Chitin-rich heteroglycan from Sporothrix schenckii sensu stricto potentiates fungal clearance in a mouse model of sporotrichosis and promotes macrophages phagocytosis.

BACKGROUND: Fungal cell wall polysaccharides maintain the integrity of fungi and interact with host immune cells. The immunomodulation of fungal polysaccharides has been demonstrated in previous studies. However, the effect of chitin-rich heteroglycan extracted from Sporothrix schenckii sensu stricto on the immune response has not been investigated. RESULTS: In this study, chitin-rich heteroglycan was extracted from S. schenckii sensu stricto, and immunomodulation was investigated via histopathological analysis of skin lesions in a mouse model of sporotrichosis and evaluation of the phagocytic function and cytokine secretion of macrophages in vitro. The results showed that the skin lesions regressed and granulomatous inflammation was reduced in infected mice within 5 weeks. Moreover, heteroglycan promoted the fungal phagocytosis by macrophages and modulated the cytokine secretion. Heteroglycan upregulated TNF-α expression early at 24 h and IL-12 expression late at 72 h after incubation, which might result from moderate activation of macrophages and contribute to the subsequent adaptive immune response. CONCLUSIONS: Chitin-rich heteroglycan extracted from S. schenckii sensu stricto potentiated fungal clearance in a mouse model of sporotrichosis. Moreover, chitin-rich heteroglycan promoted fungus phagocytosis by macrophages and modulated cytokines secretion. These results might indicate that chitin-rich heteroglycan could be considered as an immunomodulator used in the treatment of sporotrichosis.

From Secretion in Pichia pastoris to Application in Apple Juice Processing: Exo-Polygalacturonase from Sporothrix schenckii 1099-18.

BACKGROUND: Polygalacturonases are a group of enzymes under pectinolytic enzymes related to enzymes that hydrolyse pectic substances. Polygalacturonases have been used in various industrial applications such as fruit juice clarification, retting of plant fibers, wastewater treatment drinks fermentation, and oil extraction. OBJECTIVES: The study was evaluated at the heterologous expression, purification, biochemical characterization, computational modeling, and performance in apple juice clarification of a new exo-polygalacturonase from Sporothrix schenckii 1099-18 (SsExo-PG) in Pichia pastoris. METHODS: Recombinant DNA technology was used in this study. Two different pPIC9K plasmids were constructed with native signal sequence-ssexo-pg and alpha signal sequence-ssexo-pg separately. Protein expression and purification performed after plasmids transformed into the Pichia pastoris. Biochemical and structural analyses were performed by using pure SsExo-PG. RESULTS: The purification of SsExo-PG was achieved using a Ni-NTA chromatography system. The enzyme was found to have a molecular mass of approximately 52 kDa. SsExo-PG presented as stable at a wide range of temperature and pH values, and to be more storage stable than other commercial pectinolytic enzyme mixtures. Structural analysis revealed that the catalytic residues of SsExo- PG are somewhat similar to other Exo-PGs. The KM and kcat values for the degradation of polygalacturonic acid (PGA) by the purified enzyme were found to be 0.5868 µM and 179 s-1, respectively. Cu2+ was found to enhance SsExo-PG activity while Ag2+ and Fe2+ almost completely inhibited enzyme activity. The enzyme reduced turbidity up to 80% thus enhanced the clarification of apple juice. SsExo-PG showed promising performance when compared with other commercial pectinolytic enzyme mixtures. CONCLUSION: The clarification potential of SsExo-PG was revealed by comparing it with commercial pectinolytic enzymes. The following parameters of the process of apple juice clarification processes showed that SsExo-PG is highly stable and has a novel performance.

Comparative proteomics in the three major human pathogenic species of the genus Sporothrix.

Sporotrichosis is a subcutaneous mycosis of humans and other mammals, caused by dimorphic species of the genus Sporothrix. In Brazil, human disease is broadly linked to transmission by infected cats and is mainly caused by Sporothrix brasiliensis, Sporothrix schenckii and Sporothrix globosa. In this study, we used a nanoscale liquid chromatography coupled with tandem mass spectrometry approach to provide the yeast proteomic profiles of S. brasiliensis, S. schenckii and S. globosa. From a total of 247 identified proteins, 137 were found as differentially expressed. Functional classification revealed that most are related to carbohydrate and amino acid metabolism as well as stress response. Our data indicate that S. brasiliensis metabolism is distinct of that of S. schenckii and S. globosa, mainly regarding amino acid metabolism and cell wall remodeling, which are induced in the former. Enzymes belonging to glycolytic pathway are, on the other hand, up-regulated in S. schenckii and S. globosa. These findings may explain the previously described more virulent character of S. brasiliensis. Besides complementing genomic comparisons already published, this first comparative proteomic study provided information that indicates new aspects of Sporothrix species metabolism as well as offers information that may be useful in the development of prospective functional studies.

Formulation and Evaluation of a Novel Itraconazole-Clotrimazole Topical Emulgel for the Treatment of Sporotrichosis.

In recent years, the development of new pharmaceutical formulations for the treatment of sporotrichosis has become a relevant research field. In this work, we aimed to develop an emulgel containing itraconazole and clotrimazole to ensure therapeutic effectiveness against Sporothrix brasiliensis. The topical use of a formulation that combines both drugs represents an interesting option for the complementary treatment of sporotrichosis. The emulgel formulation was prepared and evaluated for its zeta potential, viscosity, in vitro antifungal activity and stability at different storage conditions. The results showed that the newly developed emulgel displayed promising physicochemical characteristics, as well as a good in vitro inhibitory activity against S. brasiliensis yeasts. The results obtained in this work suggest that the emulgel containing itraconazole and clotrimazole might highly be efficient and a complementary therapy to oral administration in the treatment of sporotrichosis.

Proteins Potentially Involved in Immune Evasion Strategies in Sporothrix brasiliensis Elucidated by Ultra-High-Resolution Mass Spectrometry.

Sporothrix brasiliensis is the prevalent agent of a large zoonotic outbreak in Brazil. With the involvement of several thousands of cases, this is the largest cohort of human and animal sporotrichosis on record in the world. Infections are characterized by local cutaneous dissemination in humans without underlying disease. S. brasiliensis has shown a high degree of virulence in a mouse model compared to the remaining Sporothrix species, including the ancestral species, Sporothrix schenckii The present paper investigates a genomic and expressed-proteome comparison of S. brasiliensis to S. schenckii Using bottom-up proteomics, we found 60 proteins exclusively expressed in S. brasiliensis No significant genomic differences were found among the genes coding for this protein set. A comparison with literature data identified nine proteins that are known to be involved in virulence and immune evasion in other species, several of which had not yet been reported for the Sporothrix species analyzed.IMPORTANCE Sporotrichosis is an important disease in Brazil that is caused by fungi of the genus Sporothrix and affects cats and humans. Our work investigated the proteins differentially expressed by S. brasiliensis in order to find out why this species is more virulent and pathogenic than S. schenckii We verified a set of proteins that may be related to immune escape and that can explain the high virulence.

A method for amplification of unknown flanking sequences based on touchdown PCR and suppression-PCR.

Thermal asymmetric staggered PCR is the most widely used technique to obtain the flanking sequences. However, it has some limitations, including a low rate of positivity, and complex operation. In this study, a improved method of it was made based on suppression-PCR and touchdown PCR. The PCR fragment obtained by the amplification was used directly for sequencing after gel purification. Using this improved method, the positive rate of amplified flanking sequences of the ATMT mutants reached 99%. In addition, the time from DNA extraction to flanking sequence analysis was shortened to 2 days with about 6 dollars each sample.

Immune Response Induced by an Immunodominant 60 kDa Glycoprotein of the Cell Wall of Sporothrix schenckii in Two Mice Strains with Experimental Sporotrichosis.

Cell wall (CW) components of fungus Sporothrix schenckii are the major inductors antigens of immune responses. The immunodominant 60 kDa glycoprotein (gp60) has been shown to be associated with the virulence of this fungus but its role in experimental sporotrichosis is unknown. In this work, the immunological effects of CW-purified gp60 were investigated in a model of experimental subcutaneous sporotrichosis in normal and gp60-preimmunized C57BL/6 and BALB/c mice strains which were then infected with S. schenckii conidia. Results showed that both mice strains use different cytokine profiles in order to fight S. schenckii infection; C57BL/6 mice seem to use a Th17 response while BALB/c mice tend to depend on a Th1 profile. Preimmunization with gp60 showed a downregulatory effect on the immune response since cytokines levels were diminished in both strains. There were no significant differences in the magnitude of dorsoplantar inflammation between gp60-preimmunized and nonimmunized mice of both strains. However, skin lesions due to the infection in gp60-preimmunized mice were more severe in BALB/c than in C57BL/6 mice, suggesting that the antigen exerts a higher downregulatory effect on the Th1 response.

A cell wall protein-based vaccine candidate induce protective immune response against Sporothrix schenckii infection.

Sporotrichosis is a subcutaneous mycosis caused by several closely related thermo-dimorphic fungi of the Sporothrix schenckii species complex, affecting humans and other mammals. In the last few years, new strategies have been proposed for controlling sporotrichosis owning to concerns about its growing incidence in humans, cats, and dogs in Brazil, as well as the toxicity and limited efficacy of conventional antifungal drugs. In this study, we assessed the immunogenicity and protective properties of two aluminum hydroxide (AH)-adsorbed S. schenckii cell wall protein (ssCWP)-based vaccine formulations in a mouse model of systemic S. schenckii infection. Fractioning by SDS-PAGE revealed nine protein bands, two of which were functionally characterized: a 44kDa peptide hydrolase and a 47kDa enolase, which was predicted to be an adhesin. Sera from immunized mice recognized the 47kDa enolase and another unidentified 71kDa protein, whereas serum from S. schenckii-infected mice recognized both these proteins plus another unidentified 9.4kDa protein. Furthermore, opsonization with the anti-ssCWP sera led to markedly increased phagocytosis and was able to strongly inhibit the fungus' adhesion to fibroblasts. Immunization with the higher-dose AH-adjuvanted formulation led to increased ex vivo release of IL-12, IFN-γ, IL-4, and IL-17, whereas only IL-12 and IFN-γ were induced by the higher-dose non-adjuvanted formulation. Lastly, passive transference of the higher-dose AH-adjuvanted formulation's anti-ssCWP serum was able to afford in vivo protection in a subsequent challenge with S. schenckii, becoming a viable vaccine candidate for further testing.

Proteomics-Based Characterization of the Humoral Immune Response in Sporotrichosis: Toward Discovery of Potential Diagnostic and Vaccine Antigens.

BACKGROUND: Sporothrix schenckii and associated species are agents of human and animal sporotrichosis that cause large sapronoses and zoonoses worldwide. Epidemiological surveillance has highlighted an overwhelming occurrence of the highly pathogenic fungus Sporothrix brasiliensis during feline outbreaks, leading to massive transmissions to humans. Early diagnosis of feline sporotrichosis by demonstrating the presence of a surrogate marker of infection can have a key role for selecting appropriate disease control measures and minimizing zoonotic transmission to humans. METHODOLOGY: We explored the presence and diversity of serum antibodies (IgG) specific against Sporothrix antigens in cats with sporotrichosis and evaluated the utility of these antibodies for serodiagnosis. Antigen profiling included protein extracts from the closest known relatives S. brasiliensis and S. schenckii. Enzyme-linked immunosorbent assays and immunoblotting enabled us to characterize the major antigens of feline sporotrichosis from sera from cats with sporotrichosis (n = 49), healthy cats (n = 19), and cats with other diseases (n = 20). PRINCIPAL FINDINGS: Enzyme-linked immunosorbent assay-based quantitation of anti-Sporothrix IgG exhibited high sensitivity and specificity in cats with sporotrichosis (area under the curve, 1.0; 95% confidence interval, 0.94-1; P<0.0001) versus controls. The two sets of Sporothrix antigens were remarkably cross-reactive, supporting the hypothesis that antigenic epitopes may be conserved among closely related agents. One-dimensional immunoblotting indicated that 3-carboxymuconate cyclase (a 60-kDa protein in S. brasiliensis and a 70-kDa protein in S. schenckii) is the immunodominant antigen in feline sporotrichosis. Two-dimensional immunoblotting revealed six IgG-reactive isoforms of gp60 in the S. brasiliensis proteome, similar to the humoral response found in human sporotrichosis. CONCLUSIONS: A convergent IgG-response in various hosts (mice, cats, and humans) has important implications for our understanding of the coevolution of Sporothrix and its warm-blooded hosts. We propose that 3-carboxymuconate cyclase has potential for the serological diagnosis of sporotrichosis and as target for the development of an effective multi-species vaccine against sporotrichosis in animals and humans.

Easy storage strategies for Sporothrix spp. strains.

The present study evaluated the maintenance of Sporothrix spp. (6 Sporothrix brasiliensis; 6 S. schenckii; 5 S. mexicana, and 3 S. globosa) in saline at 4°C, and in 10% glycerol plus either 10% lactose or 10% sucrose, at -20°C and -80°C. Viability was assessed after 3, 6, and 9 months of storage, through the recovery of strains on potato dextrose agar and analysis of macro- and micromorphological features. Conidium quantification was performed before and after storage, at 3, 6 and 9 months. 100% viability was observed, regardless of storage conditions or time period. Storage at 4°C and at -20°C did not alter the number of conidia, but lower conidium counts were observed at -80°C. This study shows that the combination of glycerol with lactose or sucrose is effective to maintain Sporothrix spp. at freezing temperatures.

Immunoproteomic analysis reveals a convergent humoral response signature in the Sporothrix schenckii complex.

Sporotrichosis is a polymorphic disease that affects both humans and animals worldwide. The fungus gains entry into a warm-blooded host through minor trauma to the skin, typically by contaminated vegetation or by scratches and bites from a diseased cat. Cellular and humoral responses triggered upon pathogen introduction play important roles in the development and severity of the disease. We investigated molecules expressed during the host-parasite interplay that elicit the humoral response in human sporotrichosis. For antigenic profiling, Sporothrix yeast cell extracts were separated by two-dimensional (2D) gel electrophoresis and probed with pooled sera from individuals with fixed cutaneous and lymphocutaneous sporotrichosis. Thirty-five IgG-seroreactive spots were identified as eight specific proteins by MALDI-ToF/MS. Remarkable cross-reactivity among Sporothrix brasiliensis, Sporothrix schenckii, and Sporothrix globosa was noted and antibodies strongly reacted with the 70-kDa protein (gp70), irrespective of clinical manifestation. Gp70 was successfully identified in multiple spots as 3-carboxymuconate cyclase. In addition, 2D-DIGE characterization suggested that the major antigen of sporotrichosis undergoes post-translational modifications involving glycosylation and amino acid substitution, resulting in at least six isoforms and glycoforms that were present in the pathogenic species but absent in the ancestral non-virulent Sporothrix mexicana. Although a primary environmental function related to the benzoate degradation pathway of aromatic polymers has been attributed to orthologs of this molecule, our findings support the hypothesis that gp70 is important for pathogenesis and invasion in human sporotrichosis. We propose a diverse panel of new putative candidate molecules for diagnostic tests and vaccine development. BIOLOGICAL SIGNIFICANCE: Outbreaks due to Sporothrix spp. have emerged over time, affecting thousands of patients worldwide. A sophisticated host-pathogen interplay drives the manifestation and severity of infection, involving immune responses elicited upon traumatic exposure of the skin barrier to the pathogen followed by immune evasion. Using an immunoproteomic approach we characterized proteins of potential significance in pathogenesis and invasion that trigger the humoral response during human sporotrichosis. We found gp70 to be a cross-immunogenic protein shared among pathogenic Sporothrix spp. but absent in the ancestral environmental S. mexicana, supporting the hypothesis that gp70 plays key roles in pathogenicity. For the first time, we demonstrate with 2D-DIGE that post-translational modifications putatively involve glycosylation and amino acid substitution, resulting in at least six isoforms and glycoforms, all of them IgG-reactive. These findings of a convergent humoral response highlight gp70 as an important target serological diagnosis and for vaccine development among phylogenetically related agents of sporotrichosis.

Isolation of the GFA1 gene encoding glucosamine-6-phosphate synthase of Sporothrix schenckii and its expression in Saccharomyces cerevisiae.

Glucosamine-6-phosphate synthase (GlcN-6-P synthase) is an essential enzyme involved in cell wall biogenesis that has been proposed as a strategic target for antifungal chemotherapy. Here we describe the cloning and functional characterization of Sporothrix schenckii GFA1 gene which was isolated from a genomic library of the fungus. The gene encodes a predicted protein of 708 amino acids that is homologous to GlcN-6-P synthases from other sources. The recombinant enzyme restored glucosamine prototrophy of the Saccharomyces cerevisiae gfa1 null mutant. Purification and biochemical analysis of the recombinant enzyme revealed some differences from the wild type enzyme, such as improved stability and less sensitivity to UDP-GlcNAc. The sensitivity of the recombinant enzyme to the selective inhibitor FMDP [N(3)-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid] and other properties were similar to those previously reported for the wild type enzyme.

Evolutionarily conserved roles of the dicer helicase domain in regulating RNA interference processing.

The enzyme Dicer generates 21-25 nucleotide RNAs that target specific mRNAs for silencing during RNA interference and related pathways. Although their active sites and RNA binding regions are functionally conserved, the helicase domains have distinct activities in the context of different Dicer enzymes. To examine the evolutionary origins of Dicer helicase functions, we investigated two related Dicer enzymes from the thermophilic fungus Sporotrichum thermophile. RNA cleavage assays showed that S. thermophile Dicer-1 (StDicer-1) can process hairpin precursor microRNAs, whereas StDicer-2 can only cleave linear double-stranded RNAs. Furthermore, only StDicer-2 possesses robust ATP hydrolytic activity in the presence of double-stranded RNA. Deletion of the StDicer-2 helicase domain increases both StDicer-2 cleavage activity and affinity for hairpin RNA. Notably, both StDicer-1 and StDicer-2 could complement the distantly related yeast Schizosaccharomyces pombe lacking its endogenous Dicer gene but only in their full-length forms, underscoring the importance of the helicase domain. These results suggest an in vivo regulatory function for the helicase domain that may be conserved from fungi to humans.

Detection of 2 immunoreactive antigens in the cell wall of Sporothrix brasiliensis and Sporothrix globosa.

The cell wall of members of the Sporothrix schenckii complex contains highly antigenic molecules which are potentially useful for the diagnosis and treatment of sporotrichosis. In this study, 2 immunoreactive antigens of 60 (Gp60) and 70 kDa (Gp70) were detected in the cell wall of the yeast morphotypes of Sporothrix brasiliensis and Sporothrix globosa.

Molecular identification of the Sporothrix schenckii complex / Identificación molecular del complejo Sporothrix schenckii

Sporothrix schenckii, an ascomycetous dimorphic organism that for over a century was recognized as the sole agent of sporotrichosis, a subcutaneous mycosis with a worldwide distribution. However, it has been proposed, based on physiologic and molecular aspects, that S. schenckii is a complex of distinct species: Sporothrix brasiliensis, Sporothrix mexicana, Sporothrix globosa, S. schenckii sensu strictu, Sporothrix luriei, and Sporothrix albicans (formerly Sporothrix pallida). Human disease has a broad range of clinical manifestations and can be classified into fixed cutaneous, lymphocutaneous, disseminated cutaneous, and extracutaneous sporotrichosis. The gold standard for the diagnosis of sporotrichosis is the culture; however, serologic, histopathologic and molecular approaches have been recently adopted for the diagnosis of this mycosis. Few molecular methods have been applied to the diagnosis of sporotrichosis to detect S. schenckii DNA from clinical specimens, and to identify Sporothrix spp. in culture. Until now, Sporothrix is the unique clinically relevant dimorphic fungus without an elucidated genome sequence, thus limiting molecular knowledge about the cryptic species of this complex, and the sexual form of all S. schenckii complex species. In this review we shall focus on the current diagnosis of the sporotrichosis, and discuss the current molecular tools applied to the diagnosis and identification of the Sporothrix complex species. 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) (AU)

Sporothrix schenckii, un organismo ascomiceto dimorfo que durante más de un siglo fue reconocido como el único agente de esporotricosis, una micosis subcutánea con una amplia distribución mundial. Sin embargo, se ha propuesto, con base en los aspectos fisiológicos y moleculares, que S. schenckii es un complejo de especies distintas: Sporothrix brasiliensis, Sporothrix mexicana, Sporothrix globosa, S. schenckii sensu stricto, Sporothrix luriei y Sporothrix albicans (antes Sporothrix pallida). La enfermedad humana tiene una gama de manifestaciones clínicas y puede clasificar e cutánea fija, linfocutánea, cutánea diseminad y esporotricosis extracutánea. El estándar de oro para el diagnóstico de esporotricosis es el cultivo del patógeno, sin embargo, los métodos serológicos, histopatológicos y, recientemente, los moleculares se han usado para el diagnóstico de esta micosis Pocos métodos moleculares han sido aplicados para el diagnóstico de la esporotricosis para detectar ADN de S. schenckii a partir de muestras clínicas y para identificar Sporothrix spp. en cultivo. Hasta ahora, Sporothrix es el único hongo dimorfo clínicamente relevante sin una secuencia genómica elucidada, limitando el conocimiento molecular de las especies crípticas de este complejo y la forma sexual de todas las especies del complejo S. schenckii. Esta revisión se centrará en el diagnóstico actual de la esporotricosis con énfasis en las herramientas moleculares vigentes aplicadas tanto al diagnóstico como en la identificación de las especies del complejo Sporothrix. Este artículo científico es parte de la serie de trabajos presentados en el «V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi» (Oaxaca, México, 2012) (AU)

Cell wall proteins of Sporothrix schenckii as immunoprotective agents.

Sporothrix schenckii is the etiological agent of sporotrichosis, an endemic subcutaneous mycosis in Latin America. Cell wall (CW) proteins located on the cell surface are inducers of cellular and humoral immune responses, potential candidates for diagnosis purposes and to generate vaccines to prevent fungal infections. This mini-review emphasizes the potential use of S. schenckii CW proteins as protective and therapeutic immune response inducers against sporotrichosis. A number of pathogenic fungi display CW components that have been characterized as inducers of protective cellular and humoral immune responses against the whole pathogen from which they were originally purified. The isolation and characterization of immunodominant protein components of the CW of S. schenckii have become relevant because of their potential in the development of protective and therapeutic immune responses against sporotrichosis. 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).

Three new resorcylic acid derivatives from Sporotrichum laxum.

Sporotrichum laxum ATCC 15155 is the producing strain of the potent anti-Helicobacter pylori natural product spirolaxine (1). Investigation of the secondary metabolites in this fungus led to the isolation of five phthalides (1, 2, 3, 6 and 9) and five resorcylic acid derivatives (4, 5, 7, 8 and 10), among which 5, 7 and 8 are new compounds. The structures were elucidated by spectroscopic analyses, and the absolute configurations of 7 and 8 were determined by Mosher's method. Addition of soy flour into the potato dextrose agar has led to the increased production of 4-10. A biosynthetic pathway consisting of a highly reducing polyketide synthase (PKS), a nonreducing PKS and a series of tailoring enzymes was proposed to produce these fungal natural products. The resorcylic acid derivatives are proposed to result from early hydrolysis of the polyketide chain or incorporation of a longer fatty acyl starter unit.

Isolation and functional characterization of Sporothrix schenckii ROT2, the encoding gene for the endoplasmic reticulum glucosidase II.

The N-linked glycosylation is a ubiquitous protein modification in eukaryotic cells. During the N-linked glycan synthesis, the precursor Glc(3)Man(9)GlcNAc(2) is processed by endoplasmic reticulum (ER) glucosidases I, II and α1,2-mannosidase, before transporting to the Golgi complex for further structure modifications. In fungi of medical relevance, as Candida albicans and Aspergillus, it is well known that ER glycosidases are important for cell fitness, cell wall organization, virulence, and interaction with the immune system. Despite this, little is known about these enzymes in Sporothrix schenckii, the causative agent of human sporotrichosis. This limited knowledge is due in part to the lack of a genome sequence of this organism. In this work we used degenerate primers and inverse PCR approaches to isolate the open reading frame of S. schenckii ROT2, the encoding gene for α subunit of ER glucosidase II. This S. schenckii gene complemented a Saccharomyces cerevisiae rot2Δ mutant; however, when expressed in a C. albicans rot2Δ mutant, S. schenckii Rot2 partially increased the levels of α-glucosidase activity, but failed to restore the N-linked glycosylation defect associated to the mutation. To our knowledge, this is the first report where a gene involved in protein N-linked glycosylation is isolated from S. schenckii.

Purification and partial biochemical characterization of a membrane-bound type II-like α-glucosidase from the yeast morphotype of Sporothrix schenckii.

The early steps of glycoprotein biosynthesis involve processing of the N-glycan core by endoplasmic reticulum α-glucosidases I and II which sequentially trim the outermost α1,2-linked and the two more internal α1,3-linked glucose units, respectively. We have demonstrated the presence of some components of the enzymic machinery required for glycoprotein synthesis in Sporothrix schenckii, the etiological agent of human and animal sporotrichosis. However, information on this process is still very limited. Here, a distribution analysis of α-glucosidase revealed that 38 and 50% of total enzyme activity were present in a soluble and in a mixed membrane fraction, respectively. From the latter, the enzyme was solubilized, purified to apparent homogeneity and biochemically characterized. Analysis of the enzyme by denaturing electrophoresis and size exclusion chromatography revealed molecular masses of 75.4 and 152.7 kDa, respectively, suggesting a homodimeric structure. Purified α-glucosidase cleaved the fluorogenic substrate 4-methylumbelliferyl-α-D: -glucopyranoside with high affinity as judged from K(m) and V(max) values of 0.3 µM and 250 nmol of MU/min/mg protein, respectively. Analysis of linkage specificity using a number of glucose α-disaccharides as substrates demonstrated a clear preference of the enzyme for nigerose, an α1,3-linked disaccharide, over other substrates such as kojibiose (α1,2), trehalose (α1,1) and isomaltose (α1,6). Use of selective inhibitors of processing α-glucosidases such as 1-deoxynojirimycin, castanospermine and australine provided further evidence of the possible type of α-glucosidase. Accordingly, 1-deoxynojirimycin, a more specific inhibitor of α-glucosidase II than I, was a stronger inhibitor of hydrolysis of 4-methylumbelliferyl-α-D: -glucopyranoside and nigerose than castanospermine, a preferential inhibitor of α-glucosidase I. Inhibition of hydrolysis of kojibiose and maltose by 1-deoxynojirimycin and castanoespermine was significantly lower than that of nigerose. Taken together, these properties are consistent with a type II-like α-glucosidase probably involved in N-glycan processing. To our knowledge, this is the first report of such an activity in a truly dimorphic fungus.

Protein profiling of the dimorphic pathogenic fungus, Sporothrix schenckii.

Sporotrichosis is a common cutaneous mycosis caused by the dimorphic fungus Sporothrix schenckii, which exhibits a temperature-dependent dimorphic switch. At 25°C, it grows in a mycelial phase, while at 37°C, it forms unicellular yeast cells. The formation of yeast cells was thought to be a requisite for the pathogenicity of S. schenckii. To identify fragments that might be related to morphogenesis, whole-cell proteins from the mold and early yeast stages of S. schenckii were analyzed using 2DE. Among thousands of protein molecules displayed, more than 300 showed a differential expression between the two phases. In particular, 24 yeast-specific proteins were identified using MALDI-TOF/MS. One of the most interesting proteins was a hybrid histidine kinase, DRK1, a global regulator of dimorphism and virulence in Blastomyces dermatitidis and Histoplasma capsulatum that was abundant in the yeast phase. Our study introduced a new approach to study dimorphism in S. schenckii, and the data may help us better understand the molecular mechanisms of phase transition.