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Morphological commitment in Candida albicans
Abstract
Stationary phase yeast cells of the dimorphic fungus albicans can reinitiate growth under appropriate conditions either as yeasts through bud formation or as hyphae through germ tube formation and elongation. Stationary phase yeast cells resuspended in fresh medium at 37 degrees C form germ tubes and those resuspended at 25 degrees C form buds. Temperature shift experiments have been used to observe when cells become committed to germ tube formation and yeast budding growth under conditions favorable to each form. The two commitment processes appear to be independent and, once initiated, occur at characteristic rates with commitment to germ tube formation preceding commitment to yeast bud formation. The rate of commitment to germ tube formation was consistent with a random process or first-order kinetics. A relationship between cell volume and commitment to yeast growth and bud emergence was consistent with observations of cell volume distribution both in stationary phase cultures and between budded and unbudded cells during resumption of growth at 25 degrees C.
... The opportunistic human pathogen Candida albicans exhibits a very similar commitment phenomenon. Stationary-phase cells are totipotent, whereas logarithmic phase cells are committed [2,3]. Interestingly, C. albicans cells became committed both when temperature was the controlling variable (25°C, yeast; 37°C, mycelia [2]) and when pH was the controlling variable (pH 4.5, yeast; pH 6.5, mycelia [3]). ...
... Stationary-phase cells are totipotent, whereas logarithmic phase cells are committed [2,3]. Interestingly, C. albicans cells became committed both when temperature was the controlling variable (25°C, yeast; 37°C, mycelia [2]) and when pH was the controlling variable (pH 4.5, yeast; pH 6.5, mycelia [3]). Commitment has also been observed in the polymorphic fungus Wangiella dermatitidis [4]. ...
... Commitment has also been observed in the polymorphic fungus Wangiella dermatitidis [4]. So far, the commitment phenomenon has been studied by correlating the time of commitment with the morphological appearance of buds, germ tubes, septa, and nuclear division [1][2][3] and the onset of DNA synthesis [5]. A new approach to the molecular basis of commitment is provided by our recent discovery [6] of the involvement of Ca(II) and the Ca(II)-binding protein calmodulin in the regulation of dimorphism in C. ulmi. ...
The commitment phenomenon exhibited by Ceratocystis ulmi is a manifestation of the requirement for a Ca(II)-calmodulin interaction for mycelial growth. Under otherwise identical conditions, addition of CaCl2(10 mM) to committed yeasts caused them to germinate, while addition of the calmodulin antagonists chlorpromazine (80 μM), dibucaine (750 μM), or trifluoperazine (60 μM) to committed mycelia caused them to bud.
... The relatively constant values for the OD normalized supernatants (Fig. 3F) are consistent with farnesol's function as a QSM. Similarly, the sharp increases in intracellular farnesol during growth (Fig. 3E) provide a reasonable explanation for a longstanding conundrum in fungal dimorphism known as the commitment phenomenon (Mitchell and Soll 1979;Chaffin and Wheeler 1981;Muthukumar and Nickerson 1985). Totipotent cells can differentiate as either yeasts or mycelia depending on the environmental conditions, often temperature, they are experiencing. ...
... Is farnesol involved in the mechanisms by which serum triggers GTF and/or the mechanisms by which repeated cell washes reduce the concentration of serum required for GTF (Ahmad Hussin et al. 2016)? (5) What is the role of Zn(II) in cell growth, farnesol production, and the availability of totipotent cells for studying yeast/mycelia dimorphism (Mitchell and Soll 1979;Chaffin and Wheeler 1981). And (6) more speculatively, is farnesol involved in the white-opaque transition (Soll et al. 2003;Dumitru et al. 2007), or the conversion of commensal cells to pathogenic cells (Hube 2004)? ...
The dimorphic fungus Candida albicans is a commensal and opportunistic fungal pathogen of humans. It secretes at least four small lipophilic molecules, farnesol and three aromatic fusel alcohols. Farnesol has been identified as both a quorum sensing molecule (QSM) and a virulence factor. Our gas chromatography (GC)-based assay for these molecules exhibits high throughput, prevention of analyte loss by avoiding filtration and rotary evaporation, simultaneous cell lysis and analyte extraction by ethyl acetate, and the ability to compare whole cultures with their cell pellets and supernatants. Farnesol synthesis and secretion were separable phenomena and pellet:supernatant ratios for farnesol were high, up to 12:1. The assay was validated in terms of precision, specificity, ruggedness, accuracy, solution stability, detection limits (DL), quantitation limits (QL), and dynamic range. The DL for farnesol was 0.02 ng/µl (0.09 µM). Measurement quality was assessed by the relative error of the whole culture versus the sum of pellet and supernatant fractions (WPS). C. albicans strain SC5314 grown at 30 °C in complex and defined media (YPD and mRPMI) was assayed in biological triplicate 17 times over 3 days. Farnesol and the three aromatic fusel alcohols can be measured in the same assay. The levels of all four are greatly altered by the growth medium chosen. Significantly, the three fusel alcohols are synthesized during stationary phase, not during growth. They are secreted quickly without being retained in the cell pellet and may accumulate up to mM concentrations.
Key points
• Quantitative analysis of both intra- and extracellular farnesol, and aromatic fusel oils.
• High throughput, whole culture assay with simultaneous lysis and extraction.
• Farnesol secretion and synthesis are distinct and separate events.
... Since our publication in 2001, farnesol and its many roles in the biology of C. not know: 1/if there is a receptor/sensor for farnesol; 2/if there is a transport system for farnesol; 3/how farnesol synthesis is regulated [opaque and anaerobic cells turn off synthesis whereas tup1 and nrg1 mutants elevate synthesis 17-19 fold (Kebaara et al., 2008); and 4/what the relationship is between farnesol and commitment. Commitment was defined by Mitchell and Soll (1979) and Chaffin and Wheeler (1981) for C. albicans and Muthukumar and Nickerson (1985) for Ceratocystis ulmi as the point in the events leading to germination or budding at which a cell may no longer choose. These organisms remain committed to a given morphology even in the absence of the original inducing conditions, i.e. resuspension in a growth medium which promotes the alternate morphology. ...
Candida albicans excretes E,E-farnesol as a virulence factor and quorum sensing molecule (QSM) that prevents the yeast to hyphal conversion. Polke et al (2016) identified eed1Δ/Δ as the first farnesol hypersensitive mutant of C. albicans. eed1Δ/Δ also excretes 10X more farnesol and while able to form hyphae, it cannot maintain hyphae. This mutant enables new research into unanswered questions, including the existence of potential farnesol receptors and transporters, regulation of farnesol synthesis, and relationships among farnesol, germ tube formation and hyphal maintenance. The eed1 farnesol hypersensitivity can be explained by higher internal concentrations of farnesol or lower thresholds for response. One possibility invokes misexpression of a transporter. Saccharomyces cerevisiae and C. albicans have transporters for farnesylated peptides, like the a-factor pheromone, which could potentially also transport farnesol for virulence and quorum sensing. Significantly, these transporters are repressed in MATa/MATα C. albicans. An evolutionary pressure for C. albicans to become diploid could derive from its use of farnesol. Alternatively, maintenance of hyphal growth may increase the farnesol response threshold. Finally, Dpp1p, Dpp2p and Dpp3p are non-specific pyrophosphatases responsible for farnesol synthesis. Changes in expression of these enzymes do not explain differences in farnesol levels implicating involvement of additional factors like a scaffolding molecule. This article is protected by copyright. All rights reserved.
... Timing is also important because of the commitment phenomenon. This is the point at which a switch in the environmental stimulus no longer causes the expected switch in morphology (Chaffin and Wheeler, 1981; Mitchell and Soll, 1979; Mosel et al., 2005). It is relevant to farnesol's mode of action because, while farnesol blocks the yeast to filament switch, it does not block the elongation of preexisting filaments (Mosel et al., 2005). ...
Candida albicans is a polymorphic fungus that causes a range of disease in humans, from mucosal infections to systemic disease. Its ability to cause disease is linked to conversion between yeast and filamentous forms of growth, and the first quorum-sensing molecule discovered in an eukaryote, farnesol, blocks this transition. In C. albicans, farnesol also kills mating-competent opaque cells, inhibits biofilm formation, protects the cells from oxidative stress, and can be a virulence factor or protective agent in disseminated and mucosal mouse models of infection, respectively. While much emphasis has been placed on determining its effect on C. albicans morphology, the molecular response to farnesol is not completely understood. The overall theme for this dissertation was to better understand the C. albicans molecular response to farnesol under quorum sensing conditions. Due to the duplicitous nature of the farnesol response in C. albicans, i.e., its ability to kill cells or simply alter morphology, we clearly defined the environmental conditions in which farnesol acts as a quorum sensing molecule or as a toxic agent towards C. albicans. This clarification enabled a subsequent two-pronged approach to study the molecular response to farnesol during morphological regulation. A direct approach was used to investigate the role of a likely candidate, Tup1, a negative regulator of hyphal development, in farnesol signaling. Secondly, a screening approach was utilized to identify new farnesol resistant mutants that may participate in the farnesol response. From the mutants identified, Czf1 (C. albicans zinc finger) was selected for further characterization and was shown to play a vital role in the morphological response to farnesol as well as farnesol tolerance. Overall, this study identified two new factors involved in farnesol signaling, and highlights the power of farnesol as a tool with which to unravel the complex signaling networks present in C. albicans.
... In agreement with previous studies [5,13] there was no evidence for commitment to bud formation prior to the time at which cell evagination took place, since 80-90% of the yeast cells were still able to form germ tubes after 180 min preincubation at the lower temperature or pH. About 80% of the cells had formed an evagination by this time. ...
The rates of germ tube formation from growing and non-growing yeast cells of Candida albicans were investigated using a protocol for dimorphism regulated by temperature and pH. Stationary-phase cells formed germ tubes less rapidly than yeast cells that were preincubated in fresh growth medium prior to induction of dimorphism by an upshift in temperature or pH. On the basis of experiments using inhibitors of macromolecular biosyntheses it is suggested that the accelerated growth kinetics required de novo RNA and protein biosynthesis, but not DNA synthesis. The results suggest that metabolically active yeast cells are better able to undergo dimorphism than non-growing cells.
The dimorphic yeast Candida albicans is capable of growing in culture as an ellipsoidal bud or as an elongate hypha. The growth phenotype depends on environmental conditions and the growth history of the cells (Evans et al., 1974, 1975; Chaffin and Sogin, 1976; Shepherd and Sullivan, 1976; Soll and Bedell, 1978; Mitchell and Soll, 1979a; Odds, 1979; Bell and Chaffin, 1980; Buffo et al., 1984; Soll and Herman, 1983). Although most noted for genital infections, C. albicans can invade a variety of tissues and is one of the most pervasive fungal pathogens in man (Odds, 1979). It is a commensal inhabitant of the human body, increasing in concentration and invading tissue usually in response to changes in the physiology of the host. Although both growth forms are found in infected tissue (Mackenzie, 1964; Odds, 1979), it seems likely that the elongating hypha penetrates tissue, leaving in its path lateral colonies of budding cells that in turn give rise to new penetrating hyphae.
Modes of cell envelope expansion were monitored in developing cells of Candida albicans 73/055 to which polystyrene beads were attached. Eight different conditions of culture medium, pH and temperature were used to promote growth in a variety of morphological forms. The cells were observed microscopically during growth in Sykes-Moore perfusion chambers, and sequential measurements of distances between the bead and the parent cell, and the bead and the apical tip were used to distinguish apical envelope expansion from general envelope expansion. Morphology index (Mi) was determined at each time point as an estimate of each cell's morphology. Calculations based on the measurements showed that general envelope expansion was inversely proportional to Mi, but that general expansion greater than 20% occurred only in cells with a final Mi less than 2.0, indicating that regulation of apical and general envelope expansion alone may be insufficient to determine the different morphologies seen in cells with higher Mi. The rate of expansion of the perimeter of cells was linearly proportional to the final Mi. This observation suggests that commitment to morphological development in C. albicans may in part involve commitment to a rate of envelope expansion, which itself helps determine the final morphology of a cell.
It has been reported that intravenous fat emulsions, because of their isotonicity and neutral pH, support microbial growth, but traditional parenteral nutrition solutions, being hypertonic and more acidic, are not as supportive. To date, few studies have documented microbial growth in total nutrient admixtures (TNA) containing dextrose, amino acids, fat, electrolytes, vitamins, and trace elements.
This study was undertaken to analyze the growth of Staphylococcus aureus, Candida albicans and four gram-negative enteric bacilli in three different nutrient admixtures, with and without the inclusion of 5% fat emulsion. The composition of the admixtures was either 5, 10, or 25% dextrose; either 0 or 5% fat; and 3% amino acids, electrolytes, vitamins, and trace elements. All admixtures were innoculated with 100 colony-forming units per milliliter, incubated at room (25°C) or refrigerated (4°C) temperature, with samples withdrawn at 0, 3, 6, 12, 24, and 48 hours and plated in triplicate.
Only C. albicans demonstrated any significant growth regardless of fat content. The pH of the admixtures was similar (acidic), and all solutions were hypertonic and found to inhibit bacterial growth.
Conclusions suggest that TNA, when formulated with normal concentrations of additives, is no more likely to support growth of contaminant organisms than the traditional solutions. This contradicts the notion that the addition of fat to total parenteral nutrition will enhance the ability of these admixtures to support microbial growth.
To characterize germ tube-specific antigens of Candida albicans, rabbit antiserum prepared to Formalin-treated yeast possessing germ tubes was adsorbed with stationary-phase blastospores. By immunofluorescence and enzyme-linked immunosorbent assay, this antibody did not react with blastospores but detected germ tube-specific antigens in hyphal forms. Germ tube-specific antigens appeared 30 min after placing blastospores in appropriate conditions for germ tube formation. Hyphae, formed by allowing yeast to germinate for 24 h, still retained germ tube-specific antigens, but blastospores budding off these hyphae were unstained, as were log-phase blastospores. Germ tube-specific antigens were sensitive to heat, sodium metaperiodate oxidation, dithiothreitol reduction, and proteolysis with pronase, trypsin, or chymotrypsin, whereas antigens common to blastospores and germ tubes were stable to boiling, treatment with proteolytic enzymes, and dithiothreitol reduction. Thus, surfaces of germ tubes can be distinguished from those of blastospores not only immunologically, but also by the sensitivity of germ tube-specific antigens to proteolytic treatments.
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.
The mean size and percentage of budded and unbudded cells of Candida albicans grown in batch culture over a wide range of doubling times have been measured. Cell volume decreased with increased doubling time and a nonlinear approach to an asymptotic minimum was observed. When cells were separated by age according to bud scars, each age showed a similar decrease. During each cell division cycle, size increased slowly during both budded and unbudded periods so that each generation was significantly larger than the preceding. There was no difference in size between the parent portion of budded cells and unbudded cells of the same age. Time-lapse photomicroscopy of cells growing on solid medium showed that cells divide asymmetrically with larger parents having a shorter subsequent cycle time than the smaller daughter, although the time utilized for bud formation was similar. When cells were shifted from a medium supporting a low growth rate and small size to a medium supporting a faster growth rate and larger size, both budded and unbudded cells increased significantly in size. As the doubling time increased, both the budded and unbudded portions of parental and daughter cycles increased.
Changes in the identity and quantity of proteins synthesized during morphogenesis may result from alterations in gene expression in the dimorphic yeast Candida albicans. Stationary phase yeast cells, upon resuming growth at 25 degrees C, form budding yeast and at 37 degrees C form germ tubes. In order to identify proteins associated with morphogenesis, we compared cytoplasmic proteins synthesized during germ tube and bud formation. Proteins synthesized during this period were labeled at four intervals with either [3H]leucine or [35S]methionine and separated by two-dimensional polyacrylamide gel electrophoresis. This study shows that, of the 230 proteins resolved on each gel, 5 were specific to the yeast morphology and 2 proteins showed reduction in net synthesis in the mycelial phase. There were, however, no mycelium-specific proteins at any labeling period. The majority of proteins were common to both morphologies and showed no major shift in number during resumption of growth. The observations reported here suggest that differential gene expression occurs during morphogenesis of C. albicans.
Candida albicans from a patient with dental caries grew on minimal medium consisting of agar supplemented with magnesium chloride and sodium phosphate. Hyphal growth was observed when the yeast was cultured between 26 degrees C and 28 degrees C under aerobic conditions, and typical chlamydospores were formed. However, when the yeast was cultured at the same temperature under anaerobic conditions, curly hyphae developed on the surface of the medium, but no chlamydospores were formed. This phenomenon was also observed if the culture was started under aerobic conditions but was continued under anaerobic conditions.
Production of farnesol by Candida albicans is the first quorum- sensing system discovered in a eukaryote (29). In C. albicans, accumulated farnesol affects both dimorphism (29, 50) and biofilm formation (62). Fungal dimorphism is defined (64) as an environmentally controlled reversible interconversion of morphology, particularly yeast and mycelial morphologies. Interest in this shift derives from the dimorphic character of many fungi that are pathogenic toward plants and animals (64). Numerous chemical and environmental parameters can shift the yeast-mycelium dimorphism, including temperature, pH, glucose levels, nitrogen source, carbon dioxide levels, transition metals, chelating agents, and inoculum size or initial cell density (64). Of these, the inoculum size effect is probably the least well studied. For fungi such as Ceratocystis ulmi (28, 42) and C. albicans (29), cells develop as budding yeasts when inoculated at ≥106 cells per ml and as mycelia when inoculated at <106 cells per ml. We believe the inoculum size effect is a general phenomenon in dimorphic fungi (Table 1). In keeping with the precedent established by homoserine lactone-based signaling in gram-negative bacteria (22), the inoculum size effect in fungi is also called quorum sensing (29) and the extracellular cell density-dependent signals are called quorumsensing molecules (QSMs). Thus, the chemical identity of the respective QSMs is of interest. Apart from C. ulmi (28) and C. albicans (29), it is a “leap of faith” on our part that the other cell density phenomena listed in Table 1 are mediated by QSMs.
Candida albicans is a dimorphic fungus that can interconvert between yeast and filamentous forms. Its ability to regulate morphogenesis is
strongly correlated with virulence. Tup1, a transcriptional repressor, and the signaling molecule farnesol are both capable
of negatively regulating the yeast to filamentous conversion. Based on this overlap in function, we tested the hypothesis
that the cellular response to farnesol involves, in part, the activation of Tup1. Tup1 functions with the DNA binding proteins
Nrg1 and Rfg1 as a transcription regulator to repress the expression of hypha-specific genes. The tup1/tup1 and nrg1/nrg1 mutants, but not the rfg1/rfg1 mutant, failed to respond to farnesol. Treatment of C. albicans cells with farnesol caused a small but consistent increase in both TUP1 mRNA and protein levels. Importantly, this increase corresponds with the commitment point, beyond which added farnesol no
longer blocks germ tube formation, and it correlates with a strong decrease in the expression of two Tup1-regulated hypha-specific
genes, HWP1 and RBT1. Tup1 probably plays a direct role in the response to farnesol because farnesol suppresses the haploinsufficient phenotype
of a TUP1/tup1 heterozygote. Farnesol did not affect EFG1 (a transcription regulator of filament development), NRG1, or RFG1 mRNA levels, demonstrating specific gene regulation in response to farnesol. Furthermore, the tup1/tup1 and nrg1/nrg1 mutants produced 17- and 19-fold more farnesol, respectively, than the parental strain. These levels of excess farnesol are
sufficient to block filamentation in a wild-type strain. Our data are consistent with the role of Tup1 as a crucial component
of the response to farnesol in C. albicans.
In this analysis we have examined in detail the effects of low concentrations of zinc on the growth and dimorphism of Candida albicans. Evidence is presented that micromolar concentrations of zinc added to growth cultures grown at 25 degrees C (i) cause a twofold increase in the final concentration of spheres at sationary phase, (ii) result in an asynchronous block in the budding cycle at stationary phase, (iii) completely suppress mycelium formation in two independently isolated human strains which produce low but significant levels of mycelia at stationary phase, and (iv) completely suppress mycelium formation in cultures of mutant M10, in which over 60% of the cells form mycelia at stationary phase. In contrast, micromolar concentrations of zinc do not inhibit mycelium formation induced by releasing cells from stationary-phase cultures into fresh medium at 37 degrees C. In addition, if zinc is present in the growth medium of the initial culture at 25 degrees C, the average time of subsequent mycelium formation after release into fresh medium at 37 degrees C is halved. It is demonstrated that the above effects are specific to zinc. The possibility of alterante pathways for mycelium formation is suggested, and the medical implications of this possibility are discussed.
The kinetics of bud formation at 25 "C and germ tube formation at 37 "C were examined in populations of Candida aZbicuns released from stationary phase by dilution into fresh nutrient medium. Evidence is presented for four separate isolates and three different defined media that: (i) cells do not deplete the media of growth-limiting nutrients when they enter stationary phase; (ii) cells accumulate as singlets when they enter stationary phase, pre-sumably at a point early in the cell cycle; (iii) daughter cells do not separate from mother cells during the first three to four cell divisions following release from stationary phase at 25 "C; and (iv) released cells cannot be induced to form germ tubes by an increase in tem-perature once they have formed their first bud at 25 "C. The relationships between the lack of cell separation, the inducibility of tube formation and stationary phase are discussed.
Cells of the pathogenic yeast Candida albicans accumulate as unbudded singlets at stationary phase in defined medium at 25 °C. When released into fresh medium at 37 °C and pH 6.5, these cells will synchronously form elongate pseudomycelia, and when released into fresh medium at either 25 °C, pH 6.5, or 37 °C, pH 4.5, they will synchronously form buds. Using pH and temperature shift experiments, we have examined when cells become committed to pseudomycelium formation and bud formation under conditions conducive to each growth form respectively. It is demonstrated that in either case commitment occurs long after release from stationary phase, at approximately the same time the first evagination is visible on the cell's surface. In addition, it is demonstrated that once a released cell has formed a bud, it and its progeny lose the capacity to form pseudomycelia until they re-enter stationary phase; on the other hand, elongating pseudomycelia retain the capacity to form buds. The possible relationships of the commitment events to septation and to the cell cycle are discussed.
The filamentous growth cycle in C. albicans was resistant to changes in environment brought about either by the serial transfer of growing cells to fresh nutrients or by sudden changes of temperature after the first h of growth. In further experiments older culture filtrates, exhausted of their ability to induce mycelial growth, were reactivated by addition of fresh nutrients or water.
The data provided evidence against the existence of both a mycelial stimulatory and inhibitory compound in the growth medium. It is concluded that although the environment initially dictates what proportion of blastospores are committed to filamentation it has no further effect on the process.
Yeast cell growth and cell division are normally coordinated. The mechanism of this coordination can be examined by attempting to dissociate the two processes. We have arrested division (with the use of temperature-sensitive cell division cycle mutants) and observed the effect of this arrest on growth, and we have limited growth (by nutritional deprivation) and observed the effect of this limitation on division. Mutants blocked at various stages of the cell cycle were able to continue growth, as evidenced by increases in cell volume, mass, and protein content. Cells that had initiated cell cycles were able to complete their cycles and arrest in G1 even when growth was very severely restricted. Under these conditions the daughter cells produced were abnormally small. Such abnormally small cells did not initiate new cell cycles (i.e., did not bud or complete any of the three known gene-controlled steps (cdc28, cdc4, cdc7) in G1) after nutrients were restored until growth to a critical size had occurred. We have also prepared abnormally large cells (by arresting division temporarily with the appropriate mating pheromone); when such cells were allowed to bud, the buds produced were much smaller than the mother cells when cytokinesis occurred. We propose that the normal coordination of cell growth with cell division is a consequence of the following two relationships. (1) Growth, rather than progress through the DNA-division cycle, is normally rate-limiting for cell proliferation. (2) A specific early event in G1, at or before the event controlled by the cdc28 gene product, cannot be completed until a critical size is attained.
Homogenous cell populations of increasing cell volume may have been isolated from exponential and stationary culture of Candida albicans by centrifugation on a sucrose gradient. Observations of the yeast-mycelial transition using these populations showed the following. (i) No fraction from early logarithmic phase cells was unable to undergo morphological transition. (ii) The time of initiation of germ tube production was correlated with cell size in stationary-phase cultures. (iii) The rate of appearance of germ tubes was nearly identical in all fractions measured. (iv) Addition of N-acetyl-D-glucosamine to homogeneous cell populations decreased the time of initial appearance of germ tubes but did not affect the rate of appearance after initiation.
Synchronous cultures of yeast and hyphal phases of Candida albicans showed exponential increases in RNA content and stepwise exponential increases in DNA content. The periods of DNA synthesis in the two phases coincided with one another and with the budding peaks of the yeast phase. Hyphae grown in synchronous cultures also showed an exponential increase in length. The hyphal phase was therefore normal. Hyphal nuclear division occurred after hyphal DNA synthesis. Germination was a unique event for a hypha and unlike yeast bud formation, preceded the first period of DNA synthesis. The exponential increase in RNA and DNA in asynchronous cultures of hyphae in serum paralleled the exponential increase in the numbers of cells in asynchronous cultures of yeasts in defined media. Thus there are no factors in serum which inhibit the normal exponential growth of C. albicans.
A chemically defined medium composed of 6 amino acids, biotin, inorganic salts and glucose for the growth of yeast and mycelial phases of Candida albicans at 25 degrees C and 37 degrees of C respectively was developed based on the aminopeptidase(s) profile of the fungus. This medium has proved successful in maintaining the growth characteristics of both phases during serial transfers. The relative pathogenicity, virulence, invasiveness and immunogenicity of the yeast and mycelial phases are discussed.
SUMMARY The mycelial and blastospore forms of Candida albicans have been grown under conditions in which the only environmental variable was temperature. The activity of phosphoglucose isomerase, phosphofructokinase and the first enzyme of the hexose-monophosphate pathway and the pathways for chitin and mannan synthesis has been determined in extracts at different times in the cell cycle. Phosphofructo- kinase has been partially purified from both forms and the effect of adenosine phosphates on its activity determined. In this yeast, concentrations of glucose-6- phosphate, fructose-6-phosphate, ATP, ADP and AMP differed in the two growth forms and at different times in the growth cycle. ~-Glutamine-~-fructose-6- phosphate aminotransferase activity at concentrations of fructose-6-phosphate found in the cell was appreciably greater in mycelium than in blastospores. The metabolism of (14C)glucose through the hexose monophosphate pathway was low after 4 and I 8 h growth, and considerably increased at 10 h. The results support the concepts of a requirement for NADPH for cell division and suggest that control over the synthesis of chitin and mannan may, in part, be provided through control of the activity of phosphofructokinase by adenosine phosphates.
Five liquid media were tested for their ability to promote filamentation in Candida albicans. Three isolates, including one atypical variant, all developed mycelium in the early stages of growth. The proportion of mycelium produced was highest in the complex media with slightly alkaline pH values (7·5 to 8·6). The time at which mycelial development reached a peak and gave way to the production of budding yeast cells was directly proportional to the minimum doubling time of C. albicans in each medium. The principal effect of the medium was initially to induce filamentation in a greater or lesser number of blastospores.
Fünf flüssige Medien wurden hinsichtlich ihrer Fähigkeit, Pilzfäden bei C. albicans zu bilden, geprüft. Drei Isolate einschließlich einer atypischen Art waren zur Mycelentwicklung in frühen Wachstumsstadien befähigt. Der Anteil des produzierten Mycels war im komplexen Medium mit einem leicht alkalischen pH Wert am größten. Der Zeitpunkt, an dem die Mycelentwicklung ihren Höchststand erreichte und von der Produktion von Sproßzellen abgelöst wurde, stand in jedum Medium in direkter Beziehung zur minimalen Generationszeit von C. albicans. Der Haupteffekt des Mediums war initial die Induktion von Pilzfädenbildung bei einer minderen oder größeren Anzahl von Blastosporen.
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