Minji Park

Lipolytic Enzymes Involved in the Virulence of Human Pathogenic Fungi

Abstract Pathogenic microbes secrete various enzymes with lipolytic activities to facilitate their survival within the host. Lipolytic enzymes include extracellular lipases and phospholipases, and several lines of evidence have suggested that these enzymes contribute to the virulence of pathogenic fungi. Candida albicans and Cryptococcus neoformans are the most commonly isolated human fungal pathogens, and several biochemical and molecular approaches have identified their extracellular lipolytic enzymes. The role of lipases and phospholipases in the virulence of C. albicans has been extensively studied, and these enzymes have been shown to contribute to C. albicans morphological transition, colonization, cytotoxicity, and penetration to the host. While not much is known about the lipases in C. neoformans, the roles of phospholipases in the dissemination of fungal cells in the host and in signaling pathways have been described. Lipolytic enzymes may also influence the survival of the lipophilic cutaneous pathogenic yeast Malassezia species within the host, and an unusually high number of lipase-coding genes may complement the lipid dependency of this fungus. This review briefly describes the current understanding of the lipolytic enzymes in major human fungal pathogens, namely C. albicans, C. neoformans, and Malassezia spp.

Whole genome sequencing analysis of the cutaneous pathogenic yeast Malassezia restricta and identification of the major lipase expressed on the scalp of patients with dandruff

Malassezia species are opportunistic pathogenic fungi that are frequently associated with seborrhoeic dermatitis, including dandruff. Most Malassezia species are lipid dependent, a property that is compensated by breaking down host sebum into fatty acids by lipases. In this study, we aimed to sequence and analyse the whole genome of Malassezia restricta KCTC 27527, a clinical isolate from a Korean patient with severe dandruff, to search for lipase orthologues and identify the lipase that is the most frequently expressed on the scalp of patients with dandruff. The genome of M. restricta KCTC 27527 was sequenced using the Illumina MiSeq and PacBio platforms. Lipase orthologues were identified by comparison with known lipase genes in the genomes of Malassezia globosa and Malassezia sympodialis. The expression of the identified lipase genes was directly evaluated in swab samples from the scalps of 56 patients with dandruff. We found that, among the identified lipase‐encoding genes, the gene encoding lipase homolog MRES_03670, named LIP5 in this study, was the most frequently expressed lipase in the swab samples. Our study provides an overview of the genome of a clinical isolate of M. restricta and fundamental information for elucidating the role of lipases during fungus‐host interaction.

The lysine biosynthetic enzyme Lys4 influences iron metabolism, mitochondrial function and virulence in Cryptococcus neoformans

The lysine biosynthesis pathway via α-aminoadipate in fungi is considered an attractive target for antifungal drugs due to its absence in mammalian hosts. The iron-sulfur cluster-containing enzyme homoaconitase converts homocitrate to homoisocitrate in the lysine biosynthetic pathway, and is encoded by LYS4 in the model yeast Saccharomyces cerevisiae. In this study, we identified the ortholog of LYS4 in the human fungal pathogen, Cryptococcus neoformans, and found that LYS4 expression is regulated by iron levels and by the iron-related transcription factors Hap3 and HapX. Deletion of the LYS4 gene resulted in lysine auxotrophy suggesting that Lys4 is essential for lysine biosynthesis. Our study also revealed that lysine uptake was mediated by two amino acid permeases, Aap2 and Aap3, and influenced by nitrogen catabolite repression (NCR). Furthermore, the lys4 mutant showed increased sensitivity to oxidative stress, agents that challenge cell wall/membrane integrity, and azole antifungal drugs. We showed that these phenotypes were due in part to impaired mitochondrial function as a result of LYS4 deletion, which we propose disrupts iron homeostasis in the organelle. The combination of defects are consistent with our observation that the lys4 mutant was attenuated virulence in a mouse inhalation model of cryptococcosis.

Efficacy and Safety of Cream Containing Climbazole/Piroctone Olamine for Facial Seborrheic Dermatitis: A Single-Center, Open-Label Split-Face Clinical Study

Background Seborrheic dermatitis (SD) is a multifactorial disease; Malassezia species play an important role in its pathogenesis. Objective We aimed to determine whether a cream containing climbazole/piroctone olamine (C/P cream), antifungal agents with expected efficacy against Malassezia species, could improve SD symptoms. Methods We instructed 24 patients with mild-to-moderate SD to apply the C/P cream and emollient cream on the right and left sides of the face, respectively, every morning and evening for 4 weeks. The casual sebum level (measured with Sebumeter®; Courage & Khazaka Electronic GmbH, Germany) and the extent of erythema (measured with Mexameter®; Courage & Khazaka Electronic GmbH) on the face were measured at baseline and after 4 weeks. The minimal inhibitory concentration (MIC) was determined to demonstrate the antifungal activity of the C/P cream. Results The casual sebum level and erythema were measured at week 4, and the median values demonstrated a quantitative improvement on the C/P cream-treated right side of the face compared to the emollient cream-treated left side. For the C/P cream, the MICs were 0.625, 5, 0.625, and 2.5 mg/ml for Malassezia restricta, M. globosa, M. sympodialis, and M. slooffiae, respectively. Conclusion Based on the reduced casual sebum level and extent of erythema, the antifungal activity of C/P cream against Malassezia species seems useful for the treatment of mild to moderate SD.

Characterisation and Expression Analysis of MrLip1, a Class 3 Family Lipase of Malassezia restricta

The genus Malassezia is associated with a wide range of skin diseases and is the predominant fungal genus isolated from human skin. Of the 14 Malassezia species identified, M. restricta is the most abundant fungal species found from both healthy and diseased skin. Emerging evidences have suggested that extracellular lipases of Malassezia play a critical role in its survival on the host skin surface. This study aimed to characterise the lipase 1 homologue (MrLip1) in M. restricta and to analyse its expression under different environmental conditions. The full sequence of the gene encoding MrLip1 was determined by rapid amplification of cDNA ends, and it was then heterologously expressed in Pichia pastoris. MrLip1 protein was successfully purified and used for lipase assay and specific antibody generation for use in expression analysis. The optimum pH and temperature for the activity of purified MrLip1 were pH 5.0 and 34 °C respectively. Furthermore, the expression of MrLip1 peaked at a similar pH and temperature, suggesting that the optimal conditions for MrLip1 protein activity and expression are similar to that found on the human skin surface. This study provides data to improve our understanding of the role and characteristics of lipase 1 in M. restricta.

Understanding the Mechanism of Action of the Anti-Dandruff Agent Zinc Pyrithione against Malassezia restricta

Dandruff is known to be associated with Malassezia restricta. Zinc pyrithione (ZPT) has been used as an ingredient in anti-dandruff treatments. The mechanism of ZPT has been investigated in several studies; however, a non-pathogenic model yeast, such as Saccharomyces cerevisiae was most often used. The aim of the present study was to understand how ZPT inhibits the growth of M. restricta. We analyzed the cellular metal content and transcriptome profile of ZPT-treated M. restricta cells and found that ZPT treatment dramatically increased cellular zinc levels, along with a small increase in cellular copper levels. Moreover, our transcriptome analysis showed that ZPT inhibits Fe-S cluster synthesis in M. restricta. We also observed that ZPT treatment significantly reduced the expression of lipases, whose activities contribute to the survival and virulence of M. restricta on human skin. Therefore, the results of our study suggest that at least three inhibitory mechanisms are associated with the action of ZPT against M. restricta: (i) an increase in cellular zinc levels, (ii) inhibition of mitochondrial function, and (iii) a decrease in lipase expression.

In Vitro Anti-Malassezia Activity of Castanea crenata Shell and Oil-Soluble Glycyrrhiza Extracts

Background A new shampoo with anti-Malassezia properties obtained from various plants is required to provide seborrheic dermatitis patients with a wider range of treatment options. Objective The aim of this study was to obtain in vitro susceptibility profiles of Malassezia restricta and M. globosa, the most important pathogenic organisms in the development of seborrheic dermatitis, to the plant extracts used in commercial anti-dandruff shampoos. Methods Minimal inhibitory concentrations (MICs) were determined for eight candidate plant extracts and two plant-derived natural products diluted with Leeming and Notman medium to final concentrations of 0.016 to 1 mg/ml. Results Castanea crenata shell, Camellia sinensis leaf, and oil-soluble Glycyrrhiza extracts presented relatively low MIC values (≤0.5 mg/ml) against both strains. The C. crenata shell and oil-soluble Glycyrrhiza extracts demonstrated especially high anti-Malassezia activity, suggesting their potential use in the treatment of seborrheic dermatitis. The extracts also showed fungistatic activity against other common facultative pathogenic yeasts, Cryptococcus and Candida. Conclusion C. crenata shell and oil-soluble Glycyrrhiza extracts could potentially be used as active ingredients in anti-seborrheic and anti-dandruff shampoo formulations. They could be helpful for repeated treatments and regular prophylaxis of scalp seborrheic dermatitis.

Mitochondrial Protein Nfu1 Influences Homeostasis of Essential Metals in the Human Fungal Pathogen Cryptococcus neoformans.

Abstract Mitochondrial protein Nfu1 plays an important role in the assembly of mitochondrial Fe-S clusters and intracellular iron homeostasis in the model yeast Saccharomyces cerevisiae. In this study, we identified the Nfu1 ortholog in the human fungal pathogen Cryptococcus neoformans. Our data showed that C. neoformans Nfu1 localized in the mitochondria and influenced homeostasis of essential metals such as iron, copper and manganese. Marked growth defects were observed in the mutant lacking NFU1, which suggests a critical role of Nfu1 in Fe-S cluster biosynthesis and intracellular metal homeostasis in C. neoformans.