Binding of plasma proteins to Candida species in vitro

The ability of purified human albumin, fibrinogen and transferrin to bind to Candida species was measured by immunofluorescence. The proteins all bound with high avidity to germ-tubes formed by Candida albicans, but did not bind to blastospores of C. albicans or other pathogenic Candida species, not even to parent blastospores bearing germ-tubes. The extent of binding of the proteins to C. albicans germ-tubes varied between growth media and from germ-tube to germ-tube. Strains of C. albicans that did not form germ-tubes were incapable of binding any of the proteins. There was evidence that purified fibrinogen bound to germ-tubes with higher avidity than albumin and transferrin. When germ-tubes were treated with whole human plasma or serum, indirect immunofluorescence revealed that proteins were bound all over the surface of C. albicans blastospore-germ-tube units, indicating behaviour different from that seen with the purified proteins tested alone or in mixtures. C. albicans cells grown in the presence of azole antifungal agents bound purified plasma proteins in the same way as cells untreated with the drugs. The results of this study suggest that binding of host proteins to the surface of C. albicans may not be a property related directly to virulence of the fungus in vivo.     

Binding of mouse fibrinogen to Candidaalbicans in vivo

Binding of fibrinogen to various Candida albicans strains has been investigated by immunofluorescence microscopy on kidney sections of experimentally infected mice. Fibrinogen appeared to bind to both mycelium and blastospores in situ whereas previous studies, carried out in vitro, have shown fibrinogen binding to mycelial elements only.     

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.     

Regulation of chitin synthesis during germ-tube formation in Candida albicans

The synthesis of chitin during germ-tube formation in Candida albicans may be regulated by the first and last steps in the chitin pathway: namely l-glutamine-d-fructose-6-phosphate aminotransferase and chitin synthase. Induction of germ-tube formation with either glucose and glutamine or serum was accompanied by a 4-fold increase in the specific activity of the aminotransferase. Chitin synthase in C. albicans is synthesized as a proenzyme. N-acetyl glucosamine increased the enzymic activity of the activated enzyme 3-fold and the enzyme exhibited positive co-operativity with the substrate, UDP-N-acetylglucosamine. Although chitin synthase was inhibited by polyoxin D (K _i =1.2M) this antibiotic did not affect germination. During germ-tube formation the total chitin synthase activity increased 1.4-fold and the expressed activity (in vivo activated proenzyme) increased 5-fold. These results could account for the reported 5-fold increase in chitin content observed during the yeast to mycelial transformation.Non-Standard Abbreviations GlcNac N-acetyl glucosamine - UDP-GlcNac UDP-N-acetyl glucosamine - PMSF phenylmethylsulphonylfluoride     

Human antimicrobial peptide LL-37 inhibits adhesion of Candida albicans by interacting with yeast cell-wall carbohydrates

Candida albicans is the major fungal pathogen of humans. Fungal adhesion to host cells is the first step of mucosal infiltration. Antimicrobial peptides play important roles in the initial mucosal defense against C. albicans infection. LL-37 is the only member of the human cathelicidin family of antimicrobial peptides and is commonly expressed in various tissues and cells, including epithelial cells of both the oral cavity and urogenital tract. We found that, at sufficiently low concentrations that do not kill the fungus, LL-37 was still able to reduce C. albicans infectivity by inhibiting C. albicans adhesion to plastic surfaces, oral epidermoid OECM-1 cells, and urinary bladders of female BALB/c mice. Moreover, LL-37-treated C. albicans floating cells that did not adhere to the underlying substratum aggregated as a consequence of LL-37 bound to the cell surfaces. According to the results of a competition assay, the inhibitory effects of LL-37 on cell adhesion and aggregation were mediated by its preferential binding to mannan, the main component of the C. albicans cell wall, and partially by its ability to bind chitin or glucan, which underlie the mannan layer. Therefore, targeting of cell-wall carbohydrates by LL-37 provides a new strategy to prevent C. albicans infection, and LL-37 is a useful, new tool to screen for other C. albicans components involved in adhesion.     

Agglutination of blastospores of Candida albicans by concanavalin A and its relationship with the distribution of mannan polymers and the ultrastructure of the cell wall.

Blastospores of Candida albicans were readily agglutinated by Concanavalin A (Con A) owing to the specific binding of this lectin to the mannan receptors of the cell surface. When mannan was extracted from the cell wall by neutral buffers, alkali and acid, the agglutination was decreased or lost depending on the degree of extraction. A relatively mild alkali treatment was sufficient to derange the multilayered wall organization and transform it into a uniform, medium-density structure having about the same thickness as the untreated wall. After a more drastic extraction, all the electron-dense components of the wall were lost, the residual, alkali-insoluble wall fabric being completely electron-transparent and of about the same thickness as the inner wall region of untreated cells. Thiol-reducing agents like mercaptoethanol or dithiothreitol also extracted wall materials, an effect which was enhanced by pronase. After dithiothreitol-pronase treatment, the outer wall layers were removed but the inner wall region was not apparently damaged and some electron-dense components remained. None of these treatments significantly affected blastospore agglutination by Con A--this was reduced (but not abolished) only by the sequential action of pronase and helicase, which led to sphaeroplast formation. These sphaeroplasts showed a varied amount of residual wall consisting of evenly distributed, fibrogranular components. Two main conclusions were drawn from these results: (i) mannan polymers extend throughout the wall of the blastospore of C. albicans; (ii) the layering of the wall, as seen by ordinary fixation and staining for electron microscopy, essentially reflects the distribution of the various alkali-soluble complexes, at different levels, both over and in the rigid, glucan-chitin matrix.     

CHS8-a fourth chitin synthase gene of Candida albicans contributes to in vitro chitin synthase activity, but is dispensable for growth

In silico analysis of the genome sequence of the human pathogenic fungus Candida albicans identified an open reading frame encoding a putative fourth member of the chitin synthase gene family. This gene, named CaCHS8, encodes an 1105 amino acid open reading frame with the conserved motifs characteristic of class I zymogenic chitin synthases with closest sequence similarity to the non-essential C. albicans class I CHS2 gene. Although the CaCHS8 gene was expressed in both yeast and hyphal cells, homozygous chs8 Delta null mutants had normal growth rates, cellular morphologies and chitin contents. The null mutant strains had a 25% reduction in chitin synthase activity and were hypersensitive to Calcofluor White. A chs2 Delta chs8 Delta double mutant had less than 3% of normal chitin synthase activity and had increased wall glucan and decreased mannan but was unaffected in growth or cell morphology. The C. albicans class I double mutant did not exhibit a bud-lysis phenotype as found in the class I chs1 Delta mutant of Saccharomyces cerevisiae. Therefore, C. albicans has four chitin synthases with two non-essential class I Chs isoenzymes that contribute collectively to more than 97% of the in vitro chitin synthase activity.     

The role of glucose in the pH regulation of germ-tube formation in Candida albicans

It has been reported that Candida albicans can form germ-tubes only in the narrow pH range of 6-8, and that by changing only the pH one can regulate germ-tube formation. We found that the pH minimum for germ-tube formation could be dramatically lowered by eliminating the glucose present in many induction solutions. Lee's medium lacking glucose, ethanol, N-acetyl-D-glucosamine, and proline induced germ-tubes at pH values as low as 3 under most conditions. The presence of as little as 1 mM-glucose in these induction solutions was sufficient to cause the cells to grow either as yeasts with multiple buds or as pseudohyphae when the pH was 3.7. However, when C. albicans was grown in any of the above induction solutions (with the exception of ethanol), containing 200 mM-glucose buffered at pH 5.8, not only were germ-tubes formed, but their rate of formation and length were also increased. Preincubation of the cells in a solution buffered at pH 3.7 and containing 200 mM-glucose, before exposure to induction solutions lacking glucose at pH 3.7 or at pH 5.8, did not inhibit germ-tube formation. Likewise, addition of glucose after 45 min exposure to an induction solution was without effect. Theophylline and dibutyryl cAMP did not counteract the action of glucose. Other sugars which suppressed germ-tube formation at low pH were fructose, galactose, mannose, xylose, gluconic acid and the nonmetabolizable sugar 3-O-methylglucose. These results indicate that pH does not directly regulate dimorphism in C. albicans, and that glucose or its metabolites may play an important role.     

Idiotypy of human anti-Candida albicans antibodies: recurrence, presence of a cross-reactive autoanti-idiotypic-like activity, and role in the induction of specific in vitro antibody response

Rabbit anti-idiotypic antibodies (L12) were raised against human anti-mannan of Candida albicans (CA) antibodies isolated from the serum of a normal donor. The absorbed anti-idiotypic antiserum bound to donor anti-CA mannan antibodies but not to control immunoglobulins. Binding was inhibited by CA mannan but not by other polysaccharide antigens. L12 was shown to cross-react with anti-CA mannan-isolated antibodies or with anti-CA antibody-containing sera from individuals unrelated to the donor. IgG fraction isolated from the donor serum was repeatedly absorbed on CA mannan Sepharose to remove anti-mannan antibodies. This IgG fraction (named autoanti-idiotypic fraction) blocked, in a dose-dependent fashion, the binding of rabbit anti-idiotype to donor anti-CA mannan antibodies. Moreover, this CP-depleted IgG fraction cross-reacted with public idiotypic determinants of unrelated anti-CA mannan antibodies. Finally, L12 induced sensitized lymphocytes to produce anti-CA mannan antibodies in vitro in the absence of antigen.     

Fucose-specific adhesins on germ tubes of Candida albicans

Lectin-like adhesins of hyphal-form Candida albicans were investigated by conventional fluorescence microscopy, fluorescence microscopy with image analysis, spectrofluorimetry and flow cytometry. Labelling was done with neoglycoprotein probes consisting of sugars (fucose, mannose, glucose, galactose, lactose) covalently linked to bovine serum albumin (BSA), which itself was labelled with fluorescein. The fucose probe bound to both the yeast and germ-tube portions of hyphal-form cells, not especially at the tip, but in the adjacent region of the germ-tube portion. Probes with the other sugars did not label the hyphal-form cells. Fucose-probe binding to the cells was optimal at pH 5.0 in citrate buffer, and was a time-dependent reaction requiring 30–60 min and reaching saturation concentration at 100 μg ml⁻¹. Each hyphal-form cell of C. albicans grown in 199 medium was calculated to have about 2×10⁷ fucose probe-binding sites. There appeared to be no requirement for Ca²⁺ or Mg²⁺ in binding. Binding of the fucose probe to the hyphal-form cells was higher at 37°C than at 22°C or 4°C. Fluorescence intensity of the fucose-labelled yeast forms was not increased over the hyphal-form cells. A germ-tube-deficient mutant when exposed to hyphal-form growth conditions for 2 h showed much less binding of the fucose probe than the wild-type which produced germ tubes. Confirmation of specificity and the need for a carrier molecule was obtained by showing that Fuc-BSA (without fluorescein) effectively inhibited the binding of the fucose probe, although l-fucose itself was inactive, as was Gal-BSA.     

Structures involved in the binding of human fibrinogen to Candida albicans germ tubes

Recent evidence for the interaction between human fibrinogen and Candida albicans germ tubes have led us to attempt to characterize the structures involved. Using 125I-radiolabeled proteins, fibrinogen purified by affinity chromatography and its plasmin degradation products, the binding sites on the fibrinogen molecule appeared to be located specifically in the D-domain. Conversely to the fibrinogen and the fragment D, radiolabeled fragment E, however, did not interact with cells. The binding was time-dependent, saturable and reversible. Scatchard analysis of the data obtained revealed an average of 6000 binding sites per germ tube with dissociation constant (Kd) of 5.2 X 10(-8) M. No potent competition was observed for a range of different proteins and carbohydrates. Fibrinogen fragment D binding proteins were identified using a dithiothreitol-iodoacetamide extract of the fungus. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, one component of 68 kDa was detected. Thus, the presence of fibrinogen binding proteins specifically localized on the cell wall surface of C. albicans germ tubes may constitute one of the factors involved in the development of candidosis.     

Structures involved in the binding of human fibrinogen to Candida albicans germ tubes

Abstract Recent evidence for the interaction between human fibrinogen and Candida albicans germ tubes have led us to attempt to characterize the structures involved. Using ¹²⁵I-radiolabeled proteins, fibrinogen purified by affinity chromatography and its plasmin degradation products, the binding sites on the fibrinogen molecule appeared to be located specifically in the D-domain. Conversely to the fibrinogen and the fragment D, radiolabeled fragment E, however, did not interact with cell. The binding was time-dependent, saturable and reversible. Scatchard analysis of the data obtained revealed an average of 6000 binding sites per germ tube with dissociation constant ( K _d) of 5.2 × 10⁻⁸ M. No potent competition was observed for a range of different proteins and carbohydrates. Fibrinogen fragment D binding proteins were identified using a dithiothreitol-iodoacetamide extract of the fungus. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, one compotent of 68 kDa was detected. Thus, the presence of fibrinogen binding proteins specifically localized on the cell wall surface of C. albicans germ tubes may constitute one of the factors involved in the development of candidosis.     

An analysis of the metabolism and cell wall composition of Candida albicans during germ-tube formation

The uptake of nutrients (glucose, glutamine, and N-acetylglucosamine), the intracellular concentrations of metabolites (glucose-6-phosphate, cyclic AMP, amino acids, trehalose, and glycogen) and cell wall composition were studied in Candida albicans. These analyses were carried out with exponential-phase, stationary-phase, and starved yeast cells, and during germ-tube formation. Germ tubes formed during a 3-h incubation of starved yeast cells (0.8 X 10(8) cells/mL) at 37 degrees C during which time the nutrients glucose plus glutamine or N-acetylglucosamine (2.5 mM of each) were completely utilized. Control incubations with these nutrients at 28 degrees C did not form germ tubes. Uptake of N-acetylglucosamine and glutamine was inhibited by cycloheximide which suggests that de novo protein synthesis was required for the induction of these uptake systems. The glucose-6-phosphate content varied from 0.4 nmol/mg dry weight for starved cells to 2-3 nmol/mg dry weight for growing yeast cells and germ tube forming cells. Trehalose content varied from 85 nmol/mg dry weight (growing yeast cells and germ tube forming cells) to 165 nmol/mg weight (stationary-phase cells). The glycogen content decreased during germ-tube formation (from 800 to 600 nmol glucose equivalent/mg dry weight) but increased (to 1000 nmol glucose equivalent/mg dry weight) in the control incubation of yeast cells. Cyclic AMP remained constant throughout germ-tube formation at 4-6 pmol/mg dry weight. The total amino acid pool was similar in exponential, starved, and germ tube forming cells but there were changes in the amounts of individual amino acids. The overall cell wall composition of yeast cells and germ tube forming cells were similar: lipid (2%, w/w); protein (3-6%), and carbohydrate (77-85%). The total carbohydrates were accounted for as the following fractions: alkali-soluble glucan (3-8%), mannan (20-23%), acid-soluble glucan (24-27%), and acid-insoluble glucan (18-26%). The relative amounts of the alkali-soluble and insoluble glucan changed during starvation of yeast cells, reinitiation of yeast-phase growth, and germ-tube formation. Analysis of the insoluble glucan fraction from cells labelled with [14C]glucose during germ-tube formation showed that the chitin content of the cell wall increased from 0.6% to 2.7% (w/w).     

Antigenic relationship between Candida parapsilosis and Candida albicans serotype B

We examined the antigenic relationship between Candida parapsilosis and C. albicans serotype B with respect to antigenic factors 13 and 13b, specific for the former species and common to both species, respectively. Acetolysis of C. albicans serotype B cell-wall mannan gave six oligosaccharides. Their chemical structure was determined by 1H-nuclear magnetic resonance (NMR) spectroscopy, methylation analysis, and partial acid hydrolysis. The structure of the hexasaccharide derived from C. albicans serotype B mannan was alpha-D-Manp-(1-2)-alpha-D-Manp-(1-3)-alpha-D-Manp-(1- 2)-alpha-D-Manp-(1-2)- alpha-D-Manp-(1-2)-D-Man (M6) which is identical to that from C. parapsilosis mannan. Inhibition of two precipitin reaction systems (anti-C. albicans serotype B serum and anti-C. parapsilosis serum to the respective homologous mannan), by oligosaccharides from homologous and heterologous mannans indicated that M6 from either C. albicans serotype B or C. parapsilosis was the most effective inhibitor. Moreover inhibition of the agglutination reaction between factor serum containing anti-factors 13 and 13b and C. albicans serotype B or C. parapsilosis cells by oligosaccharides from both mannans also indicated that the M6s were the most effective inhibitors. These results suggest that the M6s derived from the two species are identical in their chemical structure, although the structures of the whole mannans of the two species are not identical as demonstrated by gel diffusion precipitation patterns, and that M6s may be involved in the specificities of antigenic factors 13 and 13b. The amount of M6 is larger in C. parapsilosis cell-wall mannan, suggesting that high repeating frequency of M6 fragment may induce the antibody specific for C. parapsilosis.     

Structural and functional definition of the human chitinase chitin-binding domain

Mammalian chitinase, a chitinolytic enzyme expressed by macrophages, has been detected in atherosclerotic plaques and is elevated in blood and tissues of guinea pigs infected with Aspergillus. Its normal physiological function is unknown. To understand how the enzyme interacts with its substrate, we have characterized the chitin-binding domain. The C-terminal 49 amino acids make up the minimal sequence required for chitin binding activity. The absence of this domain does not affect the ability of the enzyme to hydrolyze the soluble substrate, triacetylchitotriose, but abolishes hydrolysis of insoluble chitin. Within the minimal chitin-binding domain are six cysteines; mutation of any one of these to serine results in complete loss of chitin binding activity. Analysis of purified recombinant chitin-binding domain revealed the presence of three disulfide linkages. The recombinant domain binds specifically to chitin but does not bind chitosan, cellulose, xylan, beta-1, 3-glucan, beta-1,3-1,4-glucan, or mannan. Fluorescently tagged chitin-binding domain was used to demonstrate chitin-specific binding to Saccharomyces cerevisiae, Candida albicans, Mucor rouxii, and Neurospora crassa. These experiments define structural features of the minimal domain of human chitinase required for both specifically binding to and hydrolyzing insoluble chitin and demonstrate relevant binding within the context of the fungal cell wall.     

Factor XIII binds to the A alpha- and B beta- chains in the D-domain of fibrinogen: an immunoblotting study

The binding sites in fibrinogen for Factor XIII were localized using an immunoblotting technique. Platelet Factor XIII bound to fibrinogen and to plasmin degradation products of fibrin(ogen) including Fragments: X, D1-D3, and D-dimer, but did not bind to Fragment E. Binding of Platelet Factor XIII was independent of calcium ions but could be inhibited by the presence of 0.5 M NaCl. Binding could also be inhibited by preincubating Factor XIII with a 100-fold molar excess of fibrinogen but not by 100-fold molar excess of Fragment E. Binding of Factor XIII to fibrinogen was specific, since several other proteins tested (ovalbumin, bovine serum albumin, alpha 2-macroglobulin, beta-galactosidase, fructose kinase, lactic dehydrogenase, triose phosphate isomerase, fumarase and pyruvate kinase) did not bind Factor XIII. Furthermore, binding was not observed either when Factor XIII was left out or when antiFactor XIII antiserum was substituted with nonimmune serum. When fibrinogen was reduced prior to electrophoresis, Factor XIII bound to the A alpha and B beta chains of fibrinogen and des A,B fibrinogen, the B beta-chain of Fragment X, but not the gamma-chains. Localization of the Factor XIII binding sites to the carboxy terminal segments of the A alpha and B beta chains in the Fragment D-domain of fibrinogen could have important physiological consequences.     

Regulation and solubilization of Candida albicans chitin synthetase.

A cytoplasmic component which inhibited the activation of chitin synthetase was studied in the dimorphic fungus Candida albicans. The inhibitor was found to be heat stable and trypsin sensitive and was only effective when incubated with a vacuolar protease, an activator of chitin synthetase, before the activation of chitin synthetase. In addition, the particulate chitin synthetase from the yeast form of C. albicans was solubilized by a sodium cholate-digitonin extraction and subsequently was purified approximately 30-fold by Sepharose column chromatography and Amicon XM 100 filtration. Activity of the soluble enzyme was increased by the addition of trypsin or phosphatidyl serine. The molecular weight of the enzyme was estimated to be 400,000.     

Identification and immunochemical characterization of a germ tube specific antigen of Candida albicans

Abstract Germ tube specific fractions of the dimorphic pathogenic fungus Candida albicans were fractionated according to their ability to link fibrinogen. These fibrinogen binding factors were used as immunogens to prepare monoclonal antibodies (mAbs) with BALB/c mice. Among the resulting mAbs, one (mAb 3D9.3) was shown by indirect immunofluorescence to be specific to the surface of the mycelial phase of the C. albicans species. No labelling of the cell wall of any other Candida species was observed. This morphological shape specificity was confirmed by immunoblotting where a polydispersed high molecular mass component was identified. The molecular mass varied with the extraction procedure used; over 210 kDa with EDTA-2ME treatment, and ranging from 110 to 220 kDa after Zymolyase digestion. This phase-specific epitope was sensitive to proteolysis with pronase E, proteinase K and trypsin, but not to periodate treatment. Further purification of this material would allow further development of new serodiagnostic assays that might be more specific for invasive disease than currently available tests.     

The cellular location of proteases in Candida albicans

Vacuoles prepared from yeast cells of Candida albicans were enriched in proteinase ycaB (EC 3.4.21.48) but not in aminopeptidase or beta-glucosidase. Proteinase ycaB, assayed in situ, increased 1.5-fold during starvation whereas aminopeptidase activity decreased by 25%. Proteinase ycaB increased a further 1.5-fold during germ-tube formation.