Carbon dioxide induces endotrophic germ tube formation in Candida albicans RUTH C. MOCK,JORDANH. POLLACK,' AND TADAYO HASHIMOTO~ Department of Microbiology, Layola University, Stritch School of Medicine, Maywood, IL 60153, U.S.A.
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Received August 9, 1989 Accepted December 5, 1989 MOCK,R. C., POLLACK, J. H., and HASHIMOTO, T. 1990. Carbon dioxide induces endotrophic germ tube formation in Candida albicans. Can. J . Microbiol. 36: 249-253. Candida albicans formed germ tubes when exposed to air containing 5 to 15% carbon dioxide (C02).The C02-mediatedgerm tube formation occurred optimally at 37°C in a pH range of 5.5 to 6.5. No germ tubes were produced at 25"C, even when the optimal concentration of C02 (10%) was present in the environment. The requirement of C02 for germ tube formation could be partially substituted by sodium bicarbonate but not by N2. Carbon dioxide was required to be present throughout the entire course of germ tube emergence suggesting that its role is not limited to an initial triggering of morphogenic change. We suggest that carbon dioxide may be a common effector responsible for the germ tube promoting activity of certain chemical inducers for C . albicans. Key words: Candida albican germ tubes, C02-induced germ tube formation, endotrophic germ tube formation. MOCK,R. C., POLLACK, J. H., et HASHIMOTO, T. 1990. Carbon dioxide induces endotrophic germ tube formation in Candida albicans. Can. J . Microbiol. 36 : 249-253. Expos6 B de l'air contenant de 5 B 10% de bioxyde de carbone (C02), le Candida albicans produit des tubes germinaux, et ce, de f a ~ o n optimale B 37°C et B des pHs variant de 5,5 B 6.5. Toutefois, B 25"C, meme si I'air ambiant contient une concentration optimale de 10% de C02, la production de tubes germinaux est nulle. L'exigence du C02 peut $tre substituke partiellement par du bicarbonate de sodium, mais non par du N2. Tout au cours de l'kmergence des tubes germinaux, la prksence de bioxyde de carbone s'est avkrke requise, ce qui suggtre que son rSle n'est pas limit6 au dkclenchement initial des changements morphogkniques. La suggestion est donc avancke que le bioxyde de carbone peut &re un effecteur de base responsable de I'activitk de certains inducteurs chimiques qui amorcent le dkveloppement des tubes germinaux chez le C. albicans. Mots clis : tubes germinaux de Candida albicans, induction de la formation de tubes germinaux par le C02, formation endotrophe de tubes germinaux. [Traduit par la revue]
Candida albicans is an opportunistic fungal pathogen, which can grow, depending on the environmental conditions, either in the yeast o r the mycelial form. At temperatures below 28"C, C. albicans usually assumes a yeast morphology. When yeast cells of C. albicans are exposed to an appropriate inducer at higher temperatures (37"C), they initiate the hyphal phase of growth by forming germ tubes. A variety of substances are known to induce germ tube formation in C. albicans. These include tissue culture media (Lee et al. 1975), body fluids (Landau et al. 1965; Taschdjian et al. 1960), L-proline (Dabrowa et al. 1976; Land et al. 1975), N-acetyl-D-glucosamine (GlcNAc) (Simonetti et al. 1974), and ethanol (Pollack and Hashimoto 1985). At present, the mechanism whereby these diverse compounds induce germ tube formation in C. albicans remains virtually unknown. While investigating the germ tube inducing capability of various intermediary metabolites of known chemical inducers, we found that C02 alone is quite capable of inducing germ tube formation in C . albicans. This paper describes C02-induced and bicarbonate-induced germ tube formation in C. albicans. A preliminary account of this work has appeared in abstract form (R. C. Mock, J. H. Pollack, and T. Hashimoto. 1988. 88th Annual Meeting of the American Society for Microbiology p. 391. (Abstr.)).
Materials and methods Organisms Candida albicans ATCC 58716 was routinely used for the experiments described in this paper. In some experiments, C . albicans ATCC
'present address: Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2153 Sheridan Road, Evanston, IL 60208, U.S.A. 'Author to whom all correspondence should be addressed. Rinted in Canada / Irnprirnd au Canada
10261 and 14053 were used for comparative purposes. Cells were maintained on Sabouraud dextrose agar (SDA, Difco) at 20°C. Growth and preparation of organisms Yeast-phase cells were prepared by growing the cells on SDA for 24 h at 37°C. The yeast cells were washed with distilled water by vacuum filtration at least 10 times as described earlier (Pollack and Hashimoto 1987). Approximately 95% of the yeast cells prepared by this procedure were cells without buds. The washed cells were used immediately. Preparation of slide microcultures Purified Noble agar (Difco) or agarose (type V, Sigma Chemical Co.), washed several days in double-distilledwater, was routinely used in all experiments. During the washing, water was frequently changed. All glassware, glass slides, and cover slips were acid-washed prior to use. Molten agar (3%) was mixed with an equal volume of buffer (Tris-maleate, 0 . 1 M, pH 5.75) prior to use. The concentration of the buffer was sufficient to prevent more than a 0.1 pH unit change during the course of all experiments. The buffered molten agar was placed on glass slides and the agar surface was flattened by placing a thin cover slip (No. 1 thickness, 22 x 22 rnrn) over the agar until it hardened. Approximately 20 pL of the cell suspension (2.0 X lo6cells/mL) was spread over the agar surface. Exposure to COz and bicarbonate Microcultures were incubated in an airtight chamber filled with a mixture of air and carbon dioxide (Airco, Carol Stream, IL). A high humidity was maintained inside of the chamber so that the surface of' the microculturesdid not dry during incubation. All microcultures were routinely incubated at 37OC for 3 h unless otherwise indicated. Controls were incubated in chambers containing only atmospheric air (COz concentration is approximately 0.03%). Some experiments were run under C02-free air by placing KOH pellets inside @e incubation chamber. To confim that the observed C02 effect on C . albicans was not due to a reduced partial pressure of oxygen, similar experiments were performed by partially replacing air with nitrogen gas. To test
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FIG. 1. Phase-contrast micrograph of C. albicans yeast cells forming germ tubes endotrophically on purified agar at 37OC in the presence of 10% co2. whether C 0 2 could be replaced by bicarbonate, cells (final concentration of 7.5 x lo5 cellslmL) were inoculated into Tris-maleate buffer (0.1 M, pH 5.75, final volume 2 mL, in a 12 X 75 mm glass tube) containing specified concentrations of sodium bicarbonate. Unless otherwise indicated, all tubes were sealed with Parafiim and incubated at 37°C. In some experiments, 20 pL of the cell suspension was sandwiched between slide and cover glass (22 mm2), without agar, and incubated in a moist chamber.
COrfree Atrnos- I % CO2 5'/.COz IO%C02 15%C02 20%COz 100% air pheric 99%air 95%air 90%air 85%air 8 0 % a i r C02 air
FIG.2. Effect of C 0 2 concentration on germ tube formation in C. albicans. All cells were incubated at 37OC on purified agarose. The data are the average of three separate experiments, and lines at the top of each bar represent standard deviations (SD).
(32% formed pseudohyphae), and no cell developed germ tubes at 25°C (85% of the cells not forming germ tubes had buds) (Fig. Determination of germ tube formation 3). Similar results were obtained when other strains (ATCC The percentage of cells forming germ tubes and buds was measured 10261 and 14053) of C. albicans were used (data not shown). by counting 200 cells in each sample with a phase-contrast microscope To see whether cells exposed to C02for a brief period at 37°C (Pollack and Hashimoto 1987, 1988). The length of germ tubes was commit themselves to germ tube formation, microcultures were determined by using an ocular micrometer. periodically transferred to chambers containing only atmosResults pheric air and the incubation was continued at 37°C. The results Candida albicans cells formed short germ tubes (20%), essentially no germ tubes (Fig. ate could partially replace the role of C02 in inducing germ tube 2) or buds were formed, suggesting that this concentration of formation in C . albicans. As shown in Fig. 5, C . albicans cells C 0 2 is inhibitory for growth. To determine whether the formed germ tubes when incubated at 37°C in Tris-maleate observed germ tube formation was due to C02or reduced partial buffer (0.1 M, pH 5.75) containing sodium bicarbonate. The pressure of oxygen, the atmospheric air in the incubation bicarbonate-induced germ tube formation in C . albicans occurred chamber was partially replaced with N2 gas (Airco, Carol optimally at a pH range of 5.5-6.0 and very poorly or not at Stream, IL). No cells formed germ tubes under semianaerobic all above pH 7.5 (data not shown). As shown in Fig. 5, the conditions created by partial (5, 10, 15, and 20%) replacement percentage of cells forming germ tubes rarely exceeded 60-70% of air with N2 (data not shown). Thus, it was C02, not reduced in test tubes even under optimal conditions, although the entire oxygen tension, that induced the germ tube formation in cell populations were fully capable of forming germ tubes in C. albicans. Under strict anaerobic conditions, neither budding bovine serum. Of the cells that did not form germ tubes, 25 to nor germ tube formation was observed. 30% formed small single buds. In contrast, 80 to 90% of the Relatively high incubation temperatures (33-37°C) were cells formed germ tubes when a thin layer of the same cells required for the maximum germ tube formation induced by suspended in the buffer containing bicarbonate were sandC02. Even with the optimal C 0 2 concentration (lo%), only wiched between slide and cover glass and incubated at 37°C 60% of the C . albicans cells could produce germ tubes at 30°C (Fig. 5). The control cells, suspended either in the same buffer
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MOCK ET AL.
Time (min)
FIG. 3. Effect of temperature on the kinetics of C02-induced germ tube formation in C albicans. A, 37°C; 0 , 30°C; 0 , 25°C.
Concentration (mM FIG.5. Germ tube formation in C. albicans in the presence of bicarbonate or NaCI. Cells were incubated in Tris-maleate buffer (0.1
M, pH 5.75, final volume 2 rnL) containing the indicated amounts of sodium bicarbonate (.,A) or NaCl(0,A) either in 12 X 75 mm sealed test tubes (A,A) or sandwiched between a glass slide and cover slip (O,.) and incubated at 37OC for 3 h.
or saline solution without bicarbonate, failed to form germ tubes (Fig. 5). Of such control cells, 80 to 90% formed small single buds.
C3
1 0 % C02for 45 mm, then transferred to C02 free air
Time ( m i n ) FIG. 4. Effect of shift from C02 to air on germ tube formation in C. albicans. Cells forming germ tubes on purified agarose under 10%C02 (90% air) were transferred to C02-free air at time indicated (thick arrows) and incubated up to 2 h. Essentially all cells continuously incubated under 10% C02 formed germ tubes, while those cells incubated in the presence of C02-free air or atmospheric air formed no germ tubes. All experiments were at 37°C.
Discussion Although the stimulatory effect of C02 (Bedell and Sol1 1979; Mardon et al. 1969; Odds and Abbott 1980; Sims 1986; Weld 1952) or bicarbonate (Pollack and Hashimoto 1988) on the hyphal phase of growth in C. albicans is well documented, this is the first report that carbon dioxide or bicarbonate alone can induce germ tube formation. Since no germ tubes were formed in the cells incubated under environmental conditions lacking C 0 2 (Fig. 2), it is unlikely that the germ tube formation observed in the cells was due to chemical inducers contaminating the agar. The limited size of the germ tubes also supports this assumption (Fig. 1). Furthermore, germ tubes were formed by sandwiching the cells, in the presence of bicarbonate, between slide and cover glass, in the absence of any agar. The agar was used only to maintain moisture and to provide direct contact between the cells and the C 0 2gas. Nutritionally, we believe that the germ tubes formed in the presence of C02 are endotrophic in nature. Whereas exogenous C 0 2 is required to initiate and maintain the hyphal form, no additional nutrients need to be added to sustain this growth form until the germ tubes are approximately 10 pm long. Cadida albicans -yeast cells are known to contain various storage carbohydrates such as glycogen, trehalose, and wall glucans (Chiew 1989). This indicates that C. albicans yeast cells may contain sufficient endogenous carbon sources, other than C02, to support the initial phase of cell growth. It is also evident from our earlier study on the effect of ethanol on C . albicans (Pollack and Hashimoto 1985) and those of Shepherd et al. (1985) that
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competent yeast cells do not require exogenous nitrogen compounds to form germ tubes. Thus, competent C . albicans yeast cells are fully capable of forming germ tubes endotrophically in response to proper inductive stimuli. It is important to note that C 0 2is required to be present in the environmentthroughout the entire course of incubation (Fig. 4). Premature removal of C 0 2 prior to the appearance of the germ tubes resulted in the abortive formation of germ tubes. Its removal subsequent to their appearance resulted in cessation of elongation and often the reversion to budding growth form. This suggests that the role of C 0 2 in germ tube formation in this fungus is not limited to an initial triggering effect but is a constant requirement for the hyphal mode of growth. Probably the most significant aspect of our observations made in this study is that it has raised an interesting possibility that some chemical inducers may promote germ tube formation in C . albicans via C 0 2 generated by their metabolism. As mentioned earlier, germ tube formation in C . albicans can be induced by a number of structurally or metabolically unrelated compounds (Odds 1985 and 1988; Shephard et al. 1985). There seems to be no common metabolic pathway or metabolic product shared by these diversely different chemical inducers unless ubiquitous metabolites such as C 0 2 are considered. In discussing the mode of action of different chemical inducers of Candida morphogenesis, Holmes and Shepherd (1987) speculated that these compounds may act in a similar manner via catabolism to a common intermediate. Candida albicans was shown to produce C 0 2 effectively from GlcNAc (HrmovB et al. 1983). Sims (1986) suggested that the internal concentration of C02 is the critical factor in the morphogenesis of C . albicans, whether achieved by raising the external pressure or internal metabolism. Alternatively, it is possible that an effective level of intracellular C 0 2 concentration could be achieved by the metabolism of the inducers themselves or by the catabolism of intracellular storage carbohydrates triggered by certain chemical inducers. The observation that immobilized GlcNAc, which was obviously nonmetabolizable, could induce germ tubes in C . albicans (Shephard and Sullivan 1983) supports the latter possibility. It is not too surprising that C 0 2 was found to be an important regulator of C . albicans dimorphism because it is a recognized mediator of morphogenesis in bacteria and other fungi (Jones and Greenfield 1982; J W. T. Wirnpemy 1969. 19th Symposium of the Society for General Microbiology, April 1969, University College, London. Edited by P. Meadow and S. J. Pirt. Cambridge University Press, Cambridge). Morphogenic events affected by C 0 2 are numerous. These include stimulating germination of streptomyces spores (Grund and Ensign 1978) and fungal conidia (Yanagita 1963; Pass and Griffin 1972), initiating fruiting body formation (Sietsma et al. 1977) and spherule formation (Klotz et al. 1984), altering of cell shape and division (StraskrabovB et al. 1980; Lumdsen et al. 1987), and initiating dimorphic changes in fungi (Bartnicki-Garcia and Nickerson 1962; Ho and Smith 1986). The precise mechanisms whereby C 0 2 regulates the morphogenic events in C . albicans and other fungi remain largely unknown. At this point, one can only speculate several possibilities based on the findings made in other biological systems. One possibility is through the regulation of pH. The critical role of the environmental pH in the dimorphism of C . albicans has been well documented (Sol1 1985; Pollack and Hashimoto 1987). The effect of C 0 2 and bicarbonate on intracellular pH and morphological changes induced by such intracellular pH
changes in various cells have been well documented (Roos and Baron 1981). Measurable alterations in the intracellular pH of C. albicans undergoing morphogenic changes have recently been noted (Stewart et al. 1987; Kaur et al. 1988). However, these changes may be the consequence rather than the cause of morphogenic changes. While there was a greater increase in the internal pH in budding cells compared with germ tube forming cells, there was no significant change in pH during the first 30-120 min of germ tube formation (Kaur et al. 1988). It is also less likely that cytoplasmic alkalinization plays a major role in C02-induced germ tube formation since yeast cells do not initiate germ tube formation in alkaline buffer solutions unless solutions are supplemented with exogenous inducers such as proline or GlcNAc (Pollack and Hashimoto 1987, 1988). The observation that the requirement for C 0 2 could be partially replaced by 5-15 rnM oxaloacetate (R. C. Mock and T. Hashimoto, unpublished data) suggests that the maintenance of the tricarboxylic acid cycle by anaplerotic reactions may be required for germ tube development. It was previously suggested that C 0 2 is incorporated into some intermediates via C02 fixation promoting the metabolism conducive to germination of fungal conidia (Yanagita 1963) and Streptomyces spores (Grund and Ensign 1978). Carbon dioxide is most probably fixed by carboxylation of either pyruvate or phosphoenolpyruvate. Carbon dioxide and bicarbonate are known to affect a number of cellular enzymes as well as microbial activities (Jones and Greenfield 1982). However, at present, no specific enzyme has been identified that is active only in the yeast or the mycelial phase of growth. It is probable that stimulation or suppression of some enzymes involved in morphogenesis by C02 may be involved in C . albicans dimorphism. For example, CAMP is implicated in Candida dimorphism, although evidence for its involvement is still equivocal (Niimi et al. 1980; Chattaway et al. 1981). Recently, stimulation of adenyl cyclase by bicarbonate has been reported for eukaryotic cells (Tajima et al. 1987). Suppression or stimulation by C 0 2 of some enzymes involved in the biosynthesis of unique wall components is another intriguing possibility. Finally, it is possible that the expression of certain genes coding key morphogenic substances is dependent on high C02 concentrations. Recently, microbial genes requiring high C 0 2 tension for their expression have been identified and cloned (Makino et al. 1988). W. J. 1962. Induction of BARTNICKI-GARCIA, S., and NICKERSON, yeast-like development in Mucor by carbon dioxide. J. Bacterial. 84: 829-840.
BEDELL, G. W., and SOLL,D. R. 1979. Effects of low concentrations of zinc on the growth and dimorphism of Candida albicans:evidence for zinc-resistant and -sensitive pathways for mycelial production. Infect. Immun. 26: 348-354. CHATTAWAY, F. W., WHEELER, P. R., and O'REILLY, J. 1981. Involvement of adenosine 3':5' cyclic monophosphate in the germination of blastospores of Candida albicans. J. Gen. Microbiol. 123: 233-240. CHIEW,Y. Y. 1989. The dynamics of carbohydrate metabolism in Candida albicans. Exp. Mycol.13: 49-60. DABROWA, N., TAXER,S. S. S., and HOWARD, D. H. 1976. Gemination of Candida albicans induced by proline. Infect. Immun. 13: 830-835. GRUND, A. D., and ENSIGN, J. C. 1978. Role of carbon dioxide in germination of spores of Streptomyces viridochromogenes. Arch. Microbiol. 118: 279-288.
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PASS,T., and GRIFFIN,G. J. 1972. Exogenous carbon and nitrogen Ho, C. S., and SMITH,M. D. 1986 Morphological alterations of Penicillium chrysogenum caused by carbon dioxide. J. Gen. requirements for conidial germination by Aspergillusflavus. Can. J. Microbial. 132: 3479-3484. Microbial. 18: 1453-1461. 1985. POLLACK, J. H., ~ ~ ~ H A S H I MT. OT O , Ethanol-induced germ tube HOLMES,A. R., and SHEPHERD,M. G. 1987. Proline-induced formation in Candida albicans. J. Gen. Microbiol. 131: 3303-33 10. germ-tube formation in Candida albicans: role of proline uptake and nitrogen metabolism. J. Gen. Microbiol. 133: 3219-3228. 1987. Role of glucose in the pH regulation of germ tube HRMOVA, M., STURD~K, E., KoS~K,M , GEMEINER, P , ~ ~ ~ P E T RL.u S , formation in Candida albicans. J. Gen. Microbiol. 133: 415-424. -1988. The requirements for bicarbonate and metabolism of the 1983. Growth of Candida albicans on artificial D-glucose derivatives. Z. Allg. Mikrobiol. 23: 303-312. inducer during germ tube formation by Candida albicans. Can. J. Microbial. 43: 1183-1 188. JONES,R. P., and GREENFIELD, P. F. 1982. Effect of carbon dioxide on Roos, A., and BORON,W. F. 1981. Intracellular pH. Physiol. Rev. yeast growth and fermentation. Enzyme Microb. Technol. 4: 210223. 61: 294-434. SHEPPARD,M. G., and SULLIVAN, P. A. 1983. Candida albicans KAUR,S., MISHRA,P., and PRASAD, R. 1988. Dimorphism-associated germ-tube formation with immobilized GlcNAc. FEMS Microbiol. changes in intracellular pH of Candida albicans. Biochim. Biophys. Acta, 972: 277-282. Lett. 17: 167-170. SHEPHERD, M G., POULTER,R. T. M., and SULLIVAN, P. A. 1985. KLOTZ, S. A., DRUTZ, D. J., HUPPERT.M., SUN, S. H., and Candida albicans: biology, genetics and pathogenicity. Annu. Rev. DEMARSH,P. L. 1984. The critical role of C 0 2 in the morphogenesis of Coccidioides immitis in cell-free subcutaneous chambers. Microbiol. 39: 579-614. J. Infect. Dis. 150: 127-134. SIETSMA, J. H., RAST,D., and WESSELS, J. G. H. 1977. The effect of LAND, G. A., MCDONALD,W. C., STJERNHOLM, R. L., and carbon dioxide on fruiting and on degradation of a cell wall glucan in FRIEDMAN, L. 1975. Factors affecting filamentation in Candida Schizophyllum commune. J. Gen. Microbiol. 102: 385-389. albicans: relationship of the uptake and distribution of proline to SIMONETTI,N., STRIPWLI, V., and CASSONE,A. 1974. Yeastmorphogenesis. Infect. Irnmun. 11: 101&1023. mycelial conversion induced by N-acetyl-D-glucosamine in C . LANDAU, J. W., DABROWA, N., and NEWCOMER, V. D. 1965. The albicans. Nature (London), 250: 344346. rapid formation in serum of filaments by C . albicans. J. Invest. SIMS,W. 1986. Effect of carbon dioxide on the growth and form of Dermatol. 44: 171-179. Candida albicans. J . Med. Microbial. 22: 203-208. LEE,K. L., BUCKLEY, H. R., and CAMPBELL. C. C. 1975. An amino SOLL,D. R. 1985. Candida albicans. In Fungal dimorphism with acid liquid synthetic medium for the development of mycelial and emphasis on fungi pathogenic for humans. Edited by P. J . Szaniszlo yeast forms of Candida albicans. Sabouraudia, 13: 148-153. and J. L. Harris. Plenum Press, New Yok. pp. 167-195. LUMSDEN, W. B., DUFFUS,J. H., and SLAUGHTER, J. C. 1987. Effect STEWART, E., GOW,N. A. R., and BOWEN,D. V. 1987. Cytoplasmic of C 0 2 on budding and fission yeasts. J. Gen. Microbiol. 133: 877dkalinization during germ tube formation in Candida albicans. J. 881. Gen. Microbiol. 134: 1079-1087. MAKINO,S., SASAKAWA, C., UCHIDA,I., TERAKAM), N., and V., PAcA, J., and KRAL~cKovA,E. 1980. Effect of STRASKABOVA, YOSHIKAWA. M. 1988. Cloning and C02-dependent expression of aeration and carbon dioxide on cell morphology of Candida utilis. the genetic region for encapsulation from Bacillus anthracis. Mol. Appl. Environ. Microbiol. 40: 855-861. Microbial. 2: 371-376. TAJIMA,Y., OKAMURA, N., and SUGITA,Y. 1987 The activating MARDON,D., BALISH,E., and PHILLIPS,A. W. 1969. Control of effects of bicarbonate on sperm motility and respiration at ejaculadimorphism in a biochemical variant of Candida albicans. J. tion. Biochim. Biophys. Acta, 924: 519-529. Bacterial. 100: 701-701. TASCHDJIAN, C. L., BURCHALL, J. J., and KOZINN,P. J. 1960. Rapid NIIMI, M., NIIMI, K., TOKUNAGA, J., and NAKAYAMA, H. 1980. identification of C . albicans by filamentation on serum and serum Changes in cyclic nucleotide levels and dimorphic transition in substitutes. AMA J. Dis. Child. 99: 212-215. Candida albicans. J. Bacterial. 142: 1010-1014. WELD, J. T. 1952. Candida albicans: rapid identification in pure ODDS,F. C. 1985. Morphogenesis in Candida albicans. CRC Crit. cultures with carbon dioxide on modified eosin - methylene blue Rev. Microbiol. 12: 45-93. medium. AMA Arch. Dermatol . Syphilol. 66: 961494. 1988. Candida and candidosis. 2nd ed. Bailliere Tindall, YANAGITA, T. 1963. Carbon dioxide fixation in germinating conidioLondon. spores of Aspergillus niger. J. Gen. Appl. Microbiol. 9: 343-351. ODDS,F. C., and ABBOTT,A. B. 1980. A simple system for the presumptive identification of Candida albicans and differentiation of strains within the species. Sabouraudia, 18: 301-317.
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Received August 9, 1989 Accepted December 5, 1989 MOCK,R. C., POLLACK, J. H., and HASHIMOTO, T. 1990. Carbon dioxide induces endotrophic germ tube formation in Candida albicans. Can. J . Microbiol. 36: 249-253. Candida albicans formed germ tubes when exposed to air containing 5 to 15% carbon dioxide (C02).The C02-mediatedgerm tube formation occurred optimally at 37°C in a pH range of 5.5 to 6.5. No germ tubes were produced at 25"C, even when the optimal concentration of C02 (10%) was present in the environment. The requirement of C02 for germ tube formation could be partially substituted by sodium bicarbonate but not by N2. Carbon dioxide was required to be present throughout the entire course of germ tube emergence suggesting that its role is not limited to an initial triggering of morphogenic change. We suggest that carbon dioxide may be a common effector responsible for the germ tube promoting activity of certain chemical inducers for C . albicans. Key words: Candida albican germ tubes, C02-induced germ tube formation, endotrophic germ tube formation. MOCK,R. C., POLLACK, J. H., et HASHIMOTO, T. 1990. Carbon dioxide induces endotrophic germ tube formation in Candida albicans. Can. J . Microbiol. 36 : 249-253. Expos6 B de l'air contenant de 5 B 10% de bioxyde de carbone (C02), le Candida albicans produit des tubes germinaux, et ce, de f a ~ o n optimale B 37°C et B des pHs variant de 5,5 B 6.5. Toutefois, B 25"C, meme si I'air ambiant contient une concentration optimale de 10% de C02, la production de tubes germinaux est nulle. L'exigence du C02 peut $tre substituke partiellement par du bicarbonate de sodium, mais non par du N2. Tout au cours de l'kmergence des tubes germinaux, la prksence de bioxyde de carbone s'est avkrke requise, ce qui suggtre que son rSle n'est pas limit6 au dkclenchement initial des changements morphogkniques. La suggestion est donc avancke que le bioxyde de carbone peut &re un effecteur de base responsable de I'activitk de certains inducteurs chimiques qui amorcent le dkveloppement des tubes germinaux chez le C. albicans. Mots clis : tubes germinaux de Candida albicans, induction de la formation de tubes germinaux par le C02, formation endotrophe de tubes germinaux. [Traduit par la revue]
Candida albicans is an opportunistic fungal pathogen, which can grow, depending on the environmental conditions, either in the yeast o r the mycelial form. At temperatures below 28"C, C. albicans usually assumes a yeast morphology. When yeast cells of C. albicans are exposed to an appropriate inducer at higher temperatures (37"C), they initiate the hyphal phase of growth by forming germ tubes. A variety of substances are known to induce germ tube formation in C. albicans. These include tissue culture media (Lee et al. 1975), body fluids (Landau et al. 1965; Taschdjian et al. 1960), L-proline (Dabrowa et al. 1976; Land et al. 1975), N-acetyl-D-glucosamine (GlcNAc) (Simonetti et al. 1974), and ethanol (Pollack and Hashimoto 1985). At present, the mechanism whereby these diverse compounds induce germ tube formation in C. albicans remains virtually unknown. While investigating the germ tube inducing capability of various intermediary metabolites of known chemical inducers, we found that C02 alone is quite capable of inducing germ tube formation in C . albicans. This paper describes C02-induced and bicarbonate-induced germ tube formation in C. albicans. A preliminary account of this work has appeared in abstract form (R. C. Mock, J. H. Pollack, and T. Hashimoto. 1988. 88th Annual Meeting of the American Society for Microbiology p. 391. (Abstr.)).
Materials and methods Organisms Candida albicans ATCC 58716 was routinely used for the experiments described in this paper. In some experiments, C . albicans ATCC
'present address: Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2153 Sheridan Road, Evanston, IL 60208, U.S.A. 'Author to whom all correspondence should be addressed. Rinted in Canada / Irnprirnd au Canada
10261 and 14053 were used for comparative purposes. Cells were maintained on Sabouraud dextrose agar (SDA, Difco) at 20°C. Growth and preparation of organisms Yeast-phase cells were prepared by growing the cells on SDA for 24 h at 37°C. The yeast cells were washed with distilled water by vacuum filtration at least 10 times as described earlier (Pollack and Hashimoto 1987). Approximately 95% of the yeast cells prepared by this procedure were cells without buds. The washed cells were used immediately. Preparation of slide microcultures Purified Noble agar (Difco) or agarose (type V, Sigma Chemical Co.), washed several days in double-distilledwater, was routinely used in all experiments. During the washing, water was frequently changed. All glassware, glass slides, and cover slips were acid-washed prior to use. Molten agar (3%) was mixed with an equal volume of buffer (Tris-maleate, 0 . 1 M, pH 5.75) prior to use. The concentration of the buffer was sufficient to prevent more than a 0.1 pH unit change during the course of all experiments. The buffered molten agar was placed on glass slides and the agar surface was flattened by placing a thin cover slip (No. 1 thickness, 22 x 22 rnrn) over the agar until it hardened. Approximately 20 pL of the cell suspension (2.0 X lo6cells/mL) was spread over the agar surface. Exposure to COz and bicarbonate Microcultures were incubated in an airtight chamber filled with a mixture of air and carbon dioxide (Airco, Carol Stream, IL). A high humidity was maintained inside of the chamber so that the surface of' the microculturesdid not dry during incubation. All microcultures were routinely incubated at 37OC for 3 h unless otherwise indicated. Controls were incubated in chambers containing only atmospheric air (COz concentration is approximately 0.03%). Some experiments were run under C02-free air by placing KOH pellets inside @e incubation chamber. To confim that the observed C02 effect on C . albicans was not due to a reduced partial pressure of oxygen, similar experiments were performed by partially replacing air with nitrogen gas. To test
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FIG. 1. Phase-contrast micrograph of C. albicans yeast cells forming germ tubes endotrophically on purified agar at 37OC in the presence of 10% co2. whether C 0 2 could be replaced by bicarbonate, cells (final concentration of 7.5 x lo5 cellslmL) were inoculated into Tris-maleate buffer (0.1 M, pH 5.75, final volume 2 mL, in a 12 X 75 mm glass tube) containing specified concentrations of sodium bicarbonate. Unless otherwise indicated, all tubes were sealed with Parafiim and incubated at 37°C. In some experiments, 20 pL of the cell suspension was sandwiched between slide and cover glass (22 mm2), without agar, and incubated in a moist chamber.
COrfree Atrnos- I % CO2 5'/.COz IO%C02 15%C02 20%COz 100% air pheric 99%air 95%air 90%air 85%air 8 0 % a i r C02 air
FIG.2. Effect of C 0 2 concentration on germ tube formation in C. albicans. All cells were incubated at 37OC on purified agarose. The data are the average of three separate experiments, and lines at the top of each bar represent standard deviations (SD).
(32% formed pseudohyphae), and no cell developed germ tubes at 25°C (85% of the cells not forming germ tubes had buds) (Fig. Determination of germ tube formation 3). Similar results were obtained when other strains (ATCC The percentage of cells forming germ tubes and buds was measured 10261 and 14053) of C. albicans were used (data not shown). by counting 200 cells in each sample with a phase-contrast microscope To see whether cells exposed to C02for a brief period at 37°C (Pollack and Hashimoto 1987, 1988). The length of germ tubes was commit themselves to germ tube formation, microcultures were determined by using an ocular micrometer. periodically transferred to chambers containing only atmosResults pheric air and the incubation was continued at 37°C. The results Candida albicans cells formed short germ tubes (20%), essentially no germ tubes (Fig. ate could partially replace the role of C02 in inducing germ tube 2) or buds were formed, suggesting that this concentration of formation in C . albicans. As shown in Fig. 5, C . albicans cells C 0 2 is inhibitory for growth. To determine whether the formed germ tubes when incubated at 37°C in Tris-maleate observed germ tube formation was due to C02or reduced partial buffer (0.1 M, pH 5.75) containing sodium bicarbonate. The pressure of oxygen, the atmospheric air in the incubation bicarbonate-induced germ tube formation in C . albicans occurred chamber was partially replaced with N2 gas (Airco, Carol optimally at a pH range of 5.5-6.0 and very poorly or not at Stream, IL). No cells formed germ tubes under semianaerobic all above pH 7.5 (data not shown). As shown in Fig. 5, the conditions created by partial (5, 10, 15, and 20%) replacement percentage of cells forming germ tubes rarely exceeded 60-70% of air with N2 (data not shown). Thus, it was C02, not reduced in test tubes even under optimal conditions, although the entire oxygen tension, that induced the germ tube formation in cell populations were fully capable of forming germ tubes in C. albicans. Under strict anaerobic conditions, neither budding bovine serum. Of the cells that did not form germ tubes, 25 to nor germ tube formation was observed. 30% formed small single buds. In contrast, 80 to 90% of the Relatively high incubation temperatures (33-37°C) were cells formed germ tubes when a thin layer of the same cells required for the maximum germ tube formation induced by suspended in the buffer containing bicarbonate were sandC02. Even with the optimal C 0 2 concentration (lo%), only wiched between slide and cover glass and incubated at 37°C 60% of the C . albicans cells could produce germ tubes at 30°C (Fig. 5). The control cells, suspended either in the same buffer
I
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MOCK ET AL.
Time (min)
FIG. 3. Effect of temperature on the kinetics of C02-induced germ tube formation in C albicans. A, 37°C; 0 , 30°C; 0 , 25°C.
Concentration (mM FIG.5. Germ tube formation in C. albicans in the presence of bicarbonate or NaCI. Cells were incubated in Tris-maleate buffer (0.1
M, pH 5.75, final volume 2 rnL) containing the indicated amounts of sodium bicarbonate (.,A) or NaCl(0,A) either in 12 X 75 mm sealed test tubes (A,A) or sandwiched between a glass slide and cover slip (O,.) and incubated at 37OC for 3 h.
or saline solution without bicarbonate, failed to form germ tubes (Fig. 5). Of such control cells, 80 to 90% formed small single buds.
C3
1 0 % C02for 45 mm, then transferred to C02 free air
Time ( m i n ) FIG. 4. Effect of shift from C02 to air on germ tube formation in C. albicans. Cells forming germ tubes on purified agarose under 10%C02 (90% air) were transferred to C02-free air at time indicated (thick arrows) and incubated up to 2 h. Essentially all cells continuously incubated under 10% C02 formed germ tubes, while those cells incubated in the presence of C02-free air or atmospheric air formed no germ tubes. All experiments were at 37°C.
Discussion Although the stimulatory effect of C02 (Bedell and Sol1 1979; Mardon et al. 1969; Odds and Abbott 1980; Sims 1986; Weld 1952) or bicarbonate (Pollack and Hashimoto 1988) on the hyphal phase of growth in C. albicans is well documented, this is the first report that carbon dioxide or bicarbonate alone can induce germ tube formation. Since no germ tubes were formed in the cells incubated under environmental conditions lacking C 0 2 (Fig. 2), it is unlikely that the germ tube formation observed in the cells was due to chemical inducers contaminating the agar. The limited size of the germ tubes also supports this assumption (Fig. 1). Furthermore, germ tubes were formed by sandwiching the cells, in the presence of bicarbonate, between slide and cover glass, in the absence of any agar. The agar was used only to maintain moisture and to provide direct contact between the cells and the C 0 2gas. Nutritionally, we believe that the germ tubes formed in the presence of C02 are endotrophic in nature. Whereas exogenous C 0 2 is required to initiate and maintain the hyphal form, no additional nutrients need to be added to sustain this growth form until the germ tubes are approximately 10 pm long. Cadida albicans -yeast cells are known to contain various storage carbohydrates such as glycogen, trehalose, and wall glucans (Chiew 1989). This indicates that C. albicans yeast cells may contain sufficient endogenous carbon sources, other than C02, to support the initial phase of cell growth. It is also evident from our earlier study on the effect of ethanol on C . albicans (Pollack and Hashimoto 1985) and those of Shepherd et al. (1985) that
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competent yeast cells do not require exogenous nitrogen compounds to form germ tubes. Thus, competent C . albicans yeast cells are fully capable of forming germ tubes endotrophically in response to proper inductive stimuli. It is important to note that C 0 2is required to be present in the environmentthroughout the entire course of incubation (Fig. 4). Premature removal of C 0 2 prior to the appearance of the germ tubes resulted in the abortive formation of germ tubes. Its removal subsequent to their appearance resulted in cessation of elongation and often the reversion to budding growth form. This suggests that the role of C 0 2 in germ tube formation in this fungus is not limited to an initial triggering effect but is a constant requirement for the hyphal mode of growth. Probably the most significant aspect of our observations made in this study is that it has raised an interesting possibility that some chemical inducers may promote germ tube formation in C . albicans via C 0 2 generated by their metabolism. As mentioned earlier, germ tube formation in C . albicans can be induced by a number of structurally or metabolically unrelated compounds (Odds 1985 and 1988; Shephard et al. 1985). There seems to be no common metabolic pathway or metabolic product shared by these diversely different chemical inducers unless ubiquitous metabolites such as C 0 2 are considered. In discussing the mode of action of different chemical inducers of Candida morphogenesis, Holmes and Shepherd (1987) speculated that these compounds may act in a similar manner via catabolism to a common intermediate. Candida albicans was shown to produce C 0 2 effectively from GlcNAc (HrmovB et al. 1983). Sims (1986) suggested that the internal concentration of C02 is the critical factor in the morphogenesis of C . albicans, whether achieved by raising the external pressure or internal metabolism. Alternatively, it is possible that an effective level of intracellular C 0 2 concentration could be achieved by the metabolism of the inducers themselves or by the catabolism of intracellular storage carbohydrates triggered by certain chemical inducers. The observation that immobilized GlcNAc, which was obviously nonmetabolizable, could induce germ tubes in C . albicans (Shephard and Sullivan 1983) supports the latter possibility. It is not too surprising that C 0 2 was found to be an important regulator of C . albicans dimorphism because it is a recognized mediator of morphogenesis in bacteria and other fungi (Jones and Greenfield 1982; J W. T. Wirnpemy 1969. 19th Symposium of the Society for General Microbiology, April 1969, University College, London. Edited by P. Meadow and S. J. Pirt. Cambridge University Press, Cambridge). Morphogenic events affected by C 0 2 are numerous. These include stimulating germination of streptomyces spores (Grund and Ensign 1978) and fungal conidia (Yanagita 1963; Pass and Griffin 1972), initiating fruiting body formation (Sietsma et al. 1977) and spherule formation (Klotz et al. 1984), altering of cell shape and division (StraskrabovB et al. 1980; Lumdsen et al. 1987), and initiating dimorphic changes in fungi (Bartnicki-Garcia and Nickerson 1962; Ho and Smith 1986). The precise mechanisms whereby C 0 2 regulates the morphogenic events in C . albicans and other fungi remain largely unknown. At this point, one can only speculate several possibilities based on the findings made in other biological systems. One possibility is through the regulation of pH. The critical role of the environmental pH in the dimorphism of C . albicans has been well documented (Sol1 1985; Pollack and Hashimoto 1987). The effect of C 0 2 and bicarbonate on intracellular pH and morphological changes induced by such intracellular pH
changes in various cells have been well documented (Roos and Baron 1981). Measurable alterations in the intracellular pH of C. albicans undergoing morphogenic changes have recently been noted (Stewart et al. 1987; Kaur et al. 1988). However, these changes may be the consequence rather than the cause of morphogenic changes. While there was a greater increase in the internal pH in budding cells compared with germ tube forming cells, there was no significant change in pH during the first 30-120 min of germ tube formation (Kaur et al. 1988). It is also less likely that cytoplasmic alkalinization plays a major role in C02-induced germ tube formation since yeast cells do not initiate germ tube formation in alkaline buffer solutions unless solutions are supplemented with exogenous inducers such as proline or GlcNAc (Pollack and Hashimoto 1987, 1988). The observation that the requirement for C 0 2 could be partially replaced by 5-15 rnM oxaloacetate (R. C. Mock and T. Hashimoto, unpublished data) suggests that the maintenance of the tricarboxylic acid cycle by anaplerotic reactions may be required for germ tube development. It was previously suggested that C 0 2 is incorporated into some intermediates via C02 fixation promoting the metabolism conducive to germination of fungal conidia (Yanagita 1963) and Streptomyces spores (Grund and Ensign 1978). Carbon dioxide is most probably fixed by carboxylation of either pyruvate or phosphoenolpyruvate. Carbon dioxide and bicarbonate are known to affect a number of cellular enzymes as well as microbial activities (Jones and Greenfield 1982). However, at present, no specific enzyme has been identified that is active only in the yeast or the mycelial phase of growth. It is probable that stimulation or suppression of some enzymes involved in morphogenesis by C02 may be involved in C . albicans dimorphism. For example, CAMP is implicated in Candida dimorphism, although evidence for its involvement is still equivocal (Niimi et al. 1980; Chattaway et al. 1981). Recently, stimulation of adenyl cyclase by bicarbonate has been reported for eukaryotic cells (Tajima et al. 1987). Suppression or stimulation by C 0 2 of some enzymes involved in the biosynthesis of unique wall components is another intriguing possibility. Finally, it is possible that the expression of certain genes coding key morphogenic substances is dependent on high C02 concentrations. Recently, microbial genes requiring high C 0 2 tension for their expression have been identified and cloned (Makino et al. 1988). W. J. 1962. Induction of BARTNICKI-GARCIA, S., and NICKERSON, yeast-like development in Mucor by carbon dioxide. J. Bacterial. 84: 829-840.
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