RESUMO
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.
Assuntos
Antifúngicos , Arthrodermataceae , Farmacorresistência Fúngica , Testes de Sensibilidade Microbiana , Saccharomyces cerevisiae , Terbinafina , Terbinafina/farmacologia , Antifúngicos/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Farmacorresistência Fúngica/genética , Arthrodermataceae/efeitos dos fármacos , Arthrodermataceae/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , FosforilaçãoRESUMO
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.
RESUMO
PURPOSE: Latent TGF-ß binding protein 4 (LTBP4) is involved in the production of elastin fibers and has been implicated in LTBP4-related cutis laxa and its complication, emphysema-like changes. Various factors have been implicated in the pathogenesis of emphysema, including elastic degeneration, inflammation, cellular senescence, mitochondrial dysfunction, and decreased angiogenesis in the lungs. We investigated the association between LTBP4 and emphysema using human lung fibroblasts with silenced LTBP4 genes. METHODS: Cell contraction, elastin expression, cellular senescence, inflammation, anti-inflammatory factors, and mitochondrial function were compared between the LTBP4 small interfering RNA (siRNA) and control siRNA. RESULTS: Under the suppression of LTBP4, significant changes were observed in the following: decreased cell contractility, decreased elastin expression, increased expression of the p16 gene involved in cellular senescence, increased TNFα, decreased GSTM3 and SOD, decreased mitochondrial membrane potential, and decreased VEGF expression. Furthermore, the decreased cell contractility and increased GSTM3 expression observed under LTBP4 suppression were restored by the addition of N-acetyl-L-cysteine or recombinant LTBP4. CONCLUSION: The decreased elastin expression, cellular senescence, inflammation, decreased antioxidant activity, mitochondrial dysfunction, and decreased VEGF expression under reduced LTBP4 expression may all be involved in the destruction of the alveolar wall in emphysema. Smoking is the most common cause of emphysema; however, genetic factors related to LTBP4 expression and other factors may also contribute to its pathogenesis.