Isolation and characterization of a glucocerebroside (monoglucosylceramide) from Sporothrix schenckii

A glucocerebroside from yeast forms of the pathogenic fungus Sporothrix schenckii has been isolated and its components characterized by thin-layer and gas-liquid chromatography and by mass spectrometry. The purified molecule contained glucose, sphingosine and α-hydroxy stearic acid (1:1:1) and it seemed homogenous since no other fatty acid was detected.     

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.     

Isolation of Sporothrix schenckii from environmental samples in Venezuela

The dimorphic fungus Sporothrix schenckii is the etiological agent of sporotrichosis, a subcutaneous mycosis frequently found in Latin America. The isolation of this fungus from the environment and other sources has been widely reported. Nevertheless, to our knowledge this fungus has not been isolated from the endemic areas of Venezuela. In studies related to a clinical case of sporotrichosis in "Colonia Tovar", produced by traumatism after manipulating soil samples, the fungus was isolated from the soil of that particular area. This is the first report of the isolation of S. schenckii from environmental sources in an endemic area of Venezuela.     

Purification and characterization of a dextranase from Sporothrix schenckii

A dextranase (EC 3.2.1.11) was purified and characterized from the IP-29 strain of Sporothrix schenckii, a dimorphic pathogenic fungus. Growing cells secreted the enzyme into a standard culture medium (20 °C) that supports the mycelial phase. Soluble bacterial dextrans substituted for glucose as substrate with a small decrease in cellular yield but a tenfold increase in the production of dextranase. This enzyme is a monomeric protein with a molecular mass of 79 kDa, a pH optimum of 5.0, and an action pattern against a soluble 170-kDa bacterial dextran that leads to a final mixture of glucose (38%), isomaltose (38%), and branched oligosaccharides (24%). In the presence of 200 mM sodium acetate buffer (pH 5.0), the K _m for soluble dextran was 0.067 ± 0.003% (w/v). Salts of Hg²⁺, (UO₂)²⁺, Pb²⁺, Cu²⁺, and Zn²⁺ inhibited by affecting both V _max and K _m. The enzyme was most stable between pH values of 4.50 and 4.75, where the half-life at 55 °C was 18 min and the energy of activation for heat denaturation was 99 kcal/mol. S. schenckii dextranase catalyzed the degradation of cross-linked dextran chains in Sephadex G-50 to G-200, and the latter was a good substrate for cell growth at 20 °C. Highly cross-linked grades (i.e., G-10 and G-25) were refractory to hydrolysis. Most strains of S. schenckii from Europe and North America tested positive for dextranase when grown at 20 °C. All of these isolates grew on glucose at 35 °C, a condition that is typically associated with the yeast phase, but they did not express dextranase and were incapable of using dextran as a carbon source at the higher temperature. Received: 29 December 1997 / Accepted: 4 March 1998     

Isolation and properties of extracellular proteinases from Sporothrix schenckii.

Sporothrix schenckii, mainly in the yeast form of the organism, produced extracellular proteinases when cultivated in liquid media containing albumin or collagen as a nitrogen source, but did not do so in brain heart infusion medium. Isolation of two extracellular proteinases from albumin-containing medium was performed by chromatography on DEAE-Sepharose CL-6B and Sephacryl S-200. Proteinase I had a molecular weight of 36,500, an optimal pH at 6.0, and a pI at 4.8. Despite its activities in weakly acidic conditions, proteinase I demonstrated chymotrypsinlike characteristics, these being indicated by strong inhibitory activity by phenylmethylsulfonyl fluoride and chymostatin and good kinetic constants for a synthetic chymotrypsin substrate, Suc-Ala-Ala-Pro-Phe-MCA. Proteinase II had a molecular weight of 39,000, an optimal pH at 3.5, and a pI at 3.8. Proteinase II showed cathepsin D-like characteristics, these being indicated by strong inhibitory activity by pepstatin, an acidic optimal pH, and good kinetic constants for hemoglobin. These two enzymes hydrolyzed natural substrates such as stratum corneum, type I collagen, and elastin although not type IV collagen. Proteinase production and cell growth in collagen-containing medium and the enzymatic digestion of skin constituents by isolated proteinases suggested that these two proteinases cooperatively enable the organism to invade skin and to obtain peptides from insoluble proteins.     

Purification and characterization of a guanosine diphosphatase activity from calf liver microsomal salt wash proteins

A potent guanosine diphosphatase activity that hydrolyzes GDP to 5'-GMP + Pi has been isolated and purified from the salt wash proteins of calf liver microsomes. The purified enzyme, a monomeric protein of approximate Mr 46,000, possesses nucleotide substrate specificity since, among the nucleoside diphosphates and triphosphates tested, only GDP and UDP are hydrolyzed by the enzyme. The relative affinity of the enzyme for GDP is, however, much higher than for UDP. The effect of the enzyme on the binary complex formed between eukaryotic initiation factor 2 (eIF-2) and GDP has also been investigated. The enzyme neither hydrolyzes GDP bound to eIF-2 nor catalyzes the exchange of eIF-2-bound GDP with GTP even in the presence of Met-tRNAf. The enzyme, therefore, is presumably not involved in recycling of eIF-2 in eukaryotic polypeptide chain initiation reaction. The possible biological function of the enzyme in maintaining the cellular pool of GTP-GDP is discussed.     

Melanins Protect Sporothrix brasiliensis and Sporothrix schenckii from the Antifungal Effects of Terbinafine

Terbinafine is a recommended therapeutic alternative for patients with sporotrichosis who cannot use itraconazole due to drug interactions or side effects. Melanins are involved in resistance to antifungal drugs and Sporothrix species produce three different types of melanin. Therefore, in this study we evaluated whether Sporothrix melanins impact the efficacy of antifungal drugs. Minimal inhibitory concentrations (MIC) and minimal fungicidal concentrations (MFC) of two Sporothrix brasiliensis and four Sporothrix schenckii strains grown in the presence of the melanin precursors L-DOPA and L-tyrosine were similar to the MIC determined by the CLSI standard protocol for S. schenckii susceptibility to amphotericin B, ketoconazole, itraconazole or terbinafine. When MICs were determined in the presence of inhibitors to three pathways of melanin synthesis, we observed, in four strains, an increase in terbinafine susceptibility in the presence of tricyclazole, a DHN-melanin inhibitor. In addition, one S. schenckii strain grown in the presence of L-DOPA had a higher MFC value when compared to the control. Growth curves in presence of 2×MIC concentrations of terbinafine showed that pyomelanin and, to a lesser extent, eumelanin were able to protect the fungi against the fungicidal effect of this antifungal drug. Our results suggest that melanin protects the major pathogenic species of the Sporothrix complex from the effects of terbinafine and that the development of new antifungal drugs targeting melanin synthesis may improve sporotrichosis therapies.     

Isolation of ergosterol peroxide and its reversion to ergosterol in the pathogenic fungus Sporothrix schenckii

Ergosterol peroxide, a presumed product of the H_{_2}O_{_2}-dependent enzymatic oxidation of ergosterol, has been isolated from yeast forms of the pathogenic fungus Sporothrix schenckii. The substance, which may have a role in fungal virulence, has been characterized mainly using spectroscopic methods (¹H and ¹³C nuclear magnetic resonance and high resolution mass spectra). The purified compound showed a molecular formula of C_{_28}H_{_44}O_{_3}, displaying characteristic features of epidioxy sterols and was reverted to ergosterol when submitted to S. schenckii enzymatic extract. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro inhibitory activity of sertraline against clinical isolates of Sporothrix schenckii

BackgroundSertraline (SRT) is an antidepressant that has proven its activity in vitro against Cryptococcus, Coccidioides, Trichosporon and other fungi. Disseminated sporotrichosis, although rare, has a high mortality and its treatment is difficult and prolonged, often relying in combining two or more antifungals.AimsIn our study we evaluate the antifungal activity of SRT, alone and in combination with itraconazole (ITC), voriconazole (VRC) and amphotericin B (AMB), against 15 clinical isolates of Sporothrix schenckii.MethodsWe used the broth microdilution method as described by the CLSI to test the susceptibility to antifungals, and the checkerboard microdilution method to evaluate drug interactions.ResultsThe minimum inhibitory concentration (MIC) with SRT was in the range of 4–8 μg/ml, while for AMB, VRC and ITC were 0.5–4 μg/ml, 0.5–8 μg/ml and 0.125–2 μg/ml, respectively. In addition, SRT showed synergy with ITC in one strain, mainly additivity with VRC, and indifference with AMB in others.ConclusionsThe MIC values with SRT for the isolates studied show the potential role of this drug as an adjuvant in the treatment of sporotrichosis, especially in disseminated or complicated cases.     

The nature of the secondary conidia of Sporothrix schenckii

Observations of secondary conidium production in an African strain ofS. schenckii revealed that secondary conidia are produced (1) acropetally on short sterigmata, (2) sympodially on short conidial appendages, and/or (3) sympodially or acropetally on short sporogenous cells which arise directly from the primary conidium. The secondary conidia therefore appear also to be sympodulospores.

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Zusammenfassung Beobachtungen von sekundärer Konidienbildung in einer afrikanischen Kultur vonS. schenckii zeigten, daß sekundäre Konidien gebildet werden:1) acropetal an kurzen Sterigmata; 2) sympodial oder acropetal an kurzen, sporentragenden Zellen, und 3) sympodial oder acropetal an kurzen sporentragenden Zellen, die direkt vom primären Konidium entspringen. Die sekundären Konidien erscheinen deshalb auch Sympodulosporen zu sein.

     

申克孢子丝菌毒力因子及宿主抗感染免疫的研究进展

申克孢子丝菌是一种重要的双相型真菌,其引起的孢子丝菌病是一种常见的侵袭性皮肤感染.研究该菌毒力因子及宿主对其抗感染免疫对于深入了解其致病性及防治该病具有重要意义.该文介绍了申克孢子丝菌毒力因子及宿主对其抗感染免疫方面的研究进展.     

Sporothrix brunneoviolacea and Sporothrix dimorphospora, two new members of the Ophiostoma stenoceras-Sporothrix schenckii complex

Sporothrix inflata is a saprobic member of the Ophiostoma stenoceras-Sporothrix schenckii species complex, reported mainly from soil. Ophiostoma bragantinum, an ascomycete described from Brazil, has been proposed as its possible teleomorph. Previous studies revealed that Sporothrix inflata is phenotypically and genetically variable, suggesting the existence of cryptic species. During a continued survey on the biodiversity of microfungi from different countries, seven isolates morphologically similar to S. inflata were obtained from soil samples collected in Spain and USA. In this study their phenotypic features and phylogenetic relationships were assessed. DNA sequence data of two nuclear loci revealed that these isolates correspond to two unnamed clades in S. inflata s.l., one of which also included the type strain of Humicola dimorphospora, a species that traditionally has been considered a synonym of S. inflata. These two groups are proposed herein as Sporothrix brunneoviolacea sp. nov. and Sporothrix dimorphospora comb. nov. S. brunneoviolacea is characterized phenotypically by the production of a diffusible violet-brown pigment in culture and mostly globose, pigmented, lateral blastoconidia. On the other hand S. dimorphospora lacks diffusible pigments and shows mostly subglobose to obovoid pigmented lateral blastoconidia. In contrast to the type strain of S. inflata S. brunneoviolacea and S. dimorphospora assimilate raffinose. The phylogenetic analysis suggested that the proposed anamorph-teleomorph connection between S. inflata and O. bragantinum might not be correct.     

Interaction with Pantoea agglomerans Modulates Growth and Melanization of Sporothrix brasiliensis and Sporothrix schenckii

Mycopathologia - Sporothrix brasiliensis and Sporothrix schenckii stand as the most virulent agents of sporotrichosis, a worldwide-distributed subcutaneous mycosis. The origin of Sporothrix...     

A guanosine diphosphatase enriched in Golgi vesicles of Saccharomyces cerevisiae. Purification and characterization

We have recently described a luminal guanosine diphosphatase activity in Golgi-like vesicles of Saccharomyces cerevisiae (Abeijon, C., Orlean, P., Robbins, P. W., and Hirschberg, C. B. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6935-6939). The presumed in vivo role of this enzyme is to convert GDP into GMP. GDP is a reaction product following outer-chain mannosylation of luminal proteins and a known inhibitor of mannosyltransferases. It is hypothesized that GMP then returns to the cytosol. We have purified this enzyme to apparent homogeneity. Following solubilization from a membrane pellet using a buffer containing Triton X-100, the enzyme was purified on a concanavalin A-Sepharose column followed by Mono Q fast protein liquid chromatography (FPLC) and Superose-12 FPLC columns. After treatment with endoglycosidase H, the deglycosylated active enzyme was applied to a second Mono Q FPLC column and a phenyl-Superose FPLC column. The final enzyme activity was enriched 6500-fold over that of the Triton X-100 extract. The apparant molecular mass of the deglycosylated enzyme is 47 kDa. The purified enzyme is highly specific for guanosine diphosphate, requires Ca2+ for maximal activity, and has a broad pH optimum between 7.4 and 8.2. The apparent Km for GDP is 0.1 mM; the Vmax is 4.9 mmol/min/mg of protein. An enzyme activity with similar substrate specificity has also been detected in membranes of Schizosaccharomyces pombe.     

Dimorphic expression of cerebrosides in the mycopathogen Sporothrix schenckii

Major neutral glycosphingolipid components were extracted from Sporothrix schenckii, a dimorphic fungus exhibiting a hyphal saprophytic phase and a yeast parasitic phase responsible for chronic mycotic infections in mammalian hosts. These components, one from the mycelial form and two from the yeast form, were purified and their structures were elucidated by (1)H nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and tandem ESI-MS/MS. All three were characterized as cerebrosides (monohexosylceramides) containing (4E, 8E)-9-methyl-4,8-sphingadienine as the long-chain base attached to N-2'-hydroxyoctadecanoate and N-2'-hydroxy-(E)-Delta(3)-octadecenoate as the fatty acyl components. However, while the mycelial form expressed only beta-glucopyranosylceramide, the yeast form expressed both beta-gluco- and beta-galactopyranosylceramides in approximately equal amounts. In addition, while the glucosylceramides of both mycelial and yeast forms had similar proportions of saturated and (E)-Delta(3) unsaturated 2-hydroxy fatty acid, the galactocerebroside of the yeast form had significantly higher levels of (E)-Delta(3) unsaturation.The differences in cerebroside hexose structure represent a novel type of glycosphingolipid dimorphism not previously reported in fungi. Possible implications of these findings with respect to regulation of morphological transitions in S. schenckii and other dimorphic fungi are discussed.     

Phylogeny and molecular epidemiology of Sporothrix schenckii in Japan

Mitochondrial DNA(mtDNA) diversity was investigated in 257 clinical isolants of Sporothrix schenckii obtained from 4 districts in Japan. S. schenckii was classified into 10 types based on Hae III restriction profiles. Phylogeny of types constructed by the method of Fitch and Margoliash [1] on the estimated sequence divergence within mtDNA using the methods of Nei and Li [2], showed that S. schenckii are grouped into 2 clusters, one group consisting of types 1, 2 and 3, and the other group consisting of the other seven types. In addition, types 1, 2, and 5 were correlated with their geographic origin, whereas type 4 was present throughout Japan.