Malassezia Research Articles

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.

Diacylglycerol production by genetically modified lipase from Malassezia globosa

Diacylglycerol (DAG)-enriched oil has drawn considerable attention for the prevention of obesity and other lifestyle-related diseases. In this study, a mutant lipase of SMG1 (SMG1-F278D) from Malassezia globosa was studied for the production of DAG. The SMG1-F278D exhibited 4-fold increased esterification activity as well as superior fatty acid (FA) specificity for medium chain FAs. Molecular docking study suggested that the caprylic acid (CA) was strongly bound to the catalytic residue Ser171 present in the SMG1-F278D as compared to SMG1. Molecular Dynamics (MD) simulations were employed in order to understand the structural conformations of SMG1 and SMG1-F278D. The structure of SMG1-F278D was found to be more compact as well as less deviated from its native conformation. There was an increase in β-sheet as well as α-helix in the SMG1-F278D that may stabilize the protein structure due to point mutation. Finally, the capability of SMG1-F278D in synthesis of DAG was evaluated. Effects of reaction parameters such as substrate molar ratio of glycerol to FAs, enzyme loading and reaction temperature on the esterification were investigated. The optimal reaction conditions were achieved as 4:1 molar ratio of glycerol to FAs, enzyme loading of 100U/g (U/w, with respect to the total substrates) and temperature of 25°C. The present study provides important information about SMG1 mutant for better utilization in the oils and fats industries.

Skin microbiome and atopic dermatitis

Skin microbiome maintain homeostasis with the host, and affect skin barrier and immune function. The components of skin microbiome are diverse and specific, and are affected by multiple factors. The predominance of Staphylococcus aureus and decrease in diversity of skin microbiome are a characteristic of atopic dermatitis. The overgrowth of S. aureus can aggravate inflammatory reactions in AD. S. epidermidis, although another predominant bacterium in AD, exerts an immunoprotective role by regulating skin barrier-associated immunoreactions through the dendritic cells, interleukin (IL) -17A-producing Th17 cells/IL-17 pathway, and by suppressing the overgrowth of S. aureus. Malassezia can induce and aggravate inflammatory reactions in AD through colonization, sensitization, cross reactions, and other mechanisms. Studies on skin probiotics may provide new directions for the treatment of AD. Key words: Skin; Microbial consortia; Dermatitis, atopic; Prebiotics; Immunity; Staphylococcus aureus

Synthesis and Characterization of Silver Nanoparticles Using <i>Azadirachta indica</i> Leaf Extract and their Anti-Fungal Activity against <i>Malassezia </i>species

Fungal skin infections are caused by different types of fungi among these Malassezia species is the common cause of the dermatitis in human and animals. In the present study, the skin scraping samples were collected from human and dog. The samples were inoculated into Saboraud and potato dextrose broth to achieve the fungal growth. The fungal species were isolated and characterized by colony morphology, potassium hydroxide (KOH) and lacto phenol cotton blue staining. Genomic DNA was isolated and the 28S rRNA was amplified from fungal species using the universal primers and the amplified PCR products were subjected to sequencing. The sequence analysis of 28S rRNA reveals that two sequences were similar to Malassezia globosa and one sequence is similar to Malassezia pachydermatis which causes dermatitis in human and dog, respectively. Further study was carried out to assess the antifungal activity of the silver nanoparticles synthesized through green synthesis using Azadirachta indica leaf extract and characterized by UV-Visible spectrophotometer, Transmission electron microscope (TEM), X-Ray diffraction spectrophotometer (XRD) and Fourier transform infrared spectrophotometer (FTIR). The characterized silver nanoparticles inhibit the growth of Malassezia species by forming zone of clearance. This study suggests that the silver nanoparticles could be an alternative to treat the fungal infections.

Simplified Enzymatic Upgrading of High-Acid Rice Bran Oil Using Ethanol as a Novel Acyl Acceptor.

One of the major challenges in the upgrading of high-acid rice bran oil (RBO) is to efficiently reduce the amount of free fatty acids. Here we report a novel method for upgrading high-acid RBO using ethanol as a novel acyl acceptor in combination with a highly selective lipase from Malassezia globosa (SMG1-F278N). This process enabled an unprecedented deacidification efficiency of up to 99.80% in a short time (6 h); the immobilized SMG1-F278N used in deacidification exhibited excellent operational stability and could be used for at least 10 consecutive batches without detectable loss in activity. Scale-up was performed under optimized conditions to verify the applicability of this process, and low-acid (0.08%) RBO with a high level of γ-oryzanol (27.8 g/kg) and γ-oryzanol accumulation fold (1.5) was obtained after molecular distillation at lower temperature (120 °C). Overall, we report a simplified and efficient procedure for the production of edible RBO from high-acid RBO.

Immobilization of SMG1-F278N lipase onto a novel epoxy resin: Characterization and its application in synthesis of partial glycerides

In this study, a mutant lipase of SMG1 from Malassezia globosa (SMG1-F278N) was immobilized directly from crude fermentation broth onto a novel epoxy resin (ECR8285). The obtained immobilized SMG1-F278N was characterized and its application in synthesis of partial glycerides was evaluated. The immobilization was best achieved by an initial buffer pH of 6.0, an ionic strength of 1.5mol/L and a lipase/support ratio of 20mg/g. Several analytical approaches such as Scanning electron microscope (SEM) and Fourier transform infrared (FT-IR) spectroscopy showed that SMG1-F278N was successfully immobilized onto ECR8285. Besides, the properties of immobilized SMG1-F278N were investigated and it was shown that immobilized SMG1-F278N improved their performance in wider ranges of pH and temperature. It was interesting that the thermostability of immobilized SMG1-F278N was improved significantly relative to the free SMG1-F278N. Moreover, the immobilized SMG1-F278N was employed to synthesis of partial glycerides [diacylglycerol (DAG), monoacylglycerol (MAG)] via esterification of oleic acids and glycerol. High yield (79%) of partial glycerides was obtained and it was observed that high substrate molar ratio of glycerol to oleic acids contributes to the conversion of oleic acids as well as the accumulation of MAG. The immobilized SMG1-F278N was quite stable and can be reused for 7 cycles without significant loss in activity (2%). The obtained results demonstrated that the immobilized SMG1-F278N is a prospective enzyme in oils and fats industry.

Diverse Human Skin Fungal Communities in Children Converge in Adulthood

Understanding the skin mycobiome (fungal communities) is important because both commensal and pathogenic fungi can drive cutaneous disease depending on host status and body sites, including the scalp, feet, and groin. Interestingly, age may also affect skin fungal infections as certain dermatophytoses (i.e., tinea capitis) are more frequent in children than adults. We previously described the skin mycobiomes in healthy adults, showing lipophilic fungi Malassezia predominate in most skin sites. Because children have less sebaceous skin before puberty, we compared the fungal communities of primary clinical samples from healthy children and adults, based on sequencing of a fungal phylogenetic marker. Although Malassezia predominated on the trunk, head, and arm skin of adults (age 18-39), children (age < 14) had more diverse fungal communities, for example, Eurotiomycetes, which includes common dermatophytes. Species-level classification showed that Malassezia globosa predominated in children. Collectively, our findings indicate that prepubertal skin is colonized by diverse fungi, whereas adult skin is predominantly obligatory lipophilic Malassezia, suggesting that fungal communities on skin profoundly shift during puberty. Mycobiome shifts during puberty are likely due to alterations in sebaceous gland activation and sebum composition. This study provides a foundational framework for studies investigating interactions between fungi, skin, and pediatric dermatophytosis.

Bortezomib inhibits bacterial and fungal β-carbonic anhydrases

Inhibition of the β-carbonic anhydrases (CAs, EC 4.2.1.1) from pathogenic fungi (Cryptococcus neoformans, Candida albicans, Candida glabrata, Malassezia globosa) and bacteria (three isoforms from Mycobacterium tuberculosis, Rv3273, Rv1284 and Rv3588), as well from the insect Drosophila melanogaster (DmeCA) and the plant Flaveria bidentis (FbiCA1) with the boronic acid peptidomimetic proteosome inhibitor bortezomib was investigated. Bortezomib was a micromolar inhibitor of all these enzymes, with KIs ranging between 1.12 and 11.30μM. Based on recent crystallographic data it is hypothesized that the B(OH)2 moiety of the inhibitor is directly coordinated to the zinc ion from the enzyme active site. The class of boronic acids, an under-investigated type of CA inhibitors, may lead to the development of anti-infectives with a novel mechanism of action, based on the pathogenic organisms CA inhibition.

Pityriasis Versicolor: Clinical Spectrum and Diagnosis

Pityriasis versicolor (PV) is caused by the fungus Malassezia, especially by Malassezia globosa. The predisposing factors for PV are heat, humidity, hyperhidrosis, oral contraceptives, stress, application of oily preparations, and treatment with corticosteroids. PV is found mainly on the seborrheic areas of the trunk, shoulders, upper aspects of the arms, and neck, but it may spread to the face, scalp, submammary areas, axillae, groin, skin folds, and buttocks. It is characterized by hyperpigmented, hypopigmented, or erythematous (versicolor), round-to-oval, finely scaling, thin plaques. Other less frequent clinical variants are papular, atrophic, imbricata, and pityriasis rubra pilaris-like presentations. The diagnosis of PV is usually made clinically with the aid of Wood’s light or dermoscopy. Direct microscopic examination, culture, biopsy, and molecular studies are among the laboratory diagnostic methods. Topical therapy is the treatment of choice for most patients. Systemic imidazole therapy is usually reserved for widespread or resistant cases.

Azole Antifungal Sensitivity of Sterol 14α-Demethylase (CYP51) and CYP5218 from Malassezia globosa.

Malassezia globosa cytochromes P450 CYP51 and CYP5218 are sterol 14α-demethylase (the target of azole antifungals) and a putative fatty acid metabolism protein (and a potential azole drug target), respectively. Lanosterol, eburicol and obtusifoliol bound to CYP51 with Kd values of 32, 23 and 28 μM, respectively, catalyzing sterol 14α-demethylation with respective turnover numbers of 1.7 min−1, 5.6 min−1 and 3.4 min−1. CYP5218 bound a range of fatty acids with linoleic acid binding strongest (Kd 36 μM), although no metabolism could be detected in reconstitution assays or role in growth on lipids. Clotrimazole, fluconazole, itraconazole, ketoconazole, voriconazole and ketaminazole bound tightly to CYP51 (Kd ≤ 2 to 11 nM). In contrast, fluconazole did not bind to CYP5218, voriconazole and ketaminazole bound weakly (Kd ~107 and ~12 μM), whereas ketoconazole, clotrimazole and itraconazole bound strongest to CYP5218 (Kd ~1.6, 0.5 and 0.4 μM) indicating CYP5218 to be only a secondary target of azole antifungals. IC50 determinations confirmed M. globosa CYP51 was strongly inhibited by azole antifungals (0.15 to 0.35 μM). MIC100 studies showed itraconazole should be considered as an alternative to ketoconazole given the potency and safety profiles and the CYP51 assay system can be used in structure-activity studies in drug development.

N-Nitrosulfonamides: A new chemotype for carbonic anhydrase inhibition

A series of N1-substituted aromatic sulfonamides was obtained by applying a selective sulfonamide nitration synthetic strategy leading to Ar-SO2NHNO2 derivatives which were investigated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. Two human (h) hCA isoforms, the cytosolic hCA II and the transmembrane hCA IX, in addition to the fungal enzyme from Malassezia globosa, MgCA, were included in the study. Most of the new compounds reported selectively inhibited hCA IX over hCA II and at the same time showed effective MgCA inhibitory properties, with KIs ranging between 0.22 and 8.09μM. The N-nitro sulfonamides are a new chemotype with CA inhibitory effects. As hCA IX was recently validated as antitumor/antimetastatic drug target, its selective inhibition could be exploited for interesting biomedical applications. Moreover, due to the effective MgCAs inhibitory properties of the N-nitro sulfonamides, of considerable interest in the cosmetics field as potential anti-dandruff agents, the N-nitro sulfonamides may be considered as interesting leads for the design of more efficient compounds targeting fungal enzymes.

Evaluation of Malassezia and Common Fungal Pathogens in Subtypes of Chronic Rhinosinusitis.

Fungal hypersensitivity and fungal microbiome dysbiosis are possible etiologies of chronic rhinosinusitis. The sinus fungal microbiome is not well characterized; novel sinus-associated fungi, including Malassezia, have only recently been described. The goals for this study were to verify Malassezia as a dominant component of the sinus microbiome, to speciate sinus Malassezia, and to compare select fungal species in chronic rhinosinusitis (CRS) subtypes with known fungal association to chronic rhinosinusitis with polyps (CRSwNP) and healthy controls. Twenty-eight patients were enrolled and categorized as CRSwNP (n = 15), fungus ball (n = 3), allergic fungal rhinosinusitis (AFRS, n = 3), or healthy control (n = 7). Brush samples were taken from ethmoid or maxillary sinus mucosa and tested for DNA from 7 index fungi using quantitative polymerase chain reaction. Index fungal species were chosen based on existing data of the sinus fungal microbiome. Malassezia species were detected in 68% of patients, without variation among clinical phenotypes (p > 0.99). Malassezia restricta was more commonly detected than Malassezia globosa (p = 0.029). Presence of one Malassezia species predicted the presence of the other (p = 0.035). Aspergillus was identified in 2 of 3 of fungus ball patients (both A. fumigatus) and 2 of 3 AFRS patients (1 A. fumigatus and 1 A. flavus). Aspergillus was absent in control and CRSwNP patients (p < 0.001). This study confirmed and speciated Malassezia in healthy and diseased sinuses. Presence of Malassezia species in all groups suggests a commensal role for the fungus. Future work will determine whether Malassezia influences CRS pathogenesis. Aspergillus species were identified in fungal CRS subtypes despite negative surgical cultures, highlighting the importance of culture-independent technology.

Secreted lipases from Malassezia globosa: recombinant expression and determination of their substrate specificities.

Malassezia globosa, which is associated with skin conditions such as dandruff and seborrhoeic dermatitis, possesses 13 secreted lipases, but only MgLip1, MgMDL2 and MgLip2 have been characterized. To understand the substrate preferences of these lipases and by extension their potential role in colonizing human skin, we expressed all 13 predicted secreted lipases in Pichia pastoris and evaluated their ability to utilize mono-, di- and triolein substrates. The M. globosa family class 3 lipases were shown to be specific for mono- and diacylglycerols, but exhibited no regio-selective production of diacylglycerols, which are of special interest for industrial applications. Lipases belonging to the Lip family utilized all substrates. In a further step, five lipases previously demonstrated to be expressed on human skin were tested against the eight most common di- and triacylglycerols in human sebum. All lipases liberated free fatty acids from three to eight of these substrates, proving their ability to hydrolyse key components of human sebum. Again, only Lip family lipases showed activity on triacylglycerides. Based on the demonstrated activity and expression levels of MgLip2 in M. globosa, the Lip lipase family appears to have the highest impact for the pathogenicity of M. globosa.

MICROBIAL COMPOSITION OF VARIOUS SURFACES OF SKIN DURING DEVELOPMENT OF ATOPIC DERMATITIS BASED ON DATA FROM MALDI-TOF MASS-SPECTROMETRY IDENTIFICATION METHOD

Study transformation of skin microflora during development of atopic dermatitis. 45 patients with various forms of atopic dermatitis (AtD) were examined. Control group consisted of 26 healthy individuals. The strains were cultivated on elective nutrient media. Identification ofthe isolated strains was carried out by MALDI-TOF mass-spectrometry method. A low frequency of occurrence of taxon Staphylococcus epidermidis on face skin and high frequency of occurrence of Staphylococcus aureus on upper and lower limb skin was established for AtD patients compared with healthy individuals. The frequency of occurrence of proteolyti-cally active isolates of S. aureus in AtD patients was 3 times higher than in healthy carriers of this taxon. Taxons of microorganisms not inherent to healthy individuals such as Bacillus mycoides, Pseudomonas putida, Pseudomonas radiobacter were isolated on lower limb and neck skin of AtD patients. A high frequency of occurrence of Cryptococcus satoi, Candida albicans, Malassezia globosa fungi was noted. A decrease of barrier functions of skin during AtD facilitates contamination of patients' skin with rare bacterial taxons and fungi. One of the possible mechanisms of suppression of immune competent cell functions could be proteolytic enzymes of S. aureus.

Lid mobility in lipase SMG1 validated using a thiol/disulfide redox potential probe

Most lipases possess a lid domain above the catalytic site that is responsible for their activation. Lipase SMG1 from Malassezia globose CBS 7966 (Malassezia globosa LIP1), is a mono‐ and diacylglycerol lipase with an atypical loop‐like lid domain. Activation of SMG1 was proposed to be solely through a gating mechanism involving two residues (F278 and N102). However, through disulfide bond cross‐linking of the lid, this study shows that full activation also requires mobility of the lid domain, contrary to a previous proposal. The newly introduced disulfide bond makes lipase SMG1 eligible as a ratiometric thiol/disulfide redox potential probe, when it is coupled with chromogenic substrates. This redox‐switch lipase could also be of potential use in cascade biocatalysis.

Domestic shower hose biofilms contain fungal species capable of causing opportunistic infection.

The domestic environment can be a source of pathogenic bacteria. We show here that domestic shower hoses may harbour potentially pathogenic bacteria and fungi. Well-developed biofilms were physically removed from the internal surface of shower hoses collected in four locations in England and Scotland. Amplicon pyrosequencing of 16S and 18S rRNA targets revealed the presence of common aquatic and environmental bacteria, including members of the Actinobacteria, Alphaproteobacteria, Bacteroidetes and non-tuberculous Mycobacteria. These bacteria are associated with infections in immunocompromised hosts and are widely reported in shower systems and as causes of water-acquired infection. More importantly, this study represents the first detailed analysis of fungal populations in shower systems and revealed the presence of sequences related to Exophiala mesophila, Fusarium fujikuroi and Malassezia restricta. These organisms can be associated with the environment and healthy skin, but also with infection in compromised and immuno-competent hosts and occurrence of dandruff. Domestic showering may result in exposure to aerosols of bacteria and fungi that are potentially pathogenic and toxigenic. It may be prudent to limit development of these biofilms by the use of disinfectants, or regular replacement of hoses, where immuno-compromised persons are present.

English

Yeasts of the Malassezia genus are linked to seborrheic dermatitis (SD) in humans; however, etiological species causing this disease can vary according to their geographical location. M. globosa and M. restricta are the most often isolated microorganisms and can be found in the skin of patients with SD. Nevertheless, species identification by molecular methods and the relationship among etiological agents and the clinical severity of the disease have not been determined in Mexican patients. The goal of this study was to analyze the prevalence of Malassezia species in the skin of SD patients by molecular methods in order to establish their distribution according to the severity of the disease in Monterrey, Nuevo Leon, Mexico. Skin samples from patients with SD (n = 60) were obtained by scraping and were cultured on modified Dixon agar. The Malassezia colonies were identified by amplification of the D1/D2 regions of 26S rDNA by polymerase chain reaction (PCR) and subsequent sequencing and BLAST analysis in GenBank. The positive Malassezia culture rate was 48.3%. The most commonly isolated species were Malassezia furfur (20%), Malassezia globosa (16.7%), Malassezia sympodialis (6.7%), Malassezia restricta (3.3%) and Malassezia slooffiae (1.7%). No significant difference was found in the distribution of Malassezia species according to disease severity. Key words: Seborrheic dermatitis, Malassezia, LSU rDNA D1/D2, yeasts, Malassezia furfur; M. globosa.

Carbonic anhydrase activators: Activation of the β-carbonic anhydrase from Malassezia globosa with amines and amino acids.

The β-carbonic anhydrase (CA, EC 4.2.1.1) from the dandruff producing fungus Malassezia globosa, MgCA, was investigated for its activation with amines and amino acids. MgCA was weakly activated by amino acids such as L-/D-His, L-Phe, D-DOPA, D-Trp, L-/D-Tyr and by the amine serotonin (KAs of 12.5-29.3μM) but more effectively activated by d-Phe, l-DOPA, l-Trp, histamine, dopamine, pyridyl-alkylamines, and 4-(2-aminoethyl)-morpholine, with KAs of 5.82-10.9μM. The best activators were l-adrenaline and 1-(2-aminoethyl)piperazine, with activation constants of 0.72-0.81μM. This study may help a better understanding of the activation mechanisms of β-CAs from pathogenic fungi as well as the design of tighter binding ligands for this enzyme which is a drug target for novel types of anti-dandruff agents.

Sweat Allergy.

For many years, sweat has been recognized as an exacerbation factor in all age groups of atopic dermatitis (AD) and a trigger of cholinergic urticaria (CholU). Recently, we reported the improvement of AD symptoms by spray with tannic acid, which suppresses basophil histamine release by semipurified sweat antigens in vitro, and showering that removes antigens in sweat from the skin surface. We finally identified MGL_1304 secreted by Malassezia globosa as a major histamine-releasing antigen in human sweat. MGL_1304 is detected as a 17-kDa protein in sweat and exhibits almost the highest histamine-release ability from basophils of patients with AD and CholU among antigens derived from Malassezia species. Moreover, serum levels of anti-MGL_1304 IgE of patients with AD and CholU were significantly higher than those of normal controls. Desensitization therapy using autologous sweat or MGL_1304 purified from culture of M. globosa or its cognates might be beneficial for patients with intractable CholU due to sweat allergy.

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

BackgroundSeborrheic dermatitis (SD) is a multifactorial disease; Malassezia species play an important role in its pathogenesis.ObjectiveWe 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.MethodsWe 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.ResultsThe 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.ConclusionBased 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.