Atopic dermatitis is a chronic multifactorial inflammatory skin disease, which has had a marked increase in prevalence during the last decades. Recently, a new nomenclature was recommended where the term ‘atopic eczema/dermatitis syndrome’ (AEDS) should be used to reflect the heterogeneity in this group of patients and where those patients without measurable IgE reactivity should be classified as either ‘nonallergic AEDS’ or ‘non-IgE-associated allergic AEDS’. For nearly 20 years it has been discussed whether the opportunistic yeast Malassezia, previously designated Pityrosporum, is a contributing factor to AEDS. Today there are several reports that demonstrate specific serum IgE or positive skin prick test and/or atopy patch test reactions to Malassezia in patients with AEDS. Several IgE-binding components have been identified in extracts of Malassezia ranging in molecular mass between 10 and 100 kD. The genes for nine Malassezia allergens with molecular weights ranging from 14 to 36 kD have hitherto been identified and cloned. Six of them are now produced by recombinant techniques and used in diagnostic tests. At present the genus Malassezia is subdivided into seven different species, which all have been isolated from human skin. The respective contribution of different Malassezia spp. to AEDS and in what proportion they share allergens remains to be clarified. We summarize here data that Malassezia can play a role in eliciting and maintaining eczema in patients with AEDS.

The yeast Malassezia furfur, also known as Pityrosporum orbiculare (ovale), is part of the normal microflora of the human skin but has also been associated with different skin diseases including atopic dermatitis. More than 50% of atopic dermatitis patients have positive skin test and specific IgE to M. furfur extracts; however, the pathophysiologic role of these IgE-mediated reactions in the development of the disease remains unknown. The yeast is able to produce a wide panel of IgE-binding proteins, variably recognized by sera of individual patients. In order to assess the contribution of individual components to the disease, highly pure allergen preparations are required. We have cloned M. furfur allergens from a cDNA library displayed on the phage surface, sequenced the inserts and produced recombinant proteins in Escherichia coli. Phage displaying IgE-binding proteins were selectively enriched from the library using IgE from a M. furfur-sensitized atopic dermatitis patient as a ligand. We were able to identify five different inserts coding for IgE-binding polypeptides. Three of the sequenced cDNA encode incomplete gene products with molecular masses of 21.3 kDa (MF 7), 14.4 kDa (MF 8), and 9.7 kDa (MF 9), respectively, having no sequence similarity to known proteins. The other two cDNA encode allergens of 18.2 kDa (Mal f 5) and 17.2 kDa (Mal f 6). Mal f 5 shows significant homology to M. furfur allergens Mal f 2, Mal f 3 and an Aspergillus fumigatus allergen Asp f 3. Mal f 6 has significant homology with cyclophilin. All of the recombinant polypeptides were capable of binding serum IgE from atopic dermatitis patients in immunoblotting experiments. The availability of pure recombinant M. furfur allergens will allow the careful investigation of the role of IgE-binding proteins in atopic dermatitis.

Atopy patch test reactions to Malassezia allergens differentiate subgroups of atopic dermatitis patients.Johansson, C.; Sandström, M.H.; Bartosik, Jacek; Särnhult, T.; Christiansen, J; Zargari, A.; Bäck, Ove; Wahlgren, C.F.; Faergemann, J.; Scheynius, A.; Tengvall Linder, M. Link to publication Citation for published version (APA): Johansson, C., Sandström, M. H., Bartosik, J., Särnhult, T., Christiansen, J., Zargari, A., ... Tengvall Linder, M. (2003). Atopy patch test reactions to Malassezia allergens differentiate subgroups of atopic dermatitis patients. British Journal of Dermatology, 148(3), 479-488. DOI: 10.1046479-488. DOI: 10. /j.1365479-488. DOI: 10. -2133479-488. DOI: 10. .2003 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.• Users may download and print one copy of any publication from the public portal for the purpose of private study or research.• You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal Clinical and Laboratory InvestigationsAtopy patch test reactions to Malassezia allergens differentiate subgroups of atopic dermatitis patients SummaryBackground The yeast Malassezia is considered to be one of the factors that can contribute to atopic dermatitis (AD).Objectives To investigate the reactivity to Malassezia allergens, measured as specific serum IgE, positive skin prick test and positive atopy patch test (APT), in adult patients with AD. Methods In total, 132 adult patients with AD, 14 with seborrhoeic dermatitis (SD) and 33 healthy controls were investigated for their reactions to M. sympodialis extract and three recombinant Malassezia allergens (rMal s 1, rMal s 5 and rMal s 6).Results Sixty-seven per cent of the AD patients, but only one of the SD patients and none of the healthy controls, showed a positive reaction to at least one of the Malassezia allergens (extract and ⁄ or recombinant allergens) in at least one of the tests. The levels of M. sympodialis-specific IgE in serum correlated with the total serum IgE levels. Elevated serum levels of M. sympodialis-specific IgE were found in 55% and positive APT reactions in 41% of the AD patients with head and neck dermatitis. A relatively high proportion of patients without head and neck dermatitis and patients with low total serum IgE levels had a positive APT for M. sympodialis, despite lower proportions of individuals with M. sympodialis-specific IgE among these groups of patients.Conclusions These results support that Malassezia can play a role in eliciting and maintaining eczema in patients with AD. The addition of an APT to the test battery used in this study reveals a previously overlooked impact of Malassezia hypersensitivity in certain subgroups of AD patie...

Stp1 and Stp2 are homologous transcription factors in yeast that are synthesized as latent cytoplasmic precursors with NH2-terminal regulatory domains. In response to extracellular amino acids, the plasma membrane–localized Ssy1–Ptr3–Ssy5 (SPS) sensor endoproteolytically processes Stp1 and Stp2, an event that releases the regulatory domains. The processed forms of Stp1 and Stp2 efficiently target to the nucleus and bind promoters of amino acid permease genes. In this study, we report that Asi1 is an integral component of the inner nuclear membrane that maintains the latent characteristics of unprocessed Stp1 and Stp2. In cells lacking Asi1, full-length forms of Stp1 and Stp2 constitutively induce SPS sensor–regulated genes. The regulatory domains of Stp1 and Stp2 contain a conserved motif that confers Asi1-mediated control when fused to an unrelated DNA-binding protein. Our results indicate that latent precursor forms of Stp1 and Stp2 inefficiently enter the nucleus; however, once there, Asi1 restricts them from binding SPS sensor–regulated promoters. These findings reveal an unanticipated role of inner nuclear membrane proteins in controlling gene expression.

In yeast the homologous transcription factors Stp1 and Stp2 are synthesized as latent cytoplasmic precursors with N-terminal regulatory domains. In response to extracellular amino acids the regulatory domains are endoproteolytically excised by the plasma membrane-localized SPS sensor. The processed forms of Stp1 and Stp2 efficiently enter the nucleus and induce expression of amino acid permease genes. We recently reported that the inner nuclear membrane protein Asi1 is required to prevent unprocessed forms of Stp1 and Stp2, which ectopically enter the nucleus, from binding SPS sensor-regulated promoters. Here we show that Asi3, an Asi1 homolog, and Asi2 are integral proteins of the inner nuclear membrane that function in concert with Asi1. In cells lacking any of the three Asi proteins, unprocessed full-length forms of Stp1 and Stp2 constitutively induce SPS sensor-regulated genes. Our results demonstrate that the Asi proteins ensure the fidelity of SPS sensor signaling by maintaining the dormant, or repressed state, of gene expression in the absence of inducing signals. This study documents additional components of a novel mechanism controlling transcription in eukaryotic cells.In eukaryotes the nucleoplasm and cytoplasm are separated by the nuclear envelope. The nuclear envelope consists of two closely aligned bilayers, the inner and outer membranes, each with a unique set of resident proteins. The two nuclear membranes are joined at nuclear pore complexes, which function as channels that provide selective entry and exit routes across the nuclear envelope. Aside from rather detailed knowledge regarding the structure of nuclear pores, there is markedly little known regarding non-pore nuclear proteins (1). However, accumulating data indicate that nuclear envelope proteins participate in a variety of important processes including maintenance of nuclear architecture, chromatin organization, signaling, and gene expression (1-3). Significantly, mutations in genes encoding nuclear envelope proteins are linked to at least 15 inherited human diseases and syndromes (4, 5).In mammalian cells, nuclear lamins are involved in an extensive network of protein-protein interactions, including inner nuclear membrane proteins and transcriptional regulators, some of which bind DNA (3, 6). Although yeast cells lack lamin homologs (7), processes associated with the inner nuclear membrane have been shown to influence patterns of gene expression. For example, although silencing is not obligatorily linked to perinuclear anchoring (8), recruitment of chromatin to the nuclear periphery can facilitate gene repression (9). Evidence obtained using genome-wide approaches suggest that transcriptionally active regions of chromosomes localize to nuclear pore complexes (10, 11). Consistently, a number of actively expressed genes have been found to interact with the nucleoporin Nup2; the interactions occur at the promoter region of genes and appear to correlate with early events of gene expression (12, 13). Together findings in yeast and metazoan...

For the first time the complete cDNA encoding a major allergen and novel protein of the yeast Malassezia furfur, Ma1 f 1, has been sequenced and expressed. The amino acid sequences of nine tryptic peptides of the protein were determined. Oligonucleotides were designed from these amino acid sequences. The cDNA sequence was obtained by hybridizing these primers to mRNA and enhancement by reverse-transcnptase PCR techniques. [I, 21. However, under the influence of predisposing factors, this yeast may induce allergic reactions in atopic dermatitis. The importance of M. furjiur in the pathogenesis of this disease has been investigated during the last 1 0 years and, in 40-65 % of patients, activity against M. furjiur has been shown with skin tests and/or specific IgE serology [3-81.Numerous IgE-binding components with molecular masses between 10-100 kDa have been identified by immunochemical methods [9-121. We have previously reported the production of a specific mouse monoclonal antibody (mAb 9G9) against one major allergen of M. furjiur of approximately 37 kDa [9]. This protein is now designated as Ma1 f 1 according to the new allergen nomenclature [13]. This work reports the complete cDNA sequence of a novel protein from the yeast M. furjiur that shows the criteria of a major allergen in atopic dermatitis. Note. The novel nucleotide sequence data published here have been deposited with the EMBL sequence data bank and is available under accession number X96486. 4 days at 37°C. The yeast extract was prepared as described earlier [9]. The proteins were reduced, alkylated with 50 mM iodoacetamide and separated on a 12.5 % SDS/polyacrylamide gel. Proteins were visualized by Coomassie blue staining and the allergen verified by immunoblotting using our mAb 9G9. MATERIALS AND METHODS IsolationProteolytic digestion, fractionation of internal fragments. The stained protein band was excised from the gel and subjected to in-gel digestion followed by separation of peptides [14]. Briefly, the gel piece was washed, dried and digested with 0.5 pg modified trypsin, sequence grade (Promega, Scandinavian Diagnostic Services) at 30°C overnight. The generated fragments were extracted and isolated by narrow-bore reverse-phase liquid chromatography using a column of pRPC C2/C18 SC 2.1/10 operated in a SMART system (Pharmacia Biotech). A linear gradient of acetonitrile (0.25 %/min) in 0.06 '% trifluoroacetic acid was used at a flow rate of 100 pl/min to elute the peptides. The eluate was monitored at 215 nm and 280 nm.ELISA assay. 54 fractions of the tryptic protein digest were analyzed by the standard ELlSA technique using our specific mAb 9G9 [9]. Briefly, the peptides were coated onto an ELISA plate, blocked with phosphate-buffered saline (137 mM NaCI, 2.7 mM KCI, 10.5 mM NaHPO,, 1.7 mM KH,PO,, pH 7.4) containing 0.5 % BSA and incubated overnight at room temperature with the mAb 9G9. After washing with 0.9% NaCI/O.S% Tween 20, the wells were incubated for 4 h at room temperature with alkaline-phosphatase-conjugated rabbit anti-mouse ...

The yeast Pityrosporum orbiculare (P. orbiculare) is a member of the normal human cutaneous flora, but it is also associated with several clinical manifestations of the skin. We have previously observed IgE-binding components in P. orbiculare extracts, using sera from patients with atopic dermatitis. In the present study, we raised several monoclonal antibodies (MoAbs) against P. orbiculare to characterize some of its antigens, and used Candida albicans (C. albicans) as a control. We obtained several IgG1 MoAbs which specifically recognized P. orbiculare in ELISA. Two of these were selected for immunoblotting studies on P. orbiculare, and two patterns of reactivity emerged. Firstly, one MoAb showed a distinct band at a molecular mass of 67 kDa. In the second pattern, a sharp band at about 37 kDa appeared. In contrast, the IgM antibodies raised reacted with a 14-kDa component; but they reacted with C. albicans in addition to P. orbiculare. The IgG1 antibodies seemed to react with proteins, as their ability to react in ELISA with extract pretreated with protease was greatly reduced. In contrast, IgM MoAbs were much less affected, suggesting that they recognized nonprotein components. To determine whether these MoAbs-binding components were also recognized by human IgE, we adopted a radioimmunoassay (RIA) using the MoAbs as catcher antibodies. Both the 67-kDa and the 37-kDa components were IgE-binding proteins. P. orbiculare RAST positive sera were scored as positive in the RIA, whereas the control serum was not.

In response to discrete environmental cues, Saccharomyces cerevisiae cells adjust patterns of gene expression and protein activity to optimize metabolism. Nutrient‐sensing systems situated in the plasma membrane (PM) of yeast have only recently been discovered. Ssy1p is one of three identified components of the Ssy1p–Ptr3p–Ssy5 (SPS) sensor of extracellular amino acids. SPS sensor‐initiated signals are known to modulate the expression of a number of amino acid and peptide transporter genes (i.e. AGP1, BAP2, BAP3, DIP5, GAP1, GNP1, TAT1, TAT2 and PTR2) and arginase (CAR1). To obtain a better understanding of how cells adjust metabolism in response to extracellular amino acids in the environment and to assess the consequences of loss of amino acid sensor function, we investigated the effects of leucine addition to wild‐type and ssy1 null mutant cells using genome‐wide transcription profile analysis. Our results indicate that the previously identified genes represent only a subset of the full spectrum of Ssy1p‐dependent genes. The expression of several genes encoding enzymes in amino acid biosynthetic pathways, including the branched‐chain, lysine and arginine, and the sulphur amino acid biosynthetic pathways, are modulated by Ssy1p. Additionally, the proper transcription of several nitrogen‐regulated genes, including NIL1 and DAL80, encoding well‐studied GATA transcription factors, is dependent upon Ssy1p. Finally, several genes were identified that require Ssy1p for wild‐type expression independently of amino acid addition. These findings demonstrate that yeast cells require the SPS amino acid sensor component, Ssy1p, to adjust diverse cellular metabolic processes properly.