Cell wall anchoring to cytoplasmic membrane of Candida albicans.

Cell wall ultrastructure of the opportunistic pathogenic yeast Candida albicans was investigated by stereoscopic freeze-etching technique. Three wall layers were distinguishable by this technique. No clear periplasmic space was evident. Bilayer membrane invaginations were extensive. The outermost regions of the membrane invaginations were lined with thin, spine-like fibrils, which extended into the cell wall. We suggest that the fibrils along the invaginations are involved in anchoring the cell wall to the membrane.

Surface hydrophobic and hydrophilic protein alterations in Candida albicans.

Cell surface hydrophobicity influences pathogenesis of Candida albicans. Previous studies suggested that stationary-phase hydrophilic and hydrophobic cells, obtained by growth at 37 and 23 degrees C, respectively, may have similar hydrophobic proteins. However, whether hydrophilic and hydrophobic surface proteins differ during the growth cycle at 37 degrees C is unknown. Freeze-fracture analysis revealed surface fibrillar layer differences between hydrophobic late-lag and hydrophilic stationary-phase yeast cells grown at 37 degrees C. Hydrophilic protein differences were also observed between these populations. However, similar hydrophobic proteins were detected among the late-lag and stationary phase cells grown at 37 degrees C and hydrophobic stationary-phase cells grown at 23 degrees C. These results suggest that hydrophobic proteins remain constant but hydrophilic proteins vary during growth. Thus, conversion from surface hydrophilicity to hydrophobicity by C. albicans may only require alterations in the hydrophilic fibrillar protein components.

Hydrophobic surface protein masking by the opportunistic fungal pathogen Candida albicans.

Ultrastructural and biochemical analyses of hydrophobic and hydrophilic yeast cell surface proteins of Candida albicans were performed. Hydrophobic and hydrophilic yeast cells were obtained by growth at 23 and 37 degrees C, respectively. In addition, hydrophilic yeast cells were converted to surface hydrophobicity by treatment with tunicamycin and dithiothreitol. When freeze-etched cells were examined, the temperature-induced hydrophilic cells had long (0.198 micron), compact, evenly distributed fibrils while temperature-induced hydrophobic cells had short (0.085 micron), blunt fibrils. Hydrophobic microsphere attachment to the hydrophobic cells occurred at the basement of and within the short fibril layer. Dithiothreitol-induced hydrophobic cells had the long fibrils removed; tunicamycin-induced hydrophobic cells retained some of the long fibrils, but the fibrils were less compact and more aggregated than the untreated controls. These results suggest that the long fibrils prevent hydrophobic microsphere attachment to the hydrophobic area of the cell surface. This was confirmed by assessing the hydrophobic avidity of hydrophobic yeast cell populations differing in fibril density and arrangement. 125I-labelled surface proteins from hydrophobic and hydrophilic cells were compared after separation by hydrophobic interaction chromatography-high-performance liquid chromatography and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The yeast cell populations had hydrophilic proteins of similar molecular masses (greater than 200 kDa), but the hydrophilic cells possessed at least two additional proteins (ca. 63 and 69 to 71 kDa). Hydrophobic surface proteins appeared to be similar. However, the amount of total radiolabelled hydrophobic proteins was approximately 10-fold higher for the hydrophobic cells than for the hydrophilic cells. This result agrees with the ultrastructural observations which showed that yeast cell surface hydrophobic proteins are masked by hydrophilic high-molecular-mass surface fibrils. Taken together, the data indicate that yeast cell hydrophobicity is not determined by differences in surface hydrophobic proteins but by the presence of hydrophilic, surface fibrils.

Partial biochemical characterization of cell surface hydrophobicity and hydrophilicity of Candida albicans.

Hydrophobic yeast cells of Candida albicans are more virulent than hydrophilic yeast cells in mice. Results of experiments performed in vitro suggest that surface hydrophobicity contributes to virulence in multiple ways. Before definitive studies in vivo concerning the contribution of fungal surface hydrophobicity to pathogenesis can be performed, biochemical, physiological, and immunochemical characterization of the macromolecules responsible for surface hydrophobicity must be accomplished. This report describes our initial progress toward this goal. When hydrophobic and hydrophilic yeast cells of C. albicans were exposed to various enzymes, only proteases caused any change in surface hydrophobicity. Hydrophobic cell surfaces were sensitive to trypsin, chymotrypsin, pronase E, and pepsin. This indicates that surface hydrophobicity is due to protein. Papain, however, had no significant effect. The hydrophobicity of hydrophilic cells was altered only by papain. The proteins responsible for surface hydrophobicity could be removed by exposure to lyticase, a beta 1-3 glucanase, for 30 to 60 min. When 60-min lyticase digests of hydrophobic and hydrophilic cell walls were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a 12.5% resolving gel, each protein population contained a single unique protein that was not evident in the other protein population. However, when the cell wall surface proteins of hydrophobic and hydrophilic cells were first labeled with 125I and then removed by lyticase and analyzed by SDS-PAGE, at least four low-molecular-mass (less than 65 kilodaltons) proteins associated with hydrophobic cells were either absent or much less abundant in the hydrophilic cell digests. This result was seen for both C. albicans strains that we tested. When late-exponential-phase hydrophilic cells were treated with tunicamycin, high levels of surface hydrophobicity were obtained by stationary phase. These results indicate that the surface hydrophobicity of C. albicans reflects changes in external surface protein exposure and that protein mannosylation may influence exposure of hydrophobic surface proteins.

Isolation of hydrophobic and hydrophilic variants of Candida albicans.

We have previously demonstrated that most isolates of C. albicans are hydrophobic when grown at room temperature (RT, ca. 22-24 degrees C) and hydrophilic when grown at 37 degrees C. Variants of our standard strain LGH1095 were isolated that are hydrophobic at 37 degrees C and hydrophilic at RT. After repeated phase partitioning with cyclohexane-water cell populations that were 6-16% hydrophobic at RT and 66-80% hydrophobic at 37 degrees C were obtained. Subsequent limiting dilution experiments provided clones which were more hydrophobic at RT or hydrophilic at 37 degrees C. These were then recloned until the resultant populations were consistently under 5% cell surface hydrophobicity (CSH) at RT or over 95% at 37 degrees C. Treatment with several detergents as well as sugars did not decrease the CSH of these cells. Lipase and several proteases also had no effect. When treated with trypsin at a concentration twice that used to lower CSH of normal cells to less than 5%, the hydrophobic variant only decreased in CSH by 50%. Both variants were capable of germinating, although at different levels depending on prior growth temperature. Sensitivity to the germination inhibitor morphogenic autoregulatory substance (MARS) was similar to that of the parent strain.

Dynamic expression of cell surface hydrophobicity during initial yeast cell growth and before germ tube formation of Candida albicans.

Expression of cell surface hydrophobicity (CSH) during initial growth of Candida albicans was monitored. CSH of hydrophobic and hydrophilic yeast cells changed within 30 min upon subculture into fresh medium. Morphologic evidence of germination was preceded by expression of CSH. These results indicate that CSH expression is important in C. albicans growth.

Modification and application of a simple, surface hydrophobicity detection method to immune cells.

A simple method to assess fungal cell surface hydrophobicity involves enumeration of cell-attached, polystyrene latex microspheres. Modifications and conditions of this method for application to immune cell populations were investigated. The media used for suspending cells and microspheres appeared to influence microsphere attachment. Several tissue culture media supported high levels of microsphere attachment, but serum inhibited attachment. The concentration of microspheres also influenced the apparent level of detectable cell surface hydrophobicity. Under conditions which allow different levels of apparent cell surface hydrophobicity to be discriminated, the assay revealed that surface hydrophobicity of YAC-1 cells, which are used as standard targets in murine natural killer cell assays, varied depending on the time of harvest during growth and that phagocytic populations differed in cell surface hydrophobicity. Trypsinization experiments indicated that one hydrophobic constituent of the cell surface includes protein. These results indicate that the microsphere assay is a useful method for assessing cell surface hydrophobicity. The possibility that the assay could be used to determine quantitatively the surface free energies of immune cells and cell targets is discussed.

Temperature-modulated physiological characteristics of Candida albicans.

Despite numerous investigations on candidiasis, definitive conclusions concerning virulence factors are few because of oftentimes confusing and contradictory results. By utilizing various physiologic tests, which include germ tube induction, inhibition of germination by a morphogenic autoregulatory substance, enzyme production, susceptibility to exogenous chemicals, and cell surface hydrophobicity, we demonstrated that such variability is due, in part, to the environmental conditions in which cells were grown in preparation for analysis. Room-temperature grown cells were generally less sensitive to environmental perturbation and germinated more uniformly than cells grown at 37 degrees C. The implication of these results in relation to pathogenic studies and the epidemiology of candidiasis is suggested.