INFECIMON AND IMMUNrTy, July 1975, p. 119-127 Copyright 0 1975 American Society for Microbiology

Vol. 12, No. 1

Printed in U.SA.

Factors Affecting Filamentation in Candida albicans: Changes in Respiratory Activity of Candida albicans During Filamentation G. A. LAND,I W. C. McDONALD,2 R. L. STJERNHOLM, AND L. FRIEDMAN* Departments of Microbiology and Immunology*; Biology; and Biochemistry, Tulane University School of Medicine, New Orleans, Louisiana 70112

Received for publication 26 February 1975

Glucose metabolism and respiration of Candida albicans were compared under conditions which permitted either maximal filamentous or maximal yeast growth. Changes in metabolism were monitored by comparing the quantities of ethanol produced, CO2 evolved, and oxygen consumed. Filamenting cultures produced more ethanol and less CO2 than yeasts, with oxygen consumption in the former concomitantly slower than that of the latter. Studies involving cofactors and inhibitors associated with electron transport imply that a transfer of electrons away from flavoprotein is required for maintenance of yeast morphology. Conditions consistent with a buildup of reduced flavoprotein, however, favored filament formation. These changes were expressed metabolically as a shift from an aerobic to a fermentative metabolism. The results presented are consistent with hypotheses correlating filament production with changes in carbohydrate metabolism and an interruption of electron transfer within the cell.

Candida albicans becomes filamentous under a variety of seemingly unrelated in vitro conditions, such as partial anaerobiosis (45), presence of glucose in the medium (45; A. J. E. Barlow, and F. W. Chattaway, Proc. Int. Soc. Human Animal Mycol., 1971, p. 49-50), or increased atmospheric CO2 (31, 44). Recent investigations have focused on in vitro stimulation of filaments by available nutrients (2, 5, 14, 36) as well as the effects of host factors in vivo on Candida morphology (30, 38). In the latter, not only is the nature of the invaded tissue important (4, 29, 39), but also the pH of the tissue fluid (14; Barlow and Chattaway, 1971), 02 tension (21, 31), and immunocompetence of the host (3, 9, 24, 28). In a previous paper (27), we reported the effect of selected nutrients upon morphogenesis of C. albicans. In essence, C. albicans became filamentous in a minimal medium containing small amounts of glucose and biotin when proline was the sole nitrogen source. Conversely, the yeast phase was maintained if the same salts-glucose-biotin medium contained NH4Cl instead of proline as a nitrogen source. Relatively high concentrations of glucose, however, in combination with the same concentra'Present address: Department of Microbiology, Wadley Institutes of Molecular Medicine, Dallas, Tex. 75235. 2Present address: Department of Biology, Univ. of Texas, Arlington, Tex. 76019.

11l

tion of NH-Cl provided conditions conducive to filamentation. It seemed reasonable that both glucose and proline, among other compounds, might well be associated with filamentous growth by virtue of their activities during metabolism, i.e., both may cause mitochondrial repression and a concomitant decrease in Kreb's cycle activity. In fact, radioactively labeled proline was incorporated rapidly, although for only a short time, into Kreb's cycle intermediates of C. albicans (27). We considered that perhaps the reduction in rate of incorporation came about because high levels of reduced pyridine nucleotides, generated by proline catabolism, forced an increase in oxidative phosphorylation which eventually caused repression of the mitochondria and Kreb's cycle activity, a phenomenon known as the Crabtree effect (11). High levels of glucose also have been shown to repress mitochondria, thus Kreb's cycle activity, in Saccharomyces cerevisiae (12) and in Ehrlich's ascites tumor cells (11, 26) by the Crabtree effect. It was our hypothesis that a mechanism similar to the Crabtree effect (27) exists in C. albicans and is related to changes in morphology. The following report, therefore, further defines the possible relationship of mitochondrial function with morphogenesis, by determining respiratory activity of C. albicans during the transition from yeast to filament.

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MATERIALS AND METHODS Cultural conditions. The source of C. albicans 5865 and the cultural methods used for its maintenance have been descrihed previouslv (27). Briefly, phosthe yeast was maintained by cultivation phate-buffered saline (13) containing 10-2 M NH4C1 as a nitrogen source, 102 M glucose, and 250 Ag of biotin per liter. If 10-2 M proline replaced NH4Cl in the above glucose-salts-biotin medium used to promote yeast growth, filamentous forms were obtained. The NH4Cl medium for yeast-phase maintenance was modified in three instances: (i) to contain 1 M glucose instead of 10-2 M, in order to promote moderate filamentation, (ii) to reduce the concentration of glucose to 10-3 M, thereby facilitating the uptake of labeled glucose, and (iii) to replace glucose by various Kreb's cycle intermediates. The proline medium (for promoting filamentous growth) was modified in two instances, by reducing the glucose concentration to 10f 3 M, in order to: (i) increase the utilization of mitochondrial derepressors in one experiment and (ii) increase the uptake of labeled glucose by Candida, during experiments designed to follow CO2 evolution during germination. Both series of experiments are described in detail below. All cultures were incubated at 37 C for 6 h, as previous work had demonstrated that filamentation had certainly occurred by that time (27). Methods for enumerating the percentage of either filaments or yeasts also have been described previously *(27). Alterations in mitochondrial activity. For these studies, both solid (1.5% agar) and liquid media were used in parallel, and all cultures were incubated for 6 h at 37 C. Ethidium bromide (Boot's, Ltd., London, England), acridine orange (Sigma Chemical Co., St. Louis, Mo.), and quinacrine hydrochloride (Sigma), known inhibitors of mitochondrial synthesis in other fungi (37), were added to the basal proline medium in concentrations ranging from 102 to 1 mM. Chloramphenicol (Parke, Davis and Co.), which has been found to decrease mitochondrial protein synthesis in Saccharomyces by one-half its normal rate (10), was used in concentrations ranging from 0.1 to 4 mg per ml of medium. The organism was grown also in a proline medium containing 10-3 M glucose, rather than 10-2 M, and decreasing concentrations (1 to 10- " M) of mitochondrial derepressors, viz, iodoacetic acid (IAA, Aldrich Biochemicals, Milwaukee, Wisc.) and 2-deoxyglucose (2-DG, Aldrich Biochemicals). Both compounds are known to release repressed mitochondria (26). Changes in respiratory activity were monitored by overlaying the agar cultures with phenyltetrazolium blue (35). Oxygen uptake. The rate of oxygen consumption of the two morphological forms was determined on a YSI model 53 oxygen monitor (Yellow Springs Instrument Co., Yellow Springs, Ohio), equipped with a Clarke-type oxygen electrode and a 10-inch linear recorder (Beckman). Five-milliliter volumes of medium (either proline for filaments or the NH4Cl for yeasts) were placed aseptically into sterile 25-ml reaction chambers which were then stoppered. The chamber cultures were shaken manually until the medium was saturated with oxygen and then placed in a 37 C water bath agitating at 120 rpm throughout the experiment. The temperature inside the chambers was measured in

until it had equilibrated with that of the bath, at which time the monitor and recorder were set at 100% oxygen saturation and each chamber was inoculated with 10, yeast cells suspended in 0.1 ml of saline. Electrodes were placed in the chambers, all air bubbles were removed, and 5-min readings were taken at intervals of 30 min, from time 0 through 120 min. The average rate of oxygen consumption was determined during the 5min test interval, and data were expressed as nanomoles of O2 consumed per minute per microgram (dry weight) of cells. After each sampling, the chambers were again allowed to equilibrate with the surrounding air. Additional determinations of oxygen consumption were done in either the proline or NH4Cl medium to which had been added various Kreb's cycle intermediates, electron donors, electron acceptors, electron transport uncouplers, and amino acids (see Table 1 for concentrations of each). Trapping of metabolic CO2. To determine the rate at which glucose was metabolized to CO and water, 5 sCi of n-j-14C]glucose in sterile aqueous solution (specific activity, 8 mCi/mmol; New England Nuclear, Boston, Mass.) was added to 5 ml of proline medium (containing an inoculum of 106 yeasts/ml) in a scintillation vial (27). To allow more uptake of labeled glucose, the concentration of unlabeled glucose in the medium was reduced to 10-3 M, a concentration found neither to limit growth nor to change morphology of the fungus (27). Radiolabeled metabolic CO, was trapped by carbonate formation on a folded piece of absorbent paper (Gelman Instrument Co., Ann Arbor, Mich.; 3/8 by 11 inches [ca. 0.91 by 28 cm]) saturated with 1 ml of 2 N NaOH. The folded strip was impaled on the end of an 18-gauge needle which had been inserted through the soft top of the scintillation vial. The needle was then curved to prevent the strip from falling into the medium and the hub end of the needle was sealed with molten wax to guard against contamination. Strips from parallel cultures were removed at 2.5, 10, 15, and 30 min and then at 30-min intervals up to 120 min, placed in 15 ml of Aquasol (New England Nuclear), and counted on a Beckman LS230 liquid scintillation system. Production of ethanol. Fermentative metabolism was monitored in both yeast and filamentous cells by assaying the amount of ethanol produced at various times after inoculation into either the proline (for filaments) or NH4Cl (for yeast) medium. Cultures were harvested in triplicate at intervals of 30 min up to 6 h, by membrane filtration (Millipore Corp., 0.45 Mm pore size) at 0 C. The culture fluid was retained in teflon-sealed, screw-capped culture tubes (Kimble Glass and Plastic, Division of Owens-Illinois, Toledo, Ohio) and were stored frozen. The concentration of ethanol in each sample was determined in triplicate by gas-liquid chromatography by a modification of the method of Cecchini and O'Brien (8). Briefly, 2 Ml of the filtrate was injected into a Beckman GC-25 gas chromatograph equipped with a dual-flame ionization detector and a 7-foot (213.36 cm) stainless-steel column pacKeac with Porapak 60/80 mesh, (Waters Assoc., Rochester, Minn.). The operating conditions were as follows: column temperature, 125 C; detector, 250 C; detector line, 220 C; inlet, 190 C; helium, 50 ml/min; H2, 45 ml/min; air, 275 ml/min. At the time of

FILAMENTATION IN C. ALBICANS

VOL. 12, 1975 assaying the filtrates, known concentrations of ethanol were run to verify identity of the experimental peaks. Under these conditions, ethanol had a retention time ot 90 s. The peaks generated by chromatographing known concentrations of ethanol were cut out and weighed, and the peak weights were plotted in a standard curve. In similar fashion, the peak weights of the experimental filtrates were determined, quantitation being based on a comparison of these values with the standard curve.

RESULTS

Filamentation and mitochondrial activity, C. albicans was grown as a yeast or filament in the proline or NH4Cl medium containing vwrious inhibitors or derepressors of mitochondrial activity (Table 1). With increasing concentrations of dyes, known to intercalate with cell-free

121

mitochondrial deoxyribonucleic acid from other yeasts, C. albicans lost its ability to develop filaments (Fig. 1). Chloramphenicol in all concentrations used had a very small depressing effect on filamentation (insert, Fig. 1). The addition of IAA or 2-DG, glycolytic inhibitors, prevented filamentation of Candida grown under conditions in which it would normally occur (Fig. 2). When filamentation occurred in a solid medium containing mitochondrial inhibitors, and the culture was overlayed with tetrazolium blue, there was no reduction of the dye indicating a loss of respiratory activity (26). Cultures on solid medium, containing mitochondrial derepressors, reduced the tetrazolium overlay, suggesting some respiratory activity present in the cells. When the concentrations of IAA and 2-DG were above millimolar concentration in the

TABLE 1. Effect of various electron donors, acceptors, or electron transport uncouplers upon morphology and oxygen consumption of C. albicans Proline medium

NH4CI medium Category of compound

Metabolite or inhibitora

Filament

ju

(%b

Tricarboxylic acid intermediate or glycolytic productsd

Glycolysis inhibitorse Electron donors or acceptorse

Nucleotidese

Metabolic inhibitorse

Ethanol Acetate Citrate

a-Ketoglutarate Ascorbate Succinate Iodoacetate 2-DG CoASH NAD NADH2 NADP FAD Glutathione-SH Methylene blue AMP ADP ATP Cyclic AMP KCN Antimycin A Amytal

Quinacrine Rotenone Dicumarol HOQNO DNP

of 0, consumed Filaments '1 of 0, consumed (to:tI2o) (to tI2OY I%

0 1 2 42

0.5:0.6 0.5:0.4 0.8:0.4 0.8:0

92 6 91 87

61

0.2:0

41

29 56 0 1 82 62 62 78 56 44 0 55 66 7 61 35 0 44 0 52 89 34 46

0.7:0.1 1.2:0.1 0.5:0.2 0.9:0.4 1.8:0 0.5:0 0.9:0 1.8:0 1.1:0 0.9:0 0.2:0.2 0.5:0 0.6:0 0.5:0.3 0:0 0:0 0.5:0.4 0.5:0 0:0 0.7:0 0.9:0 0.6:0 0:0

45 49 3 6 93 89 92 98 97 94 9 99 100 42 100 62 92 100 0 100 100 98 97

1.3:0 0.8:0.4 0.8:0 0.2:0 0.7:0.1 0.9:0.1 0.2:0.1 1.2:0.4 0.6:0.2 1.8:0 0.5:0 1.2:0 1.6:0 1.5:0 2.0:0 0.1:0.1 0.6:0 0.9:0 0.9:0.5 0.4:0 0:0 1.2:0 1.7:0 0:0 1.3:0 0.9:0 1.2:0 0:0

a Abbreviations: CoASH, coenzyme A sulthydryl; NAD, nicotinamide adenine dinucleotide; NADHI, nicotinamide- adenine dinucleotide, reduced form; NADP, nicotinamide adenine dinucleotide phosphate; FAD, flavine adenine dinucleotide; AMP, adenosine 5W-monophosphate; AMP, adenosine 5'-diphosphate; ATP, adenosine 5'-triphosphate; DNP, 2,4-dinitrophenol; 2-DG, 2-deoxyglucose; KCN, potassium cyanide. b Determined after 6 h of growth. c Ratio of the number of microliters of 02 consumed at 2.5 min in culture compared to that consumed at 120 min. dGlucose replaced in the medium with intermediates in 10-2 M concentrations. eConcentration (100 ,g/ml).

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LAND ET AL.

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MILLIMOLAN CONCENTRATION OF INHIBITOR 1106 I

FIG. 1. Effect of mitochondrial inhibitors upon filamentation in C. albicans. Intercalating dyes in a 4 ~ basal medium containing 10- 2 M proline and 10- 2 M glucose; ethidium bromide (*) acridine orange (-0), and quinacrine (O). Chloramphenicol in the basal medium (a) (in-sert). Filamentation in the basal medium without inhibitors was 92% -4 0.07. O1_--*->,__,_

about one-third the oxygen used by prolinegrown filaments at 2.5 min, and maintained that rate throughout the 120-min experimental period. C. albicans growing in a proline medium with a reduced glucose concentration (10-3 M) and either IAA or 2-DG (100 ug/ml) consumed oxygen at a rate different from that of a control grown in the same medium without the glycolytic inhibitors (Fig. 3). A burst of respiratory activity appeared at 2.5 min in the cultures containing these mitochondrial derepressors, but was significantly lower (P < 0.01) than the proline control without derepressors. By 30 min, the respiratory rate of cultures with either derepressor had dropped to a rate near that of control yeasts cultivated in the NH,Cl medium. The respiratory rate of cultures with derepressors had stabilized by 60 min and paralleled that of the yeast-phase culture. Candida grown in the presence of IAA, however, demonstrated 15

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MILMOLAR

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COUCEUTKAIION OF

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FaiG.n.sEfecof mitochondrial inhibitorsoupon filaetaetatin C.in albicans. iameiumcnteralating dyes inagu basalmediu containig 10cetrt2n Mf proline an-1) gluose ethdiu (*)G broid aciinOrag.()

~

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anrqinlcine (0).um Chlorcamhnico dinnth basal mediume(U)a(ionser) Fiameintation inth0bsamedium1. yest wi,thou iunhibtos grwas92 0.07.conen DEREPRESOOOS (LOG 10 FIG. 2. Graphic representation illustrating the relationship of mitochondrial derepression to filamentation of C. albicans in a medium containing 3dMglucose and varying concentrations of proline (0) IAA (*) or 2-DG (0). MOLAR

CONCENTRATION

OF

a

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1

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f - c. X~ proline medium, C. albicans did not grow. In C 0.01 mM to 1.0 from concentrations ranging mM, the fungus grew as a yeast; and in concentrations lower than 0.01 mM it grew as a fila30 2.5 ment. 60 90 120 Oxygen consumption. The rate of oxygen MIN AFTER INOCULATION OF CELLS INTO RESPIRATION CHAMBERS consumption in filamenting Candida was decreased when compared with that of yeasts FIG. 3. Oxygen consumption of yeast phase C. al(Table 1). Yeast inocula converting to filaments in the proline medium consumed oxygen at a bicans inoculated into a proline medium (0), ammowith 100 ,ug of 2-DG per ml (0), or with 100 high rate in the first 2.5 min of incubation, but mented ug of MAA per ml (0), as determined by a Clarkdropped rapidly thereafter so that by 30 min the type oxygen electrode. The yeasts converted to filaconsumption was considerably decreased (Fig. ments in the proline medium, but underwent no 3). Oxygen consumption continued to decrease morphological change in the ammonium chloride meuntil it was no longer detectable after 60 mmn. dium or the proline medium supplemented with the Yeasts, grown in the NH,C1 medium, consumed glycolytic inhibitors.

VOL. 12, 1975

FILAMENTATION IN C. ALBICANS

123

electron transport upon morphology and oxygen consumption of C. albicans. If glucose were replaced in the NH4Cl medium with either millimolar ethanol or 10- 2 M acetate, there would be no detectable change in oxygen consumption by the yeasts grown therein. Substituting acetate for glucose in the proline medium also promoted filamentation along with the typical lack of oxygen consumption at 120 min. By using ethanol instead of glucose as a carbon source in the proline medium, on the other hand, neither supported filamentation nor caused inhibition of respiration. When a-ketoglutarate, citrate, and ascorbate were used as carbon sources in the NH4Cl medium, a moderate amount of filamentation was supported and repression of oxygen consumption was noted. Similar results were obtained when the same compounds replaced glucose in the proline medium. Two generalities can be deduced from the data presented in Table 1: (i) all cofactors including adenosine 5'-monophosphate and adenosine diphosphate, when added to the NH4Cl medium, supported filamentation rather than yeast growth, and oxygen consumption was also suppressed within 30 min after inoculation; (ii) addition of adenosine triphosphate to the NH4Cl medium, however, had no effect on either aerobic respiration or morphogenesis; i.e., the fungus grew only as yeasts. In the 5.proline medium with added adenosine 5'-triphosphate, filamentation was lowered to ap3c proximately one-half that of the control without adenosine 5'-triphosphate, and oxygen consumption was detected at 120 min, albeit quite low. Dicumarol, quinacrine, and methylene blue ,2.5were selected from the list of inhibitors in Table 1 for more thorough investigation. Adding dicumarol to the NH.Cl medium in concentrations ranging from 10-4 to 1 M, stimulated filament rather than yeast formation, and to the same degree as that seen in the filament-inducing pro2.5 120 line medium. Quinacrine, when concentrations 30 60 *0 were greater than 10-4 M, depressed filamenMIN AFTER INOCULATION OF CELLS tation in the proline medium. Oxidized methylINTO RESPIRATION CHAMBERS ene blue, an electron acceptor, was the most FIG. 4. Oxygen consumption of C. albicans in a potent inhibitor of filamentation found during basal medium containing 10-2 M NH4CI, 10- 2 M glu- the course of this study, inhibiting filamentation cose, and either mM adenine dinucleotide phosphate in the proline medium at a concentration of only (0) filaments or 100 Ag of cyclic 3',5'-AMP/ml (0) 10-6 M. or with glucose increased to 1 M (*) filaments. (Only D-[6-4C]glucose metabolism. Evolution yeasts grew in a control containing 10-2 M NH4CI of radioactive CO2 in yeasts and filaments was M was consumed and 10-2 by glucose.) No oxygen any yeasts grown in a proline medium to which was added compared to determine if ofthere were glucose to CO2 100 usg of intercalating dyes per ml: ethidium bro- changes in the metabolism mide, acridine orange, or quinacrine, all represented and water during the onset of filamentation. by (0). (Cells grown in the proline medium without The data in Fig. 5 show that within 15 min after inoculation, there was a notable difference in dyes were filamentous.) a respiratory rate significantly lower than either the 2-DG-supplemented cells or the yeast controls in the NH4Cl medium (P < 0.01). When C. albicans was grown in the NH4Cl medium, but under conditions which supported filamentation (i.e., supplemented with either molar glucose, adenosine 5'-diphosphate, or other metabolites mentioned in Table 1), oxygen consumption was found to parallel the proline-grown filamentous control (Fig. 4). Filaments induced in the NH4Cl medium in the above manner, therefore, showed a high initial respiratory rate, with respiration abating after 30 min of incubation. Cyclic 3-5' adenosine monophosphate, which has been found to influence morphogenesis in other fungi (41, 42) as well as in slime molds (23, 25), affected the respiratory pattern in similar fashion. Yeasts inoculated into the proline medium containing intercalating dyes showed no oxygen consumption during the 120-min experimental period. Proline cultures containing chloramphenicol also lacked respiratory activity. C. albicans growing on the solid proline medium containing either of the above metabolites or inhibitors, save for IAA or 2-DG, demonstrated no change in colony color after a tetrazolium dye overlay. Table 1 is a summary of the effects of various electron donors, acceptors, or uncouplers of

93

124

LAND ET AL.

INFECT. IMMUN.

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_ le

l

o

f

inoculation, but were again increased after 4 h. At the end of 6 h in culture, the filaments had produced approximately 8 times as much ethanol as the yeasts, i.e., 8 x 10- and 6 x 10-6 M,

~~~~~~~~respectively.

DISCUSSION There have been two seemingly divergent 10 lopinions concerning morphogenesis (y-m conversion) in C. albicans. The first view of Cano0 Idida dimorphism suggests morphology is the 8expression of distinct biochemical changes oc0_ lcurring in the cell wall (7; P. A. DePalma, Ph.D. thesis, Boston Univ., Boston, Mass., 39 1966). Alternatively, the Nickerson school of eO s6 thought (1, 31, 32, 33) did not feel that there rn were necessarily profound biochemical differences during y-m conversion, but simply change in the linkage of the cell wall polymers as a re4, _sult of the inactivity of a flavoprotein disulfhydryl reductase. Nickerson further theorized that the inactivity of that enzyme would lead to an increase in reduced flavoprotein within the E22fi /S - ^* mitochondria, preventing the yeast mother cell

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0. 10

|

30

2.5 15

60

;0

120

MIN AFTER INOCULATION OF CELLS INTO MEDIUM

FIG. 5. Graphic representation of the 1CO2 generated by C. albicans through the metabolism of D[6-'4C]glucose while being maintained as yeasts (0) or filaments (*). The labeled C02 was trapped as a carbonate on NaOH-impregnated strips. The difference between (202 evolution of the yeast versus the mycelial phase is significant, P < 0.01.

CO2 evolution between yeast cells grown in the NH4Cl medium and filaments cultured in the proline medium. After 30 min in culture, yeasts evolved four times more CO2 than filaments, with the difference becoming 10-fold by 120

C,

O

io-/

min.

Production of ethanol. Both yeasts and filaproduced ethanol, but the amount formed by the yeast cultures was only minimal in comparison with that of filamentous forms. During the onset of filamentation in the proline medium, ethanol (approximately 4 x 10-6 M) was detected within 30 min after inoculation, whereas yeasts incubated in the NH4C1 medium produced none (Fig. 6). It was not until 2 h after inoculation that ethanol could be be detected in culture filtrates of yeasts, cd and by that time the filtrates from the filamentous cultures had 2.5 times as much. The levels of ethanol in either culture did not change during 2 to 4 h after

r

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ments

inculationithatesof yeathn

dtiethe

l

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, 2

3 4 NOURS INCUBATION

5

6

FIG. 6. Ethanol production by yeasts (*) and filamentous forms (0) of C. albicans cultivated in an

NH4CI or a proline medium, respectively, as deter-

mined by gas chromatography. The differences in ethanol concentration at 2 and 6 h between the yeast and filamentous cultures were significant, P < 0.01.

VOL. 12, 1975

and its bud from dividing and separating. The net result of blocking yeast cell division would be the formation of a chain of cells and, ultimately, a filament (32). In a recent paper, Chattaway et al. (6) demonstrated that both theories had some merit, by presenting data showing high hexose monophosphate shunt activity correlated with yeast maintenance and changes in enzymes involved in polysaccharide biosynthesis occurring upon y-m conversion. Since the hexose monophosphate shunt would provide the necessary nicotinamide adenine dinucleotide phosphate, reduced form, necessary for yeast maintenance in the Nickerson model, changes in electron flow could then be somewhat equated with their reported biochemical differences. The major findings from our previous research (27) were: (i) a buildup of reducing power in the cell by the. addition of one of the glutamate family of amino acids to the medium enhanced filamentation; (ii) a presumable change in the metabolism of glucose from aerobic to fermentative occurred during filamentation; (iii) some apparent changes occurred in the metabolic carbohydrate pools during y-m conversion; (iv) an interruption of the normal energy flow in C. albicans had a profound morphological effect. We proposed (27) that dimorphism in Candida was the phenotypic result of a complex set of controls within the cell between cytoplasmic glycolysis and mitochondrial oxidative phosphorylation. Competition for electrons, phosphate, and phosphorylated energy or glycolytic intermediates would repress oxidative phosphorylation in the mitochondria by a mechanism similar to the Crabtree effect (11, 26, 27). As yeast converted to filament, anaerobic (fermentative) metabolism would predominate over aerobic metabolism and there would be a change in electron flow as well as a change in carbohydrate metabolism as predicted by both Nickerson (33) and Chattaway et al. (6). Polarographic studies on C. albicans grown under all conditions stimulating y-m conversion demonstrated similar pattems of oxygen consumption. The pattern consisted of a sudden burst then cessation of respiration, the latter remaining throughout the experimental period. Most growth conditions which maintained the yeast phase, on the other hand, kept up a steady rate of respiration (