Unveiling the structure of GPI-anchored protein of Malassezia globosa and its pathogenic role in pityriasis versicolor.

Glycosylphosphatidylinositols (GPI)-anchored proteins (GpiPs) are related to the cell wall biogenesis, adhesion, interactions, protease activity, mating, etc. These proteins have been identified in many organisms, including fungi such as Neurospora crassa, Candida albicans, Saccharomyces cerevisiae, and Fusarium graminearum. MGL-3153 gene of Malassezia globosa (M. globosa) encodes a protein which is homologous of the M. restricta, M. sympodialis, M. Pachydermatis, and U. maydis GpiPs. Real-time PCR assay showed that the expression of MGL_3153 gene was significantly up-regulated among M. globosa isolated from patients with pityriasis versicolor (PV) compared to a healthy individual, suggesting the contribution of this gene in the virulence of M. globosa. Accordingly, the sequence of this protein was analyzed by bioinformatics tools to evaluate the structure of that. The conservation analysis of MGL-3153 protein showed that the C-terminal region of this protein, which is responsible for GPI-anchor ligation, was highly conserved during evolution while the N-terminal region just conserved in Malassezia species. Moreover, the predicted tertiary structure of this protein by homology modeling showed that this protein almost has alpha helix structure and represented a stable structure during 150 ns of molecular dynamic simulation. Our results revealed that this protein potentially belongs to GPI-anchored proteins and may contribute to the virulence of M. globosa which warrants further investigations in this area.

The lipid profile of three Malassezia species assessed by Raman spectroscopy and discriminant analysis.

Malassezia yeasts constitute the major eukaryotic cutaneous flora of homoeothermic vertebrates. These lipophilic yeasts are able to cause, trigger, or aggravate common skin diseases under favorable conditions. Species identification and subspecies differentiation is currently based on morphological characteristics, lipid assimilation profile, and molecular tests. Mass spectrometry has been also reported as a reliable, yet costly and labor-intensive, method to classify Malassezia yeasts. Here, we introduce Raman spectroscopy as a new molecular technique able to differentiate three phylogenetically close Malassezia species (M.globosa, M.pachydermatis, and M.sympodialis) by examining their lipid metabolic profile. Using Raman spectroscopy, lipid fingerprints of Malassezia cultures on Leeming-Notman agar, were analyzed by spectral bands assignment and partial least squares discriminant analysis. Our results demonstrate differential utilization of lipid supplements among these three species and the ability of Raman spectroscopy to rapidly and accurately discriminate them by predictive modelling.

Tape mount immunostaining: a versatile method for immunofluorescence analysis of fungi.

AIM: Immunofluorescence microscopy is a powerful technique to detect surface antigens and study their distribution. Analysis of fungi is often hampered by their weak adherence to glass. We therefore established a novel immunofluorescence staining method to overcome this problem. MATERIALS & METHODS: Fungal material from colonies is bound to adhesive tape and stained with antibodies. RESULTS: The obtained samples had very good optical quality, showing low unspecific background staining and allowing analysis by confocal laser scanning microscopy. We have exemplified applying the new method to study the distribution of galactomannan on conidiophores of Aspergillus fumigatus and of ß-glucans on Malassezia pachydermatis. CONCLUSION: Tape mount immunostaining facilitates analysis of fungal surface molecules and provides a base for expeditious diagnostic procedures.

Yeast Smell Like What They Eat: Analysis of Volatile Organic Compounds of Malassezia furfur in Growth Media Supplemented with Different Lipids.

Malassezia furfur is part of the human skin microbiota. Its volatile organic compounds (VOCs) possibly contribute to the characteristic odour in humans, as well as to microbiota interaction. The aim of this study was to investigate how the lipid composition of the liquid medium influences the production of VOCs. Growth was performed in four media: (1) mDixon, (2) oleic acid (OA), (3) oleic acid + palmitic acid (OA+PA), and (4) palmitic acid (PA). The profiles of the VOCs were characterized by HS-SPME/GC-MS in the exponential and stationary phases. A total number of 61 VOCs was found in M. furfur, among which alkanes, alcohols, ketones, and furanic compounds were the most abundant. Some compounds previously reported for Malassezia (γ-dodecalactone, 3-methylbutan-1-ol, and hexan-1-ol) were also found. Through our experiments, using univariate and multivariate unsupervised (Hierarchical Cluster Analysis (HCA) and Principal Component Analysis (PCA)) and supervised (Projection to Latent Structures Discriminant Analysis (PLS-DA)) statistical techniques, we have proven that each tested growth medium stimulates the production of a different volatiles profile in M. furfur. Carbon dioxide, hexan-1-ol, pentyl acetate, isomer5 of methyldecane, dimethyl sulphide, undec-5-ene, isomer2 of methylundecane, isomer1 of methyldecane, and 2-methyltetrahydrofuran were established as differentiating compounds among treatments by all the techniques. The significance of our findings deserves future research to investigate if certain volatile profiles could be related to the beneficial or pathogenic role of this yeast.

The neutral N-linked glycans of the Basidiomycetous yeasts Pseudozyma antarctica and Malassezia furfur (Subphylum Ustilaginomycotina).

Pseudozyma antarctica and Malassezia furfur are basidiomycetous yeasts under the subphylum Ustilaginomycotina. P. antarctica is a commensal organism found in certain plant species, while M. furfur is associated with several skin diseases of animals including humans. N-linked glycans of P. antarctica and M. furfur were prepared, digested with glycosidases, and structurally analyzed using high performance liquid chromatography (HPLC) and mass spectrometry (MS). Analyses revealed the presence of neutral N-linked glycans ranging in length from Man3GlcNAc2-PA to Man9GlcNAc2-PA. The two species shared the most abundant neutral N-linked glycan: Manα1-2Manα1-6(Manα1-3)Manα1-6(Manα1-2Manα1-2Manα1-3)Manß1-4GlcNAcß1-4GlcNAc (M8A). The second and third most abundant neutral N-linked glycans for P. antarctica were Manα1-2Manα1-6(Manα1-2Manα1-3)Manα1-6(Manα1-2Manα1-2Manα1-3)Manß1-4GlcNAcß1-4GlcNAc (M9A) and Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manß1-4GlcNAcß1-4GlcNAc (M5A), respectively. In the case of M. furfur, Manα1-2Manα1-6(Manα1-3)Manα1-6(Manα1-2Manα1-3)Manß1-4GlcNAcß1-4GlcNAc (M7A) was the second most abundant, while both M8A and M9A were tied for the third most abundant. The presence of putative galactose residues in the hypermannosylated neutral N-linked glycans is also discussed. This report is the first to analyze the neutral N-linked glycans of P. antarctica and M. furfur.

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.

Natural Polyphenols Selectively Inhibit ß-Carbonic Anhydrase from the Dandruff-Producing Fungus Malassezia globosa: Activity and Modeling Studies.

Around 50 % of the worldwide population is affected by dandruff, which is triggered by a variety of factors. The yeast Malassezia globosa has been labeled as the most probable causative agent for the onset of dandruff. The ß-carbonic anhydrase (CA) of MgCA was recently validated as an anti-dandruff target, with its inhibition being responsible for in vivo growth defects in the fungus. As classical CA inhibitors of the sulfonamide type give rise to permeability problems through biological membranes, finding non-sulfonamide alternatives for MgCA inhibition is of considerable interest in the cosmetic field. We recently screened a large library of human (h) CA inhibitors for MgCA inhibition, including different chemotypes, such as monothiocarbamates, dithiocarbamates, phenols, and benzoxaboroles. Herein, we expanded the research toward new MgCA inhibitors by considering a set of natural polyphenols (including flavones, flavonols, flavanones, flavanols, isoflavones, and depsides) that exhibited MgCA inhibitory activity in the micromolar range, as well as selectivity for the fungal isozyme over off-target human isoforms. The binding mode of representative derivatives within the MgCA catalytic cleft was investigated by docking studies using a homology-built model.

Biofilm, adherence, and hydrophobicity as virulence factors in Malassezia furfur.

Malassezia species are natural inhabitants of the healthy skin. However, under certain conditions, they may cause or exacerbate several skin diseases. The ability of this fungus to colonize or infect is determined by complex interactions between the fungal cell and its virulence factors. This study aims to evaluate "in vitro" the hydrophobicity levels, the adherence on a plastic surface and the biofilm formation of 16 clinical isolates of Malassezia furfur. Cellular surface hydrophobicity (CSH) levels were determined by two-phase system. The biofilm formation was determined by tetrazolium salt (XTT) reduction assay and by Scanning Electron Microscopy (SEM). Results showed many isolates were hydrophobic, adherent, and producers of biofilm on abiotic surfaces with different capacity. SEM observations confirmed an abundant extracellular matrix after 48 h of biofilm formation. About 63% of strains with high production of biofilm showed medium to high percentage of hydrophobicity and/or adherence. In addition, it has been demonstrated a correlation between hydrophobicity, adherence, and biofilm formation in about 60% of strains examined. These important virulence factors could be responsible of this yeast changing from a commensal to a pathogenic status.

Molecular epidemiology of a Malassezia pachydermatis neonatal unit outbreak.

The non-lipid-dependent yeast Malassezia pachydermatis is predominantly zoophilic but occasionally colonizes the human skin. This yeast caused an outbreak in a neonatal iIntensive care unit (NICU). This study aimed to describe the molecular epidemiology of this M. pachydermatis outbreak. All the M. pachydermatis isolates collected at a French University Hospital from January 2012 to April 2013 were included in the study. M. pachydermatis isolates, sampled from various biological samples sites in 25 patients, were identified via MALDI-TOF mass spectrometry and typed using intergenic-spacer 1 (IGS1) nucleotide sequence polymorphisms analysis. By analyzing 90 IGS1 sequences (including 43 deposited in GenBank), we found that of the 186 M. pachydermatis isolates, 47 were viable for typing and all of them clustered within type 3; 78.7% clustered within the 3D subtype; the remaining clustered within three newly described subtypes: 3E (4.3%), 3F (8.5%) and 3 G (8.5%). No particular subtype was associated with a collection site or a particular time period. This first molecular investigation of a M. pachydermatis outbreak in neonates showed that multiple genotypes can colonize the same neonate patient by. The source of this polyclonal outbreak could not be identified. It stopped after infection control measures, including the prohibition of a lipid-rich moisturizing hand cream used by the health care staff, had been implemented.

Inhibition of the ß-carbonic anhydrase from the dandruff-producing fungus Malassezia globosa with monothiocarbamates.

A series of monothiocarbamates (MTCs) was investigated for the inhibition of the ß-class carbonic anhydrase (CAs, EC 4.2.1.1) from the fungal parasite Malassezia globosa, MgCA. These MTCs incorporate various scaffolds, among which aliphatic amine with 1-4 carbons atom in their molecule, morpholine, piperazine, as well as phenethylamine and benzylamine derivatives. All the reported MTCs displayed a better efficacy in inhibiting MgCA compared to the clinically used sulphonamide drug acetazolamide (KI of 74 µM), with KIs spanning between 1.85 and 18.9 µM. The homology model of the enzyme previously reported by us was used to rationalize the results by docking some of these MTCs within the fungal CA active site. This study might be useful to enrich the knowledge of the MgCA inhibition profile, eliciting novel ideas pertaining the design of modulators with potential efficacy in combatting dandruff or other fungal infections.

Performance of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identifying Clinical Malassezia Isolates.

The genus Malassezia comprises commensal yeasts on human skin. These yeasts are involved in superficial infections but are also isolated in deeper infections, such as fungemia, particularly in certain at-risk patients, such as neonates or patients with parenteral nutrition catheters. Very little is known about Malassezia epidemiology and virulence. This is due mainly to the difficulty of distinguishing species. Currently, species identification is based on morphological and biochemical characteristics. Only molecular biology techniques identify species with certainty, but they are time-consuming and expensive. The aim of this study was to develop and evaluate a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) database for identifying Malassezia species by mass spectrometry. Eighty-five Malassezia isolates from patients in three French university hospitals were investigated. Each strain was identified by internal transcribed spacer sequencing. Forty-five strains of the six species Malassezia furfur, M. sympodialis, M. slooffiae, M. globosa, M. restricta, and M. pachydermatis allowed the creation of a MALDI-TOF database. Forty other strains were used to test this database. All strains were identified by our Malassezia database with log scores of >2.0, according to the manufacturer's criteria. Repeatability and reproducibility tests showed a coefficient of variation of the log score values of <10%. In conclusion, our new Malassezia database allows easy, fast, and reliable identification of Malassezia species. Implementation of this database will contribute to a better, more rapid identification of Malassezia species and will be helpful in gaining a better understanding of their epidemiology.

Comparison of lipid profiles of Malassezia pachydermatis strains isolated from dogs with otitis externa and without clinical symptoms of disease.

Malassezia pachydermatis can cause infections of the skin and mucous membranes, especially in animals. It becomes a problem also in medicine. It is considered that metabolic disorders as well as hormonal and immunological status of the host promote diseases caused by M. pachydermatis. Here we consider whether specific features of fungi could also favour infections. We checked whether there are differences in lipid profiles between strains obtained from dogs with otitis externa and strains obtained from healthy dogs. Lipid profiles were determined using thin layer chromatography and gas chromatography-mass spectrometry. All analyses were carried out on 32 strains derived from dogs with otitis externa and 31 strains isolated from dogs without symptoms of disease. The results show that strains isolated from dogs without symptoms of otitis externa are characterised by a higher content of fatty acids. They contain significantly more behenic and lignoceric acids on medium without addition of lipids, and more oleic acid and total monounsaturated fatty acids on medium with lipids supplementation. These strains have also a higher content of esters of ergosterol and triglycerides. Data obtained show differences which may be specific features of M. pachydermatis-specific strains related to the ability of infection, which could be not directly related of the host condition.

Novel inhibitor against Malassezia globosa LIP1 (SMG1), a potential anti-dandruff target.

Compelling evidence have demonstrated the role of lipase activity in the pathogenicity of Malassezia globosa toward dandruff and seborrheic dermatitis (D/SD). As a representative secreted lipase from M. globosa CBS 7966, Malassezia globosa LIP1 (SMG1) is considered a potential anti-dandruff target. In this study, homology modeling, docking-based virtual screening and in vitro lipase-based assay were integrated to identify the first hit compound against SMG1, with an IC50 of 20 µM against synthetic lipase substrate, and of 0.19 µM when using natural lipase substrate. Evaluation of similar compounds, along with docking, offered information on the binding patterns of the hit compound. This work is expected to serve as a starting point for the rational design of more potent inhibitors against SMG1.

Conversion of a Mono- and Diacylglycerol Lipase into a Triacylglycerol Lipase by Protein Engineering.

Despite the fact that most lipases are believed to be active against triacylglycerides, there is a small group of lipases that are active only on mono- and diacylglycerides. The reason for this difference in substrate scope is not clear. We tried to identify the reasons for this in the lipase from Malassezia globosa. By protein engineering, and with only one mutation, we managed to convert this enzyme into a typical triacylglycerol lipase (the wild-type lipase does not accept triacylglycerides). The variant Q282L accepts a broad spectrum of triacylglycerides, although the catalytic behavior is altered to some extent. From in silico analysis it seems that specific hydrophobic interactions are key to the altered substrate specificity.

Pityriazepin and other potent AhR ligands isolated from Malassezia furfur yeast.

Malassezia furfur yeast strains isolated from diseased human skin preferentially biosynthesize indole alkaloids which can be detected in the human skin and are highly potent activators of the aryl hydrocarbon receptor (AhR) and AhR-dependent gene expression. Chemical analysis of an EtOAc extract of a M. furfur strain obtained from diseased human skin and grown on l-tryptophan agar revealed several known AhR active tryptophan metabolites along with a previously unidentified compound, pityriazepin. While its structure resembled that of the known alkaloid pityriacitrin, the comprised pyridine ring had been transformed into an azepinone. The indoloazepinone scaffold of pityriazepin is extremely rare in nature and has only been reported once previously. Pityriazepin, like the other isolated compounds, was found to be a potent activator of the AhR-dependent reporter gene assay in recombinant cell lines derived from four different species, although significant species differences in relative potency were observed. The ability of pityriazepin to competitively bind to the AhR and directly stimulate AhR DNA binding classified it as a new naturally-occurring potent AhR agonist. M. furfur produces an expanded collection of extremely potent naturally occurring AhR agonists, which produce their biological effects in a species-specific manner.

Chemical organization of the cell wall polysaccharide core of Malassezia restricta.

Malassezia species are ubiquitous residents of human skin and are associated with several diseases such as seborrheic dermatitis, tinea versicolor, folliculitis, atopic dermatitis, and scalp conditions such as dandruff. Host-Malassezia interactions and mechanisms to evade local immune responses remain largely unknown. Malassezia restricta is one of the most predominant yeasts of the healthy human skin, its cell wall has been investigated in this paper. Polysaccharides in the M. restricta cell wall are almost exclusively alkali-insoluble, showing that they play an essential role in the organization and rigidity of the M. restricta cell wall. Fractionation of cell wall polymers and carbohydrate analyses showed that the polysaccharide core of the cell wall of M. restricta contained an average of 5% chitin, 20% chitosan, 5% ß-(1,3)-glucan, and 70% ß-(1,6)-glucan. In contrast to other yeasts, chitin and chitosan are relatively abundant, and ß-(1,3)-glucans constitute a minor cell wall component. The most abundant polymer is ß-(1,6)-glucans, which are large molecules composed of a linear ß-(1,6)-glucan chains with ß-(1,3)-glucosyl side chain with an average of 1 branch point every 3.8 glucose unit. Both ß-glucans are cross-linked, forming a huge alkali-insoluble complex with chitin and chitosan polymers. Data presented here show that M. restricta has a polysaccharide organization very different of all fungal species analyzed to date.

Antimycotics suppress the Malassezia extract-induced production of CXC chemokine ligand 10 in human keratinocytes.

Malassezia, a lipophilic yeast, exacerbates atopic dermatitis. Malassezia products can penetrate the disintegrated stratum corneum and encounter subcorneal keratinocytes in the skin of atopic dermatitis patients. Type 1 helper T (Th1) cells infiltrate chronic lesions with atopic dermatitis, and antimycotic agents improve its symptoms. We aimed to identify Malassezia-induced chemokines in keratinocytes and examine whether antimycotics suppressed this induction. Normal human keratinocytes were incubated with a Malassezia restricta extract and antimycotics. Chemokine expression was analyzed by enzyme-linked immunosorbent assays and real-time polymerase chain reaction. Signal transducer and activator of transcription (STAT)1 activity was examined by luciferase assays. The tyrosine-phosphorylation of STAT1 was analyzed by western blotting. The M. restricta extract increased the mRNA and protein expression of Th1-attracting CXC chemokine ligand (CXCL)10 and STAT1 activity and phosphorylation in keratinocytes, which was suppressed by a Janus kinase inhibitor. The antimycotics itraconazole, ketoconazole, luliconazole, terbinafine, butenafine and amorolfine suppressed M. restricta extract-induced CXCL10 mRNA and protein expression and STAT1 activity and phosphorylation. These effects were similarly induced by 15-deoxy-Δ-(12,14) -prostaglandin J2 (15d-PGJ2 ), a prostaglandin D2 metabolite. Antimycotics increased the release of 15d-PGJ2 from keratinocytes. The antimycotic-induced suppression of CXCL10 production and STAT1 activity was counteracted by a lipocalin-type prostaglandin D synthase inhibitor. The antimycotics itraconazole, ketoconazole, luliconazole, terbinafine, butenafine and amorolfine may suppress the M. restricta-induced production of CXCL10 by inhibiting STAT1 through an increase in 15d-PGJ2 production in keratinocytes. These antimycotics may block the Th1-mediated inflammation triggered by Malassezia in the chronic phase of atopic dermatitis.

Increased hydrophobicity in Malassezia species correlates with increased proinflammatory cytokine expression in human keratinocytes.

Malassezia cells stimulate cytokine production by keratinocytes, although this ability differs among Malassezia species for unknown reasons. The aim of this study was to clarify the factors determining the ability to induce cytokine production by human keratinocytes in response to Malassezia species. M. furfur NBRC 0656, M. sympodialis CBS 7222, M. dermatis JCM 11348, M. globosa CBS 7966, M. restricta CBS 7877, and three strains each of M. globosa, M. restricta, M. dermatis, M. sympodialis, and M. furfur maintained under various culture conditions were used. Normal human epidermal keratinocytes (NHEKs) (1 × 10(5) cells) and the Malassezia species (1 × 10(6) cells) were co-cultured, and IL-1α, IL-6, and IL-8 mRNA levels were determined. Moreover, the hydrophobicity and ß-1,3-glucan expression at the surface of Malassezia cells were analyzed. The ability of Malassezia cells to trigger the mRNA expression of proinflammatory cytokines in NHEKs differed with the species and conditions and was dependent upon the hydrophobicity of Malassezia cells not ß-1,3-glucan expression.

Fatty acid profile and influence of oxythiamine on fatty acid content in Malassezia pachydermatis, Candida albicans and Saccharomyces cerevisiae.

Malassezia pachydermatis and Candida albicans are fungi involved in the skin diseases and systemic infections. The therapy of such infections is difficult due to relapses and problems with pathogen identification. In our study, we compare the fatty acids profile of M. pachydermatis, C. albicans and S. cerevisiae to identify diagnostic markers and to investigate the effect of oxythiamine (OT) on the lipid composition of these species. Total fatty acid content is threefold higher in C. albicans and M. pachydermatis compared with S. cerevisiae. These two species have also increased level of polyunsaturated fatty acids (PUFA) and decreased content of monounsaturated fatty acids (MUFA). We noted differences in the content of longer chain (>18) fatty acids between studied species (for example a lack of 20 : 1 in S. cerevisiae and 22 : 0 in M. pachydermatis and C. albicans). OT reduces total fatty acids content in M. pachydermatis by 50%. In S. cerevisiae, OT increased PUFA whereas it decreased MUFA content. In C. albicans, OT decreased PUFA and increased MUFA and SFA content. The results show that the MUFA to PUFA ratio and the fatty acid profile could be useful diagnostic tests to distinguish C. albicans, M. pachydermatis and S. cerevisiae, and OT affected the lipid metabolism of the investigated species, especially M. pachydermatis.

Total synthesis and bioactivity of the marine alkaloid pityriacitrin and some of its derivatives.

We report herein the chemical synthesis and biological evaluation of ß-carboline alkaloid pityriacitrin and some of its new derivatives. Using tryptophan or 5-hydroxytryptophan and 5-substituted indole-3-glyoxals as the starting materials, pityriacitrin and some of its derivatives were synthesized via the acid-catalyzed Pictet-Spengler reaction and fully characterized by (1)H and (13)C NMR, mass spectroscopy and IR determinations. Biological studies revealed that pityriacitrin has a weak antiproliferative activity against a panel of breast and prostate cancer cell lines, whereas some of its derivatives exhibited stronger and potent activity, which was associated with induction of both cell apoptosis and necrosis.