Departamento de Microbiología y Genética, Instituto de Microbiología Bioquímica/CSIC, Universidad de Salamanca, 37071 Salamanca, Spain1
Author for correspondence: Angel Domínguez. Tel: +34 923 294677. Fax: +34 923 224876. e-mail: ado{at}gugu.usal.es
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ABSTRACT |
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Keywords: Yarrowia lipolytica, Candida albicans, budding, yeasthypha transition, hydroxyurea
Abbreviations: GlcNAc, N-acetylglucosamine; HU, hydroxyurea; DAPI, 4',6-diamidino-2-phenylindole; SEM, scanning electron microscopy
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INTRODUCTION |
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Two yeasts, Yarrowia lipolytica and Candida albicans, are currently being used in our laboratory as dimorphic models for analysing the yeasthypha transition. Y. lipolytica is a heterothallic yeast, amenable to genetic analysis, in which most of the natural isolates are haploid (Barth & Gaillardin, 1996 ). We have developed a simple system for inducing the yeasthypha transition, replacing the carbon source glucose by N-acetylglucosamine (GlcNAc; Rodríguez & Domínguez, 1984
) and have isolated a homeo-gene, HOY1, that is required for hypha formation and for which no clear counterpart has been described in either S. cerevisiae or C. albicans (Torres-Guzmán & Domínguez, 1997
).
C. albicans is the most frequently isolated fungal pathogen in humans and its growth mode is determined by environmental conditions (high temperature, high ratio of CO2 to O2, serum, nutrient-poor media, etc.). C. albicans is diploid, has no known sexual cycle (Odds, 1988 ; Edwards, 1990
) and several pathways regulating its cell morphology have been described (Saporito-Irwin et al., 1995
; Leberer et al., 1996
; Braun & Johnson, 1997
; Stoldt et al., 1997
; Lo et al., 1997
). However, data concerning the budding patterns in both yeasts are scanty. Chaffin (1984)
reported that in C. albicans the buds emerge primarily at one pole of the mother cell when cells are grown in the yeast form at 28 °C and pH 7·4, while bud sites not adjacent to the previous ones are found in yeast cells growing at 37 °C and pH 4·5. The same work also describes that the selection of sites for germ tube formation seems random after the cells are grown to stationary phase at 28 °C. In a more recent paper, the isolation of CaRSR1, a gene analogous to the S. cerevisiae BUD1 gene, has been described (Yaar et al., 1997
). CaRSR1 is required for normal bud site selection, germ tube emergence (Lees broth) and hyphal elongation. In this study, we show that C. albicans and Y. lipolytica display different budding patterns, which also differ from that described for S. cerevisiae. Furthermore, the behaviour of both yeasts also differs with respect to germ tube emission in the absence of DNA synthesis. Taken together, all our data suggest that several experimental models would be necessary if general conclusions are to be drawn about the developmental pathways involved in the yeasthypha transition in fungi.
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METHODS |
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Flow cytometry and light microscopy.
Cells were fixed in 70% ethanol and processed for flow cytometry (Lew et al., 1992 ) or with 4',6-diamidino-2-phenylindole (DAPI) for nuclear staining (Sherman et al., 1986
). A BectonDickinson FACScan was used for flow cytometry. Cells were photographed using a phase-contrast Zeiss axiophot photomicroscope equipped with a 35 mm camera using Ilford FP4 Plus film (125 ASA).
Scanning electron microscopy (SEM).
Cells were harvested, washed in 0·1 M sodium phosphate buffer, pH 7·4, prefixed with glutaraldehyde (5% glutaraldehyde in 0·1 M sodium phosphate buffer, pH 7·4) for 1 h, washed twice in buffer and placed in 1% osmium tetraoxide for 1 h at 4 °C. The material was subsequently washed in distilled water and dehydrated in a graded acetone series. The dehydrated cells, immersed in absolute acetone, were mounted on specimen holders, air-dried, coated with gold in a SEM Coating System (Bio-Rad) and examined under a Zeiss DSM 940 scanning electron microscope.
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RESULTS |
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Bud site selection during the yeast mode of growth
Observations of Calcofluor-stained cells (not shown) and SEM observations of all the strains tested revealed that the first bud site in an ellipsoidal cell almost always (95% of the cells) formed close to or at the tip of the cell in both haploid and diploid strains of Y. lipolytica and in diploid strains of C. albicans. This bud site formation was similar to what has been reported for S. cerevisiae. When we examined cells with two or more bud scars, a different picture emerged. In C. albicans, which is a diploid organism (Odds, 1988 ), SEM clearly showed that most the new bud sites (93%, Table 1
) were adjacent to the previous ones (axial pattern, Fig. 1a
and b
) in agreement with the results described by Chaffin (1984)
. In Y. lipolytica (whose cells are more ellipsoid) haploid (MatA and MatB) or diploid (MatA/MatB) cells, the second bud was almost invariably (in more than 95% of the cells, Table 1
) formed opposite the first bud site (bipolar pattern, Fig. 2a
and b
). In the case of S. cerevisiae, it has been described that haploid cells (a cells and
cells) and diploid cells that are homozygous at the mating type locus (a/a and
/
cells) bud axially, while diploid (a/
) cells show a different budding pattern. Diploid daughters bud opposite the previous bud site (distal budding) while diploid mother cells mainly bud bipolarly, i.e. either adjacent to or opposite the previous bud site (Madden et al., 1992
). Thus, our results with Y. lipolytica and C. albicans show clear differences with those described for S. cerevisiae.
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Similar experiments were carried out with Y. lipolytica. Fig. 7(a), lane 1 (and Fig. 8a
), shows the DNA content of an exponentially growing culture (yeast form of Y. lipolytica haploid strain SA-1), with the typical distribution of cells in the G1 and G2 phases. Cultures synchronized prior to the induction of germ tube emission, with all the cells in the G1 phase of the cell cycle, are represented in Fig. 7(a)
, lane 2 (an image of the culture is shown in Fig. 8b
). A constant increase in the DNA content, indicating that all the cells have more than one nucleus, is evident (Fig. 7a
, lanes 4 and 5) in the control culture, which also developed a normal yeasthypha transition (Fig. 8c
and d
). DNA replication did not occur in the presence of HU (Fig. 7b
); in this case the drug completely blocked the yeasthypha transition (Fig. 8e
). The observation that some cells were able to bud is in agreement with the results described for S. cerevisiae by Slater (1973)
and Hartwell (1976)
, and very few of them had a more cylindrical shape (Fig. 8e
).
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DISCUSSION |
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We analysed several strains of C. albicans and Y. lipolytica and in the latter case the behaviour of haploid and diploid strains also. Although some subtle differences among strains of different backgrounds and in the same strain under the different conditions of the yeasthypha transition were observed, we believe that the major features of our descriptions can be generalized.
Regarding bud formation during the yeast mode of growth, in both Y. lipolytica and C. albicans the first bud generally appeared on one pole of the cell, as has been described for S. cerevisiae (Madden et al., 1992 ). Also, and in agreement with the results obtained for S. cerevisiae, no cases of overlapping bud sites (overlapping bud scars) have ever been observed in axially budding or bipolar budding cells (Chant & Pringle, 1995
; Figs 1
, 2
, 3
and 4
).
On comparing budding patterns, we observed that diploid Y. lipolytica cells clearly budded in a bipolar way (like S. cerevisiae), while C. albicans followed an axial budding pattern. Surprisingly, haploid cells of Y. lipolytica also budded with a bipolar pattern, again differing from the behaviour of S. cerevisiae haploid cells. Our findings indicate that at least the three developmental models shown in Fig. 9 are necessary to understand the budding patterns of vegetatively growing yeast cells.
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Regarding germ tube emission, again, we observed similar types of behaviour for all the strains tested under all the conditions assayed. In S. cerevisiae, pseudohyphal growth requires a polar budding pattern (Gimeno et al., 1992 ) and hence haploid filament formation depends on a switch from an axial to a bipolar mode of bud site selection (Roberts & Fink, 1994
). Our results are essentially in agreement with this kind of behaviour although we stress that since Y. lipolytica haploid and diploid cells show a bipolar budding pattern such a switch is not necessary. In Y. lipolytica the second bud site and germ tube emission always occur on the pole distal to the preceding bud scar, independently of starvation and refeeding. This implies that nutritionally starved cells do not lose any specific component involved in bipolar budding-pattern signalling. For the time being we can only suggest that either axial genes involved in bud site selection, such as BUD3, BUD4, BUD10, etc. (Chant et al., 1995
; Roemer et al., 1996
), are not functional in Y. lipolytica, or that the gene products involved in the bipolar pattern act in this yeast in a dominant way. Experiments to test such possibilities are currently under way. In C. albicans, germ tube emergence is preceded by a switch from an axial to a different mode (lateral or polar) of bud site selection, resembling the behaviour of S. cerevisiae haploid cells during both invasive and pseudohyphal growth (Roberts & Fink, 1994
).
HU inhibited DNA synthesis in C. albicans and Y. lipolytica (Figs 5 and 7
). However, the addition of HU to cultures undergoing the yeasthypha transition produced a different effect in both yeast species: whereas in C. albicans germ tube emission occurs in an apparently normal way up to 90 min after the addition of HU (in agreement with the results described by Shepherd et al., 1980
), in Y. lipolytica the morphogenetic switch is completely abolished (in agreement with our previous results with asynchronous cultures, Rodríguez et al., 1990
). One possible interpretation of this latter result could be that some preformed mRNA molecules with a short half life would be degraded during the long incubation times (46 h) necessary for germ tube induction in synchronized cells of Y. lipolytica in comparison with the time required by C. albicans (12 h).
While it is clear that dimorphism in fungi is controlled by multiple signalling pathways and that some of these are conserved among distantly related fungi, this study and several others suggest that several different models should be used if we are to understand how fungal cells integrate the information from different pathways to effect the dimorphic switch.
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ACKNOWLEDGEMENTS |
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Received 25 February 1999;
revised 8 June 1999;
accepted 28 June 1999.