Draft Genome Sequence of the Ectomycorrhizal Fungus Astraeus odoratus from Northern Thailand.

We report the draft genome sequence of Astraeus odoratus, an edible ectomycorrhizal fungus from northern Thailand. The assembled genome has a size of 45.1 Mb and 13,403 annotated protein-coding genes. This reference genome will provide a better understanding of the biology of mushroom-forming ectomycorrhizal fungi in the family Diplocystidiaceae.

Exploring the antifungal activity and mechanism of action of Zingiberaceae rhizome extracts against Malassezia furfur.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhizomes from members of Zingiberaceae have long been used in Thai traditional medicine to treat cutaneous fungal infections, including Malassezia-related skin disorders. Alpinia galanga, Curcuma longa, Zingiber cassumunar, and Zingiber officinale are particularly popular in folk remedies. AIM OF THE STUDY: On account of the application background in traditional medicine, the present study aims to screen and determine the composition and possible mechanism of the rhizome extracts of selected Zingiberaceae and corresponding fractions against M. furfur. MATERIALS AND METHODS: All solvent extracts (ethanol, methanol, and n-hexane) obtained from each plant were screened for anti-Malassezia activity by agar disc diffusion assay. The MIC and MFC values of the potent rhizome extract and its bioactive fraction isolated by TLC were determined using broth dilution assay followed by chemical characterization using GC-MS. The anti-Malassezia mechanism was investigated by macroscopic and microscopic observation of cells grown in the yeast phase and hyphal phase. RESULTS: The primary screening results showed that the n-hexane extract from A. galanga possessed the most significant anti-Malassezia activity. The MIC and MFC values of this extract were in a range of 0.04-0.08 mg/mL and 0.04-0.16 mg/mL, respectively. The TLC purification of the n-hexane extract from A. galanga gave a total of nine fractions, of which only a single exhibited anti-Malassezia activity. The GC-MS analysis of the rhizome extract and the derivative fraction revealed that the major constituents were (2,6-dimethylphenyl)borate followed by a trace content of 1,8-cineol and hydrocarbons. For the antifungal mechanism of the fraction, treatments of the fraction led to morphological changes in cell size and shape, exerted massive vacuoles in yeast form, and inhibited the transition to hyphae but not likely affected chitin contents of the cell wall of M. furfur. CONCLUSIONS: According to the results, the n-hexane extract of A. galanga rhizome exhibits promising anti-Malassezia potential. The inhibitory effect on virulent hyphal growth supports that A. galanga is a valuable source of natural antifungal agents for further pharmaceutical research.

Ambient pH regulates secretion of lipases in Malassezia furfur.

Malassezia is a lipophilic cutaneous commensal yeast and associated with various skin disorders. The yeast also causes bloodstream infection via intravascular catheters and can be detected even in human gut microbiota. Ambient pH is one of the major factors that affect the physiology and metabolism of several pathogenic microorganisms. Although dynamic changes of pH environment in different parts of the body is a great challenge for Malassezia to confront, the role that ambient pH plays in Malassezia is largely unknown. In this study, we investigated the impact of ambient pH on physiology and expression of lipases in M. furfur grown under different pH conditions. The yeast was able to grow in media ranging from pH 4 to 10 without morphological alteration. Elevation in pH value enhanced the extracellular lipase activity but decreased that of intracellular lipase. The qPCR results revealed that a set of functional lipase genes, LIP3-6, were constitutively expressed regardless of pH conditions or exposure time. Based on the data, we conclude that the external pH plays a promotional role in the secretion of lipases but exerts less effect on transcription of the genes and morphology in M. furfur.

Differential Expression of Extracellular Lipase and Protease Activities of Mycelial and Yeast Forms in Malassezia furfur.

Malassezia furfur is a dimorphic yeast that is part of the human skin microflora. This fungus is a pathogen of a certain skin diseases, such as pityriasis versicolor, and in rare cases causes systemic infection in neonates. However, the role of dimorphism in the pathogenicity remains unclear. A modified induction medium (IM) was successfully able to induce mycelial growth of M. furfur under both solid and liquid condition. Filamentous elements with branching hyphae were observed when cultured in the IM. Furthermore, addition of bovine fetus serum into the liquid IM did not promote hyphal formation; on the contrary, it retrograded hyphae to the yeast form. Plate-washing assay showed that M. furfur hyphae did not possess the ability of invasive growth. Secretory proteins from both yeast and hyphal forms were isolated, and lipase and protease activities were analyzed. Intriguingly, the hyphal form showed higher activities than those of the yeast form, particularly the protease activity.

Identification of the haemolytic activity of Malassezia species.

Malassezia species are part of the normal skin flora and are associated with a number of human and animal skin diseases. However, the mechanisms that mediate infection and host-fungal interactions are poorly understood. The haemolytic activity of several microorganisms is considered a factor that contributes to pathogenicity of the organism to humans and animals. This virulence factor was previously identified in several pathogenic fungi that cause systemic mycoses, such as Aspergillus and Candida. In this study, the haemolytic activity of six major Malassezia species, including M. furfur, M. globosa, M. pachydermatis, M. restricta, M. slooffiae and M. sympodialis, was investigated. The haemolytic activity of these species was tested on tryptone soya agar with 5% sheep blood. All the examined Malassezia species produced a halo zone of complete haemolysis. A quantitative analysis of the haemolytic activity was performed by incubating sheep erythrocytes with the extraction from culture of each Malassezia species. Interestingly, M. globosa and M. restricta showed significantly high haemolytic activity compared with the other Malassezia species. In addition, M. globosa also exhibited stable haemolytic activity after treatment at 100 °C and in the presence of some proteases, indicating that this haemolytic factor is different from those of other fungi.

Purification and characterization of a secretory lipolytic enzyme, MgLIP2, from Malassezia globosa.

Malassezia globosa is a lipid-dependent yeast that is found on the human skin and is associated with various skin disorders, including dandruff and seborrhoeic dermatitis (SD). Despite its important role in skin diseases, the molecular basis for its pathogenicity is poorly understood. The current hypothesis is that dandruff and SD are linked to fatty acid metabolism and secretory lipolytic enzymes, which hydrolyse sebaceous lipids and release irritating free fatty acids. A previous genomic analysis of M. globosa identified a family of 13 homologous genes predicted to encode secreted lipases. We have also reported that M. globosa had significantly higher extracellular lipase activity compared with other species. To identify the major secretory lipases of this yeast during its growth, we successfully purified and characterized an extracellular lipase MgLIP2. Based on MALDI-TOF MS, the peptide mass fingerprint of a tryptically digested protein MgLIP2 corresponded to ORF MGL_4054 of M. globosa. This lipase showed high esterase activity against 4-nitrophenyl palmitate and 1-naphthyl palmitate but not 1-naphthyl acetate. This enzyme had optimal activity at 30 °C and pH 5.0. Furthermore, the activity significantly increased in the presence of Triton X-100 and was partially inhibited by PMSF but was unaffected by univalent and divalent metal ions.

The lipolytic enzymes activities of Malassezia species.

Malassezia yeasts are part of the cutaneous microflora commonly found on animals and human and may sometimes cause various opportunistic skin diseases. As most of Malassezia species show lipid-dependency, lipolytic enzymes such as lipase and phospholipase are necessary for them to obtain useful lipids from the environment. Consequently, these enzymes are thought to play an important role in the growth and pathogenicity of Malassezia. Here we analyze and compare extracellular lipase and phospholipase activities of several Malassezia species cultivated under common growth conditions. M. globosa showed the highest lipase activity of all of the Malassezia species included in our studies. The lipid-independent M. pachydermatis also showed high lipase and phospholipase activity. These results indicate that this Malassezia species are capable of utilizing lipids well in contrast to the other lipid-dependent species of the genus. Our data suggest that lipase may be a pathogenic factor in the skin disease associated with Malassezia and provide an explanation as to why M. globosa is an important pathogenic species in several human skin diseases despite its slow rate of growth.