Heat-shock Proteins Induced During the Mycelial ... - Semantic Scholar
Journal of General Microbiology (1987), 133, 3375-3382.
Printed in Great Britain
3375
Heat-shock Proteins Induced During the Mycelial-to-yeast Transitions of Strains of Histoplasma capsulatum By G L E N M O R E S H E A R E R , J R , ? C L A I R E H . B I R G E , P A T R I C I A D . Y U C K E N B E R G J G E O R G E S. KOBAYASHI A N D G E R A L D MEDOFF* Divisions of Infectious Diseases, Dermatology and Laboratory Medicine, Department of Medicine and the Department of Microbiology and Immunology, Washington University School of Medicine, St Louis, Missouri 63110, USA (Receiued 6 May 1987; revised 27 July 1987) Heat-shock proteins (hsp) were elicited when mycelia of the Downs strain and the more virulent G184A and G222B strains of Histoplasma capsulatum were shifted up to temperatures which induced the mycelial-to-yeast transition (34-40 "C). The classes of the major hsp which increased in synthesis in each strain were similar. However, the pattern of synthesis of these proteins at the different temperatures in Downs differed from those in the G184A and G222B strains: hsp synthesis in Downs peaked at 34 "C, whereas in G184A and G222B it was highest at 37 "C.
INTRODUCTION
The dimorphic fungal pathogen Histoplasma capsulatum exists in a mycelial form in soil and as a yeast in infected hosts (Maresca et al., 1977, 1981 ; Sacco et al., 1981 ; Schwarz, 1971). Reversible transitions between these phases can be induced in culture by temperature shifts between 25 "C (mycelial growth) and 37 "C (yeast growth). Because the mycelial-to-yeast transition is heat-induced, it is possible that its regulation involves a heat-shock response. Consistent with this possibility, the transition of mycelia to yeast at 37 "C is accompanied by rapid and marked changes in several different metabolic processes, particularly an uncoupling of oxidative phosphorylation and a decline in intracellular ATP levels, followed by a slow recovery phase (Maresca et al., 1981). We studied the induction of heat-shock proteins (hsp) in H. capsulatum when the temperature of incubation of mycelia was shifted from 25 "C to 37 "C, and discovered that hsp 70 synthesis increased soon after the shift-up in temperature (Lambowitz et al., 1983). The initial studies on the phase transition of H. capsulatum were done on Downs, a strain with low virulence in mice. When we compared the mycelial-to-yeast transition in more virulent strains of H. capsulatum (Medoff et al., 1986), we found that the changes were similar to those of the Downs strain, but less extreme. The increased sensitivity of the Downs strain to elevated temperatures may be incidental, or it may be a key factor accounting for its lower virulence for mice compared to the other strains of H. capsulatum. Here we characterize and compare more completely the hsp elicited when mycelia of Downs and the more virulent G184A and G222B strains of H . capsulatum are switched from 25 "C to temperatures of 34 "C to 40 "C.
t Present address : Department of
CA 92717, USA.
Microbiology and Molecular Genetics, University of California, Irvine,
Present address: Bio-Rad Laboratories, 1414 Harbour Way South, Richmond, CA 94801, USA. Abbreviation: hsp, heat-shock protein(s).
0001-4172 0 1987 SGM
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G . S H E A R E R AND O T H E R S METHODS
Organisms.The H . capsulutum strains are from the permanent culture collection of this laboratory (Lambowltz et al., 1983; Maresca er ul., 1977, 1981). Cells were grown in shaking flasks in a defined medium consisting of standard F-12 nutrient base (Gibco) supplemented with, per litre, 18 g glucose, 0.084 g cystine and 6 g HEPES; pH 7-5. Exponential-phase cells (mycelia grown for 48-72 h at 25 "C and yeast grown for 24-48 h at 37 "C) were used in all experiments. Hear shock. Cultures of mycelia in the mid-exponential phase of growth were incubated in a shaking water bath at 37 "C. After the designated periods of time at this elevated temperature, 10 pCi (370 kBq) ~-[~~S]methionine (Amersham) was added to the culture for 20 min. In experiments where mycelia and yeast were exposed to different temperatures (25, 30, 34,40 "C)for 90 min, the cells were cooled in a 25 "C water bath for 5 min after exposure before being pulse-labelled at 25 "C. Characteri:ation of proteins by one-dimensional electrophoresis. Cells were harvested by rapid filtration on glassfibre filters and washed twice with 10 ml distilled water at 0 "C and twice with 10 ml acetone at 0 "C. The filters were dried at 37 "C for 10 min; proteins were solubilized by heating the dry flters in 1 ml SDS (Sigma) sample buffer (Laemmli, 1970) at 100 "C for 5 min. Radioactivity incorporated into proteins was estimated by liquid scintillation counting of solubilized samples. Samples containing about 300000 d.p.m. were applied to each lane. SDS-PAGE was done using the buffer system of Laemmli (McAlister & Finkelstein, 1980), with the following modifications: the resolving gel was prepared to yield 12.5% (w/v) acrylamide and 0.42% acrylaid crosslinker (FMC Corp.) in place of bisacrylamide. Slab gels, 0-75 x 130 x 170 mm, were bonded to Gel Bond PAG film (FMC Corp.). Electrophoresis was carried out at 20 mA constant current until the bromophenol blue tracking dye reached the bottom of the slab (approx. 3 h). The standards (Amersham) used to estimate molecular mass were myosin (200 kDa), phosphorylase b (92.5 kDa), bovine serum albumin (69 kDa), ovalbumin (46 kDa), carbonic anhydrase (30 kDa) and lysozyme (14 kDa). Hsp at different temperatures were quantified by densitometry measurements on the onedimensional gels. Characterization ofproteins by two-dihensional electrophoresis. Cells were quickly washed twice with 10 ml buffer A (10 mM-Tris pH 7.4, 1 mM-phenylmethylsulphonyl fluoride) at 0 "C, resuspended in 5 ml buffer A in a 30 ml Corex tube containing 5 g 0-5 p m glass beads and broken by vigorous vortexing for 4 rnin at 0 "C. Unbroken cells were removed by centrifugation at l000g for 10min. Proteins were precipitated by adding 10:; (w/v) triGhloroacetic acid to the supernatant and incubating at 0 "C for 15 min. The precipitate was washed three times with I5 ml 80% (vp) acetone at 0 "C and once with 15 ml 100% acetone at 0 "C. Proteins were solubilized by tritrating with 200 p1 Laemmli SDS sample buffer (Laemmli, 1970) and mixing with a"glass rod. Insoluble material was removed by centrifugation at 10000g for 10 min. Electrofocusing was performed by the method of O'Farrell (1975) modified by the substitution of the zwitterionic detergent 343cholamidopropyl)dimethylammonio-I -propanesulphonate (CHAPS; Sigma) for NP-40. Ampholines (Bio-Rad) were mixtures of pH 3-7 and pH 3-10 in a 10 :1 ratio. After electrofocusing, the tube gels were immediately frozen at - 70 "C. Before use, the tube gels were equilibrated in Laemmli SDS sample buffer for 20 min and sealed with 0.57; agarose to 3 12.5% polyacrylamide slab gel and electrophoresed as described above. Flwrogruphy. g a b gels bonded to Gel Bond PAG film were processed in DMSO-PPO by the method of Bonner & Lasky (1974). The gels were then soaked in 2% (v/v) glycerol for 60 rnin and dried for 3 h at 60 "C. The dried gels were exposed to Kodak X-AR film at -70 "C until the desired density was reached. Immunoblotring. Non-radioactive samples were prepared and processed by one- and two-dimensional electrophoresis as described above. In the second dimension the samples were run on 12.5% polyacrylamide gels crosslinked with 0.33% bisacrylamide without Gel Bond PAC film. Proteins were electrophoretically transferred to nitrocellulose paper at 0-2 A for 16 h at 4 "C in a buffer consisting of 25 mM-Tris, 192 mM-glycine and 20% (v/v) methanol. The nitrocellulose was rinsed in distilled water and dried at 25 "C. Prior to addition of the primary monoclonal mouse antibody to hsp 70 of Drosophilu melanoguster (a kind gift of Dr Susan Lindquist, University of Chicago) the blots were washed for 30 rnin in 5% (w/v) non-fat dry milk (Carnation Co.) solution in phosphatebuffered saline (PBS). Blots were reacted with the primary antibody for 60 min, then with secondary antibody (Sigma; rabbit anti-mouse, 1 :loo) for 60 rnin and finally with peroxidase-conjugated goat anti-rabbit (Cappel; 1 : 1O00) for 60 min. Blots were washed twice with 504 nonfat milk in PBS for 10 rnin and once with PBS for 10 min between each antibody reaction. Antigens were visualized by incubating blots with 4-chloro-1-naphthol and hydrogen peroxide (50 mi PBS + 30 p1300,',H z 0 2added to 30 mg 4-chloro-l-naphthol+ 10 ml methanol at 0 "C). RESULTS
When mycelia of the H. capsulutum Downs strain growing at 25 "C were shifted to 37 "C for 30-1 20 min, overall protein synthesis was sustained. The synthesis of at least six proteins which were also present in mycelia at 25 "C increased sharply (Fig. 1a, lanes A-E; note bands with apparent molecular masses of 92,83,78,70,3 1 and 24 kDa), peaking after 60-1 20 min at 37 "C
Heat-shock proteins in Histoplasma capsdatum
3377
Fig. 1 . Heat-shock of mycelia of (a)H . cupsulutum,Downs strain, and (b)H . cupsulutum, G 184A strain. Fluorograms of one-dimensionalgels with approximately 300000 d.p.m. per lane (a) and approximately 240000 d.p.m. per lane (b).Lanes A, mycelia at 25 "C. Lanes B-E, mycelia at 37 "C for 30,60,90 and 120 min, respectively. Lanes F, yeast at 37 "C.
and then decreasing. Synthesis of these proteins was retained in yeast cells at 37°C and the pattern of protein synthesis in yeast was essentially the same as in mycelia at 37 "C, but the levels were lower (Fig. la, lane F); thus in the Downs strain, yeast cells at 37 "Chad the same pattern of synthesis of hsp as mycelia at 37 "C. The protein patterns in one-dimensional gels of extracts of strains G184A and G222B were similar to those of Downs at 25 "C and 37 "C (Fig. 16 shows results for G184A). However, smaller amounts of the hsp were formed at 25 "C in G184A and G222B, and the relative levels of increase of the proteins at 37 "C were greater. Although these proteins were also present in yeast cells of G184A and G222B at 37 "C, unlike the results with Downs, synthesis was at the low levels found in mycelia at 25 "C before shifting them to 37 "C. The patterns of synthesis of the six major hsp in relation to time were very similar in Downs, G184A and G222B, rising and reaching a plateau after 60-120 min at 37 "C. Fig. 2 shows the profiles of proteins synthesized in mycelia of Downs, G 184A and G222B at 25 "C (lanes A, E and I), and 90 min after shifting the temperature to 34 "C (lanes B, F and J), 37 "C(lanes C, G and K) or 40 "C (lanes D, H and L). In Downs mycelia, the level of synthesis of the six proteins was highest between 34 "C and 37 "C (lanes B and C) and decreased at 40 "C (lane D). In contrast, the level of synthesis of the six proteins in G 184A and G222B was highest at 37 "C (lanes G and K) and decreased sharply at 40 "C (lanes H and L).
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G . S H E A R E R A N D OTHERS
Flg. 2. Heat-shock 01 rnyceliii of If. ~ i ~ s u l ~ rDUWIIS u m (lanos A D), G184A (lanes F-H) and (32228 (lanes I-L) at 25 "C (lanes A, E and I) and 90 min after shifting the temperature to 34 "C(lanes B, F and I), 37 "C (lanes C, G and H) or 40 "C (lanes D, H and L). Fluorogram of one-dimensional gel with approximately 300000 d.p.m. per gel.
The differences in the responses of the strains were more apparent in the two-dimensional gels. Fig. 3 shows the profiles of proteins synthesized in Downs mycelia at 25 'C ( a )and 90 min after shifting the temperature to 34 "C(b),37 "C (c) or 40 "C( d ) .Synthesis of many polypeptides decreased at the elevated temperatures; some increased, especially the six hsp. Synthesis appeared to peak at 34 "C and decreased at 37 "C and 40 "C.Fig. 4 shows the results with G 184A for comparison. In contrast to Downs, the hsp of both G 184A and G222B were present in low amounts at 25 "C and therefore the increases seen at the higher temperatures were more dramatic. Another difference between Downs and G 184A and G222B was the temperature at which hsp synthesis peaked: 37 "C in G184A and G222B, but 34 "C in Downs. A mouse monoclonal antibody to the hsp 70 of Drosophila melanogaster reacted with a spot corresponding to the hsp 70 of H . capsulatum on Western blots of the gels shown in Figs 3 and 4 (data not shown). Therefore, the hsp 70 of the H . cupsufatum strains cross-reacted well with the hsp 70 of D .melunogaster. Hsp 70 synthesis in Downs, G 184A and G222B was quantified by densitometry of one-dimensional gels containing cell extracts obtained after exposure of mycelia to several different temperatures for 90 min (Fig. 5). N o significant changes in protein synthesis occurred when mycelia were incubated at 30 "C, a temperature at which the mycelial-to-yeast transition did not take place. Significant increases in the hsp 70 occurred at 34 "C, the lowest temperature at which the mycelial-to-yeast transition consistently occurred. The level of synthesis of hsp 70 in Downs was highest at 34 "C and waned at 37 "C and 40 "C. In contrast, hsp 70 synthesis in G184A and G222B was highest at 37 "C and decreased at 40 "C.
Heat-shock proteins in Histoplasma cupsulatum
3379
Fig. 3. Heat-shock of mycelia of H . C C Z ~ S U ~ Q CDowns U~, strain. Fluorograms of two-dimensional gels with approximately 300000 d.p.m. per gel. (a) Mycelia at 25 "C;(b) mycelia after 90 min at 34 "C; (c) mycelia after 90 min at 37 "C; ( d )mycelia after 90 min at 40 "C.
DISCUSSION
Hsp have been implicated in at least two types of physiological processes. First, eukaryotic and prokaryotic cells respond in a programmed way to sudden elevations in temperature and synthesize a group of hsp which range in molecular mass from approximately 90 to 20 kDa (Schlesinger et a/., 1982). Second, synthesis of members of the hsp family also appears to be regulated in some developmental phases of Drosophila melunoguster (Ashburner & Bonner, 1979), Succharomyces cerevisiue (McAlister & Finkelstein, 1986) and Trypanosoma (Van der Ploeg et ul., 1985). Dimorphic fungi like H. cupsuluturn are an especially interesting case in which a shift-up in temperature results in both a heat-shock response and a developmentally regulated phase transition from the mycelial form to yeast cells. The molecular masses and patterns of mobility in one- and two-dimensional gels of the proteins that show increased synthesis in mycelia of H . capsuiatum after the temperature shift-up are very similar to those of the hsp described in D. melanogaster (Ashburner & Bonner, 1979)and
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G . SHEARER A N D OTHERS
Fig. 4. Heat-shock of H . cupsulurum, G 184A strain. Fluorograms of two-dimensional gels with approximately 300000 d.p.m. per gel. (a) Mycelia at 25 "C; (b)mycelia after 90 min at 34 "C;(c) mycelia after 90 min at 37 "C; ( d ) mycelia after 90 min at 40 "C.
S. cererisiue (McAlister & Finkelstein, 1980). As in those organisms, increased synthesis of these proteins in H. cupsulurum can be correlated with an increase in specific mRNA synthesis (B. Maresca, personal communication). Furthermore, 70 kDa protein from H. capsuiaturn reacts with an antibody raised to the 70 kDa hsp of D.melunoguster. Therefore, we conclude that subjecting mycelia of H. capsufutum to an increase in temperature, from 25 "C to 37 "C, results in the synthesis of typical hsp. Downs is the least virulent and the most sensitive to elevated temperatures of the three strains we studied (Medoff et ul., 1986). Because of these differences in virulence, we were interested in any variability in hsp synthesis among the strains. We found that synthesis of hsp appears to be higher in Downs mycelia at 25 "C and yeast at 37 "Cthan in the corresponding phases of G 184A and G222B incubated at these temperatures. Synthesis of hsp peaked at a lower temperature (34 "C) in Downs than in the more virulent strains (37 "C). Similar results were found when hsp 70 mRNA synthesis was measured (B. Maresca, personal communication). One possible explanation for the differences among the strains is that Downs is more sensitive to temperatures above 34°C than the other two strains (Medoff et ul., 1986) because of the differences in hsp synthesis. Another is that the pattern of hsp synthesis in Downs reflects a
Heat-shock proteins in Histoplasma capsulatum
338 1
n
30
34
37
Temperature of incubation ("C) Fig. 5. Patterns of induction of hsp 70 in the Downs, G184A and G222B strains of H . capsulatum, after 90 min at the temperatures indicated. Extracts of the strains were run on separate gels; each lane contained extracts of the same strain which had been incubated at the different temperatures. The intensity of each of the protein bands on the gel lanes was quantified by densitometry and the hsp 70 was expressed as a percentage of the total additive intensity of all of the bands in each lane.
more general defect which results in decreased growth rate and a heat stress response at lower temperatures than in the more virulent strains (Medoff et al., 1986). Either of these possibilities or both may be the correct explanation for the differences in hsp expression. Further characterization of the hsp genes and their regulation and expression in Downs, and comparison with the other strains of H. capsulatum, will be necessary in order to differentiate between these two possibilities. We were unable to separate the induction of increased synthesis of hsp from the mycelial-toyeast phase transition; those temperatures which led to one phenomenon also resulted in the other. Therefore, the temperature shift of mycelia of H. capsulatum growing at 25 "C to 34-40 "C is truly both a heat shock and a trigger for the mycelial-to-yeast transformation. Van der Ploeg et al. (1985) have recently shown that a heat-shock response is induced in the parasitic protozoa Trypanosomabrucei and Leishmania major that shift between a poikilothermic insect vector at 22-28 "C and b homeothermic mammalian host at 37 "C. These authors hypothesized that hsp genes are implicated in the differentiation of these vector-borne parasites. Our results on H. capsulatum are consistent with theirs. In addition we have found an induction of hsp in the temperature-induced phase transitions of BIastomyces dermatitidis (Shearer et al., 1985) and Paracoccidioides brasiiiensis, two other dimorphic fungal pathogens. Therefore the induction of hsp may be a general phenomenon in all organisms that shuttle from a low temperature saprobic phase to a pathogenic form adapted to grow at the higher body temperatures of the host. Because hsp are seemingly required for recovery from heat shock,they are evidently part of the normal mycelial-to-yeast transition of these fungi. Whether they are
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G . SHEARER AND OTHERS
more intrinsic to the process of phase transition remains to be proven. Examining for their presence in pharmacological non-temperature-dependent inductions of the mycelial-to-yeast transition (Maresca e t a / . , 1977; Saccoet a/.,1981),or in the yeast-to-mycelial transition induced by a shift-down in temperature, might elucidate this. This work was supported by Public Health Service grants A1 16228, A107015 and AI07172. We thank Elizabeth Keath, Milton Schlesinger and David Schlessinger for helpful suggestions and review of this manuscript. REFERENCES
ASHBURNER, M . & BONNER,J . J. ( 1979). The induction of gene activity in Drosophila by heat shock. Cell 17, 24 1 - 254.
BONNER,W . M. & LASKEY.R. A . (1974). A film detection method for tritium labeled proteins and nucleic acid in polyacrylamide gels. European Journal q f Biochemistry 46, 83-88. LAEMMLI, U . K . (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Naruru, London 227, 68M85. LAMBOWITZ,A . M.,KOBAYASHI, G. S., PAINTER,A. & MEDOFF,G. (1983). Possible relationship of morphogenesis in pathogenic fungus, Histoplasma capsulatum, to heat shock response. Nature, London 303, 806-808.
B., MEDOFF,G., ~ H L E S S I N G ED., R , KoG. S. & MEDOFF,J . (1977). Regulation of dimorphism in the pathogenic fungus Histoplasma capsulatum. Nature, b n d o n 266,447-448. ARESCA, B., hMBOWITZ, A. M., KUMAR, v. B., GRANT,G. A., KOBAYASHI,G. S. & MEWFF, G. (198 1). Role of cysteine in regulating morphogenesis and mitochondria] activity in the dimorphic fungus Histoplasma capsulatum. Proceedings of the National Academy of Sciences ofthe United Stares of America ARESCA.
BAYASHI,
78, 4 5 9 m . MCALISTER,L. & FINKELSTEIN, D. B. (1980). Heat shock proteins and thermal resistance in yeast. Biochemical and Biophysical Research Communicarims 93, 819-824.
MEDOFF, G., MARESCA,B., LAMBOWITZ, A . M.,
KOBAYASHI,G. S., PAINTER, A., SACCO, M. & CARRATU,L. (1986). A correlation between pathogenicity and temperature sensitivity in different strains of Histoplasma capsulatum. Journal of Clinical Incestigation 78, 1633-1647. O'FARRELL, P. H. (1975). High resolution two-dimensional electrophoresis of proteins. Journal of Biological Chemistry 250, 4007-4021. SACCO, M.,MARESCA, B., KUMAR,B. V., KOBAYASHI, G. S. & MEDOFF,G. (1981).Temperature- and cyclic nucleotide-induced phase transitions of Histoplasma capsulatum. Journal of Bacteriology 144, I 17-1 20. SCHLESINGER, M. J., ALIPERTI,G., & KELLEY,G. M. (1982). The response of cells to heat shock. Trends in Biochemical Sciences 1,222-225. SCHWARZ,I . (197 1 ). The pathogenesis of histoplasmosis. In Histoplasmosis : Proceedings of the Second National Conference, pp. 244-251. Edited by L. Ajello, E. W. Chick & M.L. Furcolow. Springfield, Illinois : Charles C. Thomas Publisher. SHEARER, G., BIRGE,C., KOBAYASHI, G. S. & MEDOFF, G. (1985). Heat shack proteins in temperature induced phase transitions of Histoplasma capsulatum and Blastomyces dermatitidis. Abstracts ofthe Annual Meeting of the American Society .for Microbiology, 1985, F64, p. 375. VAN DER PLOEG, L. H. T., GIANNINI, s. H . & CANTOR, C. R. (1985). Heat shock genes: regulatory role for differentiation in parasitic protozoa. Science 228, 1443- 1 446.
Printed in Great Britain
3375
Heat-shock Proteins Induced During the Mycelial-to-yeast Transitions of Strains of Histoplasma capsulatum By G L E N M O R E S H E A R E R , J R , ? C L A I R E H . B I R G E , P A T R I C I A D . Y U C K E N B E R G J G E O R G E S. KOBAYASHI A N D G E R A L D MEDOFF* Divisions of Infectious Diseases, Dermatology and Laboratory Medicine, Department of Medicine and the Department of Microbiology and Immunology, Washington University School of Medicine, St Louis, Missouri 63110, USA (Receiued 6 May 1987; revised 27 July 1987) Heat-shock proteins (hsp) were elicited when mycelia of the Downs strain and the more virulent G184A and G222B strains of Histoplasma capsulatum were shifted up to temperatures which induced the mycelial-to-yeast transition (34-40 "C). The classes of the major hsp which increased in synthesis in each strain were similar. However, the pattern of synthesis of these proteins at the different temperatures in Downs differed from those in the G184A and G222B strains: hsp synthesis in Downs peaked at 34 "C, whereas in G184A and G222B it was highest at 37 "C.
INTRODUCTION
The dimorphic fungal pathogen Histoplasma capsulatum exists in a mycelial form in soil and as a yeast in infected hosts (Maresca et al., 1977, 1981 ; Sacco et al., 1981 ; Schwarz, 1971). Reversible transitions between these phases can be induced in culture by temperature shifts between 25 "C (mycelial growth) and 37 "C (yeast growth). Because the mycelial-to-yeast transition is heat-induced, it is possible that its regulation involves a heat-shock response. Consistent with this possibility, the transition of mycelia to yeast at 37 "C is accompanied by rapid and marked changes in several different metabolic processes, particularly an uncoupling of oxidative phosphorylation and a decline in intracellular ATP levels, followed by a slow recovery phase (Maresca et al., 1981). We studied the induction of heat-shock proteins (hsp) in H. capsulatum when the temperature of incubation of mycelia was shifted from 25 "C to 37 "C, and discovered that hsp 70 synthesis increased soon after the shift-up in temperature (Lambowitz et al., 1983). The initial studies on the phase transition of H. capsulatum were done on Downs, a strain with low virulence in mice. When we compared the mycelial-to-yeast transition in more virulent strains of H. capsulatum (Medoff et al., 1986), we found that the changes were similar to those of the Downs strain, but less extreme. The increased sensitivity of the Downs strain to elevated temperatures may be incidental, or it may be a key factor accounting for its lower virulence for mice compared to the other strains of H. capsulatum. Here we characterize and compare more completely the hsp elicited when mycelia of Downs and the more virulent G184A and G222B strains of H . capsulatum are switched from 25 "C to temperatures of 34 "C to 40 "C.
t Present address : Department of
CA 92717, USA.
Microbiology and Molecular Genetics, University of California, Irvine,
Present address: Bio-Rad Laboratories, 1414 Harbour Way South, Richmond, CA 94801, USA. Abbreviation: hsp, heat-shock protein(s).
0001-4172 0 1987 SGM
3376
G . S H E A R E R AND O T H E R S METHODS
Organisms.The H . capsulutum strains are from the permanent culture collection of this laboratory (Lambowltz et al., 1983; Maresca er ul., 1977, 1981). Cells were grown in shaking flasks in a defined medium consisting of standard F-12 nutrient base (Gibco) supplemented with, per litre, 18 g glucose, 0.084 g cystine and 6 g HEPES; pH 7-5. Exponential-phase cells (mycelia grown for 48-72 h at 25 "C and yeast grown for 24-48 h at 37 "C) were used in all experiments. Hear shock. Cultures of mycelia in the mid-exponential phase of growth were incubated in a shaking water bath at 37 "C. After the designated periods of time at this elevated temperature, 10 pCi (370 kBq) ~-[~~S]methionine (Amersham) was added to the culture for 20 min. In experiments where mycelia and yeast were exposed to different temperatures (25, 30, 34,40 "C)for 90 min, the cells were cooled in a 25 "C water bath for 5 min after exposure before being pulse-labelled at 25 "C. Characteri:ation of proteins by one-dimensional electrophoresis. Cells were harvested by rapid filtration on glassfibre filters and washed twice with 10 ml distilled water at 0 "C and twice with 10 ml acetone at 0 "C. The filters were dried at 37 "C for 10 min; proteins were solubilized by heating the dry flters in 1 ml SDS (Sigma) sample buffer (Laemmli, 1970) at 100 "C for 5 min. Radioactivity incorporated into proteins was estimated by liquid scintillation counting of solubilized samples. Samples containing about 300000 d.p.m. were applied to each lane. SDS-PAGE was done using the buffer system of Laemmli (McAlister & Finkelstein, 1980), with the following modifications: the resolving gel was prepared to yield 12.5% (w/v) acrylamide and 0.42% acrylaid crosslinker (FMC Corp.) in place of bisacrylamide. Slab gels, 0-75 x 130 x 170 mm, were bonded to Gel Bond PAG film (FMC Corp.). Electrophoresis was carried out at 20 mA constant current until the bromophenol blue tracking dye reached the bottom of the slab (approx. 3 h). The standards (Amersham) used to estimate molecular mass were myosin (200 kDa), phosphorylase b (92.5 kDa), bovine serum albumin (69 kDa), ovalbumin (46 kDa), carbonic anhydrase (30 kDa) and lysozyme (14 kDa). Hsp at different temperatures were quantified by densitometry measurements on the onedimensional gels. Characterization ofproteins by two-dihensional electrophoresis. Cells were quickly washed twice with 10 ml buffer A (10 mM-Tris pH 7.4, 1 mM-phenylmethylsulphonyl fluoride) at 0 "C, resuspended in 5 ml buffer A in a 30 ml Corex tube containing 5 g 0-5 p m glass beads and broken by vigorous vortexing for 4 rnin at 0 "C. Unbroken cells were removed by centrifugation at l000g for 10min. Proteins were precipitated by adding 10:; (w/v) triGhloroacetic acid to the supernatant and incubating at 0 "C for 15 min. The precipitate was washed three times with I5 ml 80% (vp) acetone at 0 "C and once with 15 ml 100% acetone at 0 "C. Proteins were solubilized by tritrating with 200 p1 Laemmli SDS sample buffer (Laemmli, 1970) and mixing with a"glass rod. Insoluble material was removed by centrifugation at 10000g for 10 min. Electrofocusing was performed by the method of O'Farrell (1975) modified by the substitution of the zwitterionic detergent 343cholamidopropyl)dimethylammonio-I -propanesulphonate (CHAPS; Sigma) for NP-40. Ampholines (Bio-Rad) were mixtures of pH 3-7 and pH 3-10 in a 10 :1 ratio. After electrofocusing, the tube gels were immediately frozen at - 70 "C. Before use, the tube gels were equilibrated in Laemmli SDS sample buffer for 20 min and sealed with 0.57; agarose to 3 12.5% polyacrylamide slab gel and electrophoresed as described above. Flwrogruphy. g a b gels bonded to Gel Bond PAG film were processed in DMSO-PPO by the method of Bonner & Lasky (1974). The gels were then soaked in 2% (v/v) glycerol for 60 rnin and dried for 3 h at 60 "C. The dried gels were exposed to Kodak X-AR film at -70 "C until the desired density was reached. Immunoblotring. Non-radioactive samples were prepared and processed by one- and two-dimensional electrophoresis as described above. In the second dimension the samples were run on 12.5% polyacrylamide gels crosslinked with 0.33% bisacrylamide without Gel Bond PAC film. Proteins were electrophoretically transferred to nitrocellulose paper at 0-2 A for 16 h at 4 "C in a buffer consisting of 25 mM-Tris, 192 mM-glycine and 20% (v/v) methanol. The nitrocellulose was rinsed in distilled water and dried at 25 "C. Prior to addition of the primary monoclonal mouse antibody to hsp 70 of Drosophilu melanoguster (a kind gift of Dr Susan Lindquist, University of Chicago) the blots were washed for 30 rnin in 5% (w/v) non-fat dry milk (Carnation Co.) solution in phosphatebuffered saline (PBS). Blots were reacted with the primary antibody for 60 min, then with secondary antibody (Sigma; rabbit anti-mouse, 1 :loo) for 60 rnin and finally with peroxidase-conjugated goat anti-rabbit (Cappel; 1 : 1O00) for 60 min. Blots were washed twice with 504 nonfat milk in PBS for 10 rnin and once with PBS for 10 min between each antibody reaction. Antigens were visualized by incubating blots with 4-chloro-1-naphthol and hydrogen peroxide (50 mi PBS + 30 p1300,',H z 0 2added to 30 mg 4-chloro-l-naphthol+ 10 ml methanol at 0 "C). RESULTS
When mycelia of the H. capsulutum Downs strain growing at 25 "C were shifted to 37 "C for 30-1 20 min, overall protein synthesis was sustained. The synthesis of at least six proteins which were also present in mycelia at 25 "C increased sharply (Fig. 1a, lanes A-E; note bands with apparent molecular masses of 92,83,78,70,3 1 and 24 kDa), peaking after 60-1 20 min at 37 "C
Heat-shock proteins in Histoplasma capsdatum
3377
Fig. 1 . Heat-shock of mycelia of (a)H . cupsulutum,Downs strain, and (b)H . cupsulutum, G 184A strain. Fluorograms of one-dimensionalgels with approximately 300000 d.p.m. per lane (a) and approximately 240000 d.p.m. per lane (b).Lanes A, mycelia at 25 "C. Lanes B-E, mycelia at 37 "C for 30,60,90 and 120 min, respectively. Lanes F, yeast at 37 "C.
and then decreasing. Synthesis of these proteins was retained in yeast cells at 37°C and the pattern of protein synthesis in yeast was essentially the same as in mycelia at 37 "C, but the levels were lower (Fig. la, lane F); thus in the Downs strain, yeast cells at 37 "Chad the same pattern of synthesis of hsp as mycelia at 37 "C. The protein patterns in one-dimensional gels of extracts of strains G184A and G222B were similar to those of Downs at 25 "C and 37 "C (Fig. 16 shows results for G184A). However, smaller amounts of the hsp were formed at 25 "C in G184A and G222B, and the relative levels of increase of the proteins at 37 "C were greater. Although these proteins were also present in yeast cells of G184A and G222B at 37 "C, unlike the results with Downs, synthesis was at the low levels found in mycelia at 25 "C before shifting them to 37 "C. The patterns of synthesis of the six major hsp in relation to time were very similar in Downs, G184A and G222B, rising and reaching a plateau after 60-120 min at 37 "C. Fig. 2 shows the profiles of proteins synthesized in mycelia of Downs, G 184A and G222B at 25 "C (lanes A, E and I), and 90 min after shifting the temperature to 34 "C (lanes B, F and J), 37 "C(lanes C, G and K) or 40 "C (lanes D, H and L). In Downs mycelia, the level of synthesis of the six proteins was highest between 34 "C and 37 "C (lanes B and C) and decreased at 40 "C (lane D). In contrast, the level of synthesis of the six proteins in G 184A and G222B was highest at 37 "C (lanes G and K) and decreased sharply at 40 "C (lanes H and L).
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G . S H E A R E R A N D OTHERS
Flg. 2. Heat-shock 01 rnyceliii of If. ~ i ~ s u l ~ rDUWIIS u m (lanos A D), G184A (lanes F-H) and (32228 (lanes I-L) at 25 "C (lanes A, E and I) and 90 min after shifting the temperature to 34 "C(lanes B, F and I), 37 "C (lanes C, G and H) or 40 "C (lanes D, H and L). Fluorogram of one-dimensional gel with approximately 300000 d.p.m. per gel.
The differences in the responses of the strains were more apparent in the two-dimensional gels. Fig. 3 shows the profiles of proteins synthesized in Downs mycelia at 25 'C ( a )and 90 min after shifting the temperature to 34 "C(b),37 "C (c) or 40 "C( d ) .Synthesis of many polypeptides decreased at the elevated temperatures; some increased, especially the six hsp. Synthesis appeared to peak at 34 "C and decreased at 37 "C and 40 "C.Fig. 4 shows the results with G 184A for comparison. In contrast to Downs, the hsp of both G 184A and G222B were present in low amounts at 25 "C and therefore the increases seen at the higher temperatures were more dramatic. Another difference between Downs and G 184A and G222B was the temperature at which hsp synthesis peaked: 37 "C in G184A and G222B, but 34 "C in Downs. A mouse monoclonal antibody to the hsp 70 of Drosophila melanogaster reacted with a spot corresponding to the hsp 70 of H . capsulatum on Western blots of the gels shown in Figs 3 and 4 (data not shown). Therefore, the hsp 70 of the H . cupsufatum strains cross-reacted well with the hsp 70 of D .melunogaster. Hsp 70 synthesis in Downs, G 184A and G222B was quantified by densitometry of one-dimensional gels containing cell extracts obtained after exposure of mycelia to several different temperatures for 90 min (Fig. 5). N o significant changes in protein synthesis occurred when mycelia were incubated at 30 "C, a temperature at which the mycelial-to-yeast transition did not take place. Significant increases in the hsp 70 occurred at 34 "C, the lowest temperature at which the mycelial-to-yeast transition consistently occurred. The level of synthesis of hsp 70 in Downs was highest at 34 "C and waned at 37 "C and 40 "C. In contrast, hsp 70 synthesis in G184A and G222B was highest at 37 "C and decreased at 40 "C.
Heat-shock proteins in Histoplasma cupsulatum
3379
Fig. 3. Heat-shock of mycelia of H . C C Z ~ S U ~ Q CDowns U~, strain. Fluorograms of two-dimensional gels with approximately 300000 d.p.m. per gel. (a) Mycelia at 25 "C;(b) mycelia after 90 min at 34 "C; (c) mycelia after 90 min at 37 "C; ( d )mycelia after 90 min at 40 "C.
DISCUSSION
Hsp have been implicated in at least two types of physiological processes. First, eukaryotic and prokaryotic cells respond in a programmed way to sudden elevations in temperature and synthesize a group of hsp which range in molecular mass from approximately 90 to 20 kDa (Schlesinger et a/., 1982). Second, synthesis of members of the hsp family also appears to be regulated in some developmental phases of Drosophila melunoguster (Ashburner & Bonner, 1979), Succharomyces cerevisiue (McAlister & Finkelstein, 1986) and Trypanosoma (Van der Ploeg et ul., 1985). Dimorphic fungi like H. cupsuluturn are an especially interesting case in which a shift-up in temperature results in both a heat-shock response and a developmentally regulated phase transition from the mycelial form to yeast cells. The molecular masses and patterns of mobility in one- and two-dimensional gels of the proteins that show increased synthesis in mycelia of H . capsuiatum after the temperature shift-up are very similar to those of the hsp described in D. melanogaster (Ashburner & Bonner, 1979)and
3380
G . SHEARER A N D OTHERS
Fig. 4. Heat-shock of H . cupsulurum, G 184A strain. Fluorograms of two-dimensional gels with approximately 300000 d.p.m. per gel. (a) Mycelia at 25 "C; (b)mycelia after 90 min at 34 "C;(c) mycelia after 90 min at 37 "C; ( d ) mycelia after 90 min at 40 "C.
S. cererisiue (McAlister & Finkelstein, 1980). As in those organisms, increased synthesis of these proteins in H. cupsulurum can be correlated with an increase in specific mRNA synthesis (B. Maresca, personal communication). Furthermore, 70 kDa protein from H. capsuiaturn reacts with an antibody raised to the 70 kDa hsp of D.melunoguster. Therefore, we conclude that subjecting mycelia of H. capsufutum to an increase in temperature, from 25 "C to 37 "C, results in the synthesis of typical hsp. Downs is the least virulent and the most sensitive to elevated temperatures of the three strains we studied (Medoff et ul., 1986). Because of these differences in virulence, we were interested in any variability in hsp synthesis among the strains. We found that synthesis of hsp appears to be higher in Downs mycelia at 25 "C and yeast at 37 "Cthan in the corresponding phases of G 184A and G222B incubated at these temperatures. Synthesis of hsp peaked at a lower temperature (34 "C) in Downs than in the more virulent strains (37 "C). Similar results were found when hsp 70 mRNA synthesis was measured (B. Maresca, personal communication). One possible explanation for the differences among the strains is that Downs is more sensitive to temperatures above 34°C than the other two strains (Medoff et ul., 1986) because of the differences in hsp synthesis. Another is that the pattern of hsp synthesis in Downs reflects a
Heat-shock proteins in Histoplasma capsulatum
338 1
n
30
34
37
Temperature of incubation ("C) Fig. 5. Patterns of induction of hsp 70 in the Downs, G184A and G222B strains of H . capsulatum, after 90 min at the temperatures indicated. Extracts of the strains were run on separate gels; each lane contained extracts of the same strain which had been incubated at the different temperatures. The intensity of each of the protein bands on the gel lanes was quantified by densitometry and the hsp 70 was expressed as a percentage of the total additive intensity of all of the bands in each lane.
more general defect which results in decreased growth rate and a heat stress response at lower temperatures than in the more virulent strains (Medoff et al., 1986). Either of these possibilities or both may be the correct explanation for the differences in hsp expression. Further characterization of the hsp genes and their regulation and expression in Downs, and comparison with the other strains of H. capsulatum, will be necessary in order to differentiate between these two possibilities. We were unable to separate the induction of increased synthesis of hsp from the mycelial-toyeast phase transition; those temperatures which led to one phenomenon also resulted in the other. Therefore, the temperature shift of mycelia of H. capsulatum growing at 25 "C to 34-40 "C is truly both a heat shock and a trigger for the mycelial-to-yeast transformation. Van der Ploeg et al. (1985) have recently shown that a heat-shock response is induced in the parasitic protozoa Trypanosomabrucei and Leishmania major that shift between a poikilothermic insect vector at 22-28 "C and b homeothermic mammalian host at 37 "C. These authors hypothesized that hsp genes are implicated in the differentiation of these vector-borne parasites. Our results on H. capsulatum are consistent with theirs. In addition we have found an induction of hsp in the temperature-induced phase transitions of BIastomyces dermatitidis (Shearer et al., 1985) and Paracoccidioides brasiiiensis, two other dimorphic fungal pathogens. Therefore the induction of hsp may be a general phenomenon in all organisms that shuttle from a low temperature saprobic phase to a pathogenic form adapted to grow at the higher body temperatures of the host. Because hsp are seemingly required for recovery from heat shock,they are evidently part of the normal mycelial-to-yeast transition of these fungi. Whether they are
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G . SHEARER AND OTHERS
more intrinsic to the process of phase transition remains to be proven. Examining for their presence in pharmacological non-temperature-dependent inductions of the mycelial-to-yeast transition (Maresca e t a / . , 1977; Saccoet a/.,1981),or in the yeast-to-mycelial transition induced by a shift-down in temperature, might elucidate this. This work was supported by Public Health Service grants A1 16228, A107015 and AI07172. We thank Elizabeth Keath, Milton Schlesinger and David Schlessinger for helpful suggestions and review of this manuscript. REFERENCES
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KOBAYASHI,G. S., PAINTER, A., SACCO, M. & CARRATU,L. (1986). A correlation between pathogenicity and temperature sensitivity in different strains of Histoplasma capsulatum. Journal of Clinical Incestigation 78, 1633-1647. O'FARRELL, P. H. (1975). High resolution two-dimensional electrophoresis of proteins. Journal of Biological Chemistry 250, 4007-4021. SACCO, M.,MARESCA, B., KUMAR,B. V., KOBAYASHI, G. S. & MEDOFF,G. (1981).Temperature- and cyclic nucleotide-induced phase transitions of Histoplasma capsulatum. Journal of Bacteriology 144, I 17-1 20. SCHLESINGER, M. J., ALIPERTI,G., & KELLEY,G. M. (1982). The response of cells to heat shock. Trends in Biochemical Sciences 1,222-225. SCHWARZ,I . (197 1 ). The pathogenesis of histoplasmosis. In Histoplasmosis : Proceedings of the Second National Conference, pp. 244-251. Edited by L. Ajello, E. W. Chick & M.L. Furcolow. Springfield, Illinois : Charles C. Thomas Publisher. SHEARER, G., BIRGE,C., KOBAYASHI, G. S. & MEDOFF, G. (1985). Heat shack proteins in temperature induced phase transitions of Histoplasma capsulatum and Blastomyces dermatitidis. Abstracts ofthe Annual Meeting of the American Society .for Microbiology, 1985, F64, p. 375. VAN DER PLOEG, L. H. T., GIANNINI, s. H . & CANTOR, C. R. (1985). Heat shock genes: regulatory role for differentiation in parasitic protozoa. Science 228, 1443- 1 446.
