JOURNAL OF BACTERIOLOGY, Sept. 1987, p. 4055-4060 Vol. 169, No. 90021-9193/87/094055-06$02.00/0Copyright © 1987, American Society for Microbiology

Mycelial- to Yeast-Phase Transitions of the Dimorphic FungiBlastomyces dermatitidis and Paracoccidioides brasiliensis

GERALD MEDOFF,123* AUDREY PAINTER,12 AND GEORGE S. KOBAYASHI1'2'3'4Divisions of Infectious Diseases1 and Laboratory Medicine,4 Department of Medicine,2 and Department of Microbiology

and Immunology,3 Washington University School of Medicine, St. Louis, Missouri 63110

Received 9 February 1987/Accepted 15 June 1987

The physiological changes that occur during the mycelial- to yeast-phase transitions induced by atemperature shift from 25 to 37°C of cultures of Blastomyces dermatitidis and Paracoccidioides brasiliensis canbe divided into three stages. The triggering event is a heat-related insult induced by the temperature shift whichresults in partial uncoupling of oxidative phosphorylation and declines in cellular ATP levels, respiration rates,and concentrations of electron transport components (stage 1). The cells then enter a stage in whichspontaneous respiration ceases (stage 2), and finally, there is a shift into a recovery phase during Whichtransformation to yeast morphology occurs (stage 3). Cysteine is required during stage 2 for the operation ofshunt pathways which permit electron transport to bypass blocked portions of the cytochrome system. Themycelial- to yeast-phase transitions of these two fungi are very similar to that of Histoplasma capsulatum.Therefore, these three dimorphic fungal pathogens have evolved parallel mechanisms to adjust to thetemperature shifts which induce these mycelial- to yeast-phase transitions.

Blastomyces dermatitidis, Paracoccidioides brasiliensis,and Histoplasma capsulatum are dimorphic pathogenicfungi. Although there are some differences in geographicdistribution and clinical presentation of disease caused bythese fungi, they all can cause systemic infections in hu-mans. Other similarities include the existence as mycelia innature and yeast in infected tissue, growth as mycelia incultures incubated at 25°C and as yeast at 370C, and induc-tion of reversible phase transitions by switching between 25and 37°C (7).

In previous studies (5, 5a, 8) we characterized themycelial- to yeast-phase transition of H. capsulatum inducedby the temperature shift from 25 to 37°C and also by shifts totemperatures as high as 43°C. We found that the physiolog-ical changes that occur in this fungus when the temperatureis raised can be divided into three distinct stages. Stage 1,which immediately follows the temperature shift, is charac-terized by partial or complete uncoupling of oxidative phos-phorylation, an immediate decline in ATP levels, and aprogressive decrease in respiration rates over 24 h. After 24to 40 h, the cells enter stage 2, a dormant period of 4 to 6days that is characterized by absent or low rates of respira-tion, decreased concentrations of mitochondrial electrontransport components, and inhibition of RNA and proteinsynthesis. Stage 3 is characterized by increasing concentra-tions of cytochrome components, resumption of normalrespiration, and completion of the transition to yeast mor-phology.The severity of the changes during the transition depends

on the thermal tolerance of the strain of H. capsulatum andthe level of the temperature of incubation (7). When thetemperature elevation is high enough, spontaneous respira-tion ceases in stage 2, and cysteine or other sulfhydryl-containing compounds are required for the operation ofshunt pathways which permit electron transport to bypassblocked portions of the cytochrome system.

* Corresponding author.

In this study we examined the mycelial- to yeast-phasetransitions of B. dermatitidis and P. brasiliensis, when themycelial- to yeast-phase transitions were triggered by shift-ing the incubation temperature from 25 to 37°C. As in H.capsulatum, three stages in the phase transition could bedefined, and sulfhydryl-induced shunt pathways were pres-ent and necessary for progression of the morphogenesisbeyond stage 2. Therefore, these three different dimorphicfungal pathogens have evolved parallel mechanisms to adjustto the temperature shifts which induce the mycelial- toyeast-phase transition.

MATERIALS AND METHODSCulture conditions. The strain of B. dermatitidis used in

our studies was a clinical isolate from the diagnostic mycol-ogy laboratory at Barnes Hospital, St. Louis, Mo. It hasbeen passaged in our laboratory for about 10 years. Thestrain of P. brasiliensis was originally obtained from HelenBuckley, Temple University, Philadelphia, Pa. The cellswere maintained in the mycelial phase by culture on 2%glucose-1% yeast extract at 25°C as described previously(Sa, 8). In those experiments in which the role of cysteine inmorphogenesis was assessed, the cultures were grown inAutopow medium (minimal essential medium) without addedcysteine (9). Cultures were started with a constant inoculumof cells and were grown to the mid-log phase, which oc-curred after 72 to 96 h of incubation at 25°C for mycelia and37°C for yeast. In the experiments on transforming cells,mycelial cultures were diluted 1:5 in fresh medium, dividedamong several flasks, and incubated at the higher tempera-tures. The flasks were harvested at different times, andrespiration rates were measured. Other portions of thecultures were filtered and dried at 42°C to determine dryweights.

Reagents. Oligomycin and carbonyl cyanide m-chloro-phenylhydrazone (Cl-CCP) were obtained from SigmaChemical Co. (St. Louis, Mo.) and were dissolved in abso-lute ethanol just before use. KCN was dissolved in water and

4055

4056 MEDOFF ET AL.

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FIG'. 1. (a) Oxygen electrode recordings of -respiration ofmycelial cells of B. dermatitidis at 25°C, immediately after (0') and1 min (') after the shift up to 37°C. To initiate the experiments, 0.5ml of cultures in 10-mnl tubes were switched from one water bath at25°C to another at 370C. Portions of 3 ml were then added to thecuvettes in the oxygraph at the designated time points. The numbersin parentheses are rates of oxygen consumption (microliters of 02

per hour per milligram [dry weight]). Additions were oligomycin (5,ug/ml) and Cl-CCP (0.5 mM). SHAM (0.2 mM) was added to inhibitthe alternate oxidase. The experiment was repeated four times withindependent cultures, and essentially identical results were ob-tained. (b) The same conditions as described above for panel a wereused, except that the shift up in temperature was from 25 to 43°C.

adjusted to pH 7.0. Antimycin A (Sigma) and salicylhy-droxamic acid (SHAM; Aldrich Chemical Co., Inc., Milwau-kee, Wis.) were dissolved in absolute alcohol. The inhibitorswere prepared freshly for each experiment. Ethanol, at theconcentrations used in these experiments, had no effect onoxygen uptake.

Isolation of mitochondria. Yeast, mycelia, and cells atintermediate stages of the morphological transitions wereisolated by filtration through Whatman no. 1 filter paper,washed with distilled cold water, and suspended in 0.33 Msucrose-1 mM ethylene glycol bis-N-N'-tetraacetic acid (pH7.0)-0.3% bovine serum albumin. Glass beads (1/3 volume)were added, and the cells were broken by three 15-s bursts ina Braun cell homogenizer (model MSK; Fisher ScientificCo., St. Louis, Mo.) and cooled intermittently with liquidCO2. The homogenate was centrifuged twice at 800 x g (10min) to remove the glass beads and cellular debris, and themitochondria were pelleted at 17,000 x g (30 min). Theniitochondrial pellets- were suspended in mitochondrial res-piration buffer consisting of 0.3 M suctose-8 mM NaH2PO4-5 mM MgCl2-1 mM EDTA-8 mM Tris hydrochloride (pH7.2).

-12minI- T Measurements of oxygen consumption. Cells were sus-°IOOj pended in cell respiration buffer containing 1% mannose, 1

mM CaCl2, and 1 mM dimethylglutaric acid (pH 7.2). The6) oxygen concentration was measured polarographically with

a KIC oxygraph equipped with a Clark-type oxygen elec-({/79) trode (Gilson Instruments, Middleton, Wis.). Cell respira-

tion rates were expressed as microliters of 02 per hour peryc nt \(76.V milligramn (dry weight) of cells.

c-CCP\ Oxygen consumption by mitochondria was measured in achamber containing the mitochondrial respiration buffersaturated with air. Respiration rates were expressed asmicroatoms of 0 per minute per milligram of mitochondrialprotein.

Coupling of oxidative phosphorylation. Coupling of oxida-tive phosphorylation was assayed by the ability of an

(22.\) uncoupler, Cl-CCP, to stimulate respiration in the presence

,-¢,,,\/\,S,,,~of oligomycin, an inhibitor of ATP synthetase (3). Cellrespiration assays in these experiments were carried out in

C.CCP the presence of SHAM to inhibit the alternate oxidase andforce electron flux through the cytochrome system.

Spectrophotometric measurements. Spectrophotometricmeasurements were made with a Aminco-DW2 dual-wave-length spectrophotometer (DW2; Aminco) (8). The cuvetteused for low-temperature spectra (77 K) had a path length of1 mm. Mitochondria were suspended in 0.3 M sucrose-8 mMNaH2PO4-8 mM Tris hydrochloride (pH 7.2)-i mM EDTA.Measurement of ATP levels. Cells were collected by rapid

CI.CCP filtration and immediately frozen in liquid N2. ATP was

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FIG. 2. (a) Oxygen electrode recordings of respiration ofmycelial cells of P. brasiliensis at 250C, immediately after (0') and 1min (1') after the shift up to 37°C. The methods for altering thetemperature and measuring respirations were as described in thelegend to Fig. 1. The experiment was repeated four times withindependenit cultures, and essentially identical results were ob-tained. (b) The same conditions as described above for panel b wereused, except that the temperature shift was from 25 to 41°C.

J. BACTERIOL.

I100jum 09>

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PHASE TRANSITION OF DIMORPHIC FUNGI 4057

0,0.10

1, ,I'0 2 3 4 5 30 2 3 4 5 30)

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FIG. 3. Effect of temperature shifts from 25 to 370C on intracel-

lular ATP? levels in B. dermatitidis (a) and P, brasiliensis (b). The

experimlent was carried out on three independent cultures, and

essentially identical results were obtained.

extracted with perchlorate a,nd assayed by the glucose

6-phosphate dehydrogenase-hexokinase method (2). In each

assay 10 to 15 mg (,d ry weight) of cells was used. Values were

expresse'd as micromoles of ATP per gram (dry weight).

Protein measurements. Proteins were measured by the

method describe,d by Lowry et al. (4).

RESULTS

Uncoupling of respiration. The effects of a shift up in

temperature on respiration in mycelia of B. dermatitidis and

P. brasiliensis are-shown in Fig. 1 and 2, respectively. In

these experiments, coupling between respiration and oxida-

tive phos,phorylation in whole cells was assayed by the

ability of the uncoupler Cl-CCP to stimulate respiration in

the presence of oligomycin, an inhibitor of ATP synthetase

(3). Stimulation of respiration by Cl-CCP indica,tes that

respiration is coupled to ATP synthesis, whereas lack of

stimulationl indicates uncoupled respiration. Since both B.

dermatitidis and P. brasiliensisi have alternate oxidase activ-

ity (see below), the respiration measurements were carried

out in the presence of SHAM, an inhibitor of this paFthway,to force all of the electron flow through the cytochrome

system. Respiration in B. dermatitidis at 25°C, immediately

and 1 min after a temperature shift from 25 to 37°C, is shown

in Fig. la. Respiration was tightly coupled at 25°C (about a

4.6-fold stimulation of respiration by Cl-CCP) and remained

coupled after the shift up in tempera,ture. The degree to

which respiration was stimulated by Cl-CCP, however, was

decreased' from 4.6- to 2.2-fold. This decreased stimullationby Cl-CCP suggests that partial uncoupling may have oc-

curr,ed at 37°C. Respiration did not show complete uncou-

pling of oxidative phosphorylation until the temperature was

raised to 43°C in B. dermatitidis (no stimulation by Cl-CCP

within 1 min after the shift up in temperature from 25 to

43°C) (Fig. ib).

Similar experiments on P. brasiliensis showed that the

effect on respiration of the temperature shift from 25 to 37°Cwas small since the degree to which respiration was stimu-

lated by Cl-CCP decreased from 1.8-fold to 1.5-fold (Fig. 2a).

Respiration was completely uncoupled, however, when the

cells were shifted from 25 to 41°C (Fig. 2b).

ATP levels. Intracellular ATP levels in B. dermatitidis(Fig. 3a) and P. brasiliensis (Fig. 3b) dropped to 57 and 25%of initial values, respectively, and remained low for at least30 min after the temperature of the cultures was raised from25 to 37°C. The ATP levels of yeast of both fungi incubatedat 37°C were slightly higher than mycelia at 259C. ATP levelsin both fungi decreased to undetectable levels within 5 min ofshifting the mycelia of B. dermatitidis to 43°C and themycelia of P. brasiliensis to 41°C (data not shown).

Respiration rates. Respiration rates in B. dermatitidis andP. brasiliensis following the temperature shifts from 25 to37°C are shown in Fig. 4. We were able to discern a verytransient increase in respiration in both fungi immediatelyafter the shift to 37°C, followed by the progressive decline,characteristic of stage 1 of the transition in H. capsulatum.This decline continued for 48 h in B. dermatitidis and for 24h in P. brasiliensis, at which times the cells ceased torespire. The dormant period (stage 2 of the transition) lastedfor about 4 days in B. dermatitidis cultures and 5 days in P.brasiliensis cultures. Both c,ultures then entered stage 3,which was characterized by resumption of respiration andgrowth of yeast-phase cells. Yeast forms began to appear inB. dermatitidis cultures 9 to 10 days after the shift to 37°C,and the transition was complete by about 21 days. Yeastswere present in the P. brasiliensis cultures by about day 12,and the organisms were completely converted to yeast byabout day 21.

Stimulation of respiration in stage 2 cells. In our previousstudies with H. capsulatum, (8) we showed that cysteine orother sulfhydryl-containing compounds are required to com-plete the mycelial- to yeast-phase transition at those temper-ature elevations at which spontaneous respiration of thecultures ceased during stage 2. Cysteine was also required tocomplete the mycelial- to yeast-phase transitionk of B.dermatitidis and' P. brasiliensis induced by shifting themycelia of each fungus from 25 to 37°C, This requirementwas also satisfied by ,B-mercaptoethanol, indicating the im-portance of the sulfhydryl component. Therefore, we testedwhether mitochondrial respiratory pathways in these twofungi could also be reactivated during stage 2 by addingcysteine.

Respiration in cells of B. dermatitidis during stage 2 of themycelial- to yeast-phase transition was reactivated by cys-

28 r I I

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FIG. 4. Respiration rates among the mycelial to yeast transitionsof B. dermatitidis (0) and P. brasiliensis (A). The experiments wereinitiated by shifting mycelia (10-ml cultures in 50-mi Erlenmeyerflasks) from 25 to 370C. Respiration was measured in both respira-tion buffer and fresh medium with essentially identical results.

VOL. 169, 1987

4058 MEDOFF ET AL.

FIG. 5. Effect of electron transport inhibitors on cysteine-induced respiration of stage 2 mycelial phase cells of B. dermatitidis (a) and P.brasiliensis (c) and mitochondria from B. dermatitidis (b) and P. brasiliensis (d). The 02 electrode recordings of respiration of mycelial cells(0.5 ml; 0.7 to 0.8 mg [dry weight] per ml) were done on P. brasiliensis 24 to 26 h after the temperature shift from 25 to 37°C and 48 to 50 hafter the temperature shift of B. dermatitidis. Additions were as follows: KCN, 0.5 mM; SHAM, 0.5 mM; antimycin, 1.0 ,ugIml; cysteine, 1.6mM. The numbers in parentheses are rates of oxygen consumption (in microliters of 02 per hour per milligram [dry weight]. Mitochondriawere isolated from mycelia (24 h after the temperature shift of P. brasiliensis and 48 h after the temperature shift of B. dermatitidis) and werekept at 1 mg/ml. Additions were as follows: succinate, 20 mM; KCN, 0.5 mM; SHAM, 0.5 mM; antimycin, 1 ,ug/ml; cysteine, 1.6 mM. Thevalues in parentheses are respiration rates (in microatoms of 0 per minute per milligram of mitochondrial protein).

teine (Fig. 5a). The cysteine-stimulated respiration wasconmpletely inhibited by the addition of cyanide plus SHAM,and was resistant to up to 5 p.g of antimycin per ml, even inthe presence of 0.5 mM SHAM.

Mitochondria from stage 2 cells could not utilize succinateas a substrate for respiration in the absence of cysteine (Fig.5b). As in whole cells, the cysteine-stimulated respirationwas sensitive to CN and SHAM and resistant to SHAM andantimycin. Cysteine alone did not stimulate respiration ofmitochondria (data not shown). Pyruvate (plus malate), anda-ketoglutarate were also not utilized as substrates bymitochondria without cysteine (data not shown). Respirationin the presence of cysteine and these substrates was inhib-ited by CN and SHAM and was resistant to antimycin andSHAM. In contrast, yeast, mycelia, and mitochondria fromboth phases were sensitive to 0.5 to 1 ,ug of antimycin per mlin the presence of 0.5 mM SHAM. Therefore, cysteine-induced respiration in stage 2 whole cells and mitochondriautilized the alternate (SHAM-sensitive) and cytochrome(CN-sensitive) systems for electron flux. The resistance ofthe cysteine-induced respiration to antimycin suggests thatelectron flux in this pathway bypassed the antimycin block atcytochrome b and therefore was similar to the sulfhydrylshunt pathways described in H. capsulatum (3).

Respiration in stage 2 cells of P. brasiliensis was alsoreactivated by cysteine (Fig. 5c). Cysteine by itself did notstimulate respiration in mitochondria from stage 2 cells butrequired the presence of mitochondrial substrates. The dataon stimulation of respiration of P. brasiliensis mitochondriaincubated with succinate and cysteine are shown in Fig. 5d.Mitochondria from stage 2 cells could not utilize succinate asa substrate for respiration without cysteine. Similar results

were obtained with pyruvate malate and a-ketoglutarate.The cysteine-stimulated respiration in both cells andmitochondria of P. brasiliensis was inhibited by CN andSHAM and was resistant to up to 5 ,ug of antimycin per ml inthe presence of 0.5 mM SHAM. In contrast, 0.5 to 1.0 ,ug ofantimycin per ml in the presence of 0.5 mM SHAM com-pletely blocked the cytochrome pathway in either yeast ormycelia of P. brasiliensis or mitochondria from both phases.

Electron transport components. The 77 K difference spec-tra (succinate-reduced minus oxidized and dithionite-reduced minus oxidized) of mitochondria from the mycelialphases of B. dermatitidis (Fig. 6a) and P. brasiliensis (Fig.6b) and at several different time points after the temperaturewas raised to 37°C are shown. The spectrum for succinatewith reduced minus oxidized mitochondria ofB. dermatitidisshows distinct a-peaks for cytochrome c (550 nm),cytochrome b (560 nm), and cytochrome aa3 (603 nm). Wetook advantage of the inhibitor antimycin, which blockselectron transport between cytochromes b and c (1), toassign the cytochrome c and b peaks (data not shown). Thespectrum also show P-peaks at 510 to 525 nm and Soretpeaks for cytochromes b (430 nm) and aa3 (443 nm). Theremaining spectra in Fig. 6a show the effect of the temper-ature shift to 37°C on levels of electron transport compo-nents. The calculated levels of the electron transport com-ponents in B. dermatitidis at the different times are given inTable 1. The concentrations of electron transport compo-nents declined over the first 2 to 3 days after the temperatureshift. At 2 days, cytochrome b had fallen to its lowest levelof 24% of the mycelial level at 250C, and cytochromes aa3and c fell to their lowest levels of 18 and 23%, respectively.At 3 days the concentrations of the cytochrome components

J. BACTERIOL.

PHASE TRANSITION OF DIMORPHIC FUNGI 4059

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FIG. 6. Low-temperature (77 K) difference spectra (AA) of mitochondria isolated from B. dermatitidis (a) and P. brasiliensis (b) cells atdifferent times following the shift of mycelia from 25 to 37°C. Spectra of succinate (20 mM) with anaerobic minus oxidized mitochondria areshown for mycelia at 25°C and at the different times following shift of mycelia from 25 to 37°C. Protein concentrations of B. dermatitidismitochondria (in milligrams per milliliter are as follows: mycelia at 25°C, 3.1; mycelia at 36 h, 3.3; mycelia at 72 h, 4.9; mycelia at 12 days,3.2; yeast, 2.6. Protein concentrations of P. brasiliensis mitochondria (in milligrams per milliliter) are as follows: mycelia at 25°C, 2.4; myceliaat 36 h, 2.6; mycelia at 48 h, 3.61; mycelia at 7 days, 4.0; mycelia at 12 days, 2.7; yeast, 3.8.

began to increase, and the values returned to normal as yeastbegan to appear in the cultures.The spectra for P. brasiliensis are shown in Fig. 6b. The

individual spectra are not as well defined as in those for B.dermatitidis, probably because of the difficulties we had in

TABLE 1. Concentrations of cytochromes in mitochondriaisolated from mycelia, yeast, and transforming cells of

B. dermatitidis and P. brasiliensis after temperature shifta

nmol/mg of mitochondrial protein in the indicatedcytochromes of the following strains:

Time B. dermatitidis P. brasiliensis

aa3 b c aa3 b c

0 0.28 0.21 0.30 0.09 0.16 0.1436 hours 0.10 0.08 0.11 0.02 0.05 0.072 days 0.05 0.05 0.07 NDC' 0.03 0.023 days 0.08 0.08 0.09 ND 0.05 0.045 days 0.09 0.14 0.107 days 0.12 0.16 0.14 ND 0.07 0.069 days 0.18 0.19 0.2012 days 0.20 0.20 0.25 0.08 0.15 0.14Yeast 0.21 0.25 0.27 0.13 0.22 0.19

aThe temperature shift was from 25 to 37C. Cytochrome concentrationswere calculated from low-temperature difference spectra of dithionite reducedminus oxidized mitochondria (1).

b Immediately after the mycelia were shifted to 37°C. Values are the sameas those for mycelia at 25°C.

c ND, Nondetectable.

breaking the cells of P. brasiliensis, which tended to clump.The concentrations of cytochrome components also de-creased after mycelia of P. brasiliensis were shifted from 25to 37°C (Fig. 6b and Table 1). Cytochrome aa3 becameundetectable and cytochromes b and c decreased to about 14to 19% of initial levels between 2 and 6 days after thetemperature shift. The levels began to increase at day 7, andreturned almost to normal by day 12.

DISCUSSION

In this study we have shown that the patterns of physio-logical changes during the mycelial- to yeast-phase transi-tions of the two dimorphic pathogenic fungi B. dermatitidisand P. brasiliensis induced by a temperature shift from 25 to37°C are fundamentally similar to those reported previouslyfor H. capsulatum (3, 5a, 8). In all cases, the triggering eventappeared to be a heat-related insult induced by the temper-ature shift up, which resulted in various degrees of uncou-pling of oxidative phosphorylation. The temperature shiftthen led to declines in cellular ATP levels, respiration rates,and concentrations of electron transport components (stage1). The cells then entered a stage in which spontaneousrespiration decreased or ceased (stage 2), and finally, theyshifted into a recovery phase and transformed to the yeastmorphology (stage 3). The yeast phase was completelyadapted to growth at the higher temperatures.

In previous studies with H. capsulatum (8) we showed

VOL. 169, 1987

4060 MEDOFF ET AL.

that cysteine or other sulfhydryl-containing compounds arerequired during stage 2 for the operation of shunt pathwayswhich permit electron transport to bypass blocked portionsof the cytochrome system. Both B. dermatitidis and P.brasiliensis stopped respiring in stage 2 and also requiredcysteine or other sulfhydryl-containing compounds to com-plete the mycelial- to yeast-phase transition. We found thatthe sulfhydryl shunt pathways were also present in these twofungi, and we infer that they also probably function in stage2 of the transition to activate the respiratory shunt pathways,which allow utilization of mitochondrial substrates to pro-vide energy which allows the transition to be completed.The sulfhydryl shunt pathways are also present in

Cryptococcus neoformans, Aspergillus fumigatus, and Sac-charomyces cerevisiae; these organisms do not undergo aphase transition (unpublished results). In each case the shuntpathways are detected after the fungi are exposed to tem-perature elevations which result in cessation of spontaneousrespiration. Therefore, the sulfhydryl shunt pathways maybe a general mechanism that fungi have evolved to combatheat-induced injury and death.We have previously pointed out (3) that there may be a

relationship between temperature shifts that trigger themorphological transition in H. capsulatum and heat shock.Heat shock proteins are induced in H. capsulatum in re-sponse to temperature shifts, and they are also present in B.dermatitidis and P. brasiliensis immediately after the 25 to37°C temperature change (unpublished results). Therefore,these are other examples of differentiation processes in-volved in pathogenicity which are triggered by a temperatureshift and accompanied by a heat shock response.

In all three fungi, the mycelial phase is the one adapted forgrowth in nature (7). Therefore, conversion to the yeastphase is part of a process of adaptation to new environmen-tal conditions with an elevated temperature. The conserva-tion of these mechanisms of adaptation among these threefungi suggests that H. capsulatum may be used as a modelfor the morphogenesis of other dimorphic fungal pathogensand that a common strategy for development of live vaccinesmay be possible. Infection with temperature-sensitive mu-tants which may not have the sulfhydryl shunt pathwaysmight provide effective protection. Another possibility is theuse of mycelia treated with the sulfhydryl inhibitor para-chloromercuriphenylsulfonic acid, which blocks themycelial- to yeast-phase transition of H. capsulatum (6).

These nontransforming mycelia cannot cause disease but doimmunize mice against further challenge with pathogenicyeast. Preliminary results indicate that para-chloromercuri-phenylsulfonic acid also blocks the mycelial- to yeast-phasetransition of B. dermatitidis.

ACKNOWLEDGMENTS

This study was supported by Public Health Service grantsA116228, A107015, and A107172 from the National Institutes ofHealth.We thank Alan Lambowitz and David Schlessinger for helpful

suggestions and review of the manuscript.

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1983. Possible relationship of morphogenesis in pathogenic fun-gus, Histoplasma capsulatum to heat shock response. Nature(London). 303:806-808.

4. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.1951. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 193:265-275.

5. Maresca, B., A. M. Lambowitz, V. B. Kumar, G. A. Grant, G. S.Kobayashi, and G. Medoff. 1981. Role of cysteine in regulatingmorphogenesis and mitochondrial activity in the dimorphic fun-gus Histoplasma capsulatum. Proc. Natl. Acad. Sci. USA.78:4596-4600.

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6. Medoff, G., M. Sacco, B. Maresca, D. Schlessinger, A. PainterG. S. Kobayashi, and L. Carratu. 1986. Irreversible block of themycelial-to-yeast phase transition of Histoplasma capsulatum.Science 231:476-479.

7. Rippon, J. W. 1982. Medical mycology, 2nd ed. The W. B.Saunders Co., Philadelphia.

8. Sacco, M., G. Medoff, A. M. Lambowitz, B. V. Kumar, G. S.Kobayashi, and A. Painter. 1983. Sulfhydryl induced respiratoryshunt pathways and their role in morphogenesis in the fungus,Histoplasma capsulatum. J. Biol. Chem. 258:8223-8230.

9. Schwarz, D. S., and H. W. Larsh. 1980. An effective medium forselective growth of yeast or mycelial forms of Candida albicans.Mycopathology 70:67-75.

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