cyclic adenosine 3'5‘ monophosphate (c-amp) as a factor in phase morphogenesis of blastomyces...

mykosen 27 (7) 340-345 mykosen 1 Angenommen/Accepted: 31. Oktober 1983 I 0 Grosse Verlag Berlin 1984

Cyclic Adenosine 33’ Monophosphate (c-AMP) as a Factor in Phase Morphogenesis of Blastomyces dennatitidis Zyklisches Adenosin-3~5’-Monophosphat (c-AMP) als Faktor bei der Phasenmorphogenese von Blastomyces dermatitidis Sophie Paris’ and R. G. Garrison2

‘Unite de Mycologie, lnstitut Pasteur, Paris, France *Research Service, Veterans Administration Medical Center, Kansas City, USA

Key words: Blastomyces dermatitidis - c-AMP levels - Effects of c-AMP inhibitors - Electron cytochemistry Schliisselworter: Blastomyces derrnatitidis - c-AMP-Konzentration - Wirkungen von c-AMP-Inhibitoren - Elektronencytochemie

Summary: The intracellular concentration of cyclic adenosine 33’ monophosphate (c-AMP) was found to be 1.3 times higher in the mycelium of Blastomyces dermatitidis than that found in its yeastlie growth form. Yeastlike cells of the fungus grown at the preferential temperature of 37OC reverted to mycelial phase growth in the presence of the c-AMP phosphodiesterase inhibitors theophylline or3-isobutyl-1-methyl xanthiie. The intracellular activity sites of c-AMP phosphodiesterase in converting yeastlike cells were determined by electron cytochemical means. It is believed that intracellular levels of c-AMP can critically influence phase morphogenesis of B. dermatitidis.

Zusammenfassung: Die intrazelluliire Konzentration von c-AMP im Mycelium von Blastomyces dermatitidis ist lJmal hoher als in seiner Hefe-ahnlichen Form. Hefe-ahnliche Zellen des Pil- zes, geziichtet beim Temperaturoptimum von 37OC, wechselten in die Mycelphase uber, wenn ein Inhibitor der c-AMP-Phosphodiesterase wie TheophyUii oder 3-Isobutyl-1-Methylxanthii zugegen war. Die intrazellularen Aktivitiitszentren der c-AMP-Phosphodiesterase in diesem Prozefi wurden mit elektronencytochemischen Methoden bestimmt. Es kann angenommen wer- den, dafi die intrazelullare Konzentration von c-AMP die Phasenmorphogenese von Blastomy- ces dermatitidis kritisch beeinflufit.

mykosen 27, Heft 7 (1984)

Cyclic Adenosine 3’3’ Monophosphate (c-AMP) 341

Introduction

The pathogenic dimorphic fungi are characterized by a filamentous growth phase when they occur naturally in the environment or when cultured in vitro at room temperature. On introduction into the mammalian host or in the laboratory at 37OC they convert reversibly to a parasitic yeastlike or other tissue phase. The various factors known to influence phase transition of these medically important fungi have been recently by Rippon (6) and Szaniszlo et al. (8).

It is now known that intracellular levels of cyclic adenosine 3:5’ monophosphate (c-AMP) have a profound effect on the morphogenesis of the pathogenic dimorphic fungus Histo- plasma capsulatum ( 5 ) . Levels of this nucleotide are higher in the mycelium of the fungus than in its yeastlike growth form (see ref. 8 and its citations). In the present study we docu- ment intracellular levels of c-AMP, the effects of c-AMP phosphodiesterase inhibitors, and the electron cytochemical localization of c-AMP phosphodiesterase sites in cells of the pathogenic dimorphic fungus Blastomyces dermatitidis, the etiological agent ofNorth Ame- rican blastomycosis. Our results provide additional support for a role of c-AMP in growth phase regulation of the dimorphic fungi.

Materials and Methods

Blastomyces (Ajellomyces) dermatitidis ATCC 18188 was obtained from the American Type Culture Collection, Rockville, Maryland. Stock cultures were maintained in the yeastlike phase by routine transfer to Ti-ypticase soy (Difco) agar slants at 37OC. Yeastlike cells for experimental work were generated in 250 ml flasks containing 100 ml of the same medium subjected to gyrotary shaking at 37OC for 72 h. Some growth experiments employed yeastlike cultures fortified with either 1’5, or 10 mM theophylline or 3-isobutyl-1-methyl xanthine (Sigma Chemical Co., St. Louis, Missouri) contained in shaker flasks at 37OC. The resultant growth was examined with a Leitz Orthoplan light microscope under phase contrast illumi- nation. Finely divided mycelium ofthe fungus was prepared in shaker flasks of either Sabou- raud glucose broth or a proteose peptone medium at room temperature (a. 25OC).

Some yeastlike cells or those stimulated toundergo yeast-mold transition were futed in 3 Yo glutaraldehyde in 0.05 M s-collidine-HCl buffer (PH 6.8) at 4OC for 2 h. Cyclic-AMP phosphodiesterase activity was localized by the method of Florendo et al. (1). Cells were initially incubated for 30 min. at room temperature in 60 mM tris-maleate buffer (pH 7.4) containing 2 mM MgC12, 0.25 M sucrose, and 5’-nucleotidase (5 mg/ml Crotalus atrox venom, Sigma). Cells were then further incubated for an additional 30 min at 37OC in the same buffer containing 3 mM c-AMP, 5’-nucleotidase (3 mg/ml) and 2 mM lead nitrate. Control incubations included deletions of the substrate or lead nitrate. The cells were then processed for trans- mission electron microscopy by previous described methods (2).

Cells for c-AMP assay were rapidly collected on 0.45 pm membrane filters and resuspend- ed in cold 5 % trichloroacetic acid (TCA) or in 50 mM phosphate buffer containing 5 mM theophylline. Glass beads (0.5 mm) wereadded and the cells broken at 4.000 rpm for 3 min in a Braun MSK cell homogenizer cooled with liquid C02. Homogenates were centrifuged at 32.000 x g for 1 hat 4OC. For TCA-treated cells, an aliquot of supernatant was washed 5 times with water-saturated diethyl ether (pH 1.0) at room temperature. Residual ether was removed by bubbling N2 gas through the sample. The pH was adjusted to 6.2 and c-AMP content determined. For cells broken in phosphate buffer, the supernatant was directly assayed for c-AMP. Cyclic-AMP assays were done by the radioimmunoassay of Steiner et al. (7) using the assay kit provided by Institut Pasteur Production (Pans, France). The results were expressed in pmol/mg protein. All protein determinations were done according to Lowry et al. (4) with crystalline bovine serum albumin as the standard.


342 Sophie Paris and R. G. Garrison

Fig. 1 : EffectsofS mM theophyllineonyeastlikegrowthatitspreferential temperat~reof37~C. (a)Note the germination of the yeastlike cells to form short hyphal elements. (b) Control preparation showing normal yeastlike cells. Phase contrast. Bar equals 5 pm.

Table 1 Cyclic AMP content of yeastlike and mycelial phase cells of B. dermatitidis strain ATCC 18188.

extraction pmol c-AMP/mg protein M/Y ratio mycelium yeast

method l a 30.5 22.7 1.3 method 2b 34.4 26.9 1.3 culture filtrate 0 0 - (a) medium plus cells treated with TCA (b) filltered cells broken in Podbuffer plus 5 mM theophylline

Results and Discussion

Table 1 summarizes our estimates of the intracellular content of c-AMP in cells of the two growth forms of B. dermatitidis as determined by two methods. Slightly higher yields of c-AMP were found when broken cell preparations in phosphate buffer were employed for the assay procedure. However, in each assay it was found that the mycelium contained 1.3 times the amount of c-AMP when compared to that found for the yeastlike growth form.


Cyclic Adenosine 3:5' Monophosphate (c-AMP) 343

Fig. 2: Thin section of normal yeastlikecell grown for 72 hat 37OC showing localization of c-AMPphos- phodiesterase activity at the nuclear membrane (Nm), mitochondria1 membranes (Mi). endodasmic reticulum (ER), and in membranous, vacuolar-like areas (V). Bar equals 0.25 pm.

Averages ofthe two determinations indicated themycelium to contain 32.4 and theyeastlike form 24.8 pmol c-AMP/mg protein. These relative levels of c-AMP differed in magnitude from that reported for the two growth forms of H. capsulatum; average c-AMP concentra- tions in pmol/mg protein for this fungus were reported as 46.9 for the mycelium and 9.8 for the yeastlike phase or about 5 times greater in the mycelial growth form (5 ) .

Figure 1 a shows a typical light micrograph of yeastlike cells of B. dermatitidis treated with 5 mM theophylline (or 3-isobutyl-1-methyl xanthine). Within 72 h incubation at 37OC (the preferential incubation temperature for yeastlike growth), the c-AMP phosphodiesterase inhibitors induced a germination of the yeastlike cells to form short hyphal elements; a control preparation of untreated yeastlike cells is illustrated in Figure 1 b. This shift to a hyphal morphology (even at 37OC) is believed likely a consequence of inhibition of c-AMP phosphodiesterase activity with a concomitant rise in intracellular c-AMP content. We interpret these results to suggest that intracellular levels of c-AMP are critical to phase morphogenesis of B. dermatitidis even though the observed difference in c-AMP content between the two growth forms of the fungus was small (1.3 times). This low ratio may be a reflection of the strain of the fungus employed for assay.

It is generally agreed that B. dermatitidis exhibits a yeast-mold dimorphism which is primarily regulated by temperature (3,8). Consequently it was of interest to ascertain the effects of incubation temperature on the nature ofactivity sites of c-AMPphosphodiesterase in the yeastlike cell. Thin sections of cells grown at 37OC showed a disposition of what is believed to be electron opaque reaction product (lead phosphate) along the various cyto- membranes of the nucleus, mitochondria, endoplasmic reticulum, but not at the plasma


344 Sophie Paris and R. G. Garrison

Fig. 3: Thin section ofa yeastlike cell 24 h after lowering of the incubation temperature to 25OC. Locali- zation of c-AMP phosphodiesterase occurs at the nuclear membrane (Nm), mitochondria1 membranes (Mi), and endoplasmic reticulum (ER). Note the decrease in relative enzymic activity in the vacuolar- like areas. Bar equals 0.30 pm.

membrane. Conspicuous deposits of electron opaque reaction product were sequestered in scattered, membranous, vacuolar-like regions of the cytoplasm (Fig. 2). These vacuolar-like inclusions appeared to diminish in both size and enzymic activity levels when the temperature of incubation of the cells was lowered to 25OC (Fig. 3). Such events were detected within 24 h of the thermal induction of yeast-mold transition and beforegross morphological evidence for a shift to mycelium was apparent. The exact nature of these vacuoles is not clear, but they resemble closely the lysosomal-like vacuoles seen in thin sections of converting yeastlike cells of H. capsulatum (2).

It has been known for some time that cyst(e)ine is necessary for invitro mold to yeast transition and subsequent maintainance of the yeastlike growth form of H. capsulatum. Maresca et al. ( 5 ) found that the intracellular c-AMP content is higher in the mycelial phase than that in the yeastlike phase and that when c-AMP phosphodiesterase inhibitors were added to yeastlike cultures at 37OC the cultures reverted to the mycelial growth form. These workers postulated that although the specific role of cyst(e)ine in phase morphogenesis of H. capsulatum is not understood, it is possible that they might satisfy a necessary requirement for -SH groups that might then determine the intracellular content of c-AMP. It was suggested that at 37OC the cells are able to bring cyst(e)ine to critical membrane sites. There it would activate c-AMP phosphodiesterase thus lowering intracellular c-AMP and favoring the yeastlike phase. Such membrane sites would likely be similar in identity to those illustrated in Figures 2 and 3. When the temperature is lowered to ca. 25OC, transport of cyst(e)ine decreases and c-AMP phosphodiesterase activity falls. This then results in an increase in c-AMPlevels and a


Cyclic Adenosine 3 3 ’ Monophosphate (c-AMP) 345

shift to the mycelial phase. In general our results support the concept that relative intracellular c-AMP levels may function in some manner as a phase determinant at least in some species of the pathogenic dimorphic fungi.

In the case of B. dermatitidis, it is not clear what role ifany nutritional factors might play in regulation of intracellular c-AMP content. It is possible that c-AMP phosphodiesterase in this species is thermally regulated being repressed at the lower temperature but derepressed when the cell is placed in a thermal environment favoring yeastlike phase morphology.

Acknow/edgements:This study was supported by VAproject 3046-01 (R.G.G)and Ministerede I’Indu- strie et de la Recherche 81. L. 0527 (S.P).

References

1. Florendo, N. T., R. J. Barrnett & P. Greengard (1971): Cyclic 3’3’ nucleotide phosphodiesterase: cytochemical localization in cerebral cor- tex. Science 173,745-747.

2. Garrison,R. G.&J.Tally(1981): Electroncyto- chemical evidence for lysosomal-like equiva- lents in Histoplasma capsulatum. Mycopathol.

3. Levin, S. & Z. J. Ordal(l949): Factors influen- cing the morphology of Blastomyces dermatitidis. J. Bacteriol. 52, 687-694.

4. Lowry, 0. H., N. J. Rosebrough, A. L. Farr & R. J. Randall (1951): Protein measurement with Folin phenol reagents. J. Biol. Chem. 193,

5 . Maresca,B., D. Schessinger,G. Medoff&G. S . Kobayashi (1977): Regulation of dimorphism in the pathogenic fungus Histoplasma capsulatum. Nature (Lond.) 266,447-448.

73,183-190.

265-275.

6. Rippon, J. W (1980): Dimorphism in pathogenic fungi. Crit. Rev. Microbiol. 8,49-97.

7. Steiner, A. L., C. W. Parker & D. M. Kipnes (1972): Radioimmunoassay of cyclic nucleotides. I. Preparation of antibodies and iodinated cyclic nucleotides. J. Biol. Chem. 247, 1106- 1113.

8. Szaniszlo, P. I., C. W Jacobs & P. A. Geis (1983): Dimorphism: morphological and bio- chemical aspects. In: Fungi pathogenic for humans and animals, Part A, D. H. Howard (ed.), Marcel Dekker, New York, p. 323-436.

Address: Dr. R. G. Garrison, Research Service, Veterans Administration Medical Center, 4801 Linwood Blvd., Kansas City, Missouri 64128, USA.


cyclic adenosine 3'5‘ monophosphate (c-amp) as a factor in phase morphogenesis of blastomyces...

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