Cell wall receptor for yeast killer toxin: involvement of (1 leads to 6)-beta-D-glucan

The linear (1 --> 6)-beta-d-glucans pustulan and luteose were effective competitive inhibitors of killer toxin action. Affinity chromatography of killer toxin on a pustulan-Sepharose column showed that toxin bound directly to a (1 --> 6)-beta-linked polysaccharide. Other polysaccharides found in yeast cell walls, including (1 --> 3)-beta-d-glucan, mannan, chitin, and glycogen, were not effective as inhibitors of toxin. Fractionation of yeast cell walls was attempted to identify the toxin receptor in sensitive Saccharomyces cerevisiae. The receptor activity was retained among the insoluble glucans in alkali-washed cells; yeast mannan and alkali-soluble glucan had little receptor activity. A minor fraction of receptor activity was removed from alkali-washed cells by hot acetic acid extraction, a procedure which solubilized some (1 --> 6)-beta-d-glucan and glycogen. The major fraction (>70%) of receptor activity remained with the acid-insoluble (1 --> 6)-beta-and (1 --> 3)-beta-glucans. Zymolyase, an endo-(1 --> 3)-beta-d-glucanase, solubilized a substantial fraction of the receptor activity in the acid-insoluble glucans. The receptor activity in yeast cell walls was periodate and (1 --> 6)-beta-d-glucanase sensitive, but was resistant to (1 --> 3)-beta-d-glucanase and alpha-amylase. The acid-soluble glucan fractions of a sensitive strain and a krel-l receptor-defective toxin-resistant mutant were examined. The krel-l strain had a reduced amount (ca. 50%) of (1 --> 6)-beta-d-glucan compared with the sensitive parent strain. A sensitive revertant of the krel-l strain regained the parental level of glucan. These results implicate (1 --> 6)-beta-d-glucan as a component of the yeast cell wall receptor for killer toxin.     

The first description of a (1--> 6)-beta-D-glucan in prokaryotes: (1 --> 6)-beta-D-glucan is a common component of actinobacillus suis and is the basis for a serotyping system

The chemical and antigenic properties of the cell-surface lipopolysaccharides (LPSs) and capsular polysaccharides (CPSs) of seven representative strains of Actinobacillus suis from healthy and diseased pigs were investigated. Four strains produced a linear (1 --> 6)-beta-D-glucan homopolymer, beta-D-Glcp-(1-[ --> 6)-beta-D-Glcp-(1-]n -->, as a LPS-O-chain (O1) and as a CPS (K1). Polyclonal antisera prepared against a (1 --> 6)-beta-D-glucan-containing strain showed a positive reaction against both LPSs and CPSs derived from the above strains (designated serotype O1/K1). One strain carried the (1 --> 6)-beta-D-glucan solely as a LPS-O-chain (serotype O1) and two strains did not express the (1 --> 6)-beta-D-glucan, but, instead, produced a different O-chain (designated serotype 02); these three strains expressed their own characteristic CPSs. (1 --> 6)-beta-D-Glucan structures are common cell wall components of yeast, fungi and lichens, but, to our knowledge, this is the first time a (1 --> 6)-beta-D-glucan has been described in a prokaryotic organism. Conformational and nuclear magnetic resonance analyses showed that the beta-D-Glcp-(1 --> 6)-beta-D-Glcp linkage was flexible and two distinct glycosidic conformers are described. Cross-reactive antibodies to the A. suis (1 --> 6)-beta-D-glucan could be detected in sera from a variety of species and in sera from specific pathogen free pigs. This cross-reactivity may arise from immuno-stimulation of organisms present in the surrounding environment that contain (1 --> 6)-beta-D-glucan, which may also explain the high incidence of false positive results in previous serological tests for A. suis. In addition, these (1 --> 6)-beta-D-glucan background antibodies may be protective against A. suis infection. The characterization herein of (1 --> 6)-beta-D-glucan is the foundation for the development of a serotyping system for A. suis.     

(1-->6)-Beta-D-glucan as the cell wall binding site for Debaryomyces hansenii killer toxin

AIMS: The aims of this study were to characterize the cell wall binding site of Debaryomyces hansenii killer toxin to provide a simple purification method and to determine some characteristics of this toxin. METHODS AND RESULTS: Various linear (1-->6)-beta-D-glucans of different origins were effective competitive inhibitors of the toxin action. Periodate oxidation and 1H-NMR was used to determine the receptor nature. Affinity chromatography on pustulan-Sepharose column was used to purify D. hansenii killer toxin, probably a 23-kDa protein. The killer toxin character was cureless. CONCLUSIONS: The investigation revealed that the killer toxin was mainly adsorbed by (1-->6)-beta-D-glucans. This is a low molecular weight protein, probably encoded by chromosomal genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The specificity of the killer toxin for its receptor provides an effective means to purify the killer toxin. This study is the first to identify the cell wall binding site of this killer toxin, a toxin with properties of industrial relevance.     

Effect of salt on the killer phenotype of yeasts from olive brines.

The killer properties of yeasts isolated from olive brines were examined in the absence and presence of sodium chloride in concentrations of up to 6% (wt/vol). An apparent enhancement of the killing action as the salt concentration increased, as well as changes in the spectra of activity against selected target strains, was observed in a few strains. Culture filtrates from killer strains grown at different NaCl concentrations (0, 3, or 6% [wt/vol]) were tested against sensitive yeasts cultivated under the same conditions. While the sensitivity of the target strain greatly increased in the presence of salt, no significant effect on toxin production was noticed.     

Simultaneous production of single cell protein and killer toxin by Wickerhamomyces anomalus HN1-2 isolated from mangrove ecosystem

The yeast Wickerhamomyces anomalus (the previous name was Pichia anomala) HN1-2 isolated from the mangrove ecosystem was found to be able to produce high level of both killer toxin and single cell protein. When the killer yeast cells were grown by batch cultivation in 5-l fermentor, crude protein in the cells, cell mass, reducing sugar, and diameter of the inhibition zone reached 56.0 g per 100 g of cell dry weight, 7.3 g per liter, 9.5 g per liter, and 19.0 mm, respectively within 12 h and this yeast synthesized a large amount of the essential amino acids, such as lysine (7.8%), methionine (1.8%), and leucine (9.0%). The crude killer toxin produced by the killer yeast isolate HN1-2 could kill the cells of Lodderomyces elongisporus, Candida albicans, Metschnikowia bicuspidata, Pichia guilliermondii, Saccharomyces cerevisiae, Yarrowia lipolytica, and Kluyveromyces aestuarii, which were widely distributed in natural marine environments. The results also showed that the undesirable yeast could be avoided during cell growth of the killer yeast.     

Purification and characterization of the cold-active killer toxin from the psychrotolerant yeast Mrakia frigida isolated from sea sediments in Antarctica

The psychrotolerant yeast Mrakia frigida 2E00797 isolated from sea sediments in Antarctica was found to be able to produce killer toxin against Metschnikowia bicuspidata, Candida tropicalis and Candida albicans. In the present study, the killer toxin was purified and characterized. The molecular weight of the purified killer toxin was estimated to be 55.6 kDa and the purified killer toxin shared 35.1% sequence homology with a protein kinase. The purified killer toxin's optimal temperature and pH for killing activity were 16 °C and 4.5, respectively, and it was stable in the temperature range from 10 to 25 °C at pH 4.5. The toxin's highest killing activity was observed in the presence of 3.0 g/100 ml NaCl. The purified killer toxin was able to actively kill whole cells of M. bicuspidata but could not kill the protoplast of the sensitive yeast. Of the eight yeast species tested in this study, the killer toxin was able to kill C. tropicalis and C. albicans in addition to M. bicuspidata.     

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.     

Aerosolization of particulate (1-->3)-beta-D-glucan from moldy materials

Mold-damaged building materials may contain biologically active agents, such as (1-->3)-beta-D-glucan, allergens, and mycotoxins, which have been associated with adverse health effects. The release of these components from contaminated surfaces into the air is not well understood. The purpose of this study was to characterize the release of particulate (1-->3)-beta-D-glucan from the surface of artificially mold-contaminated materials. Aspergillus versicolor and Stachybotrys chartarum were grown on malt extract agar (MEA), white ceiling tiles, and a wall-papered gypsum board for 1 and 6 months. The (1-->3)-beta-D-glucan on the surfaces of moldy materials and in air samples collected from these materials was analyzed by the Limulus amebocyte lysate assay. The aerosolization ratio was defined as the amount of (1-->3)-beta-D-glucan in the air divided by the amount on the surface. The results showed that the aerosolization of particulate (1-->3)-beta-D-glucan was influenced mainly by the type of material and the fungal species. For A. versicolor, the aerosolization ratios of particulate (1-->3)-beta-D-glucan released from the three types of material were not significantly different. However, the ratios for S. chartarum released from ceiling tiles and gypsum board were significantly higher than the ratios for this organism released from MEA (P < 0.001) and were comparable to those for A. versicolor. These findings indicate that the use of MEA in aerosolization experiments is likely to underestimate the release of S. chartarum particles from building materials. These results provide important background information for design of future laboratory or animal experiments, as well as for interpretation of field measurement data.     

Stimulation of TNF-alpha release by fungal cell wall polysaccharides

Carboxymethylated derivatives were prepared from the (1-->3)-beta-D-glucan isolated from the cell wall of baker's yeast Saccharomyces cerevisiae and from the chitin-glucan complex of the mycelium of the industrial filamentous fungus Aspergillus niger. The polysaccharides were applied to peritoneal mouse macrophages and after a 2-h incubation the release of TNF-alpha by the stimulated macrophages was measured using an enzyme-linked immunosorbent assay. As the third polysaccharide stimulant, a water-soluble derivative of chitin was assayed and the observed cytokine release was compared with the control experiment. In three concentrations of the polysaccharides applied, carboxymethyl glucan revealed a dramatic increase in the TNF-alpha release, while addition of carboxymethyl chitin-glucan resulted only in a moderate enhancement, and carboxymethyl chitin was inactive. The results indicate that fungal polysaccharides, especially (1-->3)-beta-D-glucan, are potent macrophage stimulators and activators of TNF-alpha release, which implies their potential application in antitumor therapy.     

Molecular structure of the cell wall receptor for killer toxin KT28 in Saccharomyces cerevisiae.

The adsorption of the yeast killer toxin KT28 to susceptible cells of Saccharomyces cerevisiae was prevented by concanavalin A, which blocks the mannoprotein receptor. Certain mannoprotein mutants of S. cerevisiae that lack definite structures in the mannan of their cell walls were found to be resistant to KT28, whereas the wild-type yeast from which the mutants were derived was susceptible. Isolated mannoprotein from a resistant mutant was unable to adsorb killer toxin. By comparing the resistances of different mannoprotein mutants, information about the molecular structure of the receptor was obtained. At least two mannose residues have to be present in the side chains of the outer chain of the cell wall mannan, whereas the phosphodiester-linked mannose group is not essential for binding and the subsequent action of killer toxin KT28.     

An immunoassay for the measurement of (1-->3)-beta-D-glucans in the indoor environment

An inhibition enzyme immunoassay was developed for quantitation of (1-->3)-beta-D-glucans in the indoor environment. Immunospecific rabbit antibodies were produced by immunization with bovine serum albuminconjugated laminarin.The laminarin calibration curve ranged from 40 to 3000 ng/ml.Another (1-->3)-beta-D-glucan (curdlan) showed a similar inhibition curve, but was less reactive on a weight basis. Pustulan, presumed to be (1-->3)-beta-D-glucan, also showed immunoreactivity in the assay. Control experiments indicated that this was due to (1-->3)-beta-D-glucan structures. Other non-(1-->3)-beta-D-glucan polysaccharides did not react. (1-->3)-beta-Dglucan was detectable in dust from a variety of occupational and environmental settings. We conclude that the new assay offers a useful method for indoor (1-->3)-beta-Dglucan exposure assessment.     

Killer-toxin-resistantkre12 mutants ofSaccharomyces cerevisiae: genetic and biochemical evidence for a secondary K1 membrane receptor

TheSaccharomyces cerevisiae killer toxin K1 is a secreted α/β-heterodimeric protein toxin that kills sensitive yeast cells in a receptor-mediated two-stage process. The first step involves toxin binding to β-1,6-d-glucan-components of the outer yeast cell surface; this step is blocked in yeast mutants bearing nuclear mutations in any of theKRE genes whose products are involved in synthesis and/or assembly of cell wall β-d-glucans. After binding to the yeast cell wall, the killer toxin is transferred to the cytoplasmic membrane, subsequently leading to cell death by forming lethal ion channels. In an attempt to identify a secondary K1 toxin receptor at the plasma membrane level, we mutagenized sensitive yeast strains and isolated killer-resistant (kre) mutants that were resistant as spheroplasts. Classical yeast genetics and successive back-crossings to sensitive wild-type strain indicated that this toxin resistance is due to mutation(s) in a single chromosomal yeast gene (KRE12), renderingkrel2 mutants incapable of binding significant amounts of toxin to the membrane. Sincekrel2 mutants showed normal toxin binding to the cell wall, but markedly reduced membrane binding, we isolated and purified cytoplasmic membranes from akrel2 mutant and from an isogenicKre12+ strain and analyzed the membrane protein patterns by 2D-electrophoresis using a combination of isoelectric focusing and SDS-PAGE. Using this technique, three different proteins (or subunits of a single multimeric protein) were identified that were present in much lower amounts in thekre12 mutant. A model for K1 killer toxin action is presented in which the gene product ofKRE12 functions in vivo as a K1 docking protein, facilitating toxin binding to the membrane and subsequent ion channel formation.     

Killer toxin of Hanseniaspora uvarum

The yeast Hanseniaspora uvarum liberates a killer toxin lethal to sensitive strains of the species Saccharomyces cerevisiae. Secretion of this killer toxin was inhibited by tunicamycin, an inhibitor of N-glycosylation, although the mature killer protein did not show any detectable carbohydrate structures. Culture supernatants of the killer strain were concentrated by ultrafiltration and the extracellular killer toxin was precipitated with ethanol and purified by ion exchange chromatography. SDS-PAGE of the electrophoretically homogenous killer protein indicated an apparent molecular mass of 18,000.Additional investigations of the primary toxin binding sites within the cell wall of sensitive yeast strains showed that the killer toxin of Hanseniaspora uvarum is bound by -1, 6-d-glucans.     

(1-->3)-beta-D-glucan in patients with pulmonary aspergilloma

To elucidate the role of (1-->3)-beta-D-glucan in pulmonary aspergilloma, serum concentrations of (1-->3)-beta-D-glucan were measured repeatedly for as long as 10 months in eight patients. In four patients with inactive disease, concentrations of (1-->3)-beta-D-glucan were in the normal range.The concentrations of (1-->3)-beta-D-glucan increased in two patients, although the disease was inactive. This increase might show the earliest stage of the invasive process of the disease. In two other patients with active disease, (1-->3)-beta-D-glucan increased. Other parameters, such as galactomannan, immunodiffusion and a radio-allergosorbent test, as well as inflammatory m arkers such as C-reactive protein and the leukocyte count, did not show any consistent tendency in regard to the activity of the disease. Thus, a (1-->3)-beta-D-glucan assay may add valuable data for evaluating the disease activity and understanding the disease process of pulmonary aspergilloma.     

Binding of yeast killer toxin to a cell wall receptor on sensitive Saccharomyces cerevisiae.

35S-labeled killer toxin protein bound to cells of sensitive Saccharomyces cerevisiae S14a. Strains that were resistant to toxin through mutation in the nuclear genes kre1 kre2 bound toxin only weakly. Non-radioactive toxin competed effectively with 35S-labeled toxin for binding to S14a, but did not compete significantly in the binding to mutant kre1-1. This implied that binding to kre1-1 was nonspecific. A Scatchard analysis of the specific binding to S14a gave a linear plot, with an association constant of 2.9 x 10(6) M-1 and a receptor number of 1.1 x 10(7) per cell. Killer toxin receptors were solubilized from the cell wall by zymolyase digestion. Soluble, non-dialyzable cell wall digest from S14a competed with sensitive yeast cells for 35S-labeled toxin binding and reduced toxin-dependent killing of a sensitive strain. Wall digest from kre1-1 competed only weakly for toxin binding with sensitive cells and caused little reduction of toxin-dependent killing. Although the abundant (1.1 x 10(7) per cell) receptor appeared necessary for toxin action, as few as 2.8 x 10(4) toxin molecules were necessary to kill a sensitive cell of S14a. The kinetics killing of S14a suggested that some component was saturated with toxin at a concentration 50-fold lower than that needed to saturate the wall receptor.     

The yeast KRE5 gene encodes a probable endoplasmic reticulum protein required for (1----6)-beta-D-glucan synthesis and normal cell growth.

Yeast kre mutants define a pathway of cell wall (1----6)-beta-D-glucan synthesis, and mutants in genes KRE5 and KRE6 appear to interact early in such a pathway. We have cloned KRE5, and the sequence predicts the product to be a large, hydrophilic, secretory glycoprotein which contains the COOH-terminal endoplasmic reticulum retention signal, HDEL. Deletion of the KRE5 gene resulted in cells with aberrant morphology and extremely compromised growth. Suppressors to the KRE5 deletions arose at a frequency of 1 in 10(7) to 1 in 10(8) and permitted an analysis of deletions which were found to contain no alkali-insoluble (1----6)-beta-D-glucan. These results indicate a role for (1----6)-beta-D-glucan in normal cell growth and suggest a model for sequential assembly of (1----6)-beta-D-glucan in the yeast secretory pathway.     

Purification, characterization and gene cloning of the killer toxin produced by the marine-derived yeast Williopsis saturnus WC91-2

As the killer toxin produced by Williopsis saturnus WC91-2 could kill many sensitive yeast strains, including the pathogenic ones, the extracellular killer toxin in the supernatant of cell culture of the marine yeast strain was purified and characterized. The molecular mass of the purified killer toxin was estimated to be 11.0kDa according to the data from SDS-PAGE. The purified killer toxin had killing activity, but could not hydrolyze laminarin. The optimal conditions for action of the purified killer toxin against the pathogenic yeast Metschnikowia bicuspidate WCY were the assay medium with 10% NaCl, pH 3-3.5 and temperature 16°C. The gene encoding the killer toxin from the marine killer yeast WC91-2 was cloned and the ORF of the gene was 378bp. The deduced protein from the cloned gene encoding the killer toxin had 125 amino acids with calculated molecular weight of 11.6kDa. It was also found that the N-terminal amino acid sequence of the purified killer toxin had the same corresponding sequence deduced from the cloned killer toxin gene in this marine yeast, confirming that the purified killer toxin was indeed encoded by the cloned gene.     

Pharmacokinetics, biological effects, and distribution of (1-->3)-beta-D-glucan in blood and organs in rabbits

The pharmacokinetics, biological effects and distribution in blood and organs of 125I-labeled (1-->3)-beta-D-glucan purified from Candida albicans were analyzed in rabbits during the 24-h period following an intravenous administration.The intravascular half-life of (1-->3)-beta- D-glucan was 1.8 min in the low-dose group (9.3 mug/kg) and 1.4 min in the high-dose group (222 mug/kg), and the mean (+/-SD) total body clearance was 1.12 +/- 0.30 and 1.17 +/- 0.16 ml/min, respectively. The rabbits remained well and (1-->3)-beta-D-glucan failed to alter blood cell counts. Less than 3% of the (125)I-(1-->3)-beta-D-glucan was initially associated with the cellular compartment, and this value decreased further during the 2-h period following administration (P = 0.0001). Over 97% of (125)I-(1-->3)-beta-D-glucan was associated with cell-free plasma, and the majority in plasma appeared to be present in the unbound form (not associated with lipoproteins or plasma proteins). The liver contained more than 80% of the (125)I-(1-->3)-beta-D-glucan detected in the six major organs analyzed.