Candida Albicans Yeast Cells Research Articles

Wall mannoproteins of the yeast and mycelial cells of Candida albicans: nature of the glycosidic bonds and polydispersity of their mannan moieties.

Zymolyase released between 20 and 25% of the total protein from purified walls of yeast (Y) and mycelial (M) cells of Candida albicans. The material released contained 92% carbohydrate (86% mannose and 6% glucose) and 7% protein. Over 85% of the carbohydrate was N-glycosidically linked to the protein and the rest (less than 15%) was linked O-glycosidically. Highly polydisperse, high molecular mass mannoproteins, resolved by electrophoresis as four defined bands in Y cells and two bands in M cells, had both types of sugar chains. A 34 kDa species found in both types of cells had a single 2.5 kDa N-glycosidically linked sugar chain and a 31.5 kDa protein moiety. Polydispersity in the high molecular mass mannoproteins was due to the N-linked sugar chains (mannan) with a molecular mass between 500 kDa and 20 kDa (average 100 kDa) in Y cells and between 400 kDa and 20 kDa (average 50 kDa) in M cells. Three mannoproteins of 34, 30 and 29 kDa secreted by protoplasts were associated with the high molecular mass mannoproteins, suggesting that this type of interaction might be related to the regeneration of the cell wall.

Cytoplasmic alkalinization during germ tube formation in Candida albicans.

Weak acids were used to measure the internal pH of yeast cells of Candida albicans that had been induced to form buds or germ tubes. Under conditions that supported germ tube formation the internal pH rose from around 6.8 to over 8.0 after 30 min in two different induction media. Internal pH measured by 31P NMR confirmed this pattern and also showed that the internal pH fell to around 7.0 prior to the outgrowth of germ tubes. Conditions which led to budding induced less cytoplasmic alkalinization. This alkalinization was brought about when cells were inoculated into media of neutral pH and at an increased temperature. Increasing the temperature of the medium augmented the alkalinization of the cytoplasm induced by raising the external pH. Strains of C. albicans defective in the ability to produce germ tubes did not show this dramatic cytoplasmic alkalinization under conditions which normally supported filamentous growth. The raising of internal pH may be due to the activation of the plasma membrane proton-pumping ATPase since diethylstilboestrol inhibited the cytoplasmic alkalinization and germ tube formation without causing irreversible loss of cell viability. The results show that the induction of the dimorphic transition in this organism is accompanied by a steep rise in internal pH. It is not known whether these changes are the cause or consequence of morphogenesis.

Nutritional factors determine germ tube formation inCandida albicans

Following a short (3 h) period of carbon starvation, exponential phase yeast cells of Candida albicans rapidly (T50 45 min) formed germ tubes in a glucose/ammonium ion solution. The presence of both a sugar (glucose, sucrose or galactose) and a nitrogen source (ammonium ion or an amino acid metabolized via glutamate) was critical for morphogenesis.

Role of fibronectin in the pathogenesis of candidal infections.

Adherence of Candida albicans yeast cells to mammalian cells in vitro is promoted by a cell-surface adhesin that is probably a mannan or a mannoprotein. This observation is based on results of studies that utilized either lectins or antibodies to Candida that block attachment. Selective enzymatic degradation of cell wall constituents has been used to define the surface ligand of the candidal cell. Recently, nonadhering, spontaneous mutants of C. albicans, from both yeast and mycelial forms, that lack specific mannoproteins have been described. In addition, such mutants are avirulent, an indication that attachment plays a role in the disease process. The mammalian receptor for Candida has not been identified, but in endocarditis it could be fibronectin, both because fibronectin is present at the endocardial lesion and because those Candida species that bind to fibronectin in vitro exhibit a high disease potential; i.e., Candida species that bind to fibronectin in vitro also cause endocarditis, while those that do not bind in vitro do not cause disease. Attachment of yeast cells both to human buccal and vaginal epithelial cells is reduced significantly if yeast cells are preincubated with fibronectin, a finding that indicates a possible role for fibronectin as the epithelial surface receptor for Candida.

Cytological aspects of dimorphism in Candida albicans.

From a comparison of the growth of yeast and hyphal cells of Candida albicans at 37 degrees C, the present authors suggest that the formation of hyphae is a response to nutrient stress. The major cytological evidence for this is that the formation of a germ tube is chiefly the result of the migration of cytoplasm out of the parent yeast cell, with little biosynthesis occurring other than DNA replication and wall assembly. This explains the linear outgrowth of the germ tube rather than an autocatalytic outgrowth. It is accompanied by the enlargement of the vacuole in the parent cell. Subsequent elongation of the hyphae is accompanied by vacuolation of subapical compartments, and branching only occurs from some of the subapical compartments after they have reformed sufficient cytoplasm.

The effects of solubleSaccharomyces cerevisiaemannan on the phagocytosis ofCandida albicansby mouse peritoneal macrophagesin vitro

Saccharomyces cerevisiae mannan inhibited the phagocytic activity and phagocytic capacity of mouse peritoneal macrophages elicited by Concanavalin A (Con A) for Candida albicans yeast cells that were either unopsonized or opsonized with C3. The mannan had no effect on the phagocytosis of C. albicans opsonized with normal human serum or immunoglobulins. Phagolysosome fusion in macrophages was unimpaired by the presence of S. cerevisiae mannan, which supports previous observations that mannan does not interfere with neutrophil degranulation.

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.

Effects of a new pyrazolo [3, 4-d]pyrimidine on growth and morphology of Candida albicans.

A new pyrazolo [3, 4-d]pyrimidine derivative was synthesized and its antifungal activity evaluated in vitro against mycelial and yeast cells of Candida albicans. The most striking ultrastructural changes following treatment with 10-30 micrograms/ml (mycelia) and 25-75 micrograms/ml (yeasts) consisted in the deterioration of the organelle membranes and in aberrant thickenings of the cell wall. The complete disorganization of the cytoplasmic structures seemed to be the final event.

Rapid killing of monocytes in vitro by Candida albicans yeast cells.

To study the interaction between Candida albicans blastoconidia and human phagocytes, we incubated peripheral leukocytes with fungi for 1 h at 37 degrees C and stained the cells with fluorescent vital stains ethidium bromide (EB) and fluorescein diacetate. Fungi that had been phagocytosed showed little staining; however, some leukocytes containing blastoconidia exhibited nuclear staining with EB, even though their cell membranes showed no signs of penetration by fungi. The number of EB-positive leukocytes was related to viability of the yeast cells and the temperature at which they were maintained before use. Because efforts to quantitate EB-positive leukocytes microscopically were frustrated by cell aggregation, we labeled the leukocytes with 51Cr and measured isotope release. We determined that leukocytes incubated with viable fungi released significantly more isotope than cells incubated alone or with killed blastoconidia. Furthermore, 51Cr release correlated directly with concentration of fungi in the assay, time of incubation, and temperature at which fungi were maintained before use. Using a number of isolates of C. albicans and several other species of Candida, we found that all exhibited cytotoxic activity against leukocytes, but the level of activity varied among organisms. Finally, we depleted or enriched peripheral leukocytes for specific cell populations and determined that only monocytes released more 51Cr after incubation with viable blastoconidia. Blastoconidia can lyse phagocytic cells through germination and penetration of cell membranes within 1 to 2 h, but the cytotoxic phenomenon we describe occurs within 15 to 30 min after yeast cells have been phagocytosed. Therefore, this capacity may represent a more immediate response by blastoconidia against phagocytosis and killing by monocytes.

Abnormal formation of Candida albicans walls produced by Calcofluor white: An ultrastructural and stereologic study

The effect of Calcofluor white on the formation of the cell wall was studied in yeast and mycelial cells of Candida albicans using uv microscopy, conventional electron microscopy (including qualitative and stereologic analyses), and freeze-fracture. The uv microscopy of semithin sections showed that abnormal deposition of chitin occurs in both yeast and mycelial cells. Ultrathin sections revealed that abnormal chitin deposition in yeast cells results in a wall thickening which protrudes into the cytoplasm. The thickening appeared in the region of the yeast cell wall where formation of the bud would take place. In mycelial cells, one or more bulges were observed, mainly located at the position of the septum. In freeze-fracture replicas, abnormally deposited wall material from the bulge displayed a rougher appearance than that normally deposited. Moreover, no significant differences with respect to intramembranous particles were observed between plasma membranes of control and Calcofluor-treated cells. In contrast, stereologic analysis on ultrathin sections of control and Calcofluor-treated cells showed significant differences in the volume of the cell wall between these cell populations. These results support the previously reported hypothesis that Calcofluor white interferes with deposition of chitin but not with its synthesis.

Resistance of the stationary-phase plasma membrane ofCandida albicansto filipin-induced deformation

Yeast cells of Candida albicans were treated with the polyene antibiotic filipin which specifically binds with sterol and induces morphological lesions in membranes. The plasma membrane in the exponential phase revealed numerous filipin-induced deformations. In contrast, most areas of the plasma membrane in the stationary phase resisted filipin-induced deformation, even though the cell wall did not prevent the entrance of filipin. These facts suggest that the organisation or physicochemical properties of the plasma membrane in the stationary phase is different from that in the exponential phase.

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.

Secreted phospholipases of the dimorphic fungus,Candida albicans; separation of three enzymes and some biological properties

Several phospholipases are secreted into the culture medium by growing yeast cells of Candida albicans 3125. DEAE-Sephadex column chromatography of concentrated culture filtrate revealed three separable fractions with phospholipase activities. Analysis of products of hydrolysis showed that the enzyme activities were lysophospholipase, lysophospholipase-transacylase and a phospholipase B.

Changes in surface topography ofCandida albicansduring morphogenesis

Temporal studies of germ-tube forming yeast cells of Candida albicans by scanning and transmission electron microscopy indicate that extensive vacuolation and possibly also cell wall changes may cause walls of the parent yeast cell to collapse during specimen preparation. This collapse does not occur in cells which have been grown in conditions that suppress germ tube formation and have undergone the same preparative treatment.

Germ tube-forming cells ofCandida albicansare more susceptible to clotrimazole-induced killing than yeast cells

Yeast and germ tube-forming cells of Candida albicans were compared with respect to their susceptibility to killing induced by the imidazole antifungal clotrimazole. Cultures consisting largely of germ tube-forming cells or exclusively yeast cells were prepared by incubating cells of a germ tube-proficient strain in a proline-containing phosphate buffer at 37 degrees C or 25 degrees C, respectively. When treated with clotrimazole at 37 degrees C, the cultures of germ tube cells lost colony-forming ability much more rapidly than those of yeast cells. However, this difference was diminished in the cells preincubated at 37 degrees C but prevented from forming germ tubes by 5 mM cysteine, a suppressor of germ tube formation. In another C. albicans isolate showing a very poor capacity to form germ tubes at 37 degrees C, such a difference in killing rate was much smaller than that for the germ tube-proficient strain. Furthermore, when an isogenic pair of strains, one proficient and the other deficient in germ tube formation, were compared with each other, germ tube-forming cultures of the former were found to be more sensitive than yeast cell cultures of the latter. It is inferred from these results that the germ tube-forming cell of C. albicans is more sensitive to clotrimazole-induced killing than the yeast cell.

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.

Interactions of Candida albicans yeast cells, germ tubes and hyphae with human polymorphonuclear leucocytes in vitro.

Suspensions of Candida albicans yeast cells, germ tubes and hyphae with biomass standardized by ATP measurement were compared for their relative susceptibilities to phagocytosis and intracellular killing by human polymorphonuclear leucocytes. All three forms were ingested to a similar extent, but significantly fewer yeast cells were killed intracellularly after ingestion than were filamentous forms of the fungus. Ketoconazole pretreatment significantly enhanced the susceptibility of hyphae, but not of germ tubes, to phagocytosis and intracellular killing. The opsonic requirements of the yeasts and filamentous forms for efficient phagocytosis and killing differed.

Demonstration of a septal pore in buddingCandida albicansyeast cells

Electron microscopy of Candida albicans yeast cells grown in a peptone glucose broth at 37 degrees C revealed pores in the septum identical in appearance to those already described in the hyphal form of the fungus. The presence of septal pores in yeast cells may explain apparently synchronous post-septation events in parent and daughter cells, and emphasizes the close structural similarities between different morphological forms of C. albicans.

Effect of cobalt and morphogenic autoregulatory substance (MARS) on morphogenesis of Candida albicans

Candida albicans yeast cells produce a factor, designated morphogenic autoregulatory substance (MARS), which suppresses yeast-to-mycelial conversion under conditions that support germination. In experiments designed to characterize MARS, the effect of various metals on MARS activity was tested. Cobalt but not zinc, iron, or other metals enhanced the ability of MARS to inhibit germination. Furthermore, uptake of cobalt by cells exposed to MARS was enhanced over cells in control medium if the cells were first incubated in a medium containing MARS for several hours prior to addition of cobalt to the medium. Uptake of zinc under similar conditions was not affected by MARS. The enhanced uptake of cobalt by MARS-exposed cells appears to be related to germination because such enhancement did not occur in strains of yeasts incapable of germination even in the absence of MARS.

Effect of yeast growth conditions on yeast-mycelial transition in Candida albicans.

When grown and induced to form germ tubes in liquid defined media, yeast cells of Candida albicans must reach stationary phase before acquiring ability to carry out the yeast-mycelial transition. This study examined the effect of the carbon source utilized for yeast growth on the inducibility of stationary phase yeast. When grown to the same stationary phase cell density as glucose cultures, cultures grown on citrate were fully inducible while cultures grown on galactose and mannose showed a small reduction. Cultures grown on ethanol were reduced 80% in morphological conversion. When glucose grown cells were induced in the presence of these carbon sources, hexoses supported full induction while ethanol reduced induction 80%. Induction in the presence of carboxylic acids was similar to induction in the absence of added carbon source. When induced on the same source used in yeast growth, germ tube formation was reduced for all carbon sources except hexoses. When induced in the absence of added carbon source, yeasts grown on citrate and ethanol were inhibited 80-100%. Cultures starved for glucose were more inhibited than cultures starved for NH4Cl when induced without added carbon source. These observations suggest that the metabolic state of the stationary phase cell is an important factor in the ability to respond to conditions inducing germ tube formation.