Genomic Multiplication and Drug Efflux Influence Ketoconazole Resistance in Malassezia restricta.

Minji Park,Yang Won Lee,Won Hee Jung,Yong-Joon Cho

doi:10.3389/fcimb.2020.00191

Abstract

Malassezia restricta is an opportunistic fungal pathogen on human skin; it is associated with various skin diseases, including seborrheic dermatitis and dandruff, which are usually treated using ketoconazole. In this study, we clinically isolated ketoconazole-resistant M. restricta strains (KCTC 27529 and KCTC 27550) from patients with dandruff. To understand the mechanisms of ketoconazole resistance in the isolates, their genomes were sequenced and compared with the susceptible reference strain M. restricta KCTC 27527. Using comparative genome analysis, we identified tandem multiplications of the genomic loci containing ATM1 and ERG11 homologs in M. restricta KCTC 27529 and KCTC 27550, respectively. Additionally, we found that the copy number increase of ATM1 and ERG11 is reflected in the increased expression of these genes; moreover, we observed that overexpression of these homologs caused ketoconazole resistance in a genetically tractable fungal pathogen, Cryptococcus neoformans. In addition to tandem multiplications of the genomic region containing the ATM1 homolog, the PDR5 homolog, which encodes the drug efflux pump protein was upregulated in M. restricta KCTC 27529 compared to the reference strain. Biochemical analysis confirmed that drug efflux was highly activated in M. restricta KCTC 27529, implying that upregulation of the PDR5 homolog may also contribute to ketoconazole resistance in the strain. Overall, our results suggest that multiplication of the genomic loci encoding genes involved in ergosterol synthesis, mitochondrial iron metabolism, and oxidative stress response and overexpression of the drug efflux pumps are the mechanisms underlying ketoconazole resistance in M. restricta.

Highlights

The lipophilic yeast Malassezia restricta is the most commonly found fungus on human skin; it is implicated in skin diseases such as seborrheic dermatitis and dandruff (Clavaud et al, 2013; Findley et al, 2013; Xu et al, 2016; Park T. et al, 2017)
In the case of ketoconazole, two isolates, M. restricta KCTC 27529 and KCTC 27550, showed an Minimal inhibitory concentrations (MICs) of 3.89–7.68 μg/mL, which is 16–256 times higher than those of the other M. restricta strains, including the type strain CBS 7877, which showed an MIC of 0.03–0.24 μg/mL. These results indicate that M. restricta KCTC 27529 and KCTC 27550 are resistant to ketoconazole, an imidazole antifungal drug that inhibits ergosterol biosynthesis in fungi
We investigated whether upregulation of the tandemly multiplicated ATM1 and ERG11 homologs contributed to azole resistance in the ketoconazole-resistant strains KCTC 27529 and KCTC 27550

Summary

Introduction

The lipophilic yeast Malassezia restricta is the most commonly found fungus on human skin; it is implicated in skin diseases such as seborrheic dermatitis and dandruff (Clavaud et al, 2013; Findley et al, 2013; Xu et al, 2016; Park T. et al, 2017). Multiple tropical drugs with antifungal activity against Malassezia have been used for the treatment of skin diseases associated with M. restricta (Carrillo-Muñoz et al, 2013; Cafarchia et al, 2015; Rojas et al, 2017). Among these antifungal drugs, ketoconazole, an imidazole compound, exhibits highly effective fungistatic activity against Malassezia; the effectiveness of this azole against seborrheic dermatitis and dandruff has been demonstrated (Danby et al, 1993; Pierard-Franchimont et al, 2001, 2002). Azole antifungal drugs, including ketoconazole, inhibit a cytochrome P450 enzyme, lanosterol 14α-demethylase, which participates in the synthesis of ergosterol, a major constituent of the fungal cell membrane (Vanden Bossche et al, 1987; Yoshida and Aoyama, 1987). Cryptococcus and Aspergillus isolates that are resistant to azole antifungal drugs have been reported frequently (Smith et al, 2015; Rivero-Menendez et al, 2016)

Methods

Results

Conclusion