Cell wall proteins of Candida albicans

Proteins were solubilized from cell wall fractions of Candida albicans and separated by polyacrylamide gel electrophoresis. Cell walls were isolated from 25 and 37 degrees C growing and stationary phase yeast cultures and from germ tubes. The 42 protein bands detected by dye binding were observed in all wall extracts, regardless of the temperature, growth state, or morphology of the culture. The carbohydrate content of most bands was below the detectable limit of the periodic acid Schiff reagent. The protein complement revealed by autoradiography of radiolabeled proteins was half that detected by staining. Two bands showed greater intensity from cultures grown at 37 degrees C. The radio-labeled pattern was similar with both [35S]methionine-and [14C]leucine-labeled proteins and either pulse- or continuous-labeled proteins.     

Differential synthesis of polypeptides during morphogenesis of Mucor.

The extent of differential gene expression during morphogenesis of Mucor racemosus was investigated by two-dimensional polyacrylamide gel electrophoresis of neutral and acidic polypeptides. Cellular proteins were labeled with [35S]methionine in cells growing in either the yeast or hyphal form, or in yeast cells undergoing the transition of hyphae. The results showed that of the 400 to 500 polypeptides resolved by electrophoresis, relatively few were specific to one or the other morphological form. The major change in the patterns of proteins synthesized during morphogenesis was a change in rates of synthesis of individual polypeptides. Experiments in which morphogenesis was affected under aerobic or anaerobic conditions showed that the majority of changes in the protein patterns were associated with morphogenesis and were not a specific response to O2.     

Overexpression of the actin gene is associated with the morphogenesis of Candida albicans

A progressive increase in the synthesis of actin mRNA was observed by Northern blot analysis, when cells were induced to form germ tubes at 37 degrees C by N-acetyl-D-glucosamine. Presence of trifluoperazine, a calmodulin inhibitor, or incubation of cells at 25 degrees C, or by replacing N-acetyl-D-glucosamine with glucose which inhibited germ tube formation lowered this synthesis. Furthermore, in vitro translation of total RNA revealed an increase in the synthesis of actin (45 kDa) during germ tube formation. These results suggest for the first time that the expression of actin gene is regulated during morphogenesis of C. albicans.     

Morphogenesis of Candida albicans and cytoplasmic proteins associated with differences in morphology, strain, or temperature.

The extent of change in cytoplasmic proteins which accompanies yeast-to-mycelium morphogenesis of Candida albicans was analyzed by two-dimensional gel electrophoresis. Pure cultures of yeasts and true hyphae (i.e., without concomitant production of pseudohyphae) were grown in a synthetic low-sulfate medium. The two strains selected for this study were strain 4918, which produces pure mycelial cultures in low-sulfate medium at 37 degrees C and yeast cells at 24 degrees C, and strain 2252, which produces yeast cells exclusively at both 24 and 37 degrees C in low-sulfate medium. The proteins of both strains were labeled at both temperatures with [35S]sulfate, cytoplasmic fractions were prepared by mechanical disruption and ultracentrifugation, and the labeled proteins were analyzed by two-dimensional electrophoresis. Highly reproducible protein spot patterns were obtained which defined hundreds of proteins in each extract. Ten protein spots were identified on the two-dimensional gels of the 4918 mycelial-phase extract which were not present in the 4918 yeast-phase extract. These proteins appeared to be modifications of preexisting yeast-phase proteins rather than proteins synthesized de novo in the mycelial cells because 5 were absorbed by rabbit anti-yeast-phase immunoglobulin and each of the 10 was also present in extracts of strain 2252 grown at 24 and 37 degrees C, indicating that they were neither unique to filamentous cells nor sufficient for induction or maintenance of the mycelial morphology. Thirty-three proteins were identified in the 4918 yeast-phase extract which were not present in the 4918 mycelial-phase extract. Pulse-chase experiments revealed the synthesis of new proteins during yeast-to-mycelial conversion, but none of these was unique to mycelial cells. No differences in the major cytoplasmic proteins of any of the yeast- or mycelial-phase extracts were identified. This finding suggests that the major structural proteins of the cytoplasm are not extensively modified and argues instead that proteins unique to either phase may serve a regulatory function.     

Induction of N-acetyl-D-glucosamine catabolic enzymes and germinative response in Candida albicans

The regulation of N-acetylglucosamine catabolic enzymes was studied in both yeast and germ tube forms of the dimorphic fungus Candida albicans. The induction pattern of these enzymes was the same for yeast cells incubated at 28 degrees C and in cells incubated at 37 degrees C which formed germ tubes. However, the level of activity of these enzymes in germ tube stage is lower as compared to yeast phase cells. A strain of C. albicans that did not form germ tubes was endowed with a pronounced ability for induction of N-acetylglucosamine catabolic enzymes. This result suggests that germ tube formation and N-acetylglucosamine metabolism are mutually exclusive events.     

Effect of calcium ion uptake on Candida albicans morphology

In liquid culture using a synthetic medium, added magnesium but not calcium was required for exponential growth of Candida albicans yeast cells. However, medium without added divalent cations supported 2-3 generations of yeast growth or germ tube induction. The addition of calcium ions (1.0 mM) at any stage during the induction of germ tube formation caused reversion to a yeast mode of growth, in contrast to the effect of zinc and cobalt ions which were toxic to all growth. Inhibition of germ tube formation by calcium was not observed in the presence of either magnesium (10 microM) or manganese (100 microM). The presence of either of these ions caused inhibition of 45Ca uptake in yeast cultures. We conclude that unrestricted calcium uptake resulted in the specific inhibition of C. albicans mycelial growth, indicating a critical role for calcium in the regulation of C. albicans morphogenesis.     

Effects of culture density on the kinetics of germ tube formation in Candida albicans

The relationship between culture density or phase of growth at 24.5 degrees C and the ability of Candida albicans to form germ tubes when shifted to 37 degrees C was investigated. Evidence is presented demonstrating germ tube production from liquid synthetic medium cultures at all phases of growth. Previous studies reported that only cells from stationary phase cultures were competent to form germ tubes. Comparisons between exponential and stationary phase cultures indicate more rapid and more synchronous germ tube production from cells growing in the exponential phase.     

Candida albicans VPS11 is required for vacuole biogenesis and germ tube formation

The Candida albicans vacuole has previously been observed to undergo rapid expansion during the emergence of a germ tube from a yeast cell, to occupy the majority of the parent yeast cell. Furthermore, the yeast-to-hypha switch has been implicated in the virulence of this organism. The class C vps (vacuolar protein sorting) mutants of Saccharomyces cerevisiae are defective in multiple protein delivery pathways to the vacuole and prevacuole compartment. In this study C. albicans homologues of the S. cerevisiae class C VPS genes have been identified. Deletion of a C. albicans VPS11 homologue resulted in a number of phenotypes that closely resemble those of the class C vps mutants of S. cerevisiae, including the absence of a vacuolar compartment. The C. albicans vps11Delta mutant also had much-reduced secreted lipase and aspartyl protease activities. Furthermore, vps11Delta strains were defective in yeast-hypha morphogenesis. Upon serum induction of filamentous growth, mutants showed delayed emergence of germ tubes, had a reduced apical extension rate compared to those of control strains, and were unable to form mature hyphae. These results suggest that Vps11p-mediated trafficking steps are necessary to support the rapid emergence and extension of the germ tube from the parent yeast cell.     

Septin function in Candida albicans morphogenesis

The septin proteins function in the formation of septa, mating projections, and spores in Saccharomyces cerevisiae, as well as in cell division and other processes in animal cells. Candida albicans septins were examined in this study for their roles in morphogenesis of this multimorphic, opportunistically pathogenic fungus, which can range from round budding yeast to elongated hyphae. C. albicans green fluorescent protein labeled septin proteins localized to a tight ring at the bud and pseudohyphae necks and as a more diffuse array in emerging germ tubes of hyphae. Deletion analysis demonstrated that the C. albicans homologs of the S. cerevisiae CDC3 and CDC12 septins are essential for viability. In contrast, the C. albicans cdc10Delta and cdc11Delta mutants were viable but displayed conditional defects in cytokinesis, localization of cell wall chitin, and bud morphology. The mutant phenotypes were not identical, however, indicating that these septins carry out distinct functions. The viable septin mutants could be stimulated to undergo hyphal morphogenesis but formed hyphae with abnormal curvature, and they differed from wild type in the selection of sites for subsequent rounds of hyphal formation. The cdc11Delta mutants were also defective for invasive growth when embedded in agar. These results further extend the known roles of the septins by demonstrating that they are essential for the proper morphogenesis of C. albicans during both budding and filamentous growth.     

Gratuitous induction by N-acetylmannosamine of germ tube formation and enzymes for N-acetylglucosamine utilization in Candida albicans.

N-Acetylmannosamine did not support the growth of Candida albicans, and this sugar was not accumulated by cells. Incubation of starved yeast cells at 37 degrees C with N-acetylmannosamine plus glucose resulted in germ tube formation. Furthermore, N-acetylmannosamine alone induced the uptake system for N-acetylglucosamine and the enzymes of the N-acetylglucosamine catabolic pathway to the same extent as the natural substrate. Induction of the uptake system and the enzymes was observed at 28 degrees C without germ tube formation and at 37 degrees C with germ tube formation. N-Acetylmannosamine is thus a gratuitous inducer for enzymes of the N-acetylglucosamine pathway and germ tube formation in C. albicans.     

A G1 Cyclin Is Necessary for Maintenance of Filamentous Growth in Candida albicans

Candida albicans undergoes a dramatic morphological transition in response to various growth conditions. This ability to switch from a yeast form to a hyphal form is required for its pathogenicity. The intractability of Candida to traditional genetic approaches has hampered the study of the molecular mechanism governing this developmental switch. Our approach is to use the more genetically tractable yeast Saccharomyces cerevisiae to yield clues about the molecular control of filamentation for further studies in Candida. G1 cyclins Cln1 and Cln2 have been implicated in the control of morphogenesis in S. cerevisiae. We show that C. albicans CLN1 (CaCLN1) has the same cell cycle-specific expression pattern as CLN1 and CLN2 of S. cerevisiae. To investigate whether G1 cyclins are similarly involved in the regulation of cell morphogenesis during the yeast-to-hypha transition of C. albicans, we mutated CaCLN1. Cacln1/Cacln1 cells were found to be slower than wild-type cells in cell cycle progression. The Cacln1/Cacln1 mutants were also defective in hyphal colony formation on several solid media. Furthermore, while mutant strains developed germ tubes under several hypha-inducing conditions, they were unable to maintain the hyphal growth mode in a synthetic hypha-inducing liquid medium and were deficient in the expression of hypha-specific genes in this medium. Our results suggest that CaCln1 may coordinately regulate hyphal development with signal transduction pathways in response to various environmental cues.     

Ethanol-induced germ tube formation in Candida albicans

Ethanol is the first reported compound which can induce germ tube formation in Candida albicans without the addition of any nitrogen-containing nutrients. Conditions controlling induction of germ tubes in C. albicans by ethanol were investigated. Ethanol (17.1 mM) in buffered salts solution containing sodium bicarbonate induced 70 to 80% of yeast phase cells of C. albicans to form germ tubes. Germ tubes could be induced by ethanol (0.08 to 340 mM) at temperatures ranging from 29 to 41 degrees C (optimum 37 degrees C) and at pH values ranging from 3.0 to 8.0 (optimum 5.75). The germ tubes averaged 11 micron in length after 6 h at 37 degrees C. The percentage of cells forming germ tubes decreased as the concentration of cells in the induction solution was increased above 4 X 10(5) cells ml-1. Germ tubes first appeared 45 to 60 min after continuous exposure to ethanol at 37 degrees C and all cells which formed germ tubes did so by 2 h. Germ tube length decreased as the pH was increased but was independent of the concentration of ethanol. Oxygen was required for germ tube formation. In addition to ethanol, 1-propanol, 2-propanol, 1-butanol and acetic acid could induce germ tube formation, whereas methanol could not. These results indicate that the cells must mobilize their endogenous nitrogen and probably carbohydrate reserves in order to initiate formation of germ tubes. The evidence is inconclusive as to whether ethanol itself must be metabolized for germ tube induction to occur, although it is not thought to act by a nonspecific interaction with the cell membrane.     

Candida albicans INT1-induced filamentation in Saccharomyces cerevisiae depends on Sla2p

The Candida albicans INT1 gene is important for hyphal morphogenesis, adherence, and virulence (C. Gale, C. Bendel, M. McClellan, M. Hauser, J. M. Becker, J. Berman, and M. Hostetter, Science 279:1355-1358, 1998). The ability to switch between yeast and hyphal morphologies is an important virulence factor in this fungal pathogen. When INT1 is expressed in Saccharomyces cerevisiae, cells grow with a filamentous morphology that we exploited to gain insights into how C. albicans regulates hyphal growth. In S. cerevisiae, INT1-induced filamentous growth was affected by a small subset of actin mutations and a limited set of actin-interacting proteins including Sla2p, an S. cerevisiae protein with similarity in its C terminus to mouse talin. Interestingly, while SLA2 was required for INT1-induced filamentous growth, it was not required for polarized growth in response to several other conditions, suggesting that Sla2p is not required for polarized growth per se. The morphogenesis checkpoint, mediated by Swe1p, contributes to INT1-induced filamentous growth; however, epistasis analysis suggests that Sla2p and Swe1p contribute to INT1-induced filamentous growth through independent pathways. The C. albicans SLA2 homolog (CaSLA2) complements S. cerevisiae sla2Delta mutants for growth at 37 degrees C and INT1-induced filamentous growth. Furthermore, in a C. albicans Casla2/Casla2 strain, hyphal growth did not occur in response to either nutrient deprivation or to potent stimuli, such as mammalian serum. Thus, through analysis of INT1-induced filamentous growth in S. cerevisiae, we have identified a C. albicans gene, SLA2, that is required for hyphal growth in C. albicans.     

Involvement of calcium, calmodulin and protein phosphorylation in morphogenesis of Candida albicans

N-Acetyl-D-glucosamine-induced germ tube formation in Candida albicans at 37 degrees C was accompanied by an increase in the rate of protein phosphorylation. The calmodulin antagonist trifluoperazine and the Ca2+ ionophore A23187, which inhibited germ tube formation, also reduced the rate of phosphorylation. The rate of phosphorylation was also reduced when cells were incubated at 25 degrees C, which favoured yeast-phase growth. Two-dimensional SDS-PAGE analysis of phosphoproteins from germ-tube-forming and yeast cells revealed two germ-tube-specific and three yeast-specific phosphoproteins. Germ tubes and hyphae had more calmodulin activity than yeast cells, irrespective of the germ-tube-inducing condition used. As a first step towards understanding the inhibitory effect of trifluoperazine on germ tube formation, calmodulin from C. albicans was purified to homogeneity. It was heat stable, and displayed a pronounced Ca2(+)-induced shift in electrophoretic mobility.     

Biosynthesis of specific histones during meiotic prophase of mouse spermatogenesis

A kinetics study has demonstrated histone synthesis occurring at two distinct phases during meiotic prophase of mouse spermatogenesis. These two periods have been delineated by quantifying the synthesis of DNA and basic nuclear proteins in spermatogenic cells at discrete intervals following the intratesticular injection of [3H] thymidine and [14C] arginine, respectively. One phase of histone synthesis occurs coincident with DNA synthesis in preleptotene spermatocytes. By contrast, a second phase of histone synthesis occurs during midprophase of meiosis, independent of semiconservative DNA synthesis. The [14C] arginine incorporated into the basic nuclear proteins of pachytene spermatocytes is conserved during spermiogenesis and then subsequently discarded within the residual bodies, which are formed during late spermiogenesis. Fluorographic analyses of isotopically labeled basic nuclear proteins in pachytene spermatocytes has shown that only the somatic complement of histones are synthesized during the preleptotene period, whereas the second phase involves the synthesis of proteins H1t, H2S, and "A". In addition, several nonhistone basic nuclear proteins are synthesized concomitant with the germ cell-specific histones. Thus, the data clearly demonstrate that pachytene spermatocytes actively synthesize a number of novel chromatin-associated polypeptides.     

Strain variation and morphogenesis of yeast- and mycelial-phase Candida albicans in low-sulfate, synthetic medium.

A low-sulfate synthetic medium was developed in which pure cultures of yeast- and mycelial-phase Candida albicans could be cultivated for investigations of the molecular biology of dimorphism. The medium contained ammonium ions, phosphate buffer, salts, glucose, and biotin. Morphogenesis was found to be dependent upon the strain of C. albicans. Of six strains tested in the low-sulfate medium at 37 degrees C, three formed mixed cultures of yeasts, true mycelium and pseudomycelium, two formed pure cultures of true mycelium, and one maintained yeast growth. All six strains produced pure cultures of yeasts at 24 degrees C. The buffering capacity of the medium maintained the pH at 6.9 even at high-density cell growth. The low concentration of sulfate and the absence of amino acids in the medium provided conditions in which to radiolabel cellular constituents with [35S]sulfate. For molecular investigations, the use of two strains is suggested, one forming yeasts and one forming true mycelium in low-sulfate medium at 37 degrees C, thus providing controls for both strain variation and for molecular changes induced by environmental change but unrelated to morphogenesis.     

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).     

Proteomic analysis of the oxidative stress response in Candida albicans

An efficient oxidative stress response (OSR) is important for the facultative pathogenic yeast Candida albicans to survive within the human host. We used a large scale 2-D protein gel electrophoresis approach to analyze the stress response mechanisms of C. albicans after treatment with hydrogen peroxide and the thiol oxidizing agent, diamide. Quantitation of in vivo protein synthesis after pulse labeling of the proteins with radioactive L-[35S]-methionine resulted in characteristic proteome signatures for hydrogen peroxide and diamide with significant overlap of 21 up-regulated proteins for both stressors. Among the induced proteins were enzymes with known antioxidant functions like catalase or thioredoxin reductase and a set of oxidoreductases. 2-D gel analysis of mutants in the CAP1 gene revealed that the synthesis of 12 proteins is controlled by the oxidative stress regulator Cap1p. Stressing its importance for the C. albicans OSR, all 12 proteins were also induced after oxidative challenge by hydrogen peroxide or diamide.