorylation of TrPma1 in T. rubrum and that the activity of TrPma1 depends on TrPtk2.In addition, the molecular mechanism by which the fungal Ptk2-Pma1 pathway contributes to terbinafine tolerance remains unknown.As In S. cerevisiae, Pma1 is phosphorylated by Ptk2 and exports protons from the cell (21,22).The drug:H + antiporter major facilitator superfamily (MFS) in budding yeast requires the proton gradient that crosses the plasma membrane for drug efflux (27).TrPtk2 may phosphorylate TrPma1, thereby facilitating the formation of the proton gradient necessary for the drug efflux pump to export terbinafine.An efflux pump MDR2 has been identified as a transporter for terbinafine excretion in dermatophytes (28).Since TrPtk2 has also been reported to promote polyamine, Na + , and Li + uptake (29)(30)(31), it is possible that the TrPtk2-TrPma1 pathway contributes to the acquisition of terbina fine resistance by other mechanisms.Further functional analysis of the TrPtk2-TrPma1 pathway is necessary to better understand terbinafine resistance in dermatophytes.The increased sensitivity to terbinafine of the ΔScPtk2 strain of S. cerevisiae lacking Ptk2 demonstrated in this study will also allow S. cerevisiae to be used for further studies as a genetic analysis tool alongside studies in T. rubrum.
The terbinafine-resistant dermatophyte isolates used in this study have the L393F and F397L substitution mutations in squalene epoxidase (6).The ability of omeprazole to enhance the antifungal activity of terbinafine against these clinical isolates with the major known resistance mutations is critical for therapeutic applications.Furthermore, we found that all three squalene epoxidase inhibitors used in the present study exhibited enhanced antimicrobial activity against ΔTrPtk2 compared with the terbinafine-susceptible parent strain CBS118892.The potential enhanced antifungal activities of squalene epoxidase inhibitors other than terbinafine are important for the further analysis of the function of TrPtk2 as a new medication target and its clinical translation.

Fungal and bacterial strains and culture conditions
Agrobacterium tumefaciens EAT105 (32) was cultured at 28°C in Agrobacterium induction medium supplemented with 0.2 mM acetosyringone.Fungal strains used in this study are listed in Table 1.Trichophyton rubrum CBS118892, a clinical isolated strain from a patient nail sample, was used (12).Terbinafine-resistant T. rubrum isolates (TIMM20092, TIMM20093, and TIMM20094) ( 6) were cultured at 28°C on Sabouraud dextrose agar (SDA; 1% Bacto peptone, 4% glucose, 1.5% agar, and pH unadjusted) or 0.165 M FIG 4 (Continued) the diameter of individual samples (n = 3).(C and D) Decreased terbinafine resistance in terbinafine-resistant isolates in the presence of omeprazole.(C) Spores of TIMM20092, TIMM20093, and TIMM20094 were inoculated on SDA with 0 or 20 µg/mL omeprazole and/or 2 µg/mL terbinafine and incubated for 10 days.(D) The diameters of the mycelium on SDA with 0 or 20 µg/mL omeprazole and/or 2 µg/mL terbinafine were measured after 10 days of incubation.Since TIMM20092 cultured on SDA agar with 20 µg/mL omeprazole and 2 µg/mL terbinafine showed almost no mycelial growth, the diameter was designated as the diameter of the original spot.The data shown are mean ± SD.The dots on the graph represent the diameter of individual samples (n = 3).MOPS-buffered RPMI 1640 agar.S. cerevisiae BY4741 and YJR059W were purchased from Horizon Discovery Ltd. (California).Parent (BY4741/pYES2-HTH) and ΔScPtk2 (YJR059W/ pYES2-HTH) were cultured at 30°C on yeast extract peptone dextrose (YPD) or synthetic defined medium.Conidia of T. rubrum were prepared as described previously (33).pYES2-HTH was purchased from addgene (Massachusetts).For the spot assay, overnightcultured yeast suspension was diluted with a synthetic defined medium to an optical density of 0.2 (=1.6 × 10 6 CFU/mL).The suspension was serially diluted, and 3 µL of each suspension was plated onto a synthetic defined agar plate.The samples were incubated at 28°C for 3 days.
To construct a vector for TrPtk2 complementation (pCS2-hph-eYFP-TrPtk2), the following steps were performed.First, the antibiotic resistance gene cassette (hph) was inserted between the NotI and KpnI sites of pCS2+ N-terminal eYFP, which was generated by inserting the eYFP gene into the BamHI site of pCS2+ (kind gift from S. Kurisu at Tokushima University) (34,35).Subsequently, the tef1 promoter (Ptef1) was amplified from the T. indotineae genome by PCR with a specific primer pair (F: 5′-GC GGTCGACCCACTAAGACTCCTTCAAGCTCC-3′ and R: 5′-GCGAAGCTTGGTGACGGTGTATTTT TGTGTGG-3′) and inserted between the SalI and HindIII sites of the pCS2+ N-terminal eYFP-derived vector.Finally, the TrPtk2 gene was amplified from T. rubrum cDNA via PCR using a specific primer pair (F: 5′-GCTGTACAAGGGATCCATGGCCGGTTCGTCTACAT-3′ and R: 5′-GTTCTAGAGGCTCGATTAGTTGTAGCCATCGCCCA-3′), and the fragment was inserted between the BamHI and XhoI sites of the above vector.
To construct a vector for TrPma1-eYFP overexpression (pCS2-hph-TrPma1-eYFP), the following steps were performed.First, the antibiotic resistance gene cassette (hph) was inserted between the NotI and KpnI sites of pCS2+ C-terminal eYFP (kind gift from S. Kurisu at Tokushima University) (34,35).Subsequently, the tef1 promoter (Ptef1) was amplified from the T. indotineae genome by PCR with a specific primer pair (F: 5′-GC GGTCGACCCACTAAGACTCCTTCAAGCTCC-3′ and R: 5′-GCGAAGCTTGGTGACGGTGTATTTT TGTGTGG-3′) and inserted between the SalI and HindIII sites of the pCS2+ C-terminal eYFP-derived vector.Finally, the Trpma1 gene was amplified from T. rubrum cDNA by PCR using a specific primer pair (F: 5′-TCTTTTTGCAGGATCGCCACCATGGCCGACCACGCAGCC -3′ and R: 5′-CCTCTAGAGGCTCGAGGTGCGCTCTTCTCGTGCTG-3′), and the fragment was inserted between the BamHI and XhoI sites of the above vector.

Fluorescent microscopy observation
CBS118892 + TrPma1 eYFP, ΔTERG_07844 + TrPma1 eYFP, or eYFP-TrPtk2C strains were seeded with 1-5 × 10 6 spores on sterile cover glasses and placed in a 12-well plate.They were then incubated with 500 µL of SD liquid medium at 28°C overnight.On the second day, the SD medium was replaced with fresh medium, and the spores were further incubated at 28°C overnight.On the third day, the supernatant was removed, and the cells were cultured in RPMI 1640 with or without terbinafine for 3 h.The sample was fixed with 4% paraformaldehyde (PFA, Nacalai Tesque, Japan) at room temperature for 15 minutes.The samples were washed three times with PBS and mounted on glass slides using Aqua-Poly/Mount (Polysciences, UK).The stained cells were observed using a confocal microscope system (AX, Nikon, Japan).

Phylogenetic tree analysis
The evolutionary history was inferred using the maximum likelihood method and the Whelan and Goldman + Freq.model.The tree with the highest log likelihood (−13,606.41)was used.The percentage of trees in which the associated taxa clustered together was shown below the branches.The initial trees for the heuristic search were obtained automatically by applying the Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances that were estimated using the JTT model and then selecting the topology with the superior log likelihood value.A discrete Gamma distribution was used to model the evolutionary rate differences among the sites [5 categories (+G, parameter = 5.3335)].The tree was drawn to scale, with branch lengths measured in the number of substitutions per site.This analysis involved eight amino acid sequences.There was a total of 1,163 positions in the final data set.The evolutionary analyses were conducted in MEGA11 software.

Statistical analysis
The means of the two groups were compared using Student's t-test.For three or more groups with a single variable, one-way analysis of variance (ANOVA) with Tukey's post hoc test was conducted.For means of three or more groups with two variables, two-way ANOVA with Tukey's post hoc test was performed.Prism 9 software (GraphPad Software, Boston) was utilized for these statistical analyses.Statistical significance was defined at a P value of <0.05.n.s., not significant.*, P value <0.05.**, P value <0.01.***, P value <0.001.****, P value <0.0001.

FIG 1 ( 4 FIG 2
FIG 1 (Continued) ΔTERG_07844, and eYFP-TERG_07844C, in the presence and absence of low concentrations of terbinafine.(E) Spores of CBS118892, ΔTERG_07844 (TrPtk2), and eYFP-TERG_07844C (eYFP-TrPtk2C) were inoculated on RPMI 1640 for 14 days.(F) The diameter of the mycelium on RPMI 1640 after 14 days was measured.The data shown are mean ± SD.The dots on the graph represent the diameter of individual samples (n = 3).(G) Spores of CBS118892, ΔTERG_07844 (TrPtk2), and eYFP-TERG_07844C (eYFP-TrPtk2C) were inoculated on RPMI 1640 with 5 ng/mL of terbinafine for 14 days.(H) The diameter of the mycelium on RPMI 1640 with 5 ng/mL terbinafine after 14 days was measured.The data shown are mean ± SD.The dots on the graph represent the diameter of individual samples (n = 10).

FIG 2 (
FIG 2 (Continued) eYFP-TERG_07844C (eYFP-TrPtk2C) were inoculated on SDA with 2 mM spermine, 50 mM LiCl, 0.5 M NaCl, and 0.5 M KCl and incubated for 14 days (B).The diameter of the mycelium was measured (C).The data shown are mean ± SD.The dots on the graph represent the diameter of individual samples (n = 3 for spermine; n = 10 for others).(D) Acquired resistance of S. cerevisiae to spermine and susceptibility to terbinafine after deletion of the gene encoding Ptk2.Parent

FIG 3 7 FIG 4
FIG 3 Overexpression of TrPma1 suppresses terbinafine sensitivity of TERG_07844 deletion mutant.(A) Spores of strains were inoculated on RPMI 1640 with 5 ng/mL terbinafine, 6.4 ng/mL liranaftate, or 10 ng/mL butenafine and incubated for 14 days.(B) Spores of CBS11882 + TrPma1 eYFP and TrPma1OE-eYFP(ΔTrPtk2 + TrPma1 eYFP) were inoculated on RPMI 1640 and incubated for 2 days or on RPMI 1640 with 0 or 1 µg/mL terbinafine for 3 h.The eYFP signals of the sample were observed under confocal microscopy and shown in white.Scale bars are 10 µm.

FIG 5
FIG 5 Model of terbinafine tolerance mechanism in T. rubrum.Terbinafine tolerance in Trichophyton rubrum might be promoted by the phosphorylation of TrPma1 by TrPtk2.The tolerance of T. rubrum to terbinafine is decreased by TrPtk2 knockout and omeprazole treatment.