Study on agglutinating factors from flocculent Saccharomyces cerevisiae strains.

The lectin-like theory suggest that yeast flocculation is mediated by an aggregating lectinic factor. In this study we isolated an agglutinating factor, which corresponds to lectin, from whole cells by treating the flocculent wild-type Saccharomyces cerevisiae NCYC 625 strain and its weakly flocculent mutant [rho degrees ] with EDTA and two non-ionic surfactants (Hecameg and HTAC). The dialysed crude extracts obtained in this way agglutinated erythrocytes and this hemagglutination was specifically inhibited by mannose and mannose derivatives. However, SDS-PAGE profiles showed that the three reagents had different effects on the yeast cells. The non-ionic surfactants appeared to be the most efficient, as their extracts possessed the highest specific agglutinating activity. The products released by the wild-type strain presented a higher specific agglutinating activity than those released by the [rho degrees ] mutant. Purification of the agglutinating factor from extracts of both strains by affinity chromatography revealed two active bands of relative mass of 26 and 47 kDa on SDS-PAGE. Mass spectrometry analysis by MALDI-TOF, identified a 26 kDa band as the triose phosphate isomerase (TPI) whereas a 47 kDa band was identical to enolase. Edman degradation showed that the N-terminal sequences of these proteins were similar to TPI and enolase, respectively. The difference in the flocculation behaviour of the two strains is due to changes in the protein composition of the cell wall and in the protein structure involved in cell-cell recognition.

Sterol and fatty acid composition of Candida lusitaniae clinical isolates.

The sterol and fatty acid compositions of four amphotericin B-resistant and of two amphotericin B-susceptible Candida lusitaniae clinical isolates were determined. A flow cytofluorometric susceptibility test (FCST) with a membrane potential-sensitive cationic dye was used as a complement to the conventional method for selecting the isolates. Compared to susceptible isolates, resistant ones showed a greatly reduced ergosterol content and changes in sterol composition, consistent with a defect in Delta8-->7 isomerase. Within each group, no correlation between the sterol or fatty acid pattern or composition and both the degree of in vitro susceptibility and FCST MIC was found.

The role of glucose in the Kluyveromyces bulgaricus flocculation phenomenon: transduction by cAMP-dependent protein kinase pathway?

Yeast flocculation appears to be dependent on several culture conditions such as nitrogen or carbon sources. In 0.2% glucose medium Kluyveromyces bulgaricus flocculation intensity is weak (10% at maximum) by comparison with flocculation in 2% glucose medium (85% maximum). Addition of glucose to K. bulgaricus in exponential growth phase in 0.2% glucose medium produced a rapid increase of the flocculation percentage during the 30 min following the addition of glucose. cAMP and 2,4-dinitrophenol showed similar effects while cAMP-dependent protein kinase (PKA) inhibitors exhibited an antagonist effect. Moreover, the induction of flocculation did not seem to imply translation of new proteins: cycloheximide had no effect, although growth was inhibited. The induction of flocculation mainly implies ATP hydrolysis for activation or secretion of galactose-specific receptors as demonstrated by treatment with NaN(3). We propose a hypothesis that involves a PKA transduction signal leading to flocculation.

Isolation and biochemical characterization of cell wall tight protein complex involved in self-flocculation of Kluyveromyces bulgaricus.

Flocculation of yeasts is a cell-cell aggregation phenomenon which is driven by interactions between cell wall lectins and cell wall heteropolysaccharides. In Sabouraud medium, Kluyveromyces bulgaricus was highly flocculent. Incubation of flocculent K. bulgaricus cells with EDTA or Hecameg led to extracts showing hemagglutinating and flocculating properties. Purification of the extracts by native PAGE gave two bands which allowed flocculation of deflocculated K. bulgaricus. Both bands with specific reflocculating activity were composed of five subunits, of which only three possessed weak reflocculating activity upon deflocculated yeast. The mixture of these three proteins allow the recovery of initial specific reflocculating activity of the complex. These three proteins, denoted p28, p36 and p48, presented, in their first 15 amino acids, homologies with glycolysis enzymes, i.e., 3-phosphoglycerate mutase, glyceraldehyde-3-phosphate dehydrogenase and enolase, respectively. However, no such enzymatic activity could be detected in the crude extract issued from treatment with EDTA and Hecameg of flocculent yeast cells. When yeasts had grown in glucose poor medium, flocculation was drastically affected. The EDTA and Hecameg crude extracts showed weak reflocculating activity. After PAGE, the protein complexes did not appear in the EDTA extract, but they did appear in the Hecameg crude extract. These results suggest that: (i) self-flocculation of K. bulgaricus depends on the expression of different floc-forming protein complex, (ii) these proteins are galactose specific lectins showing homologies in their primary structure with glycolysis enzymes.

Permeability of yeast cell envelope to fluorescent galactosylated telomers derived from THAM.

The work reported herein deals with the study of cellular recognition and permeability phenomena in yeasts. Various galactosylated organic telomers derived from trishydroxymethyl-aminomethane (THAM) and bearing fluorescent moieties were synthesized in order to measure their ability to cross the yeast cell envelope. Grafting fluorescent probes on the organic telomer backbone allowed us to study their specific behaviors toward the yeasts by fluorescence microscopy. Yeasts belonging to the genera Kluyveromyces and Saccharomyces were used for this study. With Saccharomyces yeast cells bearing mannose-specific lectins or lectin-like proteins, on their outer surface, all the galactosylated or nongalactosylated organic telomers passed through the cell envelope and invaded the cytoplasm. With Kluyveromyces yeast cells bearing galactose-specific lectins, the galactosylated organic telomers were blocked at the outer surface while the nongalactosylated derivatives crossed the cell envelope. Moreover, preincubation of Kluyveromyces yeasts with galactose or methylgalactose inhibited the cell surface anchorage of the organic telomers and allowed their penetration into the cytoplasm. When assays were performed on spheroplasts of both Kluyveromyces and Saccharomyces yeasts, no fixation on the surface could be observed, and all the derivatives went through the membrane and invaded the cytoplasm.

Amphotericin B resistance and membrane fluidity in Kluyveromyces lactis strains.

The membrane fluidity of reduced-amphotericin B (AmB)-sensitivity Kluyveromyces lactis mutant strain is higher than that of the wild-type K. lactis strain. After culture of the K. lactis and K. lactis mutant cells in the presence of subinhibitory doses of AmB (10 and 125 mg/liter, respectively), the plasma membranes of both yeast strains also showed a higher fluidity than did those of control cells. High membrane fluidity was associated with changes in the structural properties of the membranes. Culture of the K. lactis and K. lactis mutant cells in the presence of AmB induced changes in membrane lipid contents. In particular, phospholipid contents were increased in both strains treated with AmB, compared with their corresponding counterparts. As a result, the sterol/phospholipid ratio decreased. The relative proportion of monounsaturated fatty acids also increased after AmB treatment. The saturated fatty acid/monounsaturated fatty acid ratio decreased in K. lactis and K. lactis mutant cells treated with AmB but also in K. lactis mutant control cells compared to that in the K. lactis wild strain. These changes in lipid composition explain the higher fluidity, which could represent a process of metabolic resistance of the yeasts to AmB.

Evidence for a lectin in Kluyveromyces sp. that is involved in co-flocculation with Schizosaccharomyces pombe.

Co-flocculation is the aggregation of yeasts belonging to different genera or species. Kluyveromyces bulgaricus and Kluyveromyces lactis 5c are self-flocculent, but they can also co-flocculate with the non-flocculent yeast Schizosaccharomyces pombe 972 h(-). This co-flocculation is inhibited by D-galactose and galactose derivatives and involves the binding of a galactose-specific proteinic receptor (or lectin) of Kluyveromyces sp. to the cell wall galactomannans of S. pombe. The proteinic receptor is strongly anchored in the cell wall, it was partially purified by affinity chromatography using immobilized S. pombe galactomannans. This galactose-specific proteinic receptor does not appear to interfere in K. bulgaricus or K. lactis self-flocculation, which is mediated by another galactose-specific lectin weakly linked at the cell wall.

Mitochondrial function in cell wall glycoprotein synthesis in Saccharomyces cerevisiae NCYC 625 (Wild type) and [rho(0)] mutants.

We studied phosphopeptidomannans (PPMs) of two Saccharomyces cerevisiae NCYC 625 strains (S. diastaticus): a wild type strain grown aerobically, anaerobically, and in the presence of antimycin and a [rho(0)] mutant grown aerobically and anaerobically. The aerobic wild-type cultures were highly flocculent, but all others were weakly flocculent. Ligands implicated in flocculation of mutants or antimycin-treated cells were not aggregated as much by concanavalin A as were those of the wild type. The [rho(0)] mutants and antimycin-treated cells differ from the wild type in PPM composition and invertase, acid phosphatase, and glucoamylase activities. PPMs extracted from different cells differ in the protein but not in the glycosidic moiety. The PPMs were less stable in mitochondrion-deficient cells than in wild-type cells grown aerobically, and this difference may be attributable to defective mitochondrial function during cell wall synthesis. The reduced flocculation of cells grown in the presence of antimycin, under anaerobiosis, or carrying a [rho(0)] mutation may be the consequence of alterations of PPM structures which are the ligands of lectins, both involved in this cell-cell recognition phenomenon. These respiratory chain alterations also affect peripheral, biologically active glycoproteins such as extracellular enzymes and peripheral PPMs.

Implication of cell wall constituents in the sensitivity of Kluyveromyces lactis strains to amphotericin B.

In Kluyveromyces lactis, the cell wall compositions of Kl (ATCC 96897), a wild sensitive strain, and Klm (ATCC 96896), a strain resistant to amphotericin B (AmB), were shown to be very different, since the walls in the latter were significantly enriched in hexosamine, but had a reduced content in phosphate and amino acid. In both strains, the cell walls limited their sensitivity to this antifungal agent. The absence of cell wall increased the sensitivity of the cells to this polyene by 5 to 10-fold. When the cells were treated with enzymes such as pronase and chitinase in order to change the cell wall structure just before inoculation, the yeasts appeared more resistant to the antibiotic. However, treatments with chymopapain and phospholipase C did not significantly change the sensitivity of the two strains to this agent. Cells treated with acid phosphatase displayed a longer lag phase than the control cells. In addition, when cultured in the presence of AmB, the cells were less sensitive to this agent. The present results reveal that both a change in the ionic charges of the cell wall and an alteration in the cell wall structure modified the sensitivity of these yeast strains to AmB.

Differential binding of lectins IL-2 and CSL to candida albicans and cancer cells.

The demonstration that interleukin 2 (IL-2) is a lectin specific for oligomannosides allows to understand a new function for this cytokine: as a bifunctional molecule when bound to its receptor ss, IL-2 associates the latter which the CD3/TCR complex, interacting with oligosaccharides of CD3 through its carbohydrate-recognition domain (Zanetta et al. , 1996, Biochem. J., 318, 49-53). This induces the tyrosine phosphorylation of the IL-2R beta by ++p56(lck) , the first step of the IL-2-dependent signaling. Since this specific association is disrupted in vitro by oligomannosides with five and six mannose residues, we made the hypothesis that pathogenic cells or microorganisms could bind IL-2, consequently disturbing the IL-2-dependent response. This study shows that the pathogenic yeast Candida albicans (in contrast with nonpathogenic yeasts) binds high amounts of IL-2 as did cancer cells. In contrast with cancer cells, yeasts do not bind the Man6GlcNAc2-specific lectin CSL, an endogenous "amplifier of activation signals" (Zanetta et al. , 1995, Biochem. J., 311, 629-636).

Cell targeting by glycosidic telomers. Specific recognition of the Kb CWL1 lectin by galactosylated telomers.

This work deals with the synthesis and lectinic recognition ability of galactosylated telomers. To investigate if telomeric carriers could exhibit cellular recognition properties, we have synthesized mono- and polygalactosylated tris(hydroxymethyl)acrylamidomethane (THAM) telomers. The affinity of such macromolecular drug carriers toward a receptor, the yeast Kb CWL1 lectin, was defined, and the influence of mono- or polygalactosylation of THAM units on the recognition phenomenon was assessed. The lectinic affinity of the compounds was estimated by measuring the inhibition of yeast aggregation. The average degree of polymerization as well as the hydrophilic-lipophilic balance of such galactosylated telomers affects their recognition ability for the lectin.

Comparative extraction procedures for a galactose-specific lectin involved in flocculation of Kluyveromyces lactis strains.

The extraction of a lectinic factor involved in yeast flocculation, from two Kluyveromyces lactis strains (a flocculent K. lactis 5c and a non-flocculent K. lactis 5a strain) was performed using EDTA and two surfactants, Hecameg and HTAC. The properties of the different extracts were tested by haemagglutination and reflocculation of deflocculated K. lactis 5c cells. Hecameg gave the highest yields of active lectinic extract but the extraction with EDTA seemed more specific. HTAC extracts showed a very low activity. The possibilities of extraction of the agglutinating factor, either by an ion chelator or by surfactants, suggest that this factor may be anchored in the cell envelope, i.e. the cell wall and the membrane, by different mechanisms. All the assays revealed a galactose-specific lectinic activity was present that in the flocculent as well as in the non-flocculent strain. This indicates that the absence of flocculation with K. lactis 5a is mainly due to a defect in the ligands of the lectin rather than to a loss of the lectinic factor itself.

Structure of the phosphopeptidomannans from flocculent and non-flocculent yeast Kluyveromyces lactis.

After extraction from whole cells, and purification by gel filtration, the chemical composition and molecular mass estimation of the cell-wall phosphopeptidomannan (PPM) showed no significant difference respectively between flocculent, weakly, very weakly and non-flocculent Kluyveromyces lactis yeast strains. However, when PPMs were tested as ligands of a lectin, extracted from the flocculent strain, the PPM isolated from the flocculent and weakly flocculent strain were recognized to a higher degree than those isolated from the non and very weakly flocculent strains. Acetolysis of PPM extracted from the four strains produced five oligosaccharide fractions corresponding to mono-, di-, tri-, penta-and hexa-saccharides. The flocculent strain was characterised by a high content of di-and penta-saccharides. The 1H NMR analysis of the oligosaccharides demonstrated that the flocculent strain contained equivalent levels of the two mannobioses: Man(alpha 1-->2)Man and Man(alpha 1-->3)Man and of the two mannotrioses Man(alpha 1-->2)Man(alpha 1-->2)Man and Man(alpha 1-->3)Man(alpha 1-->2)Man. In contrast, the non-flocculent and the very weakly flocculent strains contained a single type of mannobiose Man(alpha 1-->2)Man and one type of mannotriose Man(alpha 1-->2)Man(alpha 1-->2)Man.

Identification of 24-methylene-24,25-dihydrolanosterol as a precursor of ergosterol in the yeasts Schizosaccharomyces pombe and Schizosaccharomyces octosporus.

Study of the plasma membrane sterol composition in the yeasts Schizosaccharomyces pombe and Schizosaccharomyces octosporus revealed the presence of ergosterol, lanosterol, dehydroergosterol, fecosterol, episterol and 24-methylene-24,25-dihydrolanosterol (eburicol), a C-31 derivative. The growth of both yeasts in the presence of ketoconazole led to a decrease by 85% of the ergosterol content while the levels of lanosterol and eburicol increased. This suggests that in the biosynthetic pathway of ergosterol in Schizosaccharomyces species, the transmethylation process on the C-24 may occur directly on lanosterol and not only on zymosterol. On the other hand, it cannot be excluded that in the genus Schizosaccharomyces two routes exist from lanosterol to ergosterol: the classical one via a direct C-14, C-4 demethylation of lanosterol and the second one via the formation of a C-31 derivative followed by demethylations.

Isolation and characterization of exocellular polysaccharides produced by Bifidobacterium longum.

When grown anaerobically at pH values above 5.0, on ultrafiltered complex media containing excess lactose, Bifidobacterium longum formed up to 140 mg l-1 (glucose equiv.) exopolysaccharides. The highest yield was obtained when the cells were cultivated in a peptone/yeast extract medium with pH controlled by additions of NH4OH. Whatever the conditions under study, exopolysaccharides represented about 30% of the polysaccharides produced by B. longum after 48 h of culture. Crude pronase-treated exopolysaccharide preparations were adsorbed on ion-exchange chromatographic resin to yield an anionic heteropolysaccharide fraction. Two subfractions with apparent molecular masses of 1.2 MDa and 0.36 MDa respectively were subsequently recovered after gel filtration on Sepharose 4B. In both subfractions, glucose, galactose and small amounts of uronic acids and hexosamines were present in similar molar proportions, suggesting that the excreted polymers may be synthesized from the same base unit and may have a structure resulting from repeating subunits.

[Effect of desertomycin on the synthesis of cell wall polymers in Saccharomyces uvarum]. / Action de la désertomycine sur la synthèse des polymères pariétaux du Saccharomyces uvarum.

The desertomycin action upon Saccharomyces uvarum wall synthesis has been studied. Spheroblast regeneration was carried out in a liquid medium containing labeled glucose to monitor the synthesis of different wall components. In the presence of desertomycin, wall synthesis was affected; this was expressed as a net reduction of insoluble alkali constituents content, more precisely the insoluble acido-alkali fraction that, in yeasts, is constituted by chains of beta(1,3)-glucans linked among themselves by beta(1,6) bonds. Mannan formation was not inhibited such polymers that cannot be fixed to the glucan matrix of the wall were liberated in the regeneration medium. Because of desertomycin action, the decrease in insoluble alkali content revealed an interference with the enzymatic systems catalyzing glucan synthesis. In vitro, however, this antifungal had little effect upon glucan synthetase activity: doses 5 times superior to the subinhibiting level used in vivo caused only 30% inhibition. This result can be explained by an indirect action of desertomycin. Parietal disorders were the result of membrane structure disturbance, notably the phospholipids and localized enzymatic systems. This antifungal presents an analogical structure with macrolides with recognized membrane action.

Changes in cell wall composition of deformed ras1- cells of Schizosaccharomyces pombe.

Disruption of the Schizosaccharomyces pombe ras1 gene results in a morphological transformation to large spheres, in contrast to wild-type cells which grow as rods. Chemical analysis of isolated cell walls showed no significant changes in saccharide content but an increase in protein and phosphate contents in ras1- walls relative to parent walls. Polymers tightly bound to the cell wall were solubilized by SDS treatment. Several compounds with molar mass ranging from 22 to 130 kDa and more were resolved by gel filtration and SDS-PAGE. Among low-molar-mass species, a component moving as a band at 31 kDa was conspicuous in ras1- cell walls. It was solubilized by heating in Tris-HCl buffer and shown to have a beta-1,3-glucanase activity against laminarin. The level of the enzyme was by 30% higher in the ras1- cell wall than in the wild-type cell wall. This enzyme may participate in the remodelling of the rigid glucan network and account (at least partially) for the aberrant cell shape. The ras1- cell wall contained a high level of charged polymers, especially phosphoproteins, raising the appealing possibility that ras1- is involved in a putative kinase cascade required to sense and respond to external stimuli destined for the cell wall. Although the present study shows that ras1 loss of function and altered cell wall composition are closely linked defects, it has still to be shown that the ras1 protein is directly involved in alterations found in the mutant cell walls.

Yeast motor proteins.

Yeast accomplish a variety of intracellular motile events with the aid of mechanochemical enzymes known as motor proteins. This review covers the current state of knowledge on myosins, kinesins, dyneins, dynamins and SMC proteins present in yeast cells, and the most important developments in the study of yeast mitosis. Both topics have seen rapid progress over the past few years.

Studies on the activity of Kluyveromyces lactis S-adenosylmethionine: delta 24-sterol methyltransferase in presence of polyenic antifungal agents.

The S-adenosylmethionine: delta 24-sterol methyltransferase (24 SMT) primarily considered as a mitochondrial enzyme, was recently mainly detected in lipid particles of yeasts. It catalyses the methylation of zymosterol which is an essential reaction for the synthesis of ergosterol. We have investigated in cellular extracts of two Kluyveromyces lactis strains the action of polyenic antifungal agents on the activity of this enzyme. Low concentrations of amphotericin B, candicidin and pimaricin strongly stimulate this activity, while high concentrations inhibit it or have no effect. Whatever the doses used, nystatin and filipin had no significant influence on this activity. According to the molar ratio amphotericin B/total sterols of the enzyme preparation, the interference of amphotericin B on the 24 SMT activity may result of two mechanisms.

Recognition ability and cytotoxicity of some oligosaccharidyl substituted beta-cyclodextrins.

This paper reports a chemico-enzymatic synthesis of beta-CD derivatives. The recognition properties of these derivatives were tested using flocculating yeast and isolated lectins. It was observed that the substitution of beta-cyclodextrins with galactose end arms induces the better recognition by a cell-linked galactose-specific lectin. The physicochemical effects of the beta-CD derivatives on membranes were estimated using red blood cells and the effects on the viability of yeast and human rectal tumor cells were appreciated by measuring the mitochondrial deshydrogenase activity. The substitutions of the beta-CD ring by sugar antennae decrease the negative physicochemical effects of the beta-CD, ie their hemolytic properties. However, these substitutions induce significant modifications of the biological properties of the molecules, particularly the cytotoxicity and the growth of eukaryotic cells.