Mycelial Phase Research Articles

Comparing the sugar profiles and primary structures of alkali-extracted water-soluble polysaccharides in cell wall between the yeast and mycelial phases from Tremella fuciformis.

To gain insights into dimorphism, cell wall polysaccharides from Tremella fuciformis strains were obtained from alkali-extracted water-soluble fractions PTF-M38 (from the mycelial form), PTF-Y3 and PTF-Y8 (from the yeast form) of T. fuciformis strains were used to gain some insights into dimorphism study. Their chemical properties and structural features were investigated using gel permeation chromatography, gas chromatography, UV and IR spectrophotometry and Congo red binding reactions. The results indicated that the backbones of PTF-M38, PTF-Y3 and PTF-Y8 were configured with α-linkages with average molecular weights of 1.24, 1.08, and 1.19 kDa, respectively. PTF-M38 was mainly composed of xylose, mannose, glucose, and galactose in a ratio of 1:1.47:0.48:0.34, while PTF-Y3 and PTF-Y8 were mainly composed of xylose, mannose and glucose in a ratio of 1:1.65:4.06 and 1:1.21:0.44, respectively. The sugar profiles of PTF-M38, PTF-Y3 and PTF-Y8 were also established for further comparison. These profiles showed that all three polysaccharides contained the same sugars but in different ratios, and the carbon sources (xylose, mannose, glucose, and galactose) affected the sugar ratios within the polysaccharides.

Sporothrix schenckii complex in Iran: Molecular identification and antifungal susceptibility.

Sporotrichosis is a global subcutaneous fungal infection caused by the Sporothrix schenckii complex. Sporotrichosis is an uncommon infection in Iran, and there have been no phenotypic, molecular typing or antifungal susceptibility studies of Sporothrix species. This study aimed to identify nine Iranian isolates of the S. schenckii complex to the species level using colony morphology, carbohydrate assimilation tests, and PCR-sequencing of the calmodulin gene. The antifungal susceptibilities of these Sporothrix isolates to five antifungal agents (amphotericin B (AMB), voriconazole (VRC), itraconazole (ITC), fluconazole (FLC), and terbinafine (TRB)) were also evaluated according to the M27-A3 and M38-A2 protocols of the Clinical and Laboratory Standards Institute for yeast and mycelial phases, respectively. Five of seven clinical isolates were identified as S. schenckii, and two clinical and two environmental isolates were identified as S. globosa. This is the first report of S. globosa in Iran. There was significant agreement (73%) between the results of the phenotypic and genotypic identification methods. TRB and ITC were the most effective antifungals against the Sporothrix isolates. The minimum inhibitory concentration (MIC) values of TRB for the yeast and mycelial phases of S. schenckii differed significantly. There was also a significant difference in the minimum fungicidal concentration (MFC) values of AMB and TRB for the two phases. Considering the low efficacy of VRC and FLC and the wide MIC ranges of AMB (1-16 μg/ml and 1-8 μg/ml for yeast and mycelial forms, respectively) observed in the present study, in vitro antifungal susceptibility testing should be performed to determine appropriate therapeutic regimens.

Expression and characterization of a Talaromyces marneffei active phospholipase B expressed in a Pichia pastoris expression system

Phospholipase B is a virulence factor for several clinically important pathogenic fungi, including Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus, but its role in the thermally dimorphic fungus Talaromyces marneffei remains unclear. Here, we provide the first report of the expression of a novel phospholipase gene, designated TmPlb1, from T. marneffei in the eukaryotic expression system of Pichia pastoris GS115. Sensitive real-time quantitative reverse-transcription PCR (qRT-PCR) demonstrated that the expression of TmPlb1 increased 1.85-fold in the yeast phase compared with the mycelial phase. TmPlb1 contains an open reading frame (ORF) of 732 bp that encodes a protein of 243 amino acids. The conserved serine, aspartate and histidine catalytic triad and the G-X-S-X-G domain of TmPLB1 provide the structural basis for its molecular activity. The ORF of TmPlb1 was successfully cloned into a pPIC9K vector containing an α-mating factor secretion signal that allowed the secretory expression of TmPLB1 in P. pastoris. The heterologous protein expression began 12 h after methanol induction and peaked at 96 h. Through analysis with SDS–polyacrylamide gel electrophoresis (SDS-PAGE), western blotting and mass spectrometry, we confirmed that TmPLB1 was successfully expressed. Through Ni-affinity chromatography, TmPLB1 was highly purified, and its concentration reached 240.4 mg/L of culture medium. With specific substrates, the phospholipase A1 and phospholipase A2 activities of TmPLB1 were calculated to be 5.96 and 1.59 U/mg, respectively. The high purity and activity of the TmPLB1 obtained here lay a solid foundation for further investigation.Emerging Microbes & Infections (2016) 5, e120; doi:10.1038/emi.2016.119; published online 23 November 2016

Nail Histomycology, Onychochromobiology, and Fungal Thigmatropism

Background: Thigmotropism is a biologic feature coping with the directional growth of cells following topographical guidance cues. This mechanism is involved in the invasive phase of pathogen and opportunistic fungi. It was shown experimentally with fungal hyphae of both dermatophytes and nondermatophyte molds, as well as with the mycelial phase of the dimorphic yeast Candida albicans. Objective: To revisit histomycology in onychomycoses of a diversity of fungal origins. Method: Histopathological section of nails plates were oriented parallel to the nail direction of growth. Result: Thigmotropism in part explains the patterns of orientations and shapes of fungi invading nail plates. It is probably influenced by onychochronobiology (speed of growth of the affected nails), and it governs various clinical presentations of onychomycoses.

Mycelial development preceding basidioma formation in Moniliophthora perniciosa is associated to chitin, sugar and nutrient metabolism alterations involving autophagy

We identified and characterized two chitinases, named MpCHIT1 and MpCHIT2, from the fungus Moniliophthora perniciosa – the etiologic agent of witches’ broom disease in cacao tree (Theobroma cacao L.) – during its development, mainly in the mycelia phases preceding the basidioma formation. The expression of MpCHIT1 and MpCHIT2, together with MpCHS and MpATG8 (chitin synthase and autophagy genes, respectively), was analyzed during the M. perniciosa growth and development on bran-based solid medium as well as in liquid medium containing H2O2 or rapamycin (oxidative and nutritional related-autophagy stress agents, respectively). In order to link the expression of chitin metabolism-related genes to nutritional composition influencing fungus development, we also quantified total and reduced sugars, as well as macro- and micronutrients in the bran-based solid medium. The expression analysis showed that the MpCHS expression increased through mycelial development and then decreased in the primordium and basidioma phases, while the expression of MpCHIT1 and MpCHIT2 was higher in basidioma and primordium phases, respectively. Moreover, the expression pattern of MpCHIT1 and MpCHIT2 is distinct, the second correlated with the MpATG8 expression pattern and possibly with autophagy process, while the first may be related to the basidioma formation. The quantification of total and reduced sugars, as well as macro- and micronutrients supported the idea that the cell wall restructuration due to MpCHS, MpCHIT1 and MpCHIT2 is related to stress and fungal nutrient reallocation, allowing the formation and development of the basidioma. Experiments involving M. perniciosa growth on liquid medium containing H2O2 or rapamycin showed that MpCHIT1 and MpCHIT2 were over-expressed in response to oxidative but also to nutritional related-autophagy stresses. Interestingly, the expression level of MpCHS, MpCHIT1 and MpCHIT2 in presence of rapamycin is similar to the one observed in the primordium and basidioma from bran-based solid medium. The analysis of the overall data allowed designing a general scheme of chitin metabolism and autophagy during M. perniciosa development, focusing on the mycelium phases as crucial and environmentally influenced steps preceding the primordium and basidioma formation. These data support the idea that the nutritional environment of M. perniciosa influences its development and life cycle.

Construction and analysis of the cDNA subtraction library of yeast and mycelial phases of Sporothrix globosa isolated in China: identification of differentially expressed genes.

Species included in the Sporothrix schenckii complex are temperature-dependent with dimorphic growth and cause sporotrichosis that is characterized by chronic and fatal lymphocutaneous lesions. The putative species included in the Sporothrix complex are S. brasiliensis, S. globosa, S. mexicana, S. pallida, S. schenckii, and S. lurei. S. globosa is the causal agent of sporotrichosis in China, and its pathogenicity appears to be closely related to the dimorphic transition, i.e. from the mycelial to the yeast phase, it adapts to changing environmental conditions. To determine the molecular mechanisms of the switching process that mediates the dimorphic transition of S. globosa, suppression subtractive hybridization (SSH) was used to prepare a complementary DNA (cDNA) subtraction library from the yeast and mycelial phases. Bioinformatics analysis was performed to profile the relationship between differently expressed genes and the dimorphic transition. Two genes that were expressed at higher levels by the yeast form were selected, and their differential expression levels were verified using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). It is believed that these differently expressed genes are involved in the pathogenesis of S. globosa infection in China.

RNAseq Analysis Highlights Specific Transcriptome Signatures of Yeast and Mycelial Growth Phases in the Dutch Elm Disease FungusOphiostoma novo-ulmi

Fungal dimorphism is a complex trait and our understanding of the ability of fungi to display different growth morphologies is limited to a small number of model species. Here we study a highly aggressive dimorphic fungus, the ascomycete Ophiostoma novo-ulmi, which is a model in plant pathology and the causal agent of Dutch elm disease. The two growth phases that this fungus displays, i.e., a yeast phase and mycelial phase, are thought to be involved in key steps of disease development. We used RNAseq to investigate the genome-wide gene expression profiles that are associated with yeast and mycelial growth phases in vitro. Our results show a clear molecular distinction between yeast and mycelial phase gene expression profiles. Almost 12% of the gene content is differentially expressed between the two phases, which reveals specific functions related to each growth phase. We compared O. novo-ulmi transcriptome profiles with those of two model dimorphic fungi, Candida albicans and Histoplasma capsulatum. Few orthologs showed similar expression regulation between the two growth phases, which suggests that, globally, the genes associated with these two life forms are poorly conserved. This poor conservation underscores the importance of developing specific tools for emerging model species that are distantly related to the classical ones. Taken together, our results provide insights into transcriptome regulation and molecular specificity in O. novo-ulmi and offer a new perspective for understanding fungal dimorphism.

Mutabilis in mutabili: Spatiotemporal dynamics of a truffle colony in soil

The functioning of ectomycorrhizal (ECM) symbioses is closely related to the development of the soil mycelial phase the ECM fungi. The properties and spatiotemporal dynamics of such mycelia in ecosystems is, however, poorly understood. Here we show, using a soil colony of summer truffle (Tuber aestivum) as a model, that an ECM mycelium may only grow and colonize newly-opened soil patches when soil temperatures rise above certain threshold, in this case +10 °C, provided other requirements such as sufficient soil moisture are fulfilled. Extension rates of truffle mycelium in the fields was recorded as >0.3 μm min−1, several-fold greater than that predicted from laboratory cultures. Further, we demonstrated that there was a consistent spatial differentiation in mycelium growth patterns within the fungal colony on a decimeter scale, changing from “diffusion” type of growth at the colony margin to “colony-front” pattern further away from the colony margin. This change was clearly accompanied by shifting structure of soil microbial communities with Terrimonas sp. and another unidentified bacterium correlating with the “colony-front” mycelium growth pattern, and Sphingomonas sp. and Lysobacter brunnescens with the “diffusion” type of mycelium growth. Possible implications of the observed truffle colony differentiation are discussed for processes like fruit-body formation and dispersal of this ECM fungus. Our data indicate that the thallus of T. aestivum has to be considered as a principally variable (“mutabilis”) being in space and time, whose behavior correlates with conditions in ever changing soil environment (“in mutabili”).

Signature gene expression reveals novel clues to the molecular mechanisms of dimorphic transition in Penicillium marneffei.

Systemic dimorphic fungi cause more than one million new infections each year, ranking them among the significant public health challenges currently encountered. Penicillium marneffei is a systemic dimorphic fungus endemic to Southeast Asia. The temperature-dependent dimorphic phase transition between mycelium and yeast is considered crucial for the pathogenicity and transmission of P. marneffei, but the underlying mechanisms are still poorly understood. Here, we re-sequenced P. marneffei strain PM1 using multiple sequencing platforms and assembled the genome using hybrid genome assembly. We determined gene expression levels using RNA sequencing at the mycelial and yeast phases of P. marneffei, as well as during phase transition. We classified 2,718 genes with variable expression across conditions into 14 distinct groups, each marked by a signature expression pattern implicated at a certain stage in the dimorphic life cycle. Genes with the same expression patterns tend to be clustered together on the genome, suggesting orchestrated regulations of the transcriptional activities of neighboring genes. Using qRT-PCR, we validated expression levels of all genes in one of clusters highly expressed during the yeast-to-mycelium transition. These included madsA, a gene encoding MADS-box transcription factor whose gene family is exclusively expanded in P. marneffei. Over-expression of madsA drove P. marneffei to undergo mycelial growth at 37°C, a condition that restricts the wild-type in the yeast phase. Furthermore, analyses of signature expression patterns suggested diverse roles of secreted proteins at different developmental stages and the potential importance of non-coding RNAs in mycelium-to-yeast transition. We also showed that RNA structural transition in response to temperature changes may be related to the control of thermal dimorphism. Together, our findings have revealed multiple molecular mechanisms that may underlie the dimorphic transition in P. marneffei, providing a powerful foundation for identifying molecular targets for mechanism-based interventions.

Melanization and morphological effects on antifungal susceptibility of Penicillium marneffei.

The biosynthesis of melanin has been linked with virulence in diverse pathogenic fungi. Penicillium marneffei, a dimorphic fungus, is capable of melanization in both mycelial and yeast phases, and the pigment may be produced during infection to protect the fungus from the host immune system. To investigate the impact of yeast morphological transformation on antifungal susceptibility, P. marneffei was cultured on various media including minimal medium, 1% tryptone, brain heart infusion broth, and malt extract broth by using the standardized susceptibility protocol (the M27-A protocol, RPMI medium) for yeasts. We also investigated whether P. marneffei melanization affected its susceptibility to antifungal drugs by adding L-DOPA into culture broths. There were no differences in the minimum inhibitory concentrations of P. marneffei yeast cells previously grown in various culture broths with or without L-DOPA using the M27A protocol (into which no melanin substrate can be added due to a rapid colour change of the RPMI medium to black) for testing amphotericin B, clotrimazole, fluconazole, itraconazole and ketoconazole. However, both melanized and non-melanized P. marneffei displayed increased resistance to antifungal drugs when L-DOPA was added into a selected assay medium, 0.17% yeast nitrogen base, 2% glucose, and 1.5% agar. Hence, active melanin formation appears to protect P. marneffei by enhancing its resistance to antifungal drugs.

Effect of Histoplasma capsulatum glucans on host innate immunity

β-1,3-Glucan is important for infective forms (mycelial phase) of Histoplasma capsulatum and shares many features allotted to pathogen-associated molecular patterns. These cell wall carbohydrates interact with phagocytes by binding to Toll and lectin-like receptors, present on cell surfaces of macrophages, neutrophils, and dendritic cells. This review focuses on recent findings of the major H. capsulatum and host carbohydrate-driven interactions that account for internalization of fungal infective forms into phagocytes, and its subsequent avoidance of intracellular elimination. The yeast phase of H. capsulatum possesses different modulating factors of the macrophagic-anti-fungal mechanisms, mainly α-1,3-glucan, which is considered relevant for virulence.This manuscript is part of the series of works presented at the “V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi” (Oaxaca, Mexico, 2012).

Proteome profiling of the dimorphic fungusPenicillium marneffeiextracellular proteins and identification of glyceraldehyde-3-phosphate dehydrogenase as an important adhesion factor for conidial attachment

Despite being the most important thermal dimorphic fungus causing systemic mycosis in Southeast Asia, the pathogenic mechanisms of Penicilliummarneffei remain largely unknown. By comparing the extracellular proteomes of P.marneffei in mycelial and yeast phases, we identified 12 differentially expressed proteins among which glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and heat shock protein 60 (HSP60) were found to be upregulated in mycelial and yeast phases respectively. Based on previous findings in other pathogens, we hypothesized that these two extracellular proteins may be involved in adherence during P.marneffei-host interaction. Using inhibition assays with recombinant GAPDH (rGAPDH) proteins and anti-rGAPDH sera, we demonstrated that adhesion of P.marneffei conidia to fibronectin and laminin was inhibited by rGAPDH or rabbit anti-rGAPDH serum in a dose-dependent manner. Similarly, a dose-dependent inhibition of conidial adherence to A549 pneumocytes by rGAPDH or rabbit anti-rGAPDH serum was observed, suggesting that P.marneffei GAPDH can mediate binding of conidia to human extracellular matrix proteins and pneumocytes. However, HSP60 did not exhibit similar inhibition on conidia adherence, and neither GAPDH norHSP60 exhibited inhibition on adherence to J774 or THP-1 macrophage cell lines. This report demonstrates GAPDH as an adherence factor in P.marneffei by mediating conidia adherence to host bronchoalveolar epithelium during the early establishment phase of infection.

Histoplasma yeast and mycelial transcriptomes reveal pathogenic-phase and lineage-specific gene expression profiles

BackgroundThe dimorphic fungus Histoplasma capsulatum causes respiratory and systemic disease in mammalian hosts by expression of factors that enable survival within phagocytic cells of the immune system. Histoplasma’s dimorphism is distinguished by growth either as avirulent mycelia or as pathogenic yeast. Geographically distinct strains of Histoplasma differ in their relative virulence in mammalian hosts and in production of and requirement for specific virulence factors. The close similarity in the genome sequences of these diverse strains suggests that phenotypic variations result from differences in gene expression rather than gene content. To provide insight into how the transcriptional program translates into morphological variation and the pathogenic lifestyle, we compared the transcriptional profile of the pathogenic yeast phase and the non-pathogenic mycelial phase of two clinical isolates of Histoplasma.ResultsTo overcome inaccuracies in ab initio genome annotation of the Histoplasma genome, we used RNA-seq methodology to generate gene structure models based on experimental evidence. Quantitative analyses of the sequencing reads revealed 6% to 9% of genes are differentially regulated between the two phases. RNA-seq-based mRNA quantitation was strongly correlated with gene expression levels determined by quantitative RT-PCR. Comparison of the yeast-phase transcriptomes between strains showed 7.6% of all genes have lineage-specific expression differences including genes contributing, or potentially related, to pathogenesis. GFP-transcriptional fusions and their introduction into both strain backgrounds revealed that the difference in transcriptional activity of individual genes reflects both variations in the cis- and trans-acting factors between Histoplasma strains.ConclusionsComparison of the yeast and mycelial transcriptomes highlights genes encoding virulence factors as well as those involved in protein glycosylation, alternative metabolism, lipid remodeling, and cell wall glycanases that may contribute to Histoplasma pathogenesis. These studies lay an essential foundation for understanding how gene expression variations contribute to the strain- and phase-specific virulence differences of Histoplasma.

Identification of MicroRNA-Like RNAs in Mycelial and Yeast Phases of the Thermal Dimorphic Fungus Penicillium marneffei

Background Penicillium marneffei is the most important thermal dimorphic fungus causing systemic mycosis in China and Southeast Asia. While miRNAs are increasingly recognized for their roles in post-transcriptional regulation of gene expression in animals and plants, miRNAs in fungi were less well studied and their potential roles in fungal dimorphism were largely unknown. Based on P. marneffei genome sequence, we hypothesize that miRNA-like RNAs (milRNAs) may be expressed in the dimorphic fungus.Methodology/Principal FindingsWe attempted to identify milRNAs in P. marneffei in both mycelial and yeast phase using high-throughput sequencing technology. Small RNAs were more abundantly expressed in mycelial than yeast phase. Sequence analysis revealed 24 potential milRNA candidates, including 17 candidates in mycelial and seven in yeast phase. Two genes, dcl-1 and dcl-2, encoding putative Dicer-like proteins and the gene, qde-2, encoding Argonaute-like protein, were identified in P. marneffei. Phylogenetic analysis showed that dcl-2 of P. marneffei was more closely related to the homologues in other thermal dimorphic pathogenic fungi than to Penicillium chrysogenum and Aspergillus spp., suggesting the co-evolution of dcl-2 among the thermal dimorphic fungi. Moreover, dcl-2 demonstrated higher mRNA expression levels in mycelial than yeast phase by 7 folds (P<0.001). Northern blot analysis confirmed the expression of two milRNAs, PM-milR-M1 and PM-milR-M2, only in mycelial phase. Using dcl-1KO, dcl-2KO, dclDKO and qde-2KO deletion mutants, we showed that the biogenesis of both milRNAs were dependent on dcl-2 but not dcl-1 or qde-2. The mRNA expression levels of three predicted targets of PM-milR-M1 were upregulated in knockdown strain PM-milR-M1 KD, supporting regulatory function of milRNAs.Conclusions/SignificanceOur findings provided the first evidence for differential expression of milRNAs in different growth phases of thermal dimorphic fungi and shed light on the evolution of fungal proteins involved in milRNA biogenesis and possible role of post-transcriptional control in governing thermal dimorphism.

Study of the distribution of Malassezia species in patients with pityriasis versicolor and healthy individuals in Tertiary Care Hospital, Punjab

Purpose: Pityriasis versicolor (PV) is a chronic superficial fungal disease caused by Malassezia species. Our aim was to identify Malassezia species from PV patients and healthy individuals in Punjab. Materials and Methods: Modified Dixon agar was used as isolation culture medium. Identification was based on morphological observation and biochemical evaluation. The biochemical evaluation consisted of culture onto Sabouraud dextrose agar, catalase reaction, Tween assimilation, Cremophor EL assimilation, splitting of esculin and growth at 380C. Results: Out of 58 microscopically diagnosed cases of PV, growth was obtained from 54 (93.10%) cases. The most frequently isolated species were M. globosa, M. sympodialis and M. furfur which made up 51.79%, 31.42% and 18.51% of the isolated etiological agents respectively. However, the major isolate from the back of healthy individuals was M. sympodialis (47.61%), followed by M.obtusa (19.04%), M. globosa (14.20%), M. furfur (9.52%), M. pachydermatis (4.76%) and M. slooffiae (4.76%). Conclusions:M. globosa in its mycelial phase was the main etiological agent, but as normal flora from the back of healthy subjects, it was found in significantly less number (P = 0.01), suggesting that the higher pathogenicity of M. globosa in terms of enzymatic endowment, might be the cause of its predominance in PV lesions.

HISTOPLASMIN SURVEY IN HIV-POSITIVE PATIENTS: RESULTS FROM AN ENDEMIC AREA IN NORTHEASTERN BRAZIL

SUMMARY Background. Disseminated histoplasmosis is common in AIDS patients with advanced immunosuppression in Ceará, Northeastern Brazil. The goal of this study was to determine the prevalence of Histoplasma infection in patients with HIV/AIDS living in Fortaleza, the capital of Ceará. Methods. Intradermal tests with histoplasmin (mycelial phase) were performed in 161 HIV patients with CD4 ≥ 350 cells/mm 3 . Evidence of recent illness was evaluated with immunodiffusion (ID) tests in 76 of these individuals. Results. A total of 11.8% of patients reacted to histoplasmin and 2.63% had ID test positive to Histoplasma. The presence of mango trees (Mangifera indica) in the patient neighborhood (OR = 2.870; 95% CI = 1.081-7.617; p = 0.040) and past activity involving soil (OR = 2.834; 95% CI = 1.045-7.687; p = 0.045) or visits to a farm (OR = 3.869; 95% CI = 1.189-12.591; p = 0.033) were significantly associated with Histoplasma infection. Conclusions. Patients with HIV living in Fortaleza have an expressive prevalence of infection with Histoplasma.

Regulation of genes involved in cell wall synthesis and structure duringUstilago maydisdimorphism

The cell wall is the structure that provides the shape to fungal cells and protects them from the difference in osmotic pressure existing between the cytosol and the external medium. Accordingly, changes in structure and composition of the fungal wall must occur during cell differentiation, including the dimorphic transition of fungi. We analyzed, by use of microarrays, the transcriptional regulation of the 639 genes identified to be involved in cell wall synthesis and structure plus the secretome of the Basidiomycota species Ustilago maydis during its dimorphic transition induced by a change in pH. Of these, 189 were differentially expressed during the process, and using as control two monomorphic mutants, one yeast like and the other mycelium constitutive, 66 genes specific of dimorphism were identified. Most of these genes were up-regulated in the mycelial phase. These included CHS genes, genes involved in β-1,6-glucan synthesis, N-glycosylation, and proteins containing a residue of glycosylphosphatidylinositol, and a number of genes from the secretome. The possible significance of these data on cell wall plasticity is discussed.

Malassezia species isolated from lesional and nonlesional skin in patients with pityriasis versicolor

Objectives This study aimed to identify Malassezia species found in pityriasis versicolor (PV) patients as well as to establish whether there is any association between the various species of Malassezia isolated from lesional and from nonlesional skin of those patients. Background PV is a common, benign, superficial cutaneous fungal infection caused by Malassezia species usually characterized by hypopigmented or hyperpigmented macules and patches on the chest and back. These lesions are scaly and their color varies from red, pale, yellow brown to dark brown. Patients and methods A total of 100 patients with PV were included in the study. The samples were obtained by scraping the skin surface, both from lesional and from nonlesional skin, and then incubated on Dixon's agar. The yeast isolated were identified using conventional biochemical tests and Tween assimilation. Results Skin lesions were 100% KOH positive for Malassezia species and 10% KOH positive in healthy skin. Growth from skin lesion was obtained on Dixon's agar in 83 patients (83%). The most common species was Malassezia furfur as it was detected in 47 samples (47%), followed by Malassezia globosa (23%) and Malassezia sympodialis (13%). From healthy skin of patients, growth of Malassezia was obtained from 19 specimens, M. furfur was detected in 13%, and M. globosa was detected in only three patients (3%) and M. sympodialis in three patients (3%). Conclusion M. furfur in its mycelial phase was the predominant species involved in the etiology of PV and M. globosa was the second most common species. The most frequently isolated species from clinically healthy skin was M. furfur and was recovered from healthy skin only in yeast form. We found no difference in the distribution of Malassezia species between lesional and nonlesional skin. Furthermore, no species predominance was observed whether with hyperpigmented or hypopigmented lesions as M. fufur was the most common species in both lesions in PV patients.

Molecular and Morphological Data Support the Existence of a Sexual Cycle in Species of the Genus Paracoccidioides

The genus Paracoccidioides includes the thermodimorphic species Paracoccidioides brasiliensis and P. lutzii, both of which are etiologic agents of paracoccidioidomycosis, a systemic mycosis that affects humans in Latin America. Despite the common occurrence of a sexual stage among closely related fungi, this has not been observed with Paracoccidioides species, which have thus been considered asexual. Molecular evolutionary studies revealed recombination events within isolated populations of the genus Paracoccidioides, suggesting the possible existence of a sexual cycle. Comparative genomic analysis of all dimorphic fungi and Saccharomyces cerevisiae demonstrated the presence of conserved genes involved in sexual reproduction, including those encoding mating regulators such as MAT, pheromone receptors, pheromone-processing enzymes, and mating signaling regulators. The expression of sex-related genes in the yeast and mycelial phases of both Paracoccidioides species was also detected by real-time PCR, with nearly all of these genes being expressed preferentially in the filamentous form of the pathogens. In addition, the expression of sex-related genes was responsive to the putative presence of pheromone in the supernatants obtained from previous cocultures of strains of two different mating types. In vitro crossing of isolates of different mating types, discriminated by phylogenetic analysis of the α-box (MAT1-1) and the high-mobility-group (HMG) domain (MAT1-2), led to the identification of the formation of young ascocarps with constricted coiled hyphae related to the initial stage of mating. These genomic and morphological analyses strongly support the existence of a sexual cycle in species of the genus Paracoccidioides.

Construction of recombinant protein expressed clone of Penicillium marneffei glyceraldehyde-3-phosphate dehydrogenase

Approximately 1 kb of P. marneffei gpdA was cloned into pPICzaB plasmid and transform into TOP10 E. coli competent cells. The positive clones were checked with PCR and nucleotide sequencing with AOX1 primers. After linearized plasmid with MssI restriction enzyme, the fragment was transformed into P. pastoris competent yeast cells and selected with YPDS-zeocinTM agar. The selected recombinant yeast colonies were picked and checked with specific primers by PCR (Figure 2). Penicillium marneffei is an intracellular dimorphic fungus that can cause a fatal disseminated disease in human immunodeficiency virus-infected patients (1). The factors that affect the pathogenicity of this fungus remain unclear. Recently, gpdA gene encoding GAPDH in P. marneffei was isolated and characterized. The results from this study revealed that the expression of gpdA in conidia and mycelial phase was higher than yeast phase of P. marneffei (2). GAPDH is a molecule located at the fungal cell wall, which binds components of the extracellular matrix and is capable of mediating the adherence and internalization of fungi to cells, suggesting it may involve in fungal pathogenesis (3). In this study, we have first constructed recombinant yeast which expressed GAPDH protein of P. marneffei for further studies of protein characterization and adherence properties (4).