Structure and biological activities of beta-glucans from yeast and mycelial forms of Candida albicans

We have achieved the extraction of cell wall beta-glucan from the mycelial form of Candida albicans (C. albicans) IFO 0579 (M-CSBG) by using acetic acid, sodium hypochlorite (NaClO), and dimethylsulfoxide (DMSO) treatments. The yield of M-CSBG was significantly lower (7.5% from dried mycelial cells) than that of the yeast form from C. albicans IFO 1385 (Y-CSBG, 25.9% from dried yeast cells). The properties of M-CSBG were similar to those of Y-CSBG in terms of nuclear magnetic resonance (NMR) spectra and limulus reactivity. Molecular weight (Mw) of M-CSBG was slightly higher than that of Y-CSBG. Both Y-CSBG and M-CSBG induced the production of comparable amounts of macrophage inflammatory protein-2 (MIP-2), a chemotactic factor, from mouse peritoneal exudate cells (PEC) in vitro. These findings suggest that the structure and properties of CSBG from yeast and mycelial cells are similar to each other.     

Different levels of DNA methylation in yeast and mycelial forms of Candida albicans.

Isotope dilution gas chromatography-mass spectrometry analysis of genomic DNAs isolated from three Candida albicans isolates showed significant differences in the amounts of 5-methyldeoxycytidine (m5Cyt) in DNA from yeast-form and from mycelial-form cells; the moles percent m5Cyt were 0.11, 0.11, and 0.097 for yeast-form DNA and 0.045, 0.053, and 0.047 for mycelial-form DNA for the three isolates Sh8, 9938, and B311, respectively. The lower m5Cyt values for mycelial-form cells suggest that those cells may exhibit significantly greater gene activity than yeast-form cells.     

Comparative activities of glycolytic enzymes in yeast and mycelial forms of Candida albicans

Through use of a synthetic defined medium which allows for the exclusive growth of yeast or mycelial forms of Candida albicans the activity of several major glycolytic enzymes in these forms were examined and compared. The results indicate vast metabolic differences between the forms. These data are discussed in relationship to the phenomenon of morphogenesis in C. albicans which in turn relates to problems in immunology and pathogenics of this important opportunistic organism.     

Immune responses to yeast and mycelial forms of Candida albicans in intraperitoneally infected mice

Candida albicans E-139 produced pure mycelial and yeast cultures in a low sulphate medium at different temperatures. The influence of the morphological phase, dose and viability of the fungi on the kinetic of delayed-type hypersensitivity (DTH) and anti-mycelial and anti-yeast antibodies have been studied in mice injected intraperitoneally. The mycelial form elicited higher DTH levels than the yeast phase. This effect seems to be related to its antigenic properties. The effect of dose on the immune response depends on the viability of the fungus. The mycelial cytoplasmic antigens were more effective than the yeast ones in detecting antibodies induced during the experiments, particularly during the later stages of the observation periods, suggesting that such antigens may be useful in the serodiagnosis of Candida infections.     

Growth inhibitory effect of antibiotic tetaine on yeast and mycelial forms of Candida albicans

The mycelial (M) form of Candida albicans is more sensitive to the action of the antibiotic tetaine than the yeast (Y) form. Tetaine, at low concentrations about 1 microgram/ml also inhibits Y-M transition. It causes severe deformation of cells, agglutination and inhibits septum formation in the yeast forms. Tetaine action is reversed by dipeptides in both forms and by tripeptides in M form. N-acetyl glucosamine is a powerful antagonist of tetaine action on both morphological forms. Tetaine action on mycelial forms is slightly antagonised by N-acetyl mannosamine and very powerfully by glutamine.     

The production and growth characteristics of yeast and mycelial forms of Candida albicans in continuous culture.

The growth characteristics of Candida albicans CM145,348 have been examined under aerobic conditions in continuous culture. At different steady states the environment was controlled with respect to the concentrations of dissolved oxygen, carbon and nitrogen, the pH, and the temperature. Dry matter, substrate concentration, yield, specific oxygen uptake, specific carbon dioxide release and respiration quotient were examined as a function of the dilution rate. The morphology depended on the carbon source. Maltose produced a mycelial morphology, whereas with lactate a yeast culture was obtained. With fructose or glucose as a carbon source a mixed morphology of yeast, pseudo-mycelial and mycelial forms was produced. A larger number of different growth conditions were examined in batch culture but a mixed morphology was always obtained.     

An effective medium for the selective growth of yeast or mycelial forms of Candida albicans: Biochemical aspects of the two forms

A new synthetic medium, based on a modification of a commercially available tissue culture medium, allows Candida albicans to be grown in the yeast or mycelial form. Salient features of the system are described and comparisons with previous physiological investigations are discussed. A concise biochemical profile of these two forms of C. albicans is also presented. The results indicate vast metabolic differences between the two forms.     

Chitin synthesis in Candida albicans: comparison of yeast and hyphal forms.

Chitin synthesis was studied in both yeast and hyphae of the dimorphic fungus Candida albicans. Incorporation of N-acetyl-d-[1-(3)H]glucosamine ([(3)H]GluNAc) into an acid-alkali-insoluble fraction was 10 times greater in hyphal-phase cells. A crude preparation of chitin synthetase was obtained from sonically treated protoplasts of both forms of Candida. Enzyme activity, which was determined by using [(14)C]UDP-GLuNAc as a substrate, was exclusively associated with the 80,000 x g pellet from sonically treated protoplasts of both forms. It was determined that enzyme activity (nanomoles of [(14)C]UDP-GluNAc incorporated per milligram of protein) was approximately 2 times greater in hyphae versus yeast cells. Enzyme activity in both yeast and hyphae increased six- to sevenfold when the enzyme preparations were preincubated with trypsin. A vacuolar fraction, obtained from yeast cells but not from hyphae, stimulated enzyme activity when incubated with either yeast or hyphal enzyme preparations. Membrane fractions from protoplasts coated with [(3)H]concanavalin A before disruption were isolated by Renografin density gradient centrifugation. Chitin synthetase activity was preferentially associated with the concanavalin A-labeled fraction, suggesting that the enzyme was located on the plasma membrane. In addition, enzyme activity in protoplasts treated with cold glutaraldehyde before disruption was significantly greater than in protoplasts that were sonically disrupted and then treated with cold glutaraldehyde, indicating that the enzyme resides on the inner side of the plasma membrane.     

Incorporation of specific wall proteins during yeast and mycelial protoplast regeneration in Candida albicans

The kinectics of incorporation of two precursor mannoproteins into the regenerating cell wall of Candida albicans protoplasts have been followed at 28°C and 37°C using two monoclonal antibodies specific for protein epitopes (MAb 1B12 and 4C12) as probes. Both molecules were secreted from the beginning of the regeneration process, and their incorporation was retarded significantly. Analysis of the secreted materials by Western immunoblotting with MAb 1B12 allowed the identification of two closely migrating bands at apparent Mr higher than 170 kDa and significant amounts of a highly polydisperse material of even greater molecular mass. Some of these mannoproteinaceous species carried both N- and O-glycosidically linked mannose residues, as deduced from their drop in apparent Mr when synthesized in the presence of tunicamycin and by their reactivity with Concanavalin A. Following secretion, the molecules reacting with MAb 1B12 were incorporated into the regenerating walls by covalent binding. Then, when the regenerating walls by covalent binding. Then, when the antigen molecules were solubilized from partially regenerated walls, their mobility differed when regeneration took place at 28°C (blastoconidia) or 37°C (mycelial cells).Abbreviations used IIF indirect immunofluorescence - MAb monoclonal antibody - PBS phosphate-buffed saline - PMSF phenyl methyl sulfonyl fluoride - SDS sodium dodecyl sulfate     

Killing of yeast, germ-tube and mycelial forms of Candida albicans by murine effectors as measured by a radiolabel release microassay

Candida albicans undergoes yeast to mycelial conversion under both in vivo and in vitro conditions but the relative pathogenicity of the two forms of growth is still unknown. By adapting a recently developed 51Cr radiolabel release assay, we have quantified the killing ability of different murine effector cell populations for the hyphal form of C. albicans. Up to 50% of specific 51Cr release from the mycelial form could be detected after incubation for only 1 h, with no requirement for opsonization, provided that appropriate effector: target cell ratios were used. The specific 51Cr release correlated well with viability, as assessed by dye exclusion tests, and with pathogenicity potential in cyclophosphamide-immunodepressed mice. Comparison of the activity of different murine effectors against yeast and hyphal forms showed that hyphal forms were killed by murine effectors to a similar, if not greater, extent than yeast forms. In particular, thioglycollate-induced murine polymorphonuclear neutrophils were able to kill hyphal cells extracellularly and without an opsonic requirement.     

The isolation of plasma membrane and characterisation of the plasma membrane ATPase from the yeast Candida albicans

Plasma membrane ghosts were isolated from Candida albicans ATCC 10261 yeast cells following stabilisation of spheroplasts with concanavalin A, osmotic lysis and Percoll density gradient centrifugation. Removal of extrinsic proteins with NaCl and methyl alpha-mannoside gave increased ATPase and chitin synthase specific activities in the resultant plasma membrane fraction. Sonication of this fraction yielded unilamellar plasma membrane vesicles which exhibited ATPase and chitin synthase specific activities of 4.5-fold and 3.0-fold, respectively, over those of the plasma membrane ghosts. ATPase activity in the membrane ghosts was optimal at pH 6.4, showed high substrate specificity (for Mg X ATP) and was inhibited 80% by sodium vanadate but less than 4% by oligomycin and azide. The effects of a range of other inhibitors were also characterised. Temperature effects of ATPase activity were marked, with a maximum at 35 degrees C. Breaks in the Arrhenius plot, at 12.2 degrees C and 28.9 degrees C, coincided with endothermic heat flow peaks detected by differential scanning calorimetry. ATPase was solubilised from the plasma membranes with Zwittergent in the presence of glycerol and phenylmethylsulphonyl fluoride and partially purified by glycerol density gradient centrifugation. The solubilised enzyme hydrolysed Mg X ATP at Vmax = 20 mumol X min-1 X mg-1 in the presence of phospholipids, with optimal activity at pH 6.0--6.5.     

The detection and analysis of chitinase activity from the yeast form of Candida albicans

Chitinase activity was detected in the supernatant fraction of a high-speed centrifugation preparation of broken Candida albicans yeast cells. The enzyme showed peak activity during the rapid budding phase of growth and was found to parallel the chitin synthase activity. The optimum conditions for the hydrolysis of chitin, regenerated from acetylation of chitosan, were determined. Analysis of the kinetics of the enzyme-substrate interaction and a measurement of their binding suggests that an equilibrium binding situation exists and that the kinetics follow a Langmuir isotherm interaction.     

Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms

Neutrophils phagocytose and kill microbes upon phagolysosomal fusion. Recently we found that activated neutrophils form extracellular fibres that consist of granule proteins and chromatin. These neutrophil extracellular traps (NETs) degrade virulence factors and kill Gram positive and negative bacteria. Here we show for the first time that Candida albicans, a eukaryotic pathogen, induces NET-formation and is susceptible to NET-mediated killing. C. albicans is the predominant aetiologic agent of fungal infections in humans, particularly in immunocompromised hosts. One major virulence trait of C. albicans is its ability to reversibly switch from singular budding cells to filamentous hyphae. We demonstrate that NETs kill both yeast-form and hyphal cells, and that granule components mediate fungal killing. Taken together our data indicate that neutrophils trap and kill ascomycetous yeasts by forming NETs.     

Morphological specificity of yeast and filamentous Candida albicans forms on surface properties

Physicochemical surface properties of Candida albicans were assessed from microbial adhesion to human epithelial cells and to octane droplets. The adherence of cells demonstrated the occurrence of morphological specificity for these adhesion assays. Filamentous forms exhibited adherence third times higher compared to budding forms, while their electrophoretic mobilities were comparable. Force measurements performed on filamentous form by AFM demonstrated that such adhesion was associated with microfibrillar surface structure.     

Characterization of phosphomannan-protein complexes isolated from viable cells of yeast and mycelial forms of Candida albicans NIH B-792 strain by the action of Zymolyase-100T

The isolation of phosphomannan-protein complexes from the viable cells of yeast (Y) and mycelial (M) forms of Candida albicans NIH B-792 strain was conducted by treatment with Zymolyase-100T followed by fractional precipitation with cetyltrimethylammonium bromide. The M-form complex was found to contain smaller amount of phosphate (1.3%) than that of the Y-form complex (1.6%). Proton magnetic resonance (PMR) spectra of these complexes indicated that the content of beta-1,2-linked oligomannosyl and nonreducing terminal alpha-1,3-linked mannopyranosyl residues in the M-form complex was lower than that of the Y-form complex. With hot 10 mM HCl, the Y-form complex released a mixture of oligosaccharides ranging from mannose to mannoheptaose, while the M-form complex produced lower oligosaccharides, from mannose to mannotetraose. Upon acetolysis, the acid-modified complex of the M form gave mainly mannotetraose, while that of the Y form produced mainly mannopentaose and mannohexaose in addition to mannotetraose. The average length of branching moieties of the mannan of Y-form cells was therefore longer than that of M-form cells. These results indicate that the Y to M transformation of this C. albicans strain accompanies the suppression of enzyme activity concerning the biosynthesis of mannan such as beta-1,2- and alpha-1,3-mannosyltransferases to synthesize the phosphomannan-protein complex containing mannan moiety with incomplete structure.     

Dimorphism in Candida albicans: contribution of mannoproteins to the architecture of yeast and mycelial cell walls

Wall mannoproteins of the two (yeast and mycelial) cellular forms of Candida albicans were solubilized by different agents. Boiling in 2% (w/v) SDS was the best method, as more than 70% of the total mannoprotein was extracted. Over 40 different bands (from 15 to 80 kDal) were detected on SDS-polyacrylamide gel electrophoresis of this material. The residual wall mannoproteins were released after enzymic (Zymolyase and endogenous wall beta-glucanases) degradation of wall glucan, suggesting that they are covalently linked to this structural polymer. Four bands (of 160 kDal, 205 kDal and higher molecular mass) were observed in the material released from yeast walls but only the two smaller components were detected in the material obtained from mycelial walls. Moreover, the mannoproteins of high molecular mass, which are covalently linked in walls of normal cells, were not incorporated into walls of regenerating protoplasts, but non-covalently linked mannoproteins were retained from the beginning of the process.