Cell Wall Glycoproteins Participate in the Adhesion of <Emphasis Type="Italic">Sporothrix schenckii</Emphasis> to Epithelial Cells

Sporothrix schenckii is the etiologic agent of sporotrichosis. This fungal infection is an emerging disease potentially fatal in immunocompromised patients. The adhesion to host cells is a crucial early event related with the dissemination of pathogens. In order to clarify the mechanisms of adhesion of S. schenckii yeast cell to epithelial cells, we studied the biochemical basis of this process. The electrophoretic analysis of cell wall protein from S. schenckii coupled at ConA and stained with HRP, revealed nine different proteins with MW ≥ 180, 115, 90, 80, 58, 40, 36, 22 and 18 kDa. Using ligand-like assay with biotinylated S. schenckii surface proteins, five proteins with MW ≥ 190, 180, 115, 90 and 80 kDa which have affinity to epithelial cells were identified. The adhesion of yeast to epithelial monolayer was significantly inhibited when S. schenckii was pretreated with concanavalinA (ConA) and wheat germ agglutinin (WGA) lectins, alkali, periodate, trypsin, endoglycosidase H (EndoH), salt solutions and detergents. The ability of adhesion of S. schenckii yeast was recovered by blocking the lectin with sugar complementary. These data suggest that surface glycoprotein with mannose and glucose residue could be participate in the process of fungal adhesion to epithelial cells.     

Functional,genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in <Emphasis Type="Italic">Sporothrix schenckii</Emphasis>

Background  

Sporothrix schenckiiis a pathogenic, dimorphic fungus, the etiological agent of sporotrichosis, a subcutaneous lymphatic mycosis. Dimorphism inS. schenckiiresponds to second messengers such as cAMP and calcium, suggesting the possible involvement of a calcium/calmodulin kinase in its regulation. In this study we describe a novel calcium/calmodulin-dependent protein kinase gene inS. schenckii, sscmk1, and the effects of inhibitors of calmodulin and calcium/calmodulin kinases on the yeast to mycelium transition and the yeast cell cycle.     

Cytosolic phospholipase A2: a member of the signalling pathway of a new G protein α subunit in Sporothrix schenckii

Background  

Sporothrix schenckii is a pathogenic dimorphic fungus, the etiological agent of sporotrichosis, a lymphocutaneous disease that can remain localized or can disseminate, involving joints, lungs, and the central nervous system. Pathogenic fungi use signal transduction pathways to rapidly adapt to changing environmental conditions and S. schenckii is no exception. S. schenckii yeast cells, either proliferate (yeast cell cycle) or engage in a developmental program that includes proliferation accompanied by morphogenesis (yeast to mycelium transition) depending on the environmental conditions. The principal intracellular receptors of environmental signals are the heterotrimeric G proteins, suggesting their involvement in fungal dimorphism and pathogenicity. Identifying these G proteins in fungi and their involvement in protein-protein interactions will help determine their role in signal transduction pathways.     

Differences in virulence between isolates of feline Sporotrichosis

Sporotrichosis is a chronic subcutaneous mycosis caused by Sporothrix schenckii. This work aimed to evaluate the virulence of two different isolates of S. schenckii from cutaneous (CUT) and systemic (SYS) forms of feline sporotrichosis. A standard inoculum with 2 × 10³ yeast cells/ml was prepared from each of the isolates. The experimental infection was carried out with 0.1 ml of the inoculum from both isolates and then injected in the paw pads of Swiss albino mice of groups CUT and SYS. The clinical evolution of the disease and the diameter of the lesion at the inoculated sites were evaluated during nine weeks. Four necropsies were done to collect material from the lesions (p < 0.01). Group CUT demonstrated a more evident clinical evolution of the disease from week two to week five; large lesions in the paw pad on week four (p < 0.01); and a higher incidence of lesions in other parts of the body (p < 0.01) than group SYS (p < 0.01). S. schenckii was isolated from the inoculated site in groups SYS and CUT until days 30 and 45, respectively. Granulomas with yeast cells usually localized in the central area were observed in histopathology sections on days 15 and 30 post-inoculations. Those yeast cells decreased on day 45 being absent on day 62 when tissue repair initiated. The results showed that distinct clinical isolates of S. schenckii cause significant differences in the clinical evolution of sporotrichosis.     

Increased resistance of splenectomized mice to Sporothrix schenckii infection

The susceptibility of splenectomized mice to Sporothrix schenckii was studied, and the role of the spleen in the host defense is discussed. S. schenckii Sp-1 and ddy male mice were used. The mice were divided into 3 groups consisting of splenectomized, sham-operated and intact mice. Each mouse was inoculated intravenously with 2×10⁶ yeast cells 7 days after operation and the mice were sacrified at adequate intervals for 30 days. Then histological sections stained with H&E or by PAS were prepared from various visceral organs. Using the liver sections the number of yeast cells in a 40 mm² was counted. Furthermore, the colony forming unit in 100 mg of the liver tissue was compared to each other.In the sham-operated and intact mice many purulent lesions appeared on the 5th day. On the 8th day mononuclear cells accumulated at the foci, and on the 10th day most of the foci became granulomatous. The number of yeast cells in granulomatous lesions reached a peak on the 10th day and thereafter decreased abruptly. On the other hand, in the splenectomized mice approximately half of foci became granulomatous on the 5th day, and the number of yeast cells in the foci began to decrease after the 5th day.There were definite differences in the colony forming unit between the splenectomized and sham-operated or intact mice sacrificed 9 days after inoculation. The colony forming unit of the former is 9.3×10⁵ on the average, while that of the latter two is 5.6×10⁶ and 5.1×10⁶ on the average, respectively.In conclusion the resistance of ddy mice to S. Schenckii infection is enhanced due to splenectomy.     

Gene cloning,expression, and characterization of a novel acetaldehyde dehydrogenase from <Emphasis Type="Italic">Issatchenkia terricola</Emphasis> strain XJ-2

Acetaldehyde is a known mutagen and carcinogen. Active aldehyde dehydrogenase (ALDH) represents an important mechanism for acetaldehyde detoxification. A yeast strain XJ-2 isolated from grape samples was found to produce acetaldehyde dehydrogenase with a high activity of 2.28 U/mg and identified as Issatchenkia terricola. The enzyme activity was validated by oxidizing acetaldehyde to acetate with NAD⁺ as coenzyme based on the headspace gas chromatography analysis. A novel acetaldehyde dehydrogenase gene (ist-ALD) was cloned by combining SiteFinding-PCR and self-formed adaptor PCR. The ist-ALD gene comprised an open reading frame of 1,578 bp and encoded a protein of 525 amino acids. The predicted protein of ist-ALD showed the highest identity (73%) to ALDH from Pichia angusta. The ist-ALD gene was expressed in Escherichia coli, and the gene product (ist-ALDH) presented a productivity of 442.3 U/mL cells. The purified ist-ALDH was a homotetramer of 232 kDa consisting of 57 kDa-subunit according to the SDS-PAGE and native PAGE analysis. Ist-ALDH exhibited the optimal activity at pH 9.0 and 40°C, respectively. The activity of ist-ALDH was enhanced by K⁺, NH4⁺, dithiothreitol, and 2-mercaptoethanol but strongly inhibited by Ag⁺, Hg²⁺, Cu²⁺, and phenylmethyl sulfonylfluoride. In the presence of NAD⁺, ist-ALDH could oxidize many aliphatic, aromatic, and heterocyclic aldehydes, preferably acetaldehyde. Kinetic study revealed that ist-ALDH had a k _cat value of 27.71/s and a k _cat/K _m value of 26.80 × 10³/(mol s) on acetaldehyde, demonstrating ist-ALDH, a catalytically active enzyme by comparing with other ALDHs. These studies indicated that ist-ALDH was a potential enzymatic product for acetaldehyde detoxification.     

Factors controlling the colonial structure of <Emphasis Type="Italic">Pediastrum tetras</Emphasis> (Chlorophyceae)

Accounting for morphological plasticity in phytoplankton populations is relevant for taxonomy, systematic/evolutionary, and ecological studies. In this work, the green alga Pediastrum tetras (Ehrenberg) Ralfs was used to describe the variation in population size structure over its growth cycle and to analyze responses to changes in biotic and abiotic factors. Pediastrum cultures reached a final stable concentration in approximately 10 days. This density (8 × 10⁵ cells ml⁻¹) remained stable for at least another 13 days and the intrinsic growth rate was 0.24 ± 0.01 day⁻¹. In the exponential phase, the relative number of single cells and the proportion of large cells (with vesicles inside) within colonies increased. When density peaked, a relative increase of single cells as well as small cells in new colonies took place. Finally, during the stationary phase, the trend reversed: fewer single cells and a larger cell size (without vesicles) were observed. Results indicated that nutrient supply could affect population structure, diminishing the proportion of eight-cell colonies. Daphnia magna Straus significantly reduced the Pediastrum population density due to predation, and this led to a significant decrease in the density of the largest colonies. In addition, info-chemicals induced a slight increase in the density of the largest colonies compared to the control treatment. Our study suggests a possible trade-off in P. tetras colonial size in natural environments: during the stationary growth period in a lake, Pediastrum populations tend to increase in size for efficient use of nutrients, while they decrease in size in the presence of herbivores. Handling editor: J. Padisak     

Production,purification, and characterization of a novel killer toxin from <Emphasis Type="BoldItalic">Kluyveromyces siamensis</Emphasis> against a pathogenic yeast in crab

The yeast Kluyveromyces siamensis HN12-1 isolated from mangrove ecosystem was found to be able to produce killer toxin against the pathogenic yeast (Metschnikowia bicuspidata WCY) in crab. When the killer yeast was grown in the medium with pH 4.0 and 0.5% NaCl and at 25 °C, it could produce the highest amount of killer toxin against the pathogenic yeast M. bicuspidata WCY. The killing activity of the purified killer toxin against the pathogenic yeast M. bicuspidata WCY was the highest when it was incubated at 25 °C in the assay medium without added NaCl and pH 4.0. The molecular weight of the purified killer toxin was 66.4 kDa. The killer toxin produced by the yeast strain HN12-1 could kill only the whole cells of M. bicuspidata WCY among all the yeast species tested in this study. This is the first time to report that the killer toxin produced by the yeast K. siamensis HN12-1 isolated from the mangrove ecosystem only killed pathogenic yeast M. bicuspidata WCY.     

Production of ectoine through a combined process that uses both growing and resting cells of <Emphasis Type="Italic">Halomonas salina</Emphasis> DSM 5928<Superscript>T</Superscript>

Using ectoine-excreting strain Halomonas salina DSM 5928^T, we developed a new process for high-efficiency production of ectoine, which involved a combined process of batch fermentation by growing cells and production by resting cells. In the first stage, batch fermentation was carried out using growing cells under optimal fermentation conditions. The second stage was the production phase, in which ectoine was synthesized and excreted by phosphate-limited resting cells. Optimal conditions for synthesis and excretion of ectoine during batch fermentation in a 10 l fermentor were 0.5 mol l⁻¹ NaCl and an initial monosodium glutamate concentration of 80 g l⁻¹ respectively. The pH was adjusted to 7.0 and the temperature was maintained at 33°C. In phosphate-limited resting cells medium, monosodium glutamate and NaCl concentration was 200 g l⁻¹ and 0.5 mol l⁻¹, respectively, as well as pH was 7.0. The total concentration of ectoine produced was 14.86 g l⁻¹, the productivity and yield of ectoine was 7.75 g l⁻¹ day⁻¹ and 0.14 g g⁻¹, respectively, and the percentage of ectoine excreted was 79%. These levels of ectoine production and excretion are the highest reported to date.     

Overexpression of β-glucosidase from <Emphasis Type="Italic">Thermotoga maritima</Emphasis> for the production of highly purified aglycone isoflavones from soy flour

To produce aglycone isoflavones from soy flour, the β-glucosidase A gene (bglA) of Thermotoga maritima was overexpressed in Escherichia coli BL21-CodonPlus (DE3)-RIL. The K _m and V _max values of the purified BglA for pNPG were 0.43 mM and 323.6 U mg⁻¹, respectively, and those for salicin were 9.0 mM and 183.2 U mg⁻¹, respectively. The biochemical and kinetic characteristics of his-tagged BglA were found to be similar to those of BglA, except for the temperature stability and specific activity. Production of aglycone isoflavones from soy flour by BglA was examined by HPLC. For 3 h at 80°C, all the isoflavone glycosides approximated to the complete conversion into aglycone isoflavones, over seven times higher than that obtained from soy flour without BglA.     

Characterization of a compound bioflocculant produced by mixed culture of <Emphasis Type="Italic">Rhizobium radiobacter</Emphasis> F2 and <Emphasis Type="Italic">Bacillus sphaeicus</Emphasis> F6

A compound bioflocculant CBF-F26, produced by mixed culture of Rhizobium radiobacter F2 and Bacillus sphaeicus F6, was investigated with regard to its physicochemical and flocculating properties. It was identified as a polysaccharide bioflocculant composed of rhamnose, mannose, glucose, and galactose, respectively, in a 1.3: 2.1: 10.0: 1.0 molar ratio. The average molecular weight was determined as 4.79 × 10⁵ Da by gel-permeation chromatography. Infrared spectrum and X-ray photoelectron spectroscopy revealed the presence of carboxyl, hydroxyl and amino groups in its structure. Thermostability test suggested that CBF-F26 was thermostable and high flocculating activity was maintained. Thermogravimetric property, intrinsic viscosity and surface morphology of CBF-F26 were also studied. CBF-F26 was effective under neutral and weak alkaline conditions (pH 7.0–9.0), and flocculating activities of higher than 90% were obtained in the concentration range of 8–24 mg l⁻¹ at pH 8.0. The flocculation could be stimulated by cations Ca²⁺, Zn²⁺, Fe²⁺, Al³⁺, and Fe³⁺. In addition, the probable flocculation mechanisms were proposed.     

Fungal autolysate as a nutrient supplement for ethanol and chitosan production by <Emphasis Type="Italic">Mucor indicus</Emphasis>

Mucor indicus can be used to produce ethanol from a variety of sugars, including pentose’s. An extract of it, produced by autolysis, could replace yeast extract in culture medium with improved production of ethanol. At 10 g l⁻¹, the extract gave a higher ethanol yield (0.47 g g⁻¹) and productivity (0.71 g l⁻¹ h⁻¹) compared to medium containing yeast extract (yield 0.45 g g⁻¹; productivity 0.67 g l⁻¹ h⁻¹).     

Proteolytic activity in <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> PCC7806 is inhibited by a trypsin-inhibitory cyanobacterial peptide with a partial structure of microviridin

This paper describes the characterization of proteases in Microcystis aeruginosa PCC7806 cells being inhibited by a metabolite produced by another Microcystis strain, Microcystis Ku1. With casein and oligopeptide substrates and specific inhibitors we detected activity similar to bacterial serine endoproteases. Substrate SDS-polyacrylamide gel electrophoresis revealed the presence of nine bands of proteases (ca. 35∼125 kDa). The cyanobacterial enzymes were insensitive to endogenous trypsin-inhibitory metabolites. Microcystis Ku1 produced a metabolite, tentatively characterized as microviridin, inhibiting both cyanobacterial proteases and trypsin at an estimated IC₅₀ of, respectively, 2.2 and 9.0 μg mL⁻¹. On activity gels, inhibitors specific to animal trypsin and elastase and the putative microviridin led to an inactivation of the proteases associated with the 88 and 110 kDa bands. We hypothesize that in Microcystis populations there is a “cross-talk” between the inhibitors and the proteases, and only the colonies of identical chemotypes can possibly aggregate to form blooms.     

Responses of <Emphasis Type="Italic">Rhodotorula</Emphasis> sp. Y11 to cadmium

Some aspects of the cellular responses to cadmium were extensively investigated in the yeast Rhodotorula sp. Y11. Scanning electron microscopy indicated that accumulation of cadmium in the Y11 did not cause any visible effects on cell morphology. More than 20% yeast cells still showed viability after 15 h of cadmium accumulation under 100 mg l⁻¹ cadmium concentration, and transmission electron microscopy analysis showed that plasmolysis and thickened cell wall were not observed in all of the cells. In the presence of cadmium, the activities of superoxide dismutase (SOD) and catalase (CAT) were all greater than the control, but the increase was in a dose-independent manner. Changes in SOD and CAT activities were also dependent on the time of exposure. Therefore, it suggests that antioxidative defenses play an important role in cadmium tolerance in Rhodotorula sp. Y11. Nondenaturing polyacrylamide gels revealed only one SOD isoforms in Y11 even under exposure to cadmium.     

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.     

Purification and characterization of the cold-active alkaline protease from marine cold-adaptive <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis> FS010

An extracellular cold-active alkaline serine protease from Penicillium chrysogenum FS010 has been purified. The purification procedure involved: ammonium sulfate precipitation, DEAE ion-exchange chromatography and sephadex G-100 gel chromatography. SDS–PAGE of the purified enzyme indicated a molecular weight of 41,000 ± 1,000 Da. The protease is stable in a pH range of 7.0–9.0 and has a maximum activity at pH 9.0. Compared with other industrial proteases, the enzyme shows a high hydrolytic activities at lower temperatures and a high sensitivity at a temperature over 50°C. The isoelectric point of the enzyme is approximate to 6.0. Enzymatic activity is enhanced by the addition of divalent cations such as Mg²⁺ and Ca²⁺ and inhibited by addition of Cu²⁺and Co²⁺. PMSF and DFP are its specific inhibitors. The application of the cold-active alkaline protease is extremely extensive, and widely used in detergents, feed, food, leather and many other industries.     

Ethanol production from alkali-treated rice straw via simultaneous saccharification and fermentation using newly isolated thermotolerant <Emphasis Type="Italic">Pichia kudriavzevii</Emphasis> HOP-1

In this study, simultaneous saccharification and fermentation (SSF) was employed to produce ethanol from 1% sodium hydroxide-treated rice straw in a thermostatically controlled glass reactor using 20 FPU gds⁻¹ cellulase, 50 IU gds⁻¹ β-glucosidase, 15 IU gds⁻¹ pectinase and a newly isolated thermotolerant Pichia kudriavzevii HOP-1 strain. Scanning electron micrograph images showed that the size of the P. kudriavzevii cells ranged from 2.48 to 6.93 μm in diameter while the shape of the cells varied from oval, ellipsoidal to elongate. Pichia kudriavzevii cells showed extensive pseudohyphae formation after 5 days of growth and could assimilate sugars like glucose, sucrose, galactose, fructose, and mannose but the cells could not assimilate xylose, arabinose, cellobiose, raffinose, or trehalose. In addition, the yeast cells could tolerate up to 40% glucose and 5% NaCl concentrations but their growth was inhibited at 1% acetic acid and 0.01% cyclohexamide concentrations. Pichia kudriavzevii produced about 35 and 200% more ethanol than the conventional Saccharomyces cerevisiae cells at 40 and 45°C, respectively. About 94% glucan in alkali-treated rice straw was converted to glucose through enzymatic hydrolysis within 36 h. Ethanol concentration of 24.25 g l⁻¹ corresponding to 82% theoretical yield on glucan basis and ethanol productivity of 1.10 g l⁻¹ h⁻¹ achieved using P. kudriavzevii during SSF hold promise for scale-up studies. An insignificant amount of glycerol and no xylitol was produced during SSF. To the best of our knowledge, this is the first study reporting ethanol production from any lignocellulosic biomass using P. kudriavzevii.     

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.     

<Emphasis Type="Italic">Gaetbulibacter jejuensis</Emphasis> sp. nov., isolated from seawater

A novel marine bacterium, designated strain CNURIC014^T was isolated from coastal seawater of Jeju Island in Korea. Strain CNURIC014^T formed yellow colonies on marine agar 2216 and the cells were Gram-negative, non-motile, strictly aerobic, rod-shaped. The temperature, pH and NaCl ranges for growth were 15–37°C, pH 6.0–9.0 and 1.0–7.0% NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain CNURIC014^T was most closely related to Gaetbulibacter marinus and Gaetbulibacter saemankumensis, with a sequence similarity of 95.1% and 94.6%, respectively. The DNA G+C content of the strain was 33.1 mol% and the major respiratory quinone was menaquinone-6. The major cellular fatty acids were iso-C_15:1 (22.8%), iso-C_15:0 (18.8%), summed feature 3 (iso-C_15:0 2-OH/C_16:1 ω7c, 12.9%) and iso-C_17:0 3-OH (11.5%). On the basis of phenotypic, phylogenetic, and genotypic data, strain CNURIC014^T represents a novel species within the genus Geatbulibacter, for which the name Gaetbulibacter jejuensis sp. nov. is proposed. The type strain is CNURIC014^T(=KCTC 22615^T =JCM 15976^T).     

Optimized expression of an acid xylanase from <Emphasis Type="Italic">Aspergillus usamii</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and its biochemical characterization

The xylanase gene xyn II from Aspergillus usamii E001 was placed under the control of an alcohol oxidase promoter (AOX1) in the plasmid pPIC9K and integrated into the genome of a methylotrophic yeast, P. pastoris GS115, by electroporation. His⁺ transformants were screened for on the basis of their resistance to G418 and activity assay. A transformant, P. pastoris GSC12, which showed resistance to over 6 mg G418/ml and highest xylanase activity was selected. Recombinant xylanase was secreted by P. pastoris GSC12 24 h after methanol induction of shake-flask cultures, and reached a final yield of 3139. About 68 U/mg 120 h after the induction. The molecular mass of this xylanase was estimated to be 21 kDa by SDS-PAGE. The optimum pH and temperature were 4.2 and 50 °C, respectively. Xylanase was stable below 50 °C and within pH 3.0–7.0. Its activity was increased by EDTA and Co²⁺ ion and strongly inhibited by Mn²⁺, Li⁺ and Ag⁺ ions. The K _m and V _max values with birchwood xylan as the substrate were found to be 5.56 mg/ml and 216 μmol/mg/min, respectively. This is the first report on expression and characterization of xylanase from A. usamii in P. pastoris. The hydrolysis products consisted of xylooligosaccharides together with a small amount of xylose. This property made the enzyme attractive for industrial purposes, as relatively pure xylooligosaccharides could be obtained.