Molecular and cellular events during the yeast to mycelium transition in Sporothrix schenckii

Unbudded singlets from exponentially growing yeast cells of Sporothrix schenckii were harvested, selected by filtration and allowed to form germ tubes in a basal medium with glucose at pH 4.0 and 25 degrees C. These conditions supported only the development of the mycelial form of S. schenckii in a reproducible manner which allowed further analysis of the early cellular events occurring during the yeast-to-mycelium transition. The relationship between macromolecular synthesis (DNA and RNA synthesis) and nuclear division, hyphal growth and septum formation were investigated during germ tube formation. RNA synthesis started 0 to 3 h after the induction of germ tube formation, followed by DNA synthesis and the first nuclear division, which took place between 3 and 6 h. Germ tube formation followed nuclear division and was first evidenced 6 h after the induction of germ tube formation, but was not completed until 12 h after inoculation. Septation was first observed in these germ tubes at the mother cell-germ tube junction 6 h after induction. Addition of hydroxyurea, an inhibitor of DNA synthesis, to the medium, also inhibited nuclear division and germ tube growth, suggesting that these processes in S. schenckii are dependent upon DNA synthesis.     

Effects of zinc on macromolecular synthesis and nuclear division during the yeast to mycelium transition in Sporothrix schenckii

Zinc ions (10 mM) have been reported previously to inhibit the yeast to mycelium transition in Sporothrix schenckii. Yeast cells of this fungus were harvested, selected by filtration and allowed to form germ tubes in a basal medium with glucose in the presence of 10 mM zinc and the effects of this ion on protein, RNA and DNA synthesis and nuclear division recorded. All of these processes were affected by the addition of 10 mM zinc to the medium. Nevertheless, the inhibition of protein synthesis was observed earlier than that of RNA or DNA synthesis and was of a greater magnitude than that observed for both of these processes. Protein synthesis was inhibited within the first hour after inoculation, at which time this process begins in the control cells. RNA synthesis was inhibited during the 3 to 6 h interval after inoculation, that is, 3 h after the start of this process in the control cells. After 9 h of incubation, the inhibition of protein synthesis had reached its maximum at 70%, while that of RNA synthesis was only 52%. DNA synthesis was slightly inhibited, with maximum inhibition being observed 9 h after inoculation. Nuclear division in cells forming germ tubes in the presence of 10 mM zinc took place with a 3 h delay in relation to the control cells. These observations suggest that the inhibition of protein synthesis might be the most important mechanism by which zinc inhibits the yeast to mycelium transition in S. schenckii.     

Calcium uptake and efflux during the yeast to mycelium transition inSporothrix schenckii

A group of five children with kwashiorkor, seven with marasmic kwashiorkor and one underweight child were given an aflatoxin-free diet consisting of maize meal and milk powder. Blood specimens were collected on admission; on day 4 and 10, 24 hour urine and stool samples were collected for the first ten days. Serum, urine and stool samples were analysed for aflatoxins using high performance liquid chromatography with fluorescent detection, after various extraction and clean-up procedures. The children with kwashiorkor and marasmic kwashiorkor excreted aflatoxins in stools for up to 9 and 6 days after admission respectively. No aflatoxins were detected in the stools or urine of the underweight child. In kwashiorkor, urinary excretion ceased after 2 days, while in marasmic kwashiorkor urinary excretion persisted for 4 days. In stools, B₁ was the type of aflatoxin detected most frequently in kwashiorkor and least frequently in marasmic kwashiorkor. Aflatoxin M₂ was frequently detected in the stools of both groups of children.Estimates of the total amount of aflatoxin excreted by kwashiorkor and marasmic kwashiorkor indicate that these children were harbouring up to 4 g/kg body weight at the time of admission.These findings establish that aflatoxins accumulate in body fluids and tissues in kwashiorkor and marasmic kwashiorkor which is only slowly eliminated.     

Different protein kinase C isoforms are present in the yeast and mycelium forms of Sporothrix schenckii

Protein kinase C (PKC) plays an important role in the control of proliferation and differentiation of a wide range of cell types, and fungi are no exception. Previous results reported by us on the effects of the phorbol ester, 12-myristate-13-acetate phorbol (PMA) and other PKC effector molecules, on dimorphism in Sporothrix schenckii suggested the presence of this enzyme in the fungus and its involvement in the control of morphogenetic transitions. The work summarized here confirms the presence of PKC in yeast and mycelium extracts of S. schenckii. Different isoforms of this enzyme were found to be present in the yeast and mycelium forms of the fungus and were identified by Western blot analysis using affinity purified anti-PKC isoforms specific antibodies: the γ and ζ isoforms were detected in both the yeast and mycelium forms of the fungus, while the β isoform was only detected in the yeast form. The presence of PKC was confirmed biochemically by measuring total enzyme activity in both forms of the fungus. No significant differences were observed for the PKC activity level recorded for both the mycelium and yeast forms of the fungus (p ≤ 0.05). These data confirm the presence of PKC activity in Sporothrix schenckii and constitutes the first evidence concerning the differential expression of PKC isoforms in the mycelium and yeast forms of a dimorphic fungus, supporting the possible involvement of this important signal transduction enzyme in the control of morphogenesis in this fungus. This revised version was published online in June 2006 with corrections to the Cover Date.     

申克孢子丝菌菌丝相至酵母相转化的实验研究

目的观察申克孢子丝菌菌丝相向酵母相转化的形态学变化并初步研究连续传代后菌株转化为酵母相的百分率。方法将95株申克孢子丝菌临床株于脑心浸液琼脂培养基上连续传代至酵母相,利用显微镜及血细胞计数板计数菌丝和孢子比例并记录显微镜下形态。结果 95株申克孢子丝菌中,经1次传代即成功转化有23株,经2次传代有14株,经3次传代有10株,经4次传代有6株,4次传代后总计55.8%实验菌株转化为酵母相。结论部分申克孢子丝菌由菌丝相向酵母相转化需经过连续多次传代,连续传代增加了菌丝相至酵母相的转化率。     

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.     

Ultrastructural studies of the mycelium-to yeast transformation of Sporothrix schenckii.

Fine details of the internal and external morphology of the in vitro mycelial phase (MP) to yeastlike phase (YP) transition of the dimorphic fungal pathogen Sporothrix schenckii are shown in electron micrographs of ultrathin sections. Morphological transformation at the ultrastructural level was observed to occur by direct formation of budlike structures at the tips and along the hyphae and by oidial cell formation. Direct budding of yeast from conidiospores was not observed. Early transitional forms arising by direct blastic action from the MP possessed conspicuous electron-dense microfibrillar material at the outer limits of the cell wall. The electron density of this microfibrillar material was enhanced by staining with acidified dialyzed iron. It is believed that this extracellular material may be composed in part of an acid mucosubstance. No acid phosphatase activity was associated with this microfibrillar material. This substance was found to be a characteristic of the outer limits of the cell wall of the YP of S. schenckii. Oidial YP cell formation occurred later during the transition. The cell wall of the developing oidial YP transitional form arose from an inner layer of the converting hyphae. No consupicuous alterations of the cytoplasmic content of the parent MP cell was observed during MP-to-YP transition. It is suggested that the MP-to-YP transition of S. schenckii may be regulated by at least two mechanisms involving alterations of the biochemical and/or biophysical nature of the cell wall of the MP cell in response to the conversional stimuli.     

Effect of amphotericin B on the lipids of yeast cells of Sporothrix schenckii

Yeast cells of five strains of Sporothrix schenckii were obtained for partial analysis of lipid composition. Quantitative analysis of lipids and sterols were completed, as well as qualitative analysis of sterols by thin layer chromatography and by ultraviolet spectra. These determinations were made on cells cultured in the absence and presence of amphotericin B at sub-MIC (minimum inhibitory concentration) levels. Marked alterations in lipid content were observed in the amphotericin B-treated cells. The major alterations were the reduction of total lipid (18.7–57.6%) and sterols (48.5–96.7%) after exposure to the polyenic antibiotic. It is concluded that amphotericin B altered the lipid profiles, especially sterols of S. schenckii.     

Characterization of novel structures of mannosylinositolphosphorylceramides from the yeast forms of Sporothrix schenckii.

Novel structures of glycoinositolphosphorylceramide (GIPC) from the infective yeast form of Sporothrix schenckii were determined by methylation analysis, mass spectrometry and NMR spectroscopy. The lipid portion was characterized as a ceramide composed of C-18 phytosphingosine N-acylated by either 2-hydroxylignoceric acid (80%), lignoceric (15%) or 2,3-dihydroxylignoceric acids (5%). The ceramide was linked through a phosphodiester to myo-inositol (Ins) which is substituted on position O-6 by an oligomannose chain. GIPC-derived Ins oligomannosides were liberated by ammonolysis and characterized as: Manpalpha1-->6Ins; Manpalpha1-->3Manpalpha1-->6Ins; Manpalpha1-->6Manpalpha1-->3Manpalpha1-->3Manpalpha1-->6Ins; Manpalpha1-->2Manpalpha1-->6Manpalpha1-->3Manpalpha1-->3Manpalpha1-->6Ins. These structures comprise a novel family of fungal GIPC, as they contain the Manpalpha1-->6Ins substructure, which has not previously been characterized unambigously, and may be acylated with a 2,3 dihydroxylignoceric fatty acid, a feature hitherto undescribed in fungal lipids.     

申克孢子丝菌酵母相和菌丝相cDNA消减文库的构建

目的 筛选与申克孢子丝菌酵母相与菌丝相双相转换相关的差异表达基因,为探讨其双相转换的分子的机制奠定基础.方法 应用抑制性消减杂交技术,构建高特异性的申克孢子丝菌菌丝相（mycelium,M）和酵母相（yeast,Y）的正反cDNA消减文库,并对其差异表达的基因进行生物信息学分析.结果 M＋Y文库获得751条表达序列标签,平均长度为690.98 bp,经拼接后获得101条非冗余序列.Y＋M文库获得875条表达序列标签,平均长度为575.9 bp,拼接获得249条非冗余序列.经BLASTN比对,这些差异表达基因中,某些结构基因和功能不明的细胞分子类基因可能与双相转换有关.结论 成功构建了高特异性的申克孢子丝菌菌丝相和酵母相的正反cDNA 消减文库,为进一步筛选申克孢子丝菌的双相转换基因奠定了基础.     

Analysis of restriction profiles of Mitochondrial DNA from Sporothrix schenckii and related fungi

Restriction profiles by HaeIII of mitochondrial DNA were studied for classification and distinction of Sporothrix schenckii (100 strains), S. schenckii var. luriei (1), S. curviconia (1), S. inflata (7), Ceratocystis stenoceras (17) and C. minor (7). These 6 species showed unique restriction profiles which could be discriminated from each other. S. schenckii was further separable into 11 types, S. inflata into 4 types, C. stenoceras into 4 types and C. minor into 7 types based on restriction profile heterogeneity.     

Expression of a Pho85 cyclin-dependent kinase is repressed during the dimorphic transition in Sporothrix schenckii

Sporothrix schenckii is a pathogenic fungus that undergoes a dimorphic transition from yeast to mycelium in response to environmental conditions such as cell density, temperature, and calcium. We identified a homolog of the Pho85 cyclin-dependent kinase (Cdk) that mediates cellular responses to environmental conditions in other organisms. By Western blot, three proteins containing the PSTAIRE motif, which characterize the cyclin-dependent protein kinases, were identified in S. schenckii. The gene encoding a Pho85 homolog, PhoSs, was identified and sequenced. The phoSs gene consists of 990bp, contains one intron, and encodes a protein of 306 amino acids. The S. schenckii Pho85 homolog shares features with Cdks, including the PSTAIRE motif, an ATP binding domain, and a serine-threonine kinase domain. By quantitative competitive RT-PCR, expression of the phoSs gene was found to decrease 30-fold during the yeast to mycelium transition. The addition of extracellular calcium accelerated the dimorphic transition and restored phoSs expression. These findings suggest PhoSs may participate in the control of the yeast to mycelium transition in S. schenckii.     

Effects of hyperthermia on phagocytosis and intracellular killing of Sporothrix schenckii by polymorphonuclear leukocytes

The effects of hyperthermia on phagocytosis and killing of Sporothrix schenckii by polymorphonuclear leukocytes (PMNs) were investigated in order to clarify the mechanism of local thermotherapy for sporotrichosis. Yeast cells of S. schenckii, PMNs and serum were incubated at 37°C or 40°C for 2 or 4 hours. Rate of phagocytosis and killing rate (rate of germination) were estimated, and their processes were observed by transmission electron microscopy. There was no effect of hyperthermia on the phagocytosis rate, but the killing rate increased significantly at 40°C. Electron microscopic examination showed an increase of granularity in the yeast cytoplasm, elongation and fragmentation of the cell membrane. The ultrastructural changes were basically identical under both temperatures, but the degree of these changes was higher at 40°C than at 37°C. Although both intact and degenerated yeasts were found in the same conditions, their transient forms were few, suggesting that the PMN-killing process was completed promptly.     

Localization of divalent cations in phosphate-rich cytoplasmic granules in yeast

Phosphate-rich yeast cells (Saccharomyces cerevisiae) take up more calcium and strontium than phosphate-deficient cells. The divalent cations appear to be tightly bound in phosphate-rich cells. Electron microscopical investigations, combined with energy-dispersive X-ray microanalysis, showed that divalent cations were sequestered in cytoplasmic granules, together with a large amount of phosphorus. The major part of the divalent cations is retrieved in the polyphosphate fraction, a minor part is bound to lipids. The results suggest that polyphosphate granules may serve as an important store for divalent cations.     

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.