The pathogenesis and host defence mechanisms of oral candidosis.
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Candidiasis, Chronic Mucocutaneous↗
Biomedical subjects
M G Shepherd
Publications and source records attributed to M G Shepherd.
The proteins of dental enamel.
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Amelogenesis↗
The rational management of oral candidosis associated with dentures.
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Antifungal Agents↗
The kinetics and divalent cation inhibition of plasma membrane ATPase in the yeast Candida albicans.
The kinetics of ATP hydrolysis and cation effects on ATPase activity in plasma membrane from Candida albicans ATCC 10261 yeast cells were investigated. The ATPase showed classical Michaelis-Menten kinetics for the hydrolysis of Mg X ATP, with Km = 4.8 mM Mg X ATP. Na+ and K+ stimulated the ATPase slightly (9% at 20 mM). Divalent cations in combination with ATP gave lower ATPase activity than Mg X ATP (Mg greater than Mn greater than Co greater than Zn greater than Ni greater than Ca). Divalent cations inhibited the Mg X ATPase (Zn greater than Ni greater than Co greater than Ca greater than Mn). Free Mg2+ inhibited Mg X ATPase weakly (20% inhibition at 10 mM). Computed analyses of substrate concentrations showed that free Zn2+ inhibited Zn X ATPase, mixed (Zn2+ + Mg2+) X ATPase, and Mg X ATPase activities. Zn X ATP showed high affinity for ATPase (Km = 1.0 mM Zn X ATP) but lower turnover (52%) relative to Mg X ATP. Inhibition of Mg X ATPase by (free) Zn2+ was noncompetitive, Ki = 90 microM Zn2+. The existence of a divalent cation inhibitory site on the plasma membrane Mg X ATPase is proposed.
Adenosine Triphosphatases↗
The inactivation of ornithine transcarbamoylase by N delta-(N'-sulpho-diaminophosphinyl)-L-ornithine.
Phaseolotoxin, a tripeptide inhibitor of ornithine transcarbamoylase, is a phytotoxin produced by Pseudomonas syringae pv. phaseolicola, the causal agent of halo-blight in beans. In vivo the toxin is cleaved to release N delta-(N'-sulpho-diaminophosphinyl)-L-ornithine, the major toxic chemical species present in diseased leaf tissue. This paper reports on the interaction between N delta-(N'-sulpho-diaminophosphinyl)-L-ornithine and ornithine transcarbamoylase. N delta-(N'-Sulpho-diaminophosphinyl)-L-ornithine was found to be a potent inactivator of the enzyme, in contrast with phaseolotoxin, which previously has been reported to inhibit the enzyme reversibly. Inactivation by N delta-(N'-[35S]sulpho-diaminophosphinyl)-L-ornithine resulted in the incorporation of 35S into ethanol-precipitated protein. The stoicheiometry of 35S incorporation was approximately 1 mol/mol of active sites. Inactivation was second-order and a rate constant of 10(6) M-1 X s-1 at 0 degree C in 50 mM-Tris/HCl, pH 9.0, was obtained. Carbamoyl phosphate, a substrate of ornithine transcarbamoylase, protected the enzyme from inactivation. A dissociation constant of 3 microM for the enzyme-carbamoyl phosphate complex was calculated. L-Ornithine, the second substrate for ornithine transcarbamoylase, protected the enzyme only at high concentrations. The results are consistent with N delta-(N'-sulpho-diaminophosphinyl)-L-ornithine being a potent affinity label that binds via the carbamoyl phosphate-binding site of ornithine transcarbamoylase. Cleavage of phaseolotoxin to N delta-(N'-sulpho-diaminophosphinyl)-L-ornithine in vivo appears to be an important function in the physiology of the disease.
Binding Sites↗
Pathogenicity of morphological and auxotrophic mutants of Candida albicans in experimental infections.
The relative pathogenicities of yeast and mycelial forms of Candida albicans were determined after intravenous injection of the two forms into mice. Yeast and mycelial forms of C. albicans CMI45348 were prepared in chemostat culture. Both morphological forms were pathogenic, but the histology of kidney sections always showed a mixture of the yeast and mycelial elements of the organism. Similarly, infection of mice with prototrophic strains produced a mixture of morphological forms at the site of infection. The yeast (CA2) and mycelial (hOG301) morphological mutants of C. albicans were pathogenic, and sections from the kidneys of the infected mice showed that the mutants retained their original morphological forms. These data indicate that both the yeast and mycelial forms of C. albicans can adhere, invade, and proliferate in an infected host. Auxotrophic diploid mutants were nonpathogenic. However, construction of a prototrophic tetraploid strain from two auxotrophs restored the pathogenicity of the organism.
Animals↗
Characterization of a tetraploid derivative of Candida albicans ATCC 10261.
A morphometric analysis of Candida albicans yeast cells utilizing scanning electron microscopy showed that the cell volume and the DNA content of a tetraploid strain (derived by cell fusion) were 2.4 to 3.0 and 2.0 times, respectively, those of the progenitor diploid strain, ATCC 10261. The pathogenicities of both strains were similar.
Candida albicans↗
Morphological studies of N-acetylglucosamine induced germ tube formation by Candida albicans.
In N-acetylglucosamine induced germ tube formation by Candida albicans, multiple (up to five) protuberances appeared within 90 min at 37 degrees C on each yeast cell. The protuberances were extensions of the cytosol and contained vesiclelike structures. Usually only one protuberance subsequently developed into a germ tube. The germ tubes emanated from all aspects of the cell surface but seldom from the budding (long axis) poles. Pseudohyphae, which originated from the budding pole, exhibited a marked constriction at the site of emergence and were 0.6-2.5 microns in diameter compared with a diameter of 0.6-0.8 micron for germ tubes. The presence of septa confirmed that germ tubes are precursors of septate mycelia. Ultrathin-section transmission electron microscopy of aldehyde plus osmium fixed cells revealed electron-lucent walls with a thin electron-dense outer layer. A fibrillar border was also routinely associated with germ tubes. Poststaining with potassium permanganate revealed, in addition, a previously invisible fuzzy layer on the outer region of the cell wall which extended over bud scars and germ tubes and which coalesced at sites of contact between cells.
Acetylglucosamine↗
Candida albicans: biology, genetics, and pathogenicity.
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Adhesiveness↗
N-acetyl-D-glucosamine-induced morphogenesis in Candida albicans.
N-acetylglucosamine is a morphogenic effector in the human pathogenic yeast Candida albicans. Depending on temperature, N-acetylglucosamine induces yeast-mycelial conversion or chlamydospore formation. N-acetylglucosamine is also a carbon source for growth in the yeast form. Germ-tube formation, i.e. the intermediary of yeast-mycelial conversion, is induced at temperatures in excess of 33 degrees C; at lower temperatures the yeast or the pseudomycelial form of the organism predominates. 2-Deoxyglucose, at concentrations which do not affect yeast growth, is a potent inhibitor of N-acetylglucosamine-induced germ-tube formation. N-acetylglucosamine suffices as both the inducer and the carbon sources for morphogenesis and both transcription and translation are required for the yeast to mycelial transition. The metabolism of N-acetylglucosamine is essentially the same for yeast phase cells (28 degrees C) and germ-tube forming cells (37 degrees C): enzymes for N-acetylglucosamine uptake and catabolism are equally well induced by gene expression at 28 degrees C and 37 degrees C. During germ-tube formation, the chitin content and the activity of the regulatory enzyme chitin synthase increase. Germ-tube formation in C. albicans can also be induced gratuitously by a number of N-acetylhexosamine derivatives (N-acetylglucosamine covalently linked to agarose, N-acetylmannosamine, hyaluronic acid, colloidal chitin, and mucin). These compounds are not taken up by the yeast cells and do not support growth which suggests that germ-tube formation is triggered by a cell-surface receptor mechanism. It is proposed that, after binding to the receptor, N-acetylglucosamine produces an intracellular message which primes the cell for morphogenesis. This message would ultimately be responsible for the choice of the mode of growth, spherical versus apical, that is characteristic of yeast or mycelial form.
Acetylglucosamine↗
The inhibition of ornithine transcarbamoylase from Escherichia coli W by phaseolotoxin.
The mechanism of inhibition of ornithine transcarbamoylase by the bacterial toxin phaseolotoxin [N-delta-(phosphosulphamyl)ornithylalanylhomoarginine] was investigated. Ornithine transcarbamoylase was purified by affinity chromatography from Escherichia coli W argR- by using N-delta-(phosphonoacetyl)ornithine as the ligand. Under steady-state conditions phaseolotoxin inhibition was reversible and exhibited mixed kinetics with respect to carbamoyl phosphate. The apparent Ki and apparent K'i were 0.2 microM and 10 microM respectively. Inhibition with respect to ornithine was noncompetitive, with an apparent Ki of 0.9 microM. These data are consistent with competitive binding of phaseolotoxin to the carbamoyl phosphate-binding site of the enzyme. The toxin also appears to be able to bind to the enzyme-carbamoyl phosphate complex, although, since K'i is 50 times greater than Ki, this event is kinetically much less significant. In the presence of phaseolotoxin ornithine transcarbamoylase exhibited a transient phase of activity before a steady state. This is consistent with low rates of association and dissociation for the toxin with enzyme and the enzyme-toxin complex. Rate constants of 2.5 X 10(4)M-1 X s-1 and 5 X 10(-3)s-1 were estimated for the association and dissociation constants respectively.
Amino Acids↗
Purification and properties of a beta-1,6-glucanase from Penicillium brefeldianum.
An inducible endo-beta-1,6-glucanase was purified from Penicillium brefeldianum by DEAE-cellulose, Bio-Gel P-150 and high-pressure liquid chromatography. The final preparation was essentially free from beta-1,3-glucanase and beta-glucosidase activities. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed one protein band with an Mr of 44000. The Vmax. and Km values were calculated to be 624 units (mumol/min)/mg and 2.78 mg/ml respectively. The glucanase had lytic activity against mycelial cells of the yeast Candida albicans. The yield of purified beta-1,6-glucanase from 100 mg dry weight of freeze-dried culture filtrate varied from 60 to 180 units.
Chromatography, DEAE-Cellulose↗
Analysis of wall glucans from yeast, hyphal and germ-tube forming cells of Candida albicans.
Acid-soluble and alkali-insoluble glucan fractions were prepared from yeast, hyphal and germ-tube forming cells of Candida albicans. Alkali-insoluble glucan was also extracted from purified yeast cell walls. Paper chromatography of partial acid hydrolysates confirmed that the glucan preparations contained beta(1----3)- and beta(1----6)-chains but no mixed intra-chain beta(1----3)/(1----6) linkages. Methylation and 13C-NMR analyses showed that the acid-soluble glucan consisted of a highly branched polymer composed mainly (67.0% to 76.6%) of beta(1----6)-linked glucose residues. The alkali-insoluble glucan from yeast and hyphal cells contained from 29.6% to 38.9% beta(1----3) and 43.3% to 53.2% beta(1----6) linkages. Alkali-insoluble glucan from germ-tube forming cells consisted of 67.0% beta(1----3) and 14% beta(1----6) linkages. Branch points accounted for 6.7%, 12.3% and 17.4% of the residues in the alkali-insoluble glucan of yeast, germ-tube forming and hyphal cells, respectively.
Candida albicans↗
Metabolism of [14C]glucose by regenerating spheroplasts of Candida albicans.
Spheroplasts of Candida albicans were regenerated in [14C]glucose and buffered magnesium sulphate (0.1 M-Tris/HCl; 0.5 M-MgSO4, pH 7.2) at 35 degrees C. Uptake of glucose by spheroplasts was faster than that by intact yeast cells. After 6 h, 65% of the glucose taken up by the yeast appeared as CO2 and 30% was incorporated into the cellular material. With spheroplasts, 55% of the glucose taken up was expired as CO2, 25% was excreted into the medium as other metabolites and 20% was incorporated into the cells. The regenerating spheroplasts excreted 14C-labelled carbohydrates into the medium which were fractionated on a Sephadex G-15 column. Acid hydrolysis of the low molecular-weight fraction yielded the following sugars: mannose (75.7%), fucose (3.8%), arabinose (3%), galactose (2.1%) and an unidentified monosaccharide (14%). Spheroplasts did not incorporate mannoprotein into the regenerated wall. The wall carbohydrate from regenerated spheroplasts was fractionated on the basis of solubility in sodium hydroxide. The alkali-insoluble fraction was analysed by sequential enzyme hydrolysis; 40% of the incorporated counts were associated with beta (1----3)-linked glucan and 50% with a mixed glucan comprising beta (1----3)- and beta (1----6)-linkages and chitin.
Candida albicans↗
Isolation and structure of glucan from regenerating spheroplasts of Candida albicans.
Regenerating spheroplasts of Candida albicans formed organized glucan nets in liquid culture. The nets consisted of interwoven microfibrils about 50 nm wide, but of an undetermined length. Partial acid hydrolysis of the polysaccharide showed the presence of chains of beta(1----3)- and beta(1----6)-linked glucose residues, but no intrachain beta(1----3) and beta(1----6) linkages. Periodate oxidation and GLC of the methylated glucan indicated a highly branched polymer (9.5% branch points). Sequential enzymic degradation of the isolated nets confirmed the presence of chains of beta(1----3)- and beta(1----6)-linked glucose residues. Degradation by (1----3)-beta- and (1----6)-beta-glucanase released 23% (w/w) and 30% (w/w) respectively of the carbohydrate as glucose equivalents. The residual material was degraded by chitinase. Equal amounts of N-acetylglucosamine and glucose equivalents were detected in the chitinase hydrolysate, suggesting a possible linkage between glucan and chitin. Our data indicate that the cell wall of C. albicans contains at least two highly branched glucans with predominantly beta(1----3) or beta(1----6) linkages.
Candida albicans↗
Exo-(1----3)-beta-glucanase, autolysin and trehalase activities during yeast growth and germ-tube formation in Candida albicans.
Exo-(1----3)-beta-glucanase, beta-glucosidase, autolysin and trehalase were assayed in situ in Candida albicans during yeast growth, starvation and germ-tube formation. Cell viability, germ-tube formation, intracellular glucose-6-phosphate dehydrogenase and beta-glucosidase were unaffected in cells incubated in 0.1 M-HC1 for 15 min at 4 degrees C. However, in situ trehalase, (1----3)-beta-glucanase and autolysin activities in acid-treated cells decreased by 95, 50 and 35% respectively, indicating that these enzymes are, in part, associated with the cell envelope. Trehalase activity increased throughout yeast growth and remained elevated during the first hour of incubation for germ-tube formation. All of the in situ trehalase activity in starved yeast cells could be measured without the permeabilizing treatment. beta-Glucosidase activity declined throughout yeast growth and did not alter during germ-tube formation. Both the (1----3)-beta-glucanase and autolysin activities were optimal at pH 5 X 6, inhibited by gluconolactone and HgCl2, and maximal at 15-16 h during yeast growth. Although autolysin activity increased by 50-100% when starved yeast cells were incubated for germ-tube formation, the in situ (1----3)-beta-glucanase remained constant. When acid-treated starved yeast cells were similarly induced, in situ (1----3)-beta-glucanase increased 100% over 3 h of germ-tube formation. Yeast cells secreted (1----3)-beta-glucanase into the growth medium. This was highest in early exponential phase cultures (34% of the maximum in situ activity) and declined throughout growth. (1----3)-beta-Glucanase was also secreted into the medium during germ-tube formation and this represented 80-100% of the in situ activity in germ-tube forming cells. Both secretion of (1----3)-beta-glucanase and germ-tube formation were inhibited by 2-deoxyglucose, ethidium bromide, trichodermin and azaserine.
Amidohydrolases↗
Magnesium and the regulation of germ-tube formation in Candida albicans.
Candida albicans requires Mg2+ for germ-tube formation. Mg-deficient media, metal ion chelators and the ionophore A23187 inhibited germ-tube formation. Cell Mg content during exponential yeast-phase growth remained constant but increased throughout germ-tube formation. The onset of germ-tube formation coincided with a sharp peak in Mg concentration within the cells. Yeast-phase cells of strain CA2, which did not form germ-tubes, had a lower Mg content and failed to accumulate Mg when incubated under conditions for germ-tube formation. Mg also increased the uptake and incorporation of N-acetylglucosamine. These findings point to a central regulatory role for Mg in C. albicans morphogenesis.
Acetylglucosamine↗
The secretion of N-acetylglucosaminidase during germ-tube formation in Candida albicans.
N-Acetylglucosaminidase was induced by either N-acetylglucosamine or N-acetylmannosamine in several strains of Candida albicans. Enzyme activity was not induced in a N-acetylglucosamine non-utilizing mutant which is unable to express the first three steps in the N-acetylglucosamine catabolic pathway. The enzyme, purified 500-fold, had a specific activity of 36.8 units (mg protein)-1 and catalysed the hydrolysis of p-nitrophenyl-beta-n-acetylglucosamine, N,N'-diacetylchitobiose and N,N',N"-triacetylchitotriose. No activity was observed toward colloidal chitin, hyaluronic acid or mucin. The cellular distribution of N-acetylglucosaminidase was determined by measuring in situ enzyme activity before and after acid treatment of intact cells. N-Acetylglucosaminidase (80-88% of the total cellular activity) was rapidly secreted to the periplasm when the enzyme was induced either during yeast growth at 28 degrees C or germ-tube formation at 37 degrees C. Export of the enzyme from the periplasm into the medium was fourfold greater during germ-tube formation, and after 6 h incubation the amount of enzyme released into the medium represented 70% of cell-associated enzyme activity.
Acetylglucosamine↗