The functional pathways of nicotinamide adenine dinucleotide (NAD) biosyn- thesis and their regulation were studied in the dimorphic fungus Candida albicans.
Vol. 139, No. 3
JOURNAL OF BACTERIOLOGY, Sept. 1979, P. 883-888 0021-9193/79/09-0883/06$02.00/0
Nicotinamide Adenine Dinucleotide Metabolism in Candida albicans W. LAJEAN CHAFFIN,' RODNEY A. BARTON,2 ELAINE L. JACOBSON,t AND MYRON K. JACOBSON 2* Department of Biology, Texas Christian University, Fort Worth, Texas 76129,' and Departments of Chemistry and Biochemistry, North Texas State University/Texas College of Osteopathic Medicine, Denton, Texas 762032
Received for publication 16 July 1979
The functional pathways of nicotinamide adenine dinucleotide (NAD) biosynthesis and their regulation were studied in the dimorphic fungus Candida albicans. The presence of a functional endogenous pathway of NAD biosynthesis from tryptophan was demonstrated. In addition, nicotinamide served as an efficient salvage precursor for NAD biosynthesis but nicotinate was not utilized. The pathway for nicotinamide utilization involved nicotinate and nicotinate nucleotides as intermediates, suggesting that the failure to utilize nicotinate involves a transport defect. The mechanisms that regulate NAD levels during exponential growth operated to maintain constant NAD levels when NAD biosynthesis occurred exclusively from endogenous or salvage pathways or from a combination of the two. The regulation also operated such that the salvage pathway was preferentially utilized. The pyridine nucleotide coenzymes, nicotinamide adenine dinucleotide (NAD) and NAD phosphate (NADP), are primary components of the reduction-oxidation (redox) metabolism of cells. (In this paper, the terms NAD and NADP are used in context to represent the total pyridine nucleotide pool. NAD+ and NADP+, or NADH and NADPH, are used to denote the specific oxidized or reduced forms, respectively.) As coenzymes for numerous dehydrogenases, they are common intermediates of many diverse metabolic sequences and have been postulated to be important control parameters in growth regulation (2, 3). In the fungi, changes in growth or morphology or both have been related to changes in pyridine nucleotide pools in Neurospora crassa (4-6, 23) and Saccharomyces cerevisiae (24). Pyridine nucleotide metabolism has not been studied in the dimorphic fungus Candida albicans. In humans, C. albicans is an important opportunistic pathogen. Since morphological change in C. albicans has been related to the redox state of cell wall sulfhydryl groups (10, 17, 19, 20) and to the general redox metabolism of the cell (7, 18), the pyridine nucleotide metabolism of the organism is of particular interest. We have determined and report here the functional pathways of pyridine nucleotide biosyn-
thesis and their regulation in C. albicans. We have demonstrated the presence of a functional pathway for the endogenous synthesis of the pyridine ring of NAD from tryptophan and a salvage pathway of NAD biosynthesis from nicotinamide. We have also studied the regulation of the two pathways. In addition, we report that C. albicans appears to be unique in that it cannot utilize exogenous nicotinate as a salvage precursor for NAD biosynthesis.
MATERIALS AND METHODS Organism and culture conditions. C. albicans 3153A was maintained on 1% yeast extract-2% peptone-2% glucose agar plates. Cells were grown at 30°C on a gyratory shaker in a liquid defined medium (13). Since this medium does not contain sources of preformed pyridine ring compounds, it is referred to here as minimal medium. In some experiments, the medium was supplemented with tryptophan, nicotinamide, or nicotinate. L-[5-'H]tryptophan, [carbonyl- '4C]nicotinamide, and [carboxyl-'4C]nicotinate were from Amersham Corp. Cell numbers were determined by using a Petroff-Hauser counting chamber. Extraction of pyridine nucleotides. To determine total NAD content (NAD+ plus NADH), portions of culture containing 3 x 107 to 6 x 107 cells were harvested by centrifugation at 1,500 x g for 10 min, and the medium was removed by aspiration; alternatively, cells were harvested by filtration using a 0.45um filter. The oxidized and reduced pvridine nucleot Present address: Department of Biology, Texas Woman's tides were extracted from the cells with 2.0 ml of 0.5
University, Denton, TX 76202.
M 883
HCIO1
or 2.0 ml of 0.25 M
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as
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CHAFFIN ST AL.
described previously ( 11), except that the extracts were treated at 60°C for 10 min. Control experiments in which portions of culture were extracted directly demonstrated that centrifugation or membrane filtration does not affect the size of the NAD pool. When NAD and NADH standards are subjected to acid and alkaline extraction, respectively, as described here, over 90', of the standards are recovered. Extraction of cells in which the pyridine nucleotide pools were prelabeled wtih ['4C]nicotinamide resulted in extraction of over 95%7 of the radiolabel. The expected products of acid and alkaline extraction were confirmed by
chromatography. For analysis of radiolabeled cultures by chromatography, cell pellets were extracted with ice-cold 10% trichloroacetic acid. After centrifugation, trichloroacetic acid was removed by four extractions with diethyl ether. I'his method of extraction results in greater than 95'S extraction of radiolabel. Analysis of NAD+. NAD+ was determined by an enzymatic cycling assay (11). Duplicate analyses were done on each extract, and the results rarely differed from each other by more than 5%c. To test for the presence in the extracts of either activators or inhibitors of the cycling assay, known amounts of NAD+ were added to some assays. Neither activation nor inhibition was observed in any of the extracts. Uptake of nicotinate and nicotinamide. Uptake of radiolabeled nicotinate or nicotinamide was determined by filtration of a portion of culture using a 0.45ym membrane filter. The filtered cells were washed with a solution of physiological saline containing 1 mM nicotinate or nicotinamide, and the filter was dried and radioactivity was determined. A portion of culture was counted directly to determine percent uptake and specific radioactivitv. Chromatography of pyridine nucleotides. Extracts were chromatographed on sheets of Whatman 3MM filter paper by descending chromatography. Chromatograms were developed with the system of Witholt (25). Chromatograms were cut into 1-cm strips for liquid scintillation counting in a toluene-base(i cocktail. In some experiments, compounds were eluted from paper chromatograms with distilled water and were subjected to high-pressure liquid chromatography oIn an ISCO Model 1440 high-pressure liquid chromatograph equipped with a Whatman IPartisil PXS 10/25 SAX anion exchange column. Compounds were eluted with a linear gradient from 0.007 M KH,PO, (pH 4.0) to 0.25 M KH2PO.-0.5 M KCI (pH 4.5). Cell-free extracts. Cells were collected by cenitrifugation and washed in cold t). M T'ris-hydrochloride buffer (pH 9.0). A concentrated cell suspension was mixed with 0.45-mm glass beads. The mixture was blended in a Vortex mixer for a total of 6 min with intervals on ice to keep the mixture cold. Greater than 90% cell breakage was obtained. After centrifugation at 1,500 x g for 15 min, the supernatant (pH 7.6) was used as a crude extract. Crude cell extract (4.1 mg/ml of protein) was incubated with 82 AM [14C]nicotinamide (61 mCi/mmol) as described by Lange and Jacobson (12). Portions of the reaction were analyzed by paper chromatography as described above.
J. BAC TFRIOL. Protein determination. Portions of culture containing 3 x 10 to 6 x i0' cells were added to an equal volume of ice-cold 207 (wt/vol) trichloroacetic acid. After at least 15 min on ice, the samples were centrifuged at 1,500 x g for 10 min. The pellets were suspende(d in t).25 M NaOH and were frozen until assayed for protein (15).
RESULTS NAD levels during growth. Cultures of C. albicans 3153A grown at 30°C exclusively exhibit yeast morphology. A typical growth curve in minimal medium is shown in Fig. 1. During exponential growth, cell numbers double each 80 to 90 min until the cells reach a density of approximately 7 x 107 cells per ml, at which time cell numbers gradually increase to a final cell density of approximately 1.7 x 108. The total NAD content, expressed as picomoles of NAI) per microgram of total protein, is also shown in Fig. 1. The NAD content is constant during exponential growth and increases up to fourfold as the cells enter and reach stationary phase. Cells in the exponential phase of growth were utilized in all of the additional experiments described in this paper. Endogenous pathway of NAD biosynthesis. Since minimal medium does not contain any source of preformed pyridine ring compounds that will serve as salvage precursors for NAD biosynthesis, C. albicans must contain endogenous pathways of NAD biosynthesis. Ahmed and Moat (1) have demonstrated that aerobically grown S. ceretuisiae can convert tryptophan to nicotinate, suggesting the presence in fungi of the tryptophan pathway of NAD biosynthesis (21). We have examined C. albicans for the presence of this pathway by culturing them in the presence of L-[5- 3H]tryptophan and analyzing for conversion of radiolabel into NAD.
i
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FIG. 1. NAD levels during grouth of C. albicans. Symbol.s: A, picomoles of NAD per microgram of protein; *, cells per milliliter.
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Chromatography of the acid-soluble pools demonstrates that radiolabel comigrates with both NAD and NADP (Fig. 2). Confirmation that the radiolabel comigrating with NAD was in fact NAD was shown by enzymatic treatment and rechromatography. As expected, treatment with bacterial alkaline phosphatase did not alter the chromatographic migration. After treatment with snake venom phosphodiesterase, radioactivity migrated exclusively with nicotinamide mononucleotide, and after combined treatment with snake venom phosphodiesterase and bacterial alkaline phosphatase, radioactivity migrated with nicotinamide ribonucleoside (Rf = 0.74). These results are consistent with the presence in C. albicans of a functional endogenous pathway of NAD biosynthesis from tryptophan. Salvage pathway(s) of NAD biosynthesis. To study the utilization of the salvage precursors nicotinate and nicotinamide by C. albicans, ['4C]nicotinate or [14C]nicotinamide was added to cultures growing in minimal medium, and the uptake of radiolabel was determined after growth for four generations (Fig. 3). Whereas nicotinamide was taken up by the cells in a concentration-dependent fashion, only very small amounts of nicotinate were utilized at medium concentrations as high as 30 MM. The same result was obtained with [3H]nicotinate. In an additional experiment, cultures containing 10.1 MM [14C]nicotinamide or 3.6 MM [3H]nicotinate were grown to stationary phase. Under these conditions, 44% of the nicotinamide was taken up by the cells, but only 0.6% of the nicotinate was taken up. A chromatographic separation of the acid-soluble pools of a culture grown in medium containing [14C]nicotinamide 26
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FIG. 2. Incorporation of radiolabel from tryptophan into pyridine nucleotides. Cells were grown for four generations in medium supplemented with L -[53H]tryptophan (50 p.M, 40 mCi/mmol). Acid-soluble pools were extracted and analyzed by paper chromatography as described in the text. The location and identity of standards is shown across the top. NMN, Nicotinamide mononucleotide; Na, nicotinate; N, nicotinamide; Trp, tryptophan.
A
10 20 30 Precursor, pM FIG. 3. Uptake of nicotinate and nicotinamide by C. albicans. Cultures (10 ml) were grown in minimal medium supplemented with [carboxy-14 Cinicotinate or [carbonyl-.4 Cinicotinamide. Cultures containing nicotinate (A) contained 0.25 ,uCi at 0.4 LM, 0.75 ,uCi at 1.2 LM, and 0.5 jLCi at 4.8, 13.7, and 30.6 ,uM. Cultures containing nicotinamide (0) contained 0.25 jLCi at 0.4 pM, 0.63 ,uCi at 1.0 ELM and 0.5 ,uCi at 4.4, 14.1, and 29.6 ,uM. After growth for four generations to 6 x 10' to 7 x 10' cells per ml, 9-ml portions were analyzed for uptake of radiolabel as described in the 0
text.
is shown in Fig. 4. A small amount of nicotinamide was observed, but most of the radiolabel was associated with NAD. These results demonstrate that C. albicans contain a functional salvage pathway of NAD biosynthesis proceeding from nicotinamide. Cell-free extracts of C. albicans rapidly convert nicotinamide to nicotinate (Fig. 5, top panel). The addition of 5-phosphoribosyl-1-pyrophosphate, MgCl2, and ATP results in the formation of pyridine mononucleotides and dinucleotides (Fig. 5, bottom panel). Analysis by high-pressure liquid chromatography revealed that the material migrating just behind nicotinamide mononucleotide was nicotinate mononucleotide and that migrating just behind NAD was nicotinate adenine dinucleotide. From these data, we conclude that C. albicans utilize nicotinamide via nicotinate and nicotinate nucleotide intermediates. Regulation of pathways. The flux through the endogenous and salvage pathways was examined under conditions in which both pathways may be utilized. Cultures of C. albicans growing in minimal medium were grown for four generations in media containing [:3H]tryptophan and various levels of unlabeled nicotinamide. The cells were harvested, and total NAD levels were determined. Portions of cell extracts were also examined for conversion of [3H]tryptophan to NAD (Table 1). The presence of nicotinamide
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,J. BACTERICIL.
CHAFFIN E'T AL.
886
in the culture medium does not affect the of the NAD pool but does affect the conversion of ['H]tryptophan tO NAD in a concentration-depencX.it fashion. As expected, nicotinate does not affect the rate of conversion of radiolabel into NAD. This result demonstrates that the presence of salvage precursor does not affect total NAD levels but lowers the fltux through the
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DISCUSSION The role of the pyridine nucleotides as common intermediates of redox metabolism in numerous and diverse metabolic l)athways has led to postulations that they may be involved in growth regulation (2, 3). The regulation of growth in C. albicanis is of interest in that this organism can grow with either yeast or mycelial morphology, depending upon the conditions of growth. In hurmans this organism is an opportunistic pathogen. The yeast form is usually associated with a saprophytic existence, whereas the mycelial form is associated with a pathogenic existence. A number of studies have suggested that dimorphism in C. albican.s is related to cellular redox metabolism (i10, 17, 19, 20). Changes in pyridine nucleotide pools have previo)usly beein associated with changes in growth in the fungi. In N. cr-assa, mor-phological properties of mvcelia have been related to pyridine nucleotide levels (4, 5) and to formation and germination of spores (6, 23). In S. cereveisiae, the levels of NADP relative to NAD are reversibly adjusted in response to the carbon source of the growth medium (24). The studies of Ahnied and( Moat (1) have shown that aerobically grown S. cereuisiae can convert tryptophan to nicotinate, suggesting that the pathway for the biosynthesis of NAD from tryptophan (21) is present in the fungi. Our observation that C. albicans readily convert tryptophan to NAD in vivo provides direct evidence for the presence of this pathway of NAD biosvnthesis in the fungi. O(ur observation that C. albicans can efficiently utilize nicotinamide as a precursor for NAI) but utilizes nicotinate poorly if at all was
VOL. 139, 1979
unexpected for two reasons. First, nicotinate has been reported to serve as an efficient precursor for NAD in a wide variety of organisms (8). Although the fungi have not been widely studied, nicotinate is utilized by N. crassa (5) and nicotinate is utilized much more efficiently than nicotinamide by S. cerevisiae (R. A. Barton and M. K. Jacobson, unpublished data). Second, the only known pathway in the fungi for the utilization of nicotinamide involves nicotinate as an intermediate (22). However, a second pathway for the biosynthesis of NAD from nicotinamide via nicotinamide mononucleotide has been described in mammals (9). The data obtained in this study show that cell-free extracts rapidly convert nicotinamide to nicotinate and nicotinate nucleotide intermediates. Nicotinamide mononucleotide was not observed. Our results show that C. albicans utilize nicotinamide by the pathway originally described by Preiss and Handler (22) which involves nicotinate as an intermediate. We therefore conclude that the failure of C. albicans to utilize nicotinate must result from a defect in the transport of this compound. The results obtained in this stuidy indicate that the levels of NAD are carefully regulated in C. albicans. Constant NAD levels are maintained during exponential growth under conditions in which NAD is derived exclusively from endogenous synthesis, from a combined utilization of endogenous and salvage pathways, or primarily from salvage pathways. The mechanisms that regulate NAD biosynthesis operate in a manner in which the availability of salvage precursors greatly reduces the rate of endogenous synthesis. A similar mode of regulation of NAD biosynthesis has been observed in N. crassa (14) and in E. coli (16), although the endogenous pathway in E. coli is different from the one described here. The molecular basis of this mode of regulation awaits further study. It also remains to be seen whether the fourfold rise in NAD levels observed when cells enter stationary phase represents a continued net synthesis of NAD after cessation of protein synthesis or reflects a more complex regulatory change. The present study should provide a basis for studies of pyridine nucleotide metabolism in C. albicans under conditions that lead to morphological changes. Such studies may prove useful in assessing the role of pyridine nucleotide metabolism in growth control. The finding that C. albicans utilizes nicotinate very poorly for pyridine nucleotide synthesis, combined with the efficient utilization of nicotinate by mammals, may also provide a rationale for chemotherapy involving this organism.
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ACKNOWLEDGMENTS This work was supported by grants from the Texas Christian University Research Foundation and Research Corporation (Brown-Hazen Fund) to W.L.C., by grants BC184 from the American Cancer Society and B-633 from the Robert A. Welch Foundation, and by North Texas State University Faculty Research Funds to M.K.J. We thank Sharon Yoder and Nancy Jasheway for technical assistance. LITERATURE CITED 1. Ahmed, F., and A. G. Moat. 1966. Nicotinic acid biosynthesis in prototrophs and tryptophan auxotrophs of Saccharomyces cerevisiae. J. Biol. Chem. 241:775-780. 2. Atkinson, D. E. 1971. Adenine nucleotides as stoichiometric coupling agents in metabolism and as regulatory modifiers: the adenylate energy charge. In H. Vogel (ed.), Metabolic regulation. Academic Press Inc., New York. 3. Barnes, L. D., J. J. McGuire, and D. E. Atkinson. 1972. Yeast diphosphopyridine nucleotide specific isocitrate dehydrogenase. Regulation of activity and unidirectional catalysis. Biochemistry 11:4322-4329. 4. Brody, S. 1970. Correlation between reduced nicotinamide adenine dinucleotide phosphate levels and morphological changes in Neurospora crassa. J. Bacteriol. 101:802-807. 5. Brody, S. 1972. Regulation of pyridine nucleotide levels and ratios in Neurospora crassa. J. Biol. Chem. 247: 6013-6017. 6. Brody, S., and S. Harris. 1973. Circadian rhythms in Neurospora: spacial differences in pyridine nucleotide levels. Science 180:498-500. 7. Chattaway, F. W., R. Bishop, M. R. Holmes, F. C. Odds, and A. J. E. Barlow. 1973. Enzyme activities associated with carbohydrate synthesis and breakdown in the yeast and mycelial forms of Candida albicans. J. Gen. Microbiol. 75:97-109. 8. Chaykin, S. 1967. Nicotinamide coenzymes. Annu. Rev. Biochem. 36:149-170. 9. Dietrich, L. S., L. Fuller, I. L. Yero, and L. Martinez. 1966. Nicotinamide mononucleotide pyrophosphorylase activity in animal tissues. J. Biol. Chem. 241:188-191. 10. Falcone, G., and W. J. Nickerson. 1956. Cell wall mannanprotein of bakers yeast. Science 124:272-273. 11. Jacobson, E. L., and M. K. Jacobson. 1976. Pyridine nucleotide levels as a function of growth in normal and transformed 3T3 cells. Arch. Biochem. Biophys. 175: 627-634. 12. Lange, R. A., and M. K. Jacobson. 1977. Synthesis of pyridine nucleotides by mitochondrial fractions of yeast. Biochem. Biophys. Res. Commun. 76:424-428. 13. Lee, K. L., H. R. Buckley, and C. C. Campbell. 1975. An amino acid liquid synthetic medium for development of mycelial and yeast forms of Candida albicans. Saubouraudia 13:148-153. 14. Lester, G. 1971. End-product regulation of the tryptophan-nicotinic acid pathway in Neurospora crassa. J. Bacteriol. 107:448-455. 15. Lowry, 0. H., N. J. Rosenbrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 16. McLaren, J., D. T. C. Ngo, and B. Olivera. 1973. Pyridine nucleotide metabolism in Escherichia coli. III. Biosynthesis from alternative precursors in vivo. J. Biol. Chem. 248:5144-5159. 17. Nickerson, W. J. 1948. Enzymatic control of cell division in microorganisms. Nature (London) 162:241-245. 18. Nickerson, W. J. 1963. Symposium on biochemical bases of morphogenesis in fungi. IV. Molecular bases of form in yeasts. Bacteriol. Rev. 27:305-324.
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19. Nickerson, W. J., and C. W. Chung. 1954. Genetic block in the cellular division mechanisnm of a morphological mutant of a yeast. Am. J. Bot. 41:114-120. 20. Nickerson, W. J., and G. Falcone. 1956. Identification of protein disulfide reductase as a cellular division enzyme in yeast. Science 124:722-728. Nishizuka, Y., and 0. Hayaishi. 19633. Studies on the biosynthesis of nicotinanide adenine dinucleotide. J. Biol. Chem. 238:3369-3377. 22. Preiss, J., and P. Handler. 1958. Biosynthesis of diphosphopyTidine ntucleotide. J. Biol. Chemn. 233:49:35(:X). 21.
J. BACTERIOL. 23. Schmit, J. C., R. C. Fahey, and S. Brody. 1975. Initial biochemical events in germilination of Neuorspolra crassa conidia, p. 112-119. In P. Gerhardt, R. N. Costilow, and .J. IL. Sadoff. (ed.), Spores VI. Anmerican Societv for Microbiology, Washington, D.C. 24. Ting, H. Y., E. L. Jacobson, and M. K. Jacobson, 1977. ReguLlation of nicotinamide adenine dinucleotide phosphate levels in Saccharconv ces cereuisiae. Arch. Biochenm. Biophys. 183:98-1(04. 25. Witholt, B. 1971. A bioaUtographic procedUre forl detecting TPN, I)PN, NMN, and NR. Methods Enzymol. 18: 813-816.
