Experimental methodology to quantify Candida albicans cell surface hydrophobicity

A new method of formation of yeast cell lawns for contact angle measurement (with water, formamide and 1-bromonaphthalene) is described. The cell lawns were formed on agar layers avoiding liquid penetration. The method was validated by comparing the hydrophobicity of Candida albicans grown at different temperatures and the hydrophobicity of bacterial cell lawns built on agar layers and obtained by the usual filtration method.     

Involvement of transglutaminase in the formation of covalent cross-links in the cell wall ofCandida albicans

Activity of the enzyme glutaminyl-peptide-γ-glutamylyl-transferase (EC 2.3.2.13; transglutaminase), which forms the interpeptidic cross-link N^∈-(γ-glutamic)-lysine, was demonstrated in cell-free extracts obtained from both the yeast like and mycelial forms ofCandida albicans. Higher levels of enzymatic activity were observed in the cell wall fraction, whereas the cytosol contained only trace amounts of activity. Cystamine, a highly specific inhibitor of the enzyme, was used to analyze a possible role of transglutaminase in the organization of the cell wall structure of the fungus. Cystamine delayed protoplast regeneration and inhibited the yeast-to-mycelium transition and the incorporation of proteins into the cell wall. The incorporation of covalently bound high-molecular-weight proteins into the wall was sensitive to cystamine. Proteic epitopes recognized by two monoclonal antibodies, one of which is specific for the mycelial walls of the fungus, were also sensitive to cystamine. These data suggest that transglutaminase may be involved in the formation of covalent bonds between different cell wall proteins during the final assembly of the mature cell wall.     

Influence of fluconazole at subinhibitory concentrations on cell surface hydrophobicity and phagocytosis of Candida albicans

Cell surface hydrophobicity (CSH) status influences virulence of Candida albicans and decreases the susceptibility of yeast cells to phagocytic killing. We tested whether subinhibitory concentrations of fluconazole, which is widely used in the treatment and prophylaxis of candidiasis, affect CSH and the susceptibility of C. albicans to enzymatic digestion by glucanase and to phagocytic killing. Treatment of yeast cells with subinhibitory fluconazole concentrations resulted in greater phagocytosis. This effect was independent of CSH but may be related to increased cell wall porosity resulting from alterations in the cell envelope. The use of subinhibitory concentrations of fluconazole in patients with competent phagocytes may contribute to resistance to candidiasis regardless of yeast CSH status.     

Complex interaction between different proteinaceous components within the cell-wall structure ofCandida albicans

We have previously described a monoclonal antibody, MAb DC3:H10, which recognized an epitope preferentially expressed on the surface ofCandida albicans germ tubes. In the present study we examined the MAb-reactive material further. Immunoblot analysis of the material purified partially by Sephadex G-200 and DEAE-Sephacel chromatography reacted with antibodies to theC. albicans C3d receptor (CR2). In an ELISA, MAb DC3:H10 captured antigen that was recognized by both anti-CR2 and anti-mp58 fibrinogen binding mannoprotein polyclonal antibodies. The MAb DC3:H10 failed to compete with either of these antisera in an ELISA. Indirect immunofluo-rescence (IIF) analysis showed differences in surface distribution for the MAb DC3:H10, the CR2, and the mp 58 epitopes. Dual labeling IIF experiments showed MAb DC3:H10 binding to be unaffected by binding of fibrinogen or anti-mp58 antibody. However, the binding patterns of MAb DC3:H10 were modified in the presence of anti-CR2 antibody, suggesting a complex interaction between these cell wall components.     

The effect of mycophenolic acid on the cell cycle ofCandida albicans

Mycophenolic acid inhibited the growth ofCandida albicans. Cultures exposed to a concentration of 8.4 g ml^–1 mycophenolic acid were found to exhibit cell cycle arrest with two or more buds. Nuclear staining revealed that these were nucleate implying a possible defect in cytokinesis.The results are discussed in relation to the possible mode of action of mycophenolic acid.     

Characterization of a major cell wall antigen and potential adhesin in three strains of Candida albicans

Selected strains of Candida albicans were examined to reveal the surface antigenicity and biochemical nature of major cell wall proteins that also were shown to serve as cellular adhesins on human buccal epithelial cells. Confirmation of the adhesive properties of these cells was made by scanning electron microscopy and immunofluorescence microscopy. Particular attention was directed at the clinical isolate KM-302. By means of indirect immunofluorescence staining, the KM-302 blastoconidia absorbed rabbit anti-C. albicans ATCC-32354 serum, revealing specific localization of surface antigens on germ tubes and pseudohyphae. Extracellular polymeric material and the cell wall extract of C. albicans KM-302 blastoconidia were found to contain a major surface antigen of 49 kDa that exhibited 42% adhesion inhibition in vitro. Of considerable significance is that immunogold localization by electron microscopy showed the antigen to be almost exclusively cell wall bound. This major antigen, identified in affinity and gel filtration chromatography fractions, was composed of 4% carbohydrate and 95.7% protein and had an isoelectric point of 6.1. The major antigen also showed a high level of similarity with that of C. albicans strain SC-5314 inasmuch as the major antigen of that strain had carbohydrate and protein compositions of 4 and 95.5%, respectively. Both of these strains also possessed the same percent of adhesion inhibition of human buccal epithelial cells.Abbreviations BECs buccal epithelial cells - CWE cell wall extract - EPP extracellular polymers and proteins - FITC fluorescein isothiocyanate - mAg major antigen - OD ₆₀₀ optical density at 600 nm - PBS phosphate buffered saline - TEM transmission electron microscopy - YNB yeast nitrogen base     

Common and form-specific cell wall antigens of Candida albicans as released by chemical and enzymatic treatments

In order to investigate the antigenic properties of the proteins and mannoproteins present in the cell surface of Candida albicans, and to identify individual antigenic moieties and their distribution, a number of polyclonal antisera were obtained by immunizing rabbits with chemical and enzymatic cell wall extracts obtained from intact cells from both growth forms (yeast and mycelium) of the fungus. Prior to injection, wall moieties present in the extracts were subjected to different treatments and/or purification procedures such as adsorption onto polystyrenelatex microbeads or electrophoretic separation. When used as probes in indirect immunofluorescence assays, the different antisera gave rise to different fluorescence patterns varying in intensity and topological localization of the reactivity in C. albicans cells. When the different antisera were used as probes in Western blots of wall proteinaceous materials solubilized from both blastospores and germ tubes, differences in reactivity and specificity were readily discernible, allowing to identify a number of common and form-specific cell wall components. Of special interest was the fact that one of the antisera raised, after adsorption onto heat-killed blastospores, specifically recognized medium to low molecular weight moieties present only in the cell wall extracts from germ tubes. When this antiserum was used as probe in immunofluorescence assays, reactivity was confined to the hyphal extensions. Together, these observations seem to indicate that the different antibody preparations described in this report could represent important tools in the study of different aspects of the cell wall biology in C. albicans, including the identification and study of the distribution of common and form-specific cell wall-bound antigens.Abbreviations IIF Indirect immunofluorescence - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - PAb polyclonal antibody     

Changes in the cell wall glycoprotein composition of Candida albicans associated to the inhibition of germ tube formation by EDTA

Hyphal development in Candida albicans was blocked by EDTA. This effect was not due to a general growth inhibition since the chelator did not affect protein and DNA synthesis. Recovery of mycelial growth was observed when EDTA-grown cells were incubated at 37°C in EDTA-free medium. High-molecular-weight mannoproteins (HMWM) that are mycelium-specific wall components, and particularly a 260-kDa species (HMWM-260), were absent in the wall of cells grown under germination conditions in the presence of EDTA. Synthesis of the HMWM-260 species was not inhibited but its incorporation (secretion) into the wall structure seemed to be blocked in EDTA-treated cells.     

Growth ofCandida albicans in a minimal synthetic medium without biotin

Summary Growth ofCandida albicans strain B 311-10 was observed in a minimal synthetic biotin-free medium, using different glucose concentrations, during the first 30 hours of its development at 28 °C. The yeast's growth was observed spectrophotometrically at 675 nm reading its optical density every hour. The minimal medium of Shepherdet al. [12], with glucose (15 g/L) and biotin was modified: this vitamin was eliminated and the concentration of glucose was gradually lowered to 0.5 g/L. At 5 g/L of glucose and without biotin very good growth was obtained. Based on our results during the first 30 hours of growth, biotin has no influence on the yeast's growth. This medium would be useful for the study of the physiology ofC. albicans during the first period of its development.     

Regeneration of the cell wall in protoplasts of Candida albicans

To assess the dynamics of synthesis of the wall by regenerating Candida albicans protoplasts deposition of chitin and mannoproteins were investigated ultrastructurally using wheat germ agglutinin conjugated with either horseradish peroxidase or colloidal gold, and Concanavalin A coupled to ferritin respectively.Freshly prepared protoplasts lacked wheat germ agglutinin receptor sites but after 1–2 h of regeneration, they were detected. After 4–5 h of regeneration, the cell wall showed a discrete structure which was only labelled with wheat germ agglutinin in thin sections. At this stage of regeneration the outermost layer of the wall was labelled with clusters of Concanavalin A-ferritin particles.After 8 h regeneration, the cell wall appeared compact, and homogenously marked with wheat germ agglutinin whereas only the surface layers appeared consistently labelled with Concanavalin A-ferritin.From these observations we conclude that C. albicans protoplasts are able to regenerate in liquid medium a cell wall consisting of a network of chitin fibrils and mannoproteins at least (glucan polymers were not determined in the present cytological study). The former are the fundamental component of the inner layers at early stages of regeneration, whereas the latter molecules are predominant in the outer layers of the wall.Abbreviations WGA-HRP wheat germ agglutinin conjugated with horseradish peroxidase - WGA-Au wheat germ agglutinin conjugated with colloidal gold - Con A-ferritin Concanavalin A coupled to ferritin     

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.     

Oxygen as a possible tropic factor in hyphal growth ofCandida albicans

Hyphae ofCandida albicans elongated towards the oxygen-rich direction when exposed to gradients of oxygen concentration in thin-layer and capillary-tube cultures with corn meal (CM) agar. The thin-layer culture was prepared by covering a drop of molten CM agar containingC. albicans cells with a cover slip in Petri dishes. Cells located in the central region of the thin-layered medium neither grew nor produced hyphae. Cells in the marginal regions at first directed their hyphae in arbitrary directions after forming a small colony. Hyphae then gradually changed their direction of elongation and eventually oriented towards the nearest margin. Under anaerobiosis, cells seeded in the thin-layered medium did not grow even in the marginal regions. When exposed to air, the cells in the marginal regions rapidly began to form hyphae which elongated towards the nearest margin. To prepare an oxygen gradient in capillary-tube cultures, CM agar, and dilute and dense cell suspensions in CM agar were introduced sequentially into the capillary tubes, and the end closest to the dense cell suspension was sealed with paraffin. Among cells in the dilute layer, only that located closest to the meniscus grew well and extended hyphae towards the meniscus, where oxygen concentrations were highest. These studies suggest a positive aerotropic response in the hyphal growth ofC. albicans.     

An electron microscopy study of wall expansion during Candida albicans yeast and mycelial growth using concanavalin A-ferritin labelling of mannoproteins

Depending upon growth temperature, Candida albicans can exhibit two different morphologies, a budding yeast or a mycelium. By studying the distribution of concanavalin A-ferritin particles on the cell wall surface during bud and germ tube formation, we have elucidated the way cell wall extension occurs. Both processes initially require the localized lysis of the wall in order to allow the incorporation of the newly synthesized material. Later on, the cell wall behaves as an elastic structure, allowing extension by an intosusception process and, as a consequence, cell growth.Abbreviation Con A concanavalin A     

The influence of post-filtration washing on the in vitro assay ofCandida albicans adherence to human buccal epithelial cells

A simple in vitro assay technique was used to determine the effect of post-filtration washing on the adherence ofC. albicans (NCPF 3736) to human buccal epithelial cells (BEC). Washing was carried out with a range of volumes of phosphate buffered saline (PBS), viz. 0, 5, 10 and 20 ml, at a standard flow rate. Both the number ofC. albicans adherent to BEC and the percentage of BEC with adherentC. albicans were significantly decreased (p<0.001 for each of these measures) after washing with 5 ml PBS. Further increases in the volume of PBS did not significantly decrease either measure of adherence. These data indicate that only a small volume of PBS, 5 ml, is required to achieve the removal of non-adherentC. albicans from the surface of BEC. The result of the adherence assay is not significantly affected by increasing the volume of PBS used. It is concluded that considerable savings in time may be made through using only a small (5 ml) volume of washing buffer at a standard flow rate.Abbreviations BEC buccal epithelial cells - PBS phosphate buffered saline - MEM Eagle's minimum essential medium - NCPF National collection of pathogenic fungi     

A novel antifungal from an Actinomadurae with preferential activity against the mycelial phase ofCandida albicans

Summary Sch 40873, a novel antifungal compound isolated from the fermentation broth of anActinomadura spp. was discovered in an assay designed to detect compounds with preferential activity against the invasive mycelial form ofCandida albicans. The geometric mean MIC of Sch 40873 against sevenCandida spp. in Sabouraud dextrose broth (yeast phase) was 58 g/ml and in Eagles minimum essential medium (mycelial phase) was <0.03 g/ml. Sch 40873 demonstrated slight in vivo topical activity in a hamster vaginal model.     

Non-glucan Attached Proteins of Candida albicans Biofilm Formed on Various Surfaces

Non-glucan attached proteins of the cell surface and extracellular matrix of Candida albicans biofilms formed on two catheter surfaces and denture acrylic were examined. The SDS-PAGE protein profiles of these proteins compared with that obtained from planktonic yeast cells and germ tubes were generally similar. This observation suggested that this class of biofilm surface proteins is not composed of a unique set of extracellular proteins or that one or a few proteins dominate the non-glucan attached proteins of biofilm. However, differences were observed in the proteins obtained from biofilm formed on one catheter surface and two proteins, Grp2p and ORF19.822p, identified by mass spectrometry following two-dimensional separation. These proteins have previously been associated with drug resistance and their presence or abundance appeared to be influenced by the surface on which the biofilm was formed.     

Cytochemical and ultrastructural studies of Candida albicans

Ultrastructural modifications of the cell wall coat of Candida albicans during adherence to host cells were investigated using various cytochemical techniques. Attachment of the fungus to buccal epithelial cells appeared to involve spatial rearrangement of their cell wall surface. In particular adhering yeast developed a fibrogranular surface layer visualized by the periodic acid — thiocarbohydrazide silver proteinate technique (a polysaccharide detectron technique); Concanavalin A binding sites detected on their cell wall coat were highly increased. Attachment of yeasts to epithelial cells appeared mediated by fibrillar structures or polysaccharidic granules distributed on the cell wall coat. But free extra-cell wall material containing mannoproteins released from the yeast surface suggested additional mechanisms.Abbreviations Con A Concanavalin A - Man-fer mannosyl ferritin - PATAg Periodic acid-thiocarbohydrazide-silver proteinate     

Preliminary characterization of the material released to the culture medium byCandida albicans yeast and mycelial cells

Culture filtrate concentrates were obtained fromCandida albicans yeast and mycelial cells grown in the presence of¹⁴C-protein hydrolysate for radioactive labeling of cellular polypeptides. Both growth forms released to the medium minor but significant amounts of proteinaceous materials. The analysis of culture filtrate concentrates by means of SDS-polyacrylamide gel electrophoresis and fluorography revealed a similar and complex electrophoretic pattern, though some qualitative and quantitative differences between samples obtained from yeast and mycelial cells were observed. Materials released, mostly composed of mannoproteins as shown by their affinity towards concanavalin A, presented (i) cross-reactivity (by Western immunoblotting and ELISA) against polyclonal antisera to genuine cell wall components (among them the 58-kilodalton fibrinogen-binding mannoprotein) and (ii) high affinity for polystyrene-latex microbeads. Results presented suggest a possible common identity for the molecules shed to the medium and the cell wall protein and mannoprotein constituents.Abbreviations SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electroporesis - PAS periodic acid/Schiff method     

Investigations of mannan and glucan in the cell wall ofCandida boidinii cultivated on methanol

The polysaccharide components (mannan and glucan) in the cell wall ofCandida boidinii M 363 grown on methanol and glucose as control were investigated using electron microscopy, cytochemical and biochemical methods. An ultrastructural rearrangement of the polymers in the cell wall of yeasts cultivated on methanol in comparison to those cultivated on glucose was established. The morphological changes correlate to the quantitative changes in the polysaccharide constituents of the cell wall. The forming and the role of thiosemihydrocarbazide (TSHC) — negative zones in theCandida boidinii cell wall cultivated on methanol media are discussed.     

Influence of different levels of ammonium concentrations on cell growth,RNA and protein production byCandida albicans

Candida albicans starved cells were incubated in minimal synthetic liquid media containing different concentrations of ammonium sulphate (0.00, 0.02, 0.05, 0.10, 0.03, 0.50 g/L). Culture growth was monitored by measuring daily the optical density and by evaluating RNA and protein cellular content after 48 and 96 hours from the inoculum. The environmental availability of ammonium ion influenced the biomass production, that was maximum when its concentration was 0.10 and 0.30 g/L. In addition, an effect on cell duplication time was observed, this was particularly evident when the (NH₄)₂SO₄ concentration was 0.10 g/L. The protein content increased in relation to the increase of ammonium ion availability, with a peak in correspondence to 0.30 g/L and a drop when the greatest concentrations were employed. RNA production was inversely proportional in respect to protein production. The optimal range of ammonium sulphate concentration forC. albicans growth was 0.10–0.30 g/L; over these concentrations there was an inhibitory effect. The rate of the protein and RNA syntheses seems to indicate the growth phase and the nitrogen nutritional conditions of the cultures, respectively.