pH-Dependent Expression, Stability, and Activity of Malassezia restricta MrLip5 Lipase

Background@#The lipophilic yeasts Malassezia spp. are normally resident on the surface of the human body, and often associated with various skin diseases. Of the 18 known Malassezia spp., Malassezia restricta is the most predominantly identified Malassezia sp. found on the human skin. Malassezia possesses a large number of genes encoding lipases to degrade human sebum triglycerides into fatty acids, which are required not only for their growth, but also trigger skin diseases. Previously, we have shown that MrLIP5 (MRET_0930), one of the 12 lipase genes in the genome of M. restricta, and is the most frequently expressed lipase gene in the scalp of patients with dandruff. @*Objective@#In this study, we aimed to analyze the activity, stability, and expression of MrLip5, with particular focus on pH. @*Methods@#We heterologously expressed MrLip5 in Escherichia coli, and purified and analyzed its activity and expression under different pH conditions. @*Results@#We found that MrLip5 was most active and stable and highly expressed under alkaline conditions, which is similar to that of the diseased skin surface. @*Conclusion@#Our results suggest that the activity and expression of MrLip5 are pH-dependent, and that this lipase may play an essential role at the M. restricta-host interface during disease progression.

Antifungal Mechanism of Action of Lauryl Betaine Against Skin-Associated Fungus Malassezia restricta

Betaine derivatives are considered major ingredients of shampoos and are commonly used as antistatic and viscosity-increasing agents. Several studies have also suggested that betaine derivatives can be used as antimicrobial agents. However, the antifungal activity and mechanism of action of betaine derivatives have not yet been fully understood. In this study, we investigated the antifungal activity of six betaine derivatives against Malassezia restricta, which is the most frequently isolated fungus from the human skin and is implicated in the development of dandruff. We found that, among the six betaine derivatives, lauryl betaine showed the most potent antifungal activity. The mechanism of action of lauryl betaine was studied mainly using another phylogenetically close model fungal organism, Cryptococcus neoformans, because of a lack of available genetic manipulation and functional genomics tools for M. restricta. Our genome-wide reverse genetic screening method using the C. neoformans gene deletion mutant library showed that the mutants with mutations in genes for cell membrane synthesis and integrity, particularly ergosterol synthesis, are highly sensitive to lauryl betaine. Furthermore, transcriptome changes in both C. neoformans and M. restricta cells grown in the presence of lauryl betaine were analyzed and the results indicated that the compound mainly affected cell membrane synthesis, particularly ergosterol synthesis. Overall, our data demonstrated that lauryl betaine influences ergosterol synthesis in C. neoformans and that the compound exerts a similar mechanism of action on M. restricta.

Mon1 Is Essential for Fungal Virulence and Stress Survival in Cryptococcus neoformans

Mon1 is a guanine nucleotide exchange factor subunit that activates the Ypt7 Rab GTPase and is essential for vacuole trafficking and autophagy in eukaryotic organisms. Here, we identified and characterized the function of Mon1, an ortholog of Saccharomyces cerevisiae Mon1, in a human fungal pathogen, Cryptococcus neoformans. Mutation in mon1 resulted in hypersensitivity to thermal stress. The mon1 deletion mutant exhibited increased sensitivity to cell wall and endoplasmic reticulum stress. However, the mon1 deletion mutant showed more resistance to the antifungal agent fluconazole. In vivo studies demonstrated that compared to the wild-type strain, the mon1 deletion mutant attenuated virulence in the Galleria mellonella insect model. Moreover, the mon1 deletion mutant was avirulent in the murine inhalation model. These results demonstrate that Mon1 plays a crucial role in stress survival and pathogenicity in C. neoformans.

Azole Resistance Caused by Increased Drug Efflux in Candida glabrata Isolated from the Urinary Tract of a Dog with Diabetes Mellitus

A yeast-like organism was isolated from a urine sample of a 6-year-old neutered male miniature poodle dog with urinary tract infection, diabetes ketoacidosis, and acute pancreatitis. We identified the yeast-like organism to be Candida glabrata and found that this fungus was highly resistant to azole antifungal drugs. To understand the mechanism of azole resistance in this isolate, the sequences and expression levels of the genes involved in drug resistance were analyzed. The results of our analysis showed that increased drug efflux, mediated by overexpression of ATP transporter genes CDR1 and PDH1, is the main cause of azole resistance of the C. glabrata isolated here.

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.

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.

The Zinc Transport Systems and Their Regulation in Pathogenic Fungi

Zinc is an essential micronutrient required for many enzymes that play essential roles in a cell. It was estimated that approximately 3% of the total cellular proteins are required for zinc for their functions. Zinc has long been considered as one of the key players in host-pathogen interactions. The host sequesters intracellular zinc by utilizing multiple cellular zinc importers and exporters as a means of nutritional immunity. To overcome extreme zinc limitation within the host environment, pathogenic microbes have successfully evolved a number of mechanisms to secure sufficient concentrations of zinc for their survival and pathogenesis. In this review, we briefly discuss the zinc uptake systems and their regulation in the model fungus Saccharomyces cerevisiae and in major human pathogenic fungi such as Aspergillus fumigatus, Candida albicans, and Cryptococcus gattii.

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

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.

A Ferroxidase, Cfo1, Regulates Diverse Environmental Stress Responses of Cryptococcus neoformans through the HOG Pathway

The iron uptake and utilization pathways play a critical role in allowing human pathogens, including Cryptococcus neoformans, the causative agent of fatal meningoencephalitis, to survive within the mammalian body by competing with the host for iron. Here we show that the iron regulon is also required for diverse environmental stress responses and that in C. neoformans, it is regulated by the high-osmolarity glycerol response (HOG) pathway. Between CFO1 and CFO2, two ferroxidase genes in the iron regulon, CFO1 but not CFO2 was induced during oxidative and osmotic stress. Interestingly, we found that the HOG pathway repressed basal expression of both CFO1 and CFO2. Furthermore, when the HOG pathway was blocked, CFO2 also responded to oxidative and osmotic stress and the response of CFO1 was increased. We also established that CFO1 plays a major role in responding and adapting to diverse environmental stresses, including oxidative and genotoxic damage, osmotic fluctuations, heavy metal stress, and stress induced by cell membrane destabilizers. Therefore, our findings indicate that in C. neoformans, the iron uptake and utilization pathways are not only required for iron acquisition and survival, but also play a significant role in the environmental stress response through crosstalk with the HOG pathway.

Lipolytic Enzymes Involved in the Virulence of Human Pathogenic Fungi

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.

Evaluation of Expression of Lipases and Phospholipases of Malassezia restricta in Patients with Seborrheic Dermatitis

BACKGROUND: Malassezia species (spp.) are cutaneous opportunistic pathogens and associated with various dermatological diseases including seborrheic dermatitis, dandruff and atopic dermatitis. Almost all Malassezia spp. are obligatorily lipid-dependent, which might be caused by lack of the myristic acid synthesis. Recent genome analysis of M. restricta and M. globosa suggested that the absence of a gene encoding fatty acid synthesis might be compensated by abundant genes encoding hydrolases, which produce fatty acids, and that lipases and phospholipases may play a role in virulence of the fungus. OBJECTIVE: The current study aimed to investigate the contribution of lipases and phospholipases in virulence of the M. restricta as being the most frequently isolated Malassezia spp. from the human skin. METHODS: Swap samples of two different body sites of at least 18 patients with seborrheic dermatitis were obtained and in vivo expression of lipases and phospholipases of M. restricta was analyzed by the gene specific two-step nested RT-PCR. RESULTS: The results of the current study suggest that majority of the patients display expression of lipase RES_0242. CONCLUSION: These data imply a possible role of lipase in the host environment to produce free fatty acids for the fungus.

Evaluation of Expression of Lipases and Phospholipases of Malassezia restricta in Patients with Seborrheic Dermatitis

BACKGROUND: Malassezia species (spp.) are cutaneous opportunistic pathogens and associated with various dermatological diseases including seborrheic dermatitis, dandruff and atopic dermatitis. Almost all Malassezia spp. are obligatorily lipid-dependent, which might be caused by lack of the myristic acid synthesis. Recent genome analysis of M. restricta and M. globosa suggested that the absence of a gene encoding fatty acid synthesis might be compensated by abundant genes encoding hydrolases, which produce fatty acids, and that lipases and phospholipases may play a role in virulence of the fungus. OBJECTIVE: The current study aimed to investigate the contribution of lipases and phospholipases in virulence of the M. restricta as being the most frequently isolated Malassezia spp. from the human skin. METHODS: Swap samples of two different body sites of at least 18 patients with seborrheic dermatitis were obtained and in vivo expression of lipases and phospholipases of M. restricta was analyzed by the gene specific two-step nested RT-PCR. RESULTS: The results of the current study suggest that majority of the patients display expression of lipase RES_0242. CONCLUSION: These data imply a possible role of lipase in the host environment to produce free fatty acids for the fungus.

Identification and Functional Characterization of a Cryptococcus neoformans UPC2 Homolog

Azoles are currently the most widely used class of antifungal drugs clinically, and are effective for treating fungal infections. Target site of azoles is ergosterol biosynthesis in fungal cell membrane, which is absent in the mammalian host. However, the development of resistance to azole treatments in the fungal pathogen has become a significant challenge. Here, we report the identification and functional characterization of a UPC2 homolog in the human pathogen Cryptococcus neoformans. UPC2 plays roles in ergosterol biosynthesis, which is also affected by the availability of iron in Saccharomyces cerevisiae and Candida albicans. C. neoformans mutants lacking UPC2 were constructed, and a number of phenotypic characteristics, including antifungal susceptibility and iron utilization, were analyzed. No differences were found between the mutant phenotypes and wild type, suggesting that the role of C. neoformans UPC2 homolog may be different from those in S. cerevisiae and C. albicans, and that the gene may have a yet unknown function.

A Case of Status Epilepticus Characterized by Ictal Hemiplegia

A 39-year-old woman has had several episodes of transient right hemiplegia. On neurological examination during the ictal period she had alert consciousness with aphasia, head and eyeball deviation to the right side, and right hemiplegia. Brain MRI was normal. EEG-video monitoring of ictal period showed continuous ictal discharge in the midline frontocentral area coincided with right hemiplegia. After injection of diazepam, the ictal discharge and right hemiplegia disappeared. We report a case of status epilepticus characterized by ictal hemiplegia.

Comparison of the Concentrations of 8-MOP in both Plasma and Suction Blister Fluid after Oral Ingestion

BACKGROUND: The value of plasma concentration of 8-Methoxypsoralen(8-MOP) in the supervision of photochemotherapy has been recognized. However, plasma levels of 8-MOP were not proportionate to the degree of PUVA induced erythema and couldn't alone predict the degree of PUVA induced erythemal reaction. We made a speculation that the degree of PUVA induced erythema might correlate better with skin tissue levels of 8-MOP than plasma levels. Suction blister fluid(SBF) has been known to represent tissue fluid in the skin. So we per-formed a study of comparison of 8-MOP concentrations in both plasma and SBF. OBJECTIVE: Our purpose was to evaluate the correlation of the concentrations of 8-MOP in plasma and SBF 2 hours after oral administration of 0.6 mg/kg of 8-MOP. METHODS: Twenty six patients, aged between 16 and 50 years, undergoing suction blister surgery for vitiligo treatment, participated in this open study. Single oral doses of 0.6 mg/kg of body weight of 8-MOP were taken. Blood samples(5ml) and SBF(2ml) were collected at 2 hours after the drug administration, and 8-MOP concentration in plasma and SBF were quantitated by reverse phase high-performance liquid chromatography (HPLC). RESULTS: 8-MOP concentrations in plasma and SBF ranged from 18 to 545 ng/ml and 8 to 179 ng/ml, respectively. On the analysis of linear regression, a close-relation could not be observed between two SBF levels; measured and predicted values which were calculated from measured plasma and SBF concentrations (r²=0.583, P < 0.001). CONCLUSION: The correlation of plasma and SBF concentrations of 8-MOP is weak. So, SBF levels of psoralen are recommended for the study of PUVA erythemal reactions.