Immunochemical studies on mannans of the genusSaccharomyces

Structural and immunochemical studies were carried out on mannans isolated from various species of the genusSaccharomyces. It has been found that mannans ofSaccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces chevalieri, Saccharomyces italicus, Saccharomyces logos, Saccharomyces oviformis, Saccharomyces uvarum andSaccharomyces willianus are structurally and antigenically closely related, they gave high cross-reactions with anti-Saccharomyces cerevisiae serum. Mannotetraosa was found to be the immunodominant group of these mannans. Mannans ofSaccharomyces rouxii andSaccharomyces rosei showed different structural features and immunochemical properties.     

On the Immunochemical and Biochemical Studies of Fungi

The nature of the cross reaction of the mycelial mannan of Trichophyton rubrum and galactomannan isolated from the culture medium of Aspergillus fumigatus with antisera of Saccharomyces cerevisiae and Candida albicans is described. Cross-reactivity of polysaccharides of both T. rubrum and A. fumigatus was weak with antisera of C. albicans and S. cerevisiae, but the galactomannan of A. fumigatus showed slightly stronger activity than the mannan of T. rubrum which possesses more closely related chemical structure of the mannans of S. cerevisiae and C. albicans.     

Biochemical and Immunochemical Studies of Fungi

Crude mannans extracted from Candida albicans and Saccharomyces cerevisiae by autoclaving yeast cells in citrate buffer (pH 7.0) according to Peat's method, were fractionated repeatedly by column chromatography on DEAE-Sephadex, acetate form, yielding neutral and acidic mannans. The former fraction showed a single peak by boundary electrophoresis and ultracentrifugal analysis, while the latter contained small amounts of phosphorus and protein. Using purified mannans as controls, various serological experiments were carried out with mannan antigens extracted from C. albicans with 45% phenol water and with 3% NaOH. No remarkable differences were observed in the antigenic activity of 4 mannan antigens from C. albicans, and the purified mannan exhibited very high antigenic activity. It was found that the mannan of S. cerevisiae was antigenically less specific than that of C. albicans mannan. The difference in serological specificity between mannans of both species may reflect not only differences in mannopyranose linkages but differences in the structure of the macromolecules.     

Rapid and extensive vacuolation of the budding yeastsSaccharomyces cerevisiae andCandida albicans by amphotericin B

The effect of amphotericin B (AMPH) on vacuolation in the budding yeastsSaccharomyces cerevisiae andCandida albicans was studied. The minimum inhibitory concentration of AMPH for growth ofS. cerevisiae andC. albicans was 1 µg/ml. In untreated control cultures, mature cells had large central vacuoles in the exponential phase, which hampered the detection of vacuolation effect. Small buds in untreated exponential phase cells, however, only rarely showed vacuoles under the light microscope. Treatment with 0.2 µg/ml of AMPH for 20–30 min induced extensive vacuolation not only in mothers but also buds ofS. cerevisiae. Extensive vacuolation lasted 4 h or more, and growth rate of the cells was much reduced for 8 h or more. Vacuolation itself was not fatal: on removal of the drug most cells gradually recovered from vacuolation and eventually multiplied. A similar effect of AMPH was also observed inC. albicans but at a higher concentration (0.5 µg/ml).     

Amylolytic activity of yeasts

The total amylolytic activity of 14 selected fermentation type I members of the generaSaccharomyces, Zygosaccharomyces, Candida andTorulopsis was studied. Fractions with α-glucosidase activity, the specificty of which was tested for maltose and sucrose, were isolated on carboxymethyl-cellulose from the intracellular contents of two strains ofCandida albicans and one ofCandida stellatoidea. The fraction from the strainCandida albicans 29-3-109, which is more virulent for mice, displayed the greatest α-glucosidase activity, moderate activity was present in the strainCandida stellatoidea 29-64-1, while the lowest activity was found in the less virulent strain ofCandida albicans, 29-3-19.     

Systematics of the genusCandida Berkhout: Proton magnetic resonance spectra of the mannose-containing polysaccharides of some further species ofCandida as an aid in classification

The proton magnetic resonance spectra of the mannose-containing polysaccharides of 48Candida species were determined. Two other species formed heteropolymers for which p.m.r. spectra could not be obtained. The species were grouped as follows: 17 species formed mannose-containing polysaccharides with spectra like those ofHansenula anomala, Pichia farinosa, Pichia membranaefaciens orPichia robertsii mannans; 12, like those ofMetschnikowia andDebaryomyces mannans; 6, like those of mannans ofSaccharomyces species; 4, like that ofTorulopsis bombicola mannan; and 4, like those ofCandida obtusa andCandida tepae polysaccharides. Five formed mannans whose spectra were unlike those of the mannans of any other yeast species examined.     

Studies on the Antigenic Activities of Yeasts

Five antigenic mannans isolated from the cells of Candida albicans serotype A, C. albicans serotype B, C. tropicalis, C. stellatoidea and Saccharomyces cerevisiae were examined for their reactivities against concanavalin A, the size of the combining site has been estimated to be relatively small, up to 4 hexopyranosyl residues. In the quantitative precipitation reaction, all mannan-concanavalin A systems afforded nearly the same amounts of nitrogen or mannan precipitated, and the ratios of precipitation-inhibition with α1→2 linked manno-oligosaccharides, from biose to tetraose, were also equal regardless of the structural differences of these mannans. Furthermore, in agar-gel double diffusion analysis, all the systems gave a corresponding precipitation arc which completely fused with the adjacent ones. These behaviors of mannan-concanavalin A systems resemble those of antigen-antibody systems consisting of the same mannans and anti-C. albicans serotype B serum. It also provided evidence that the previous interpretation on the lesser serologic specificity of this serum compared to that of anti-C. albicans serotype A was due to the smaller size of combining sites for antibodies of the former than of the latter serum.     

Antimicrobial activity of 2-vinylfuran derivatives

2-Vinylfuran derivatives were found to inhibit algal and yeast growth. Experiments with a respiratory type ofSaccharomyces cerevisiae DT XII, its respiration-deficient mutant DT XII A, andCandida albicans showed that all 2-vinylfurans are inhibitors of key processes of energy metabolism (especially glycolysis). The properties determining the inhibitory activity are chemical reactivity and lipophilicity. The reactivity of the studied derivatives was characterized by second-order rate constantsk (L. mol⁻¹.s⁻¹) for reaction with mercaptoacetic acid (as a model thiol), and the lipophilicity by calculated Σπ_i. An equation correlating the structure and the activity of 2-vinylfurans was derived. The significance of reactions of 2-vinylfurans with thiols or other nucleophilic groups of cell components is stressed. The reaction centre of 2-vinylfurans in these reactions is the electrophilic exocyclic double bond. The presence of a nitro group in position 5 of the furan ring is not indispensable for biological activity of 2-vinylfurans.     

Untersuchungen über Antigengemeinschaften zwischen hefeartigen Pilzen

Zusammenfassung An einer willkürlichen Auswahl von Stämmen aus den GeneraCandida, Torulopsis, Saccharomyces undSchizosaccharomyces konnten mit Hilfe der Immunelektrophorese außer bekannten Beziehungen der Polysaccharidantigene noch einige gemeinsame Eiweißantigene nachgewiesen werden.Übereinstimmungen bestehen mitTsuchiya in Bezug auf die enge Verwandtschaft vonC. albicans, C. tropicalis undC. stellatoidea, sowie zwischenS. cerevisiae undC. robusta.Bei Berücksichtigung aller Antigene fanden wir zusätzlich Antigengemeinschaften zwischenT. glabrata undS. rouxii, sowie weitere antigene Beziehungen beiC. krusei, C. utilis, T. famata undS. cerevisiae, die auf gemeinsamen Eiweißantigenen beruhen.Es wurde der Versuch unternommen, einige Eiweißantigene ausC. albicans zu isolieren. Nach Fällung des größten Teils der Antigene mit Universalpuffer verblieben im Rückstand 2 Eiweißantigene, mit denen Kaninchen immunisiert wurden. Von den beiden induzierten Antikörpern gegen Eiweißantigen vonC. albicans reagierte der eine nur mitC. tropicalis, der andere nur mitC. stellatoidea, jedoch nicht mit anderen Hefen.

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Summary The serologic relationships of various yeast strains, arbitrarily selected from the generaCandida, Torulopsis, Saccharomyces, andSchizosaccharomyces, were investigated by means of immunoelectrophoretic technique. In addition to serologic crossings ofC. albicans, C. tropicalis, andC. stellatoidea as well as ofS. cerevisiae, andC. robusta, which had previously been reported byTsuchiya and other investigators, we have found, that protein antigens are shared by various strains, i.e.C. krusei, C. utilis, T. famata, andS. cerevisiae. Taking into account the overall picture of their antigenic patterns,T. glabrata andS. rouxii were found to stand particulary close to each other.Attempts were made to isolate some of these proteide antigens fromC. albicans. After partial precipitation of the crude homogenate by the addition ofJohnson &Lindsay's Universal Buffer followed by centrifugation, the supernatant was used to immunize rabbits. Two types of antibodies could be detected; while both reacted in the immunoelectrophoresis withC. albicans antigens, one also precipitatedC. tropicalis and the otherC. stellatoidea. None of the 10 other yeasts which have been tested showed any activity with these rabbit sera.

     

Different affinities of the α- and β-anomers ofD-glucose,D-mannose andD-xylose for the glucose uptake system of baker’s yeast

A recording technique for measuring the sugar uptake by cell suspensions using a polarimeter is described. The method makes it possible to calculate the uptake rates of the α-and β-anomers. The constitutive monosaccharide transport system ofSaccharomyces cerevisiae andSaccharomyces fragilis exhibits a higher affinity for the α-anomers ofd-glucose,d-manose andd-xylose than for the corresponding β-anomers, this resulting in a preferential uptake of the α-anomers from a mixture. The α-anomer ofd-xylose is preferred both during influx and efflux. The membrane transport ofd-xylose inSaccharomyces cerevisiae is not associated with a change of the anomer configuration. The facilitated diffusion system appears to possess a regulatory role for the utilization ofd-glucose andd-mannose in both yeast species investigated.     

Glycosidases of moulds

Selection of a large number of different strains of hyphal fungi of the genusAspergillus, capable of production of extracellular mannosidase and mannanase type enzymes, was carried out. Before cultivating the strains on liquid synthetic medium containing 0.5%Saccharomyces cerevisiae mannan as the carbon source, they were adapted by multiple passage on solid synthetic media containingd-mannose,d-mannose and α-mannan and lastly only α-mannan. The extracellular enzymatic preparations of the mould fungi were tested for their ability to hydrolyse three different substrates—Saccharomyces cerevisiae, Torulopsis ingeniosa andTorulopsis colliculosa mannan. The production of α-mannosidase was found to be specifically dependent on the character of the substrate used for cultivation of the fungus.     

TheAspergillus nidulans geneschsA andchsD encode chitin synthases which have redundant functions in conidia formation

We previously isolated three chitin synthase genes (chsA, chsB, andchsC) fromAspergillus nidulans. In the present work, we describe the isolation and characterization of another chitin synthase gene, namedchsD, fromA. nidulans. Its deduced amino acid sequence shows 56.7% and 55.9% amino acid identity, respectively, with Cal1 ofSaccharomyces cerevisiae and Chs3 ofCandida albicans. Disruption ofchsD caused no defect in cell growth or morphology during the asexual cycle and caused no decrease in chitin content in hyphae. However, double disruption ofchsA andchsD caused a remarkable decrease in the efficiency of conidia formation, while double disruption ofchsC andchsD caused no defect. Thus it appears thatchsA andchsD serve redundant functions in conidia formation.     

Systematics of the generaDebaryomyces andMetschnikowia: Proton magnetic resonance spectra of their mannans as an aid in classification

The proton magnetic resonance spectra of the mannans of a number ofDebaryomyces andMetschnikowia species (Endomycetales) were determined. The spectra of all of the mannans had several similar characteristics, regardless of the species from which they originated. Pichia vini, a species originally classified asDebaryomyces vini, formed a mannan with a spectrum almost identical with those ofMetschnikowia (Candida) reukaufii andPichia haplophila. Debaryomyces vanrijii, originally placed in the genusPichia, formed a mannan with a spectrum identical with those ofPichia robertsii andCandida (Pichia) guilliermondii mannans.The authors wish to thank Mr. M. Mazurek for the determination of the p.m.r. spectra, and Mr. N. R. Gardner and Mr. R. J. Magus for valuable technical assistance.     

Interactions ofCandida albicans with bacteria and salivary molecules in oral biofilms

The yeastCandida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells.C. albicans andCandida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind toStreptococcus gordonii NCTC 7869 while two otherCandida species (Candida krusei andCandida kefyr) do not. Adherence ofC. albicans was greatest when the yeast had been grown at 30° C to mid-exponential growth phase. For 21 strains ofC. albicans there was a positive correlation between the ability to adhere toS. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence ofC. albicans toS. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins ofC. albicans are co-expressed.     

Restoration of respiratory competence in the yeastCandida utilis after somatic fusion withSaccharomyces cerevisiae

After somatic fusion between a mitochondrial mutant ofCandida utilis andSaccharomyces cerevisiae respiratory competent strain, the complex III of the respiratory chain seems to be restored. Fusion products, FP, synthesizing normal apocytochromeb were recovered and showed normal-shaped mitochondria along the cytoplasm as in theCandida utilis original, respiratory-competent strain.     

Ketoconazole susceptibility of yeasts by the FCST method

The flow cytofluorometric susceptibility test (FCST) was extended fromCandida albicans to additional clinically relevant yeasts.Candida tropicalis, C. parapsilosis, C. lusitaniae, C. krusei, C. guilliermondii, Torulopsis glabrata, andRhodotorula rubra were also amenable to the FCST. Of the most frequently encountered yeasts,C. albicans andC. tropicalis exhibited the E_max response, which is believed to be an in vitro indicator of ketoconazole susceptibility with potential clinical relevance.Torulopsis glabrata, for which ketoconazole therapy is less effective, did not exhibit the E_max response; rather it exhibited an MIC effect, but usually at ketoconazole concentrations greater than those therapeutically achievable.     

Ultrastructure of chitin in hyphae ofCandida albicans and other dimorphic and mycelial fungi

Summary Chitin microfibrils exposed by chemical extraction of hyphal walls ofCandida albicans, Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidiodes brasiliensis, Coprinus cinereus andMucor mucedo were of variable morphology but gave identical infrared spectra and behaved as pure chitin in chromatographic analyses. The microfibrils of the four dimorphic fungi studied were shorter than those in the mouldsC. cinereus andM. mucedo but were similar to those reported for the yeastSaccharomyces cerevisiae. InC. albicans the microfibrils in the septal plates of hyphae were predominantly tangentially orientated and were longer than those in the lateral walls. Microfibrils produced by chitin synthasein vitro were very much longer than any observed from hyphal preparations.     

Extracellular polysaccharide-protein complexes produced by selected strains ofCandida albicans (Robin) Berkhout

Water-soluble extracellular polysaccharide-protein complexes of a molecular weight of about 200,000 were isolated from glucose-containing nutrient media after cultivation of slightly, moderately and highly virulent strains ofCandida albicans (Robin) Berkhout. Chemical, physico-chemical and immunological properties of these compounds were studied. The complexes contain 74–86% of mannose, 21–31% of glucose, 1–1.5% of glucosamine and 11–14% of proteins built from 17 aminoacids. The polysaccharideprotein complexes are immunologically active and differ from each other by their precipitation ability if added to specific antisera prepared against moderately highly virulentCandida albicans strains.     

In-vitro susceptibility of Candida albicans and Candida stellatoidea to sulfamethoxazole

The susceptibility of organisms of the speciesCandida to sulfamethoxazole were tested in-vitro using the disc sensitivity method.Under specified conditions a zone of growth inhibition surrounding sulfamethoxazole tablet sensi disc was consistently produced in cultures ofCandida albicans, Candida stellatoidea andSaccharomyces cervisiae.Diameters of growth inhibition zone were measured and found to be greatest in cultures ofCandida stellatoidea andSaccharomyces, but growth inhibition inCandida albicans cultures was observed to a lesser degree.The correlation between in vivo pathogenicity of organisms of the speciesCandida, and degree of growth inhibition by sulfamethoxazole sensi disc presents an interesting relationship.     

Glycine oxidation and conversion into amino acids in Saccharomyces cerevisiae and Candida albicans

Humans are exposed much more often to exogenous Saccharomyces cerevisiae (a baker’s yeast) than exogenous Candida albicans (a highly infectious yeast) but suffer no apparent complications from S. cerevisiae. We hypothesize that variations in characteristics between these two species may be due, in part, to differences in glycine metabolism. In this study, we examined differences in glycine oxidation between C. albicans and S. cerevisiae. Both C. albicans and S. cerevisiae were cultured in glycine enriched media, followed by determination of glycine oxidation and amino acid concentrations in cells. Glycine was degraded to a much greater extent in C. albicans than in S. cerevisiae. Threonine concentrations and glycine oxidation were also elevated in C. albicans. Almost all of the disappearance of glycine from incubation media was accounted for by the formation of serine, threonine, and CO₂ in S. cerevisiae, whereas these products represented only 50% of the metabolized glycine in C. albicans. The unidentified metabolites of glycine in C. albicans, presumably purines, could contribute to its infectious capacity and this warrants further study.