Stage specific proteins, like a 70 kDa species, are inhibited during yeast to mycelium phase transition. The analysis of protein patterns in specific phases of ...
Journalof Medical & VeterinaryMycology 1997,35, 205-211
Accepted 30 January 1997
Protein synthesis patterns of Paracoccidioides brasiliensis isolates in stage-specific forms and during cellular differentiation S. M. S A L E M - I Z A C C , R. S. A. JESUINO, W. A. BRITO, M. PEREIRA, M. S. S. FELIPE* &
C. M. A. SOARES Laboratdrio de Biologia Molecular, ICB Universidade Federal de Goids, Campus II, 74001-970, Goiania, Goids, Brazil; and *Laboratdrio de Biologia Molecular, tCB, Universidade de Brasilia, 70910-900, Brasilia, DF, Brazil In this paper we compared the protein synthesis patterns of Paracoccidioides brasiliensis isolates. The protein profiles were compared for both yeast and mycelial forms and similarity analysis among them was performed by calculating similarity matrices and grouping the isolates in dendrograms. The examined isolates exhibited highly variable cellular morphology at 36 °C, when typical yeast cells were expected. On the other hand, at 26 °C all the isolates showed mycelial morphology. The analysis of protein synthesis profiles made it possible to cluster the P. brasiliensis isolates into groups that correlated with the morphological data. Interestingly, growth at 36 °C strongly decreased the heterogeneity of protein synthesis patterns seen in mycelial isolates. It was possible to cluster the isolates grown at 36 °C in three groups based on their two-dimensional protein synthesis analysis. The similarity index observed among the mycelial isolates was lower than that obtained with yeast cells, suggesting a more homogeneous gene expression pattern in the host-adapted form than in the saprobic phase. Keywords
P. brasiliensis, protein synthesis patterns
Introduction Paracoccidioides brasiliensis is a thermally dimorphic
fungus, the aetiological agent of paracoccidioidomycosis, the most prevalent systemic mycosis in Brazil [1]. The infectious phase is the mycelium or its propagules. When inhaled, the inoculum differentiates to form yeast cells establishing the infection in human lungs [24]. It is known that P. brasiliensis isolates present distinct biochemical and physiological characteristics and it is believed that this fact may account for some of the differences found in the clinical manifestations of paracoccidioidomycosis [5,6]. Several biochemical aspects have been studied in P. brasiliensis isolates. Lipid composition and isoenzymatic profiles show differences among isolates [7,8]. The profiles of DNA polymorphic fragments are different among isolates suggesting genetic variability in P. brasiliensis [9]. Despite these studies, there Correspondence: Dr C. M. A. Soares, Laboratdrio de Biologia Molecular, ICB, UniversidadeFederal de Goifis,Campus II, 74001970, Goifinia,Goi/ts, Brazil. © 1997ISHAM
are no descriptions of the protein patterns associated with P. brasiliensis isolates. Molecular aspects related to cellular differentiation and consequently the infectious pathway are poorly understood. The inhibition and induction of particular proteins preceding the morphological alterations seen in dimorphism was described [10]. Stage specific proteins, like a 70 kDa species, are inhibited during yeast to mycelium phase transition. The analysis of protein patterns in specific phases of growth may constitute a way of characterizing P. brasiliensis isolates, as has been found with other pathogens [11-14], and in addition may contribute to basic biological knowledge of the fungus. The identification of molecular alterations during the initial stages of cellular differentiation may be important in the characterization of proteins that could be related to dimorphism. In this paper morphological characteristics and protein synthesis profiles of yeast and mycelial forms of P. brasiliensis isolates were compared. We also analysed the degree of change occurring in protein patterns during cellular differentiation for these isolates of P. brasiliensis.
Salem-lzacc et
al.
Materials and methods
Organisms The isolates of P. brasiliensis studied in this work were: Pb01, Pb166, Pb662, Pb2052 and Pb7455 (M. R. R. Silva collection, UFG, Goi~mia, GO, Brazil); Pbl8 and Pbll3 (C. S. Lacaz collection, FMUSP, S. Paulo, Brazil). The yeast and mould forms were cultured on solid FavaNeto's medium [15] at 36 °C and 26 °C, respectively. The yeast cells were subcultured every 10 days and moulds every 15 days. Scanning electron
microscopy
Yeast and mycelial cells underwent primary fixation by immersion in a solution containing 2.0% (v:v) glutaraldehyde and 2.0% (w:v) paraformaldehyde in 0.05 M cacodylate buffer pH 7.4, for 24 h at 5 °C. Then the cells were adhered to slides, washed two times in the cacodylate buffer and treated with 1.0% (w:v) OsO4 for 30 min at 20 °C. The cells were washed to remove excess OsO4. Dehydration was accomplished through a graded acetone series ranging from 30% to 100% (v:v). The specimens were mounted on stubs, dried by a critical point method [16] and gold coated in a Sputter Coater Balzers SCD 050. The specimens were examined in a JEOL JEM 840A scanning electron microscopy.
Phase transition Phase transition was performed as described [10]. In brief, mycelium grown on solid medium was transferred to liquid medium [17] and incubated for 48 h. After this time the cells were transferred to the differentiation temperature of 36 °C.
Incorporation of [3SS]-L-methionine The mycelium and yeast cells were transferred from solid to liquid medium. After 48 h the precursor was added (200/tCi m1-1) for 18h. In the cellular differentiation experiments the radioactive precursor was added immediately before the temperature shifts.
Analysis of proteins by two-dimensionalgel electrophoresis After 18 h in the presence of the radioactive precursor the cells were washed in cold Tris buffer (20 mM Tris HC1 pH 8'8, 2mM CaC12), containing the protease inhibitors (Sigma): TLCK (N-alpha-p-tosyl-L-lysine chloromethyl ketone) 50#g ml-1; TPCK (N-tosyl-L-phenylalanine chloromethyl ketone) 50/~g ml-1; PMSF (phenylmethylsulphonil fluoride) 4 raM; iodoacetamide 5 mM; EDTA
(ethylenediaminotetraacetic acid) 1 mM; leupeptin 20/~M; PCMB (4-chloromercuribenzoic acid) l mM, centrifuged at 10 000g, frozen in liquid nitrogen and broken by mechanical maceration. Cellular debris was removed by centrifugation and the proteins were precipitated by 10% (w:v) TCA (trichloroacetic acid) addition. The pellet was washed in 100% cold acetone and resuspended in lysis buffer containing: 9-5 M urea, 2% (w:v) NP40 (nonidet P40), 5% (v:v) fl-mercaptoethanol, ampholines 5.0-8.0 and 3-5 10.0 (ratio 4:1). Electrophoresis was performed according to O'Farrel' et al. [18]. The second dimension w a s performed in a 10% SDS-polyacrylamide gel according to the Laemmli description [19].
Fluorography Gel fluorography was performed according to Bonner and Laskey [20]. After electrophoresis the gels were treated with dimethyl sulfoxide (DMSO) for 2 h and for three additional hours in the fluorography solution 22% (w:v) PPO (2,5-diphenyl-oxazole) in DMSO. The gels were dried and exposed to a Kodak X-Omat film at - 7 0 °C. Statistical
analysis
Analysis of protein patterns was carried out by constructing matrices based on autoradiography photographs. A master image was constructed showing the proteins preferentially expressed and strongly detected in yeast and mycelial cells, considering all the isolates. The proteins were numbered in the master image. Two gels of each isolate were utilized and the spots present in both were considered in the image construction. The strong synthesis of a particular protein was designed by a score of one, while minor expression, which could not be precisely detected as an individualized spot, was scored as zero. The total scored spots in all two-dimensional autoradiographies was 40. The exposure time was about the same for all autoradiograms as we normalized the total sample radioactivity. Similarity analysis was performed utilizing the Jaccard index [21]. The dendrograms showing the patterns of similarity were constructed by the Unweighted Pair Group Method Analysis (UPGMA) using the NTSYS program [22].
Results
Morphological analysis In scanning electron microscopy, the isolates Pb01 and Pb7455 presented rounded cells, varying in size from 5-10/~m, with several budding loci, as often described for P. brasiliensis (Fig. la, b). The isolates Pbl8, Pb166, and Pb2052 showed elongated cells, with budding (Fig. © 1997 ISHAM, Journal of Medical & Veterinary Mycology 35, 205-21 I
Protein synthesisin P. brasiliensisisolates ~ 1 ~
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synthesis patterns found in the yeast cells of seven isolates: Pb01, Pb7455, Pbl8, Pb166, Pb2052, Pbll3 and Pb662 are presented in Fig. 2. Characteristically, all of them present high expression of some protein species, like these with molecular weights of 80, 72, 71, 64 and 60 kDa. Some differences among the protein patterns are evident for different isolates. Isolates Pb01 and Pb7455, with typical yeast morphology at 36 °C, presented a high expression of a group of proteins with high molecular weight above 60 kDa (Fig. 2a, b). The isolates Pbl8, Pb166 and Pb2052, which showed elongated cells at 36 °C, expressed species like the 70 and 72 kDa proteins, not synthesized in the other isolates (Fig. 2cm). The isolates Pbll3 and Pb662, with filamentous morphology at 36 °C, presented the highest expression of the more basic species, with isoelectric points ranging from 5.8 to 6.8 and molecular weights ranging from 80 to 30 kDa (Fig. 2f, g).
Comparing the protein patterns in yeast cells
(g) lOl~m
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The protein profiles in yeast cells were compared by calculating a similarity matrix. For that analysis a synthetic image was constructed for use in the comparison of the protein profiles among the isolates. The matrices for each isolate in the yeast phase was calculated using the Jaccard index, and are shown in Table 1. The similarity index varied from 88.9 to 36.0% among the isolates. The cluster analysis placed the seven isolates in three groups, group I (Pb01 and Pb7455); group II (Pb 113 and Pb662), and group III (Pb2052, Pb166 and Pbl8), having < 50% homology among them (Fig. 3). Inside each group the homology varied from 88"9% for Pb166 and Pb2052 to 65.2% for the Pb662 and Pb113 isolates.
Protein synthesis patterns in mycelium Fig. 1 Scanning electron microscopy of P. brasiliensis isolates. (a g), cells cultured at 36 °C from isolates: (a), Pb01; (b), Pb7455; (c), Pbl8; (d), Pb166; (e), Pb2052; (f), Pb113; (g), Pb662; (h), cells cultured at 26 °C, Pb01.
lc-e). Two isolates, Pbll3 and Pb662, showed, even at 36°C, in addition to few yeast forms, cells with a filamentous morphology similar to mycelium (Fig. lf, g). On the other hand, all the isolates cultured at 26 °C showed typical mycelial morphology, with a network of hyphae, as evidenced for isolate Pb01 (Fig. lh).
Protein synthesis patterns in yeast cells Initially, all the isolates were compared in relation to the protein synthesis patterns in the yeast cells. The protein © 1997 ISHAM, Journal of Medical & Veterinary Mycology ]5, 205-21 I
The protein synthesis patterns were also analysed in the mycelium. Figure 4 shows characteristic patterns for [~sS]-L-methionine incorporation found in the mycelial form of the isolates. The general protein profiles differed greatly among the mycelial forms, as shown for Pbll3 and Pb662 (Fig. 4a, b). Quantitative and qualitative differences are evident among the analysed isolates. Isolate Pbll3 showed high expression of some proteins species, like the 80, 60 and 58 kDa, that were not detected in Pb662 (Fig. 4a). In the Pb662 the most elevated synthesis was detected for acidic proteins with molecular masses ranging from 90 to 40 kDa. The comparison of protein patterns in the myceliat forms of the isolates is shown in Table 2. The similarity index ranged from 0' 154 for the comparison between isolates Pb 166 and Pb662 to 0.500 for Pb01 and Pbll3.
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Salem-lzaccet al.
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Fig. 2 Two-dimensional protein synthesis patterns in cells cultured at 36 °C from P. brasiliensis. (a), Pb01; (b), Pb7455; (c), Pbl8; (d), Pb166; (e), Pb2052; (f), Pb113; (g), Pb662. The arrows indicate proteins detected only in particular isolates.
Table 1 Similarity matrix obtained from protein synthesis patterns in yeast cells of P. brasiliensis isolates
Similarity coefficients for the isolates Isolate
Pb01
Pb7455
Pb01 Pb7455 Pb113 Pb662 Pb2052 Pb166 Pbl8
1-000 0.800 0.542 0.464 0.542 0.480 0458
1-000 0.500 0.423 0.500 0.435 0.409
Pb113
Pb662
Pb2052
Pb166
Pbl8
1-000 0.889 0.778
1.000 0.778
1.000
1-000 0.652
1-000
0.417 0.360 0.391
0-462 0-462 0-440
Comparison of protein synthesis patterns during the mycelium to yeast cellular differentiation We analysed the protein synthesis alterations during the initial stage of mycelium to yeast transition in P. brasiliensis isolates Pb01, Pb 166 and Pb662. These isolates were selected based on the clusters defined in Fig. 3. It is important to note that each one presented different morphology at 36 °C. The 35S-labelled proteins were analysed during the first 18 h after temperature shift for all the isolates (Fig. 5). The mycelium to yeast transition is characterized by induction of synthesis of certain protein species like the 71 and 72 kDa proteins, respectively, in Pb01 and Pb166. The dramatic increase in level of these species is noteworthy, as is the increased synthesis of the 70, 60 and 35 kDa proteins in Pb166. In the isolate Pb662 a marked decrease in synthesis of certain proteins was observed. For example, the 76 and 56 kDa ceased to be
The numbers in bold designate the isolates which are clustered in the dendrogram analysis shown in Fig. 3.
0.400
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0.800
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0!400 0!500 0!600 0!700 0!800 0!900 l!OOO Fig. 3 Dendrogram of the P. brasiliensis isolates based on the protein synthesis patterns of cells cultured at 36 °C.
produced at detectable levels just after the temperature transfer. ©1997 ISHAM,Journal of Medical & Veterinary Mycology 35, 205-211
Protein
(a)
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Fig. 4 Two-dimensional protein synthesis patterns in cells cultured at 26 °C from P. brasiliensis. (a), Pbl 13; (b), Pb662. The arrows indicate proteins detected in the isolates.
66-
36-
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Table 2 Similarity matrix obtained from the protein synthesis patterns of P. brasiliensis isolates in the mycelial phase Similarity coefficients for the isolates Isolate
Pb01
Pb7455
Pb113
Pb662
Pb166
PbO1 Pb7455 Pbll3 Pb662 Pb166
1"000 0-222 0"500 0"185 0'222
1-000 0.316 0.364 0.176
1.000 0.296 0'389
1"000 0.154
1.000
Discussion In this paper we characterize several P. brasiliensis isolates according to differences in cellular morphology and protein synthesis patterns in yeast and mycelial phases and during the cellular transition from mycelium to yeast. The analysed hyphal cells presented the characteristic morphology described for P. brasiliensis. With regard to the yeast phase very different morphologies were found among the isolates. The presence of filamentous cells despite the high time of incubation at 36 °C (more than 2 years of continuous subculturing) suggests incomplete transition to the yeast morphology as described by some authors [23]. The patterns of protein synthesis in mycelium were highly heterogeneous among the isolates. However, for cells at 36 °C, the heterogeneity was reduced and protein patterns permitted clustering in three different groups which correlated with morphological analysis. The greater disparity of protein patterns in the mycelial phases suggests a more heterogeneous gene expression profile in the saprobic phase. This could reflect the ability of mycelial cells to adapt to different environmental conditions. It is known that in P. brasiliensis difficulties in recognizing individual isolates exist and it is possible that strains that grow under different names may represent similar or identical isolates. The characterization of © 1997 ISHAM,Journal of Medical & Veterinary Mycology 35, 205-21 I
isolates through protein profiles has been described in several micro-organisms [11,12,14,24,25]. The clustering tendency evidenced for isolates in the yeast phase suggests the discriminatory capacity of the technique in characterizing the isolates. It is noteworthy that the yeast groups defined by proteins profiles correspond with groups defined by the RAPD technique, where the Pb01 and Pb7455 constituted a separate group, and Pb2052 and Pb662 belonged to two separate subgroups [9]. The only exception is the isolate Pbl8 which, in RAPD analysis, shared a subgroup clustering with the isolates Pb662 and Pbll3 (E. E. W. I. Madlum et al., unpublished results). The analysis of the protein synthesis patterns during the mycelium-to-yeast differentiation process showed that alterations in gene expression are distinct among the isolates. It was not possible to detect a common alteration among all the analysed isolates, which suggests that the phase transition is a very complex process in P. brasiliensis. Similarly, in Candida albicans the existence of phase-specific genes that are co-regulated in one phenotype and dissociated in others was observed during phase transition, suggesting the complexity of regulation of the transition event in dimorphic fungi [26]. The protein synthesis profiles in all the isolates during cellular differentiation (18 h) did not correlate with the final stage-specific profile. It seems that the phase transition event in P. brasiliensis is a multistage process. These data confirm previous descriptions in Pb01 [10] and isolate Ber [27]. In these isolates the yeast or mycelium protein profiles are not evidenced during the first hours of transition, corroborating the idea that the phasespecific protein patterns appears later in the cellular differentiation in P. brasiliensis. Acknowledgements This work was supported with grants from the Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico
Salem-lzacc et al.
26°C (a)
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(CNPq), Fundaqfio de Apoio /t Pesquisa (FUNAPEUFG), Pr6-Reitoria de Pesquisa e P6s-Graduaqfio (PRPPG-UFG) and Fundaqfio de Apoio /l Pesquisa do Distrito Federal (FAP/DF). The authors wish to thank Alexandre Siqueira Guedes Coelho for the helpful statistical discussions during this work. The authors also thank Jos6 Oscar Rodrigues de Moraes (UFG) and Sonia Nair Bfio (UnB) for the help with the electron microscopy and its analysis.
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~
-36 -29
Fig. 5 Two-dimensional protein synthesis patterns during cellular differentiation from 26 to 36 °C in P. brasiliensis. (a) and (b), Pb01 at 26 and 36 °C, respectively; (c) and (d), Pb166 at 26 and 36 °C, respectively; (e) and (f), Pb662 at 26 and 36 °C, respectively. The arrows indicate proteins with altered expression during the temperature shift.
4 Medoff G, Painter A, Kobayashi GS. Mycelial- to yeast-phase transitions of the dimorphic fungi Blastomyces dermatitidis and Paracoccidioides brasiliensis. J Bacteriol 1987; 169: 4055 60. 5 Restrepo A. The ecology of Paracoccidioides brasiliensis: a puzzle still unsolved. Sabouraudia 1985; 23: 323-34. 6 Franco M, Montenegro MR, Mendes RP, et al. Paracoccidiodomycosis: a recently proposed classification of its clinical forms. Rev Soc Bras Med Trop 1987; 20: 129-32. 7 Manocha MS, San-Blas G, Centeuo S. Lipid composition of Paracoccidioides brasiliensis: possible correlation with virulence of different strains. J Gen Microbiol 1980; 117:147 54. 8 Svidzinsky Tie, Camargo ZP. Isoenzyme profile of Paracoccidioides brasiliensis. J Med Vet Mycol 1995; 33:281 5. 9 Soares CMA, Mollinari-Madlum EEWI, da Silva SP, Pereira M, Felipe MSS. Characterization of Paracoccidioides brasiliensis isolates by random amplified polymorphic DNA analysis. J Clin Microbiol 1995; 33:505 7. 10 da Silva SP, Felipe MSS, Pereira M, Azevedo MO, Soares CM de A. Phase transition and stage-specific protein synthesis in the dimorphic fungus Paracoccidioides brasiliensis. Exp Mycol 1994; 18: 294-9. © 1997 ISHAM, Journal of Medical & Veterinary Mycology 35, 205-21 I
Protein s?nthesis in P. brasiliensisisolates
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©1997 ISHAM, Journal of Medical & Veterinary Mycology 35, 205-21 J
20 Bonner WM, Laskey RA. A film detection method for tritiumlabelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem 1974; 46: 83-8. 21 Sneath PHA, Sokal RR. The estimation of taxonomy resemblance. In: Kennedy D, Park RB, eds. Numerical Taxonomy. San Francisco: WH Freeman, 1973: 114-87. 22 Rohlf FJ. NTSYS-pc Numerical Taxonomy and Multivariate Analysis System. New York: Exeter Publishing Ltd, 1988. 23 Kashino SS, Calich VLG, Burger E, Singer-Vermes LM. In vivo and in vitro characteristics of six Paracoccidioides brasiliensis strains. Mycopathologia 1985; 92: 173-8. 24 Tabaqchali S, O'Farrell S, Holland D, Silman R. Method for the typing of Clostridium difficile based on polyacrylamide gel electrophoresis of [35S]methionine-labeled proteins. J Clin Microbiol 1986; 23: 197-8. 25 Mansour NS, Mikhail EM, El Masry MA, Sabry AG, Mohareb EW. Biochemical characterisation of human isolates of Blastocystis hominis. J Med Mierobiol 1995; 42: 304-7. 26 Morrow B, Ramsey H, Soll DR. Regulation of phase-specific genes in the more general switching system of Candida albicans strain 3153A. J Med Vet Mycol 1994; 32" 287 94. 27 Clemons KV, Feldman D, Stevens DA. Influence of oestradiol on protein expression and methionine utilization during morphogenesis of Paracoccidioides brasiliensis. J Gen Microbiol 1989; 135: 1607-17.
