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. 2024 May 28;27(6):110139.
doi: 10.1016/j.isci.2024.110139. eCollection 2024 Jun 21.

Targeting dermatophyte Cdc42 and Rac GTPase signaling to hinder hyphal elongation and virulence

Masaki Ishii  1 , Yasuhiko Matsumoto  2 , Tsuyoshi Yamada  3   4 , Hideko Uga  1 , Toshiaki Katada  1 , Shinya Ohata  1
Affiliations

Targeting dermatophyte Cdc42 and Rac GTPase signaling to hinder hyphal elongation and virulence

Masaki Ishii et al. iScience. .

Abstract

The development of antifungal drugs requires novel molecular targets due to limited treatment options and drug resistance. Through chemical screening and establishment of a novel genetic technique to repress gene expression in Trichophyton rubrum, the primary causal fungus of dermatophytosis, we demonstrated that fungal Cdc42 and Rac GTPases are promising antifungal drug targets. Chemical inhibitors of these GTPases impair hyphal formation, which is crucial for growth and virulence in T. rubrum. Conditional repression of Cdc24, a guanine nucleotide exchange factor for Cdc42 and Rac, led to hyphal growth defects, abnormal cell morphology, and cell death. EHop-016 inhibited the promotion of the guanine nucleotide exchange reaction in Cdc42 and Rac by Cdc24 as well as germination and growth on the nail fragments of T. rubrum and improved animal survival in an invertebrate infection model of T. rubrum. Our results provide a novel antifungal therapeutic target and a potential lead compound.

Keywords: Microbiology; Mycology; cell biology; molecular biology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Mammalian Rac and Rac/Cdc42 inhibitors suppress conidial germination in Trichophyton rubrum (A) Structures of Rac inhibitors EHT1864 and NSC23766 and Rac/Cdc42 inhibitor AZA1. (B) Effects of mammalian small GTPase inhibitors on conidial germination in T. rubrum were observed. Conidia were stained with calcofluor white. n = 3 each. ∗∗, p < 0.01; ∗∗∗, p < 0.001. Mean ± SD. The lower panel showed representative fungal cell. (C) Effects of mammalian small GTPase inhibitors on mycelial growth in T. rubrum were observed. (D) Effects of mammalian Rac and Rac/Cdc42 inhibitors EHT1864, AZA1, and NSC23766 on conidial germination in T. rubrum were observed. Conidia were stained with calcofluor white. n = 3 each. ∗∗, p < 0.01; ∗∗∗∗, p < 0.0001. Mean ± SD.
Figure 2
Figure 2
XP_003237709.1 and XP_003234353.1 are TrRac and TrCdc42 in T. rubrum (A) Phylogenetic tree of fungal Rac and Cdc42 proteins, as prepared using the neighbor-joining method. The percentage of replicate trees clustering taxa together in bootstrap test (500 replicates) is indicated at branches. Evolutionary distances were calculated using Poisson correction method in terms of amino acid substitutions per site. (B) Fluorescence-based guanine nucleotide exchange assay method. (C) SDS-PAGE analysis of purified recombinant TrRac proteins stained with stain-free fluorescence stain. (D) TrRac guanine nucleotide exchange assay was performed. 1 μM recombinant TrRac and 0.1 μM bodipy GTP were mixed with or without 100 mM ammonium sulfate (AS), and fluorescence intensity was measured. Mean ± SD. (E) SDS-PAGE analysis of purified recombinant TrCdc42-His proteins stained with stain-free fluorescence stain. (F) TrCdc42-His guanine nucleotide exchange assay was performed. 1 μM recombinant TrCdc42-His and 0.1 μM bodipy GTP were mixed with or without 100 mM AS, and fluorescence intensity was measured. Mean ± SD.
Figure 3
Figure 3
T. rubrum Cdc24 stimulates guanine nucleotide exchange of Rac and Cdc42 (A) Phylogenetic tree analysis of fungal Cdc24 and human Tiam1 using the neighbor-joining method. The percentage of replicate trees clustering taxa together in bootstrap test (500 replicates) is indicated at branches. Evolutionary distances were calculated using Poisson correction method in terms of amino acid substitutions per site. (B) Protein domains of Cdc24 shown in an image. (C) SDS-PAGE analysis of purified recombinant DH-PH domain of TrCdc24 proteins stained with stain-free fluorescent stain. (D) Guanine nucleotide exchange of TrRac with or without TrCdc24DH-PH was observed. 1 μM TrRac and 0.1 μM bodipy GTP were incubated with or without 0.5, 1, and 2 μM TrCdc24DH-PH. Mean ± SD. (E) Guanine nucleotide exchange of TrCdc42-His with or without TrCdc24DH-PH was observed. 1 μM TrCdc42-His and 0.1 μM bodipy GTP were incubated with or without 0.5, 1, and 2 μM TrCdc24DH-PH. Mean ± SD.
Figure 4
Figure 4
Repression of Trcdc24 shows mycelial growth inhibition (A) Schematic representation of the Trcdc24 locus in the genome of T. rubrum CBS118892 WT and Pctr4Trcdc24 mutant. (B) Southern blot analysis of genome DNA samples from T. rubrum CBS118892 WT and Pctr4Trcdc24. (C) Quantification of Trcdc24 mRNA in total RNA of T. rubrum CBS118892 and Pctr4Trcdc24 cultured in medium containing 0, 0.1, 1, or 10 μM CuSO4 or 20 μM BCS, a copper ion chelator. n = 5 each. ∗∗∗∗, p < 0.0001. Mean ± SD. (D) Mycelial growth of T. rubrum CBS 118892 and P ctr4Trcdc24 on an agar plate with 0, 0.1, 1, or 10 μM CuSO4 or 20 μM BCS.
Figure 5
Figure 5
Repression of Trcdc42 shows mycelial growth inhibition in a TrRac deletion background (A) Schematic representation of the Trrac locus in the genome of T. rubrum CBS118892 WT and Trrac deletion mutant (ΔTrrac). (B) PCR analysis conducted on genomic DNA samples from T. rubrum CBS118892 WT, Pctr4Trrac, and ΔTrrac/P ctr4Trcdc42 using primers 1 and 2 as depicted in A. (C) PCR analysis was conducted on genomic DNA samples from T. rubrum CBS118892 WT, Pctr4Trrac, P ctr4Trcdc42, and ΔTrrac/P ctr4Trcdc42 using primers 3 and 4 as depicted in A. (D) Mycelial growth of T. rubrum CBS 118892, P ctr4Trcdc24, ΔTrrac, P ctr4Trcdc42, ΔTrrac/P ctr4Trcdc42 on an agar plate with 0 or 10 μM CuSO4. (E) Schematic representation of the Trcdc42 locus in the genome of T. rubrum CBS118892 WT and Pctr4Trcdc42 mutant. (F) PCR analysis was conducted on genomic DNA samples from T. rubrum CBS118892 WT, P ctr4Trcdc42, and ΔTrrac/P ctr4Trcdc42 using primers 5 and 6 as depicted in A. (G) Quantification of Trcdc42 mRNA in total RNA of T. rubrum CBS 118892 and Pctr4Trcdc42 cultured in medium containing 0 or 10 μM CuSO4. n = 3 each. ∗∗∗, p < 0.001. Mean ± SD.
Figure 6
Figure 6
Repression of Trcdc24 shows abnormal cell formation, actin localization, cell death, and reduced cell viability (A) Morphology of Pctr4Trcdc24 cells was observed for 3 days under cdc24 repression condition (with 10 μM CuSO4) or non-repression condition (without CuSO4). Fungal cells were stained with calcofluor white before observation. (B) Polarity of WT and Pctr4Trcdc24 cells cultured with or without 10 μM CuSO4. Fungal cells were stained with calcofluor white. The polarity index was calculated on day 1 (n = 50–60 each), day 2 (n = 50–60 each), and day 3 (n = 60 each). ∗, p < 0.05; ∗∗∗∗, p < 0.0001. Mean ± SD. (C) Cell area was measured on day 3. The dots on the graph represent the value of the individual cell area. n = 60 each. Mean ± SD. (D) Actin (green) and DNA (magenta) were visualized under Trcdc24 repression condition (with 10 μM CuSO4) or non-repression condition (without CuSO4) in Pctr4Trcdc24 cells. (E) WT and Pctr4Trcdc24 cells were stained with calcofluor white and PI. (F) The survival rate of WT and Pctr4Trcdc24 cells was observed. n = 3 each. ∗∗, p < 0.01; ∗∗∗, p < 0.001. Mean ± SD. (G) The area of Pctr4Trcdc24 cells was measured on day 7. The dots on the graph represent the value of the individual cell area. The numbers of W/O CuSO4, viable, and dead cells were 60, 27, and 42, respectively. ∗∗, p < 0.01. Mean ± SD.
Figure 7
Figure 7
Therapeutic efficacy of EHop-016 against an invertebrate infection model of dermatophytosis (A) Structure of the fungal Cdc42 and Rac inhibitor EHop-016. (B) Inhibitory effect of EHop-016 on guanine nucleotide exchange of TrCdc42-His by TrCdc24DH-PH. 0 (n = 5), 32 (n = 3), and 100 (n = 3) μM of EHop-016 were added. ∗∗, p < 0.01. Mean ± SD. (C) Inhibitory effect of EHop-016 on conidial germination was observed. n = 3 each. ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001. Mean ± SD. (D) Therapeutic effect of EHop-016 in a silkworm infection assay with T. rubrum. (E) Inhibitory effect of EHop-016 on the growth of T. rubrum on human nails. After 36 days of incubation at 28°C, the nail fragments were observed. On the solvent-treated nail (Viecle), a fungal lawn was observed, but no fungal lawn was observed on the EHop-016-treated nail. (F) Model. TrCdc42 and TrRac are activated to their GTP-bound forms by TrCdc24. Subsequently, the activated GTPases bind to effector molecules, like TrCla4, to enhance the polymerization of actin. This, in turn, promotes conidial germination, hyphal growth, and viability, ultimately leading to an enhanced infection by the fungus. The employment of a fungal Cdc42 and Rac inhibitor, EHop-016, demonstrated the potential to mitigate the fungal infection.
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