cytochemical study of the yeast and mycelial cell walls ofparacoccidioides brasiliensis

EXPERIMENTALMYCOLOGY lo, 228-242 (1986)

Cytochemical Study of the Yeast and Mycelial Ceil Walls of Paracoccidioides brasiliensis

SOPHIE -PARIS,* MARIE-CHRISTINE PREVOST,? JEAN-PAUL LATGI&$ AND ROBERT G. GARRISON§

*Unite de Mycologic, fStation centrale de rnicroscopie klectronique, ZUnite’ de Lutte Biologique, Institut Pasteur, 2.5-28 rue du Dr. Roux, 75724 Paris, Cedex 15, France, and §The Research Service, Veterans

Administration Medical Center, Kansas City, Missouri 64128

Accepted for publication June 30, 1986

PARIS,S.,PREVOST,M-C.,LATGB, J-F!, ANDGARRISON, R. G. 1986.Cytochemicalstudy ofthe yeast and mycelial cell walls of Paracoccidioides brasiliensis. Experimental Mycology 10, 228-242. Cell wall structure and macromolecular organization of the various growth forms of Paracoccidioides brasiliensis were investigated using chemical, enzymatic, and cytochemical methods. The wall of yeast-like cells was not sensitive to periodic acid and p(1 --f 3) glucanase treatments, but bound calcofluor suggesting that it is composed of an ol(l + 3) glucan and of chitin. The fibrillar outer layer of bud cell initials and of abscision areas, were characterized by the presence of a strongly periodic acid- and concanavalin A-reactive substance that was sensitive to the lytic action of protease, presumably composed of mannan and proteins. The outermost layer of the mycelium was sensitive to protease, and periodic acid and peanut agglutinin positive indicating that it was composed of galactomannan and protein. The inner layer is mainly composed of a p(1 -+ 3), (1 -+ 6) glucan (sensitive to snail enzyme but not to an exo @(I -+ 3) glucanase), and chitin. Septa were brightly fluorescent with calcofluor. Our results are compared with models previously proposed by other authors. 0 1986 Academic press, hc.

INDEX DESCRIPTORS: Yeast; mycelial cell wall; Paracoccidioides brasiliensis; cytochemistry.

Paracoccidioides brasiliensis (Splen- dore) de Almeida is the etiological agent of the chronic mycotic disease, South Amer- ican blastomycosis. The parasitic form of the fungus is a unicellular, synchronous, multilateral budding yeast-like cell. This parasitic form can be obtained in vitro at 37°C. When the yeast-like form is trans- ferred to a lower temperature (23°C) it con- verts to the saprophytic mycelial form. The morphological intermediate between the yeast and mycelial forms was defined by San-Blas et al. (1980) as the mycelial bud.

The various factors known to influence the dimorphism of the fungus have been re- viewed by San-Blas and San-Blas (1985) and Szaniszlo et al. (1983). The cell wall is the main determinant of morphology and has been studied by both electron microscopy and biochemical analyses. Carbonell and Gil (1982) showed that the yeast-like

cell wall was thicker and different in sub- structure from that of its homologous my- Celia1 counterpart. Studies on the biochemistry of the walls of both yeast-like and my- Celia1 forms have demonstrated that the wall of the yeast-like form contains a(1 + 3) glucan whereas the mycelial form contains p(1 + 3) glucan (Kanetsuna et al., 1969, 1972; Kanetsuna and Carbonell, 1970). Synthesis of the a(1 + 3) glucan is stopped in favor of the p-linked glucan when the temperature of incubation is shifted from 37 to 23°C (Kanetsuna et aZ., 1972). The study of cell wall composition of a nondimorphic mutant confirmed the importance of this glucan in the dimorphic nature of P. brasikensis (San-Blas et al., 1981). But besides the glucan, chitin may also have a morphogenetic role (Kanet- suna, 1981).

Several models proposed for the role of

228 0147-5975/86 $3.00 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

CYTOCHEMISTRY OF Parncoccidioides brasiliensis 229

the cell wall in the dimorphism of P. brasiliensis take into account the chemical composition of cell walls without cytochemical localization of the various polysaccharides (Kanetsuna, 1972, 1981; San-Blas and San- Blas, 1985). A number of techniques are now available for the subcellular localization of a variety of structural polysaccharide or polysaccharide-containing substances at both the light and electron microscopic level. In the present report, we describe results of a cytochemical investigation which links together chemical composition and morphology of the cell wall of the various growth forms (yeast-like cell, yeast bud, mycelial bud, and mycelium) of P. brasiliensis at the substructural level. Such studies provide additional insights on the role of the cell wall in dimorphism of this biologically interesting and medically important fungal organism.

MATERIALS AND METHODS

Organism and Growth Conditions

Strain MTC of Paracoccidioides brasiliensis (obtained from A. Restrepo, Me- dellin, Colombia) was used throughout this StUdy.

Yeast-like form.’ Yeast-like cells were grown in PGY broth (Bactopeptone, 5 g; yeast extract, 5 g; glucose, 10 g; distilled water, 1 liter (San-Bias et al., 1983) for 3 days at 36°C with shaking. The exponen- tially growing cells of this preculture were then inoculated in fresh PGY medium and incubated at 36” for 1 day (Flores-Carreon et al., 1979).

MyceEial form. Yeast-like cells were al- lowed to transform to mycelium by growing early log phase yeast-like cells in PGY medium at 23 t 1°C for 2-4 days with shaking (Flores-Carreon et al., 1979).

1 Abbreviations used: PATAg, periodic acid-thio- carbohydrozide silver proteinate technique; CFW, calcofluor white; FITC, fluorescein isothiocyanate; Cork A, concanavalin A; PNA, peanut agglutinin; WGA, wheat germ agglutinin; PBS, phosphate-buff- ered saline; M, mycelial form: Y, yeast-like form.

Electron Microscopy

The preparation of the material for electron microscopy was done as previously described by Latge et al. (1982).

Periodic acid- thiocarboh~ldrazide- silver proteinate technique. Ultrathin sections of cells were collected on gold grids and processed by the PATAg techn Thiery (1967). Controls included t tions not treated with periodic acid or this- carbohydrazide, or by oxidation with 0.1% hydrogen peroxide.

Enzymatic Treatments

Washed cells were incubated I h at 3 in the enzymatic solution with 5 m thiothreitol (DTT, Sigma). Protea type I) was used a .5 mg/ml in 50 m

7.4. Snail digestive ique Franqaise, Vil-

leneuve-la-Garenne, France) 5% was luted in 50 mM acetate phosphate buffer, pH 6.0. The activity of snail enzymes was tested, under the same conditions, on laminarin (linear p(I + 3) glucan) (linear p(1 + 6)glucan), a 6)mannan. Cell walls were inc at 37°C with an exoP(l + 3) (laminarinase Sigma, 1 mg/ml) acetate buffer, p

Fluorescence Microscopy

Calcofluor white. The cells were fixe with a 3% formaldehyde-sa~i~e so~~ti~~ and suspended in an aqueous solution of 0.1% (w/v) calcofluor white 2 (Toinzart et Matignon, Vitry-sur-Seine, France) or Primulin (Sigma, IO0 Fg/ml) for 5 min at room temperature, then centrifuged and washed once with distilled water according to Sloat and Pringle (1978).

Fluorescein isoth~ocya~ate-co~~~~ated lectins. T conjugated lectins, Con A, PNA, and GA, were obtruded from In- dustrie Biologique Frangaise, France. After several washings in 0.15 iba Na~~-~.~~ M PBS, pH 7.4, cells were incubated for 1 h in

230 PARIS ET AL.

a solution of lectin in PBS (100 pg/ml). Cells were then washed three times with PBS. Controls were made with cr-methyl- mannoside (0.2 M) for Con A, lactose (0.2 J4) for PNA, and chitobiose (0.2 M) for WGA.

Wall Isolation and Purification

The cultures (M and Y forms) were centrifuged and washed three times in cold distilled water. Rupture of the cells was performed using a MSK Braun homogeneizer (Melsungen Apparatebau) with l-mm diam- eter glass beads. Long breaking times (Y = 10 min and M = 15 min) are needed to have all cells broken. These long disruption times resulted in the presence of glass frag- ments in the wall preparation which gave high ash values especially for mycelium wall extracts. Washing of the cell walls was performed as previously reported (Latge et al., 1984) and then lyophilized. The purity of the walls was judged by the inability to detect ribose by gas chromatography.

Analytical Methods

Total hexoses (neutral sugars) were determined by the anthrone and by the phenol technique (Herbert et al., 1971) using glucose as the standard. Total amino sugars were determined by a modification of the Elson-Morgan method with p-di- methylaminobenzaldehyde after hydrolysis of the cell walls (3 days at 20°C in 70% H,S04, then 8 h at 100°C in 1.5 N H,SO,) according to Tracey (1955). Hexoses and chitin were expressed in equivalents of glucose and N-acetylglucosamine, respectively. After alkaline hydrolysis (5 min at 100°C in 1 N NaOH), proteins were as- sayed according to the method of Lowry (as modified by Layne, 1957) using bovine serum albumin as a standard. Hydrolysis of the cell wall material was performed in 0.5 N chlorhydric methanol for 24 h at 80°C. Monosaccharides were identified as trifluoroacetylated methylglucosides by gas chromatography (Zanetta et al., 1972). The

conditions were as follows: column size 3m x 2 mm; phase OV 210 on Chromosorb

WHP; gas vector nitrogen 8 ml min-l; temperature programmed from 100 to 205°C at 1” min-‘.

RESULTS

Chemical studies indicated that Y and M walls have a different composition (Table 1). Yeast cell walls contained more hexoses than mycelial cell walls. Glucose, galactose, and mannose were found in the two phases. Both Y and M had glucose as the main hexose but the mycelial phase had higher amounts of mannose and galactose compared to the yeast phase. As the mannose/galactose ratios were similar in yeast (1.6) and in mycelium (1.4), the deficit in glucose in mycelium seems to be replaced by both galactose and mannose and not only by one of them. The amount of chitin was about the same in the two phases but chitin might be underestimated as no ki- netic estimates of the hydrolysis were done.

To better understand the ontogeny of the wall components during morphogenesis, the four stages of P. bvasiliensis (mature yeast-like cell, bud cell initial, mycelial bud, and mycelium), are considered sepa- rately.

1. Yeast-like Cell Wall

Figure la shows portions of a medial longitudinal thin section of the cell wall of a mature yeast-like cell after conventional chemical fixation and heavy metal staining regimens. The thick cell wall was homog- enously electron translucent and poorly contrasted except at the site of bud initials. With the PATAg stain, the cell wall appeared composed of several zones of very light and variable PATAg staining inten- sities which vary in number with age of the cell (Figs. lb, c). Aging of the cell resulted in the appearence of a slightly PATAg reactive median layer (Fig. lc). The Y cell wall was not sensitive to the lytic action of the

CYTOCHEMISTRY OF Paracoccidioides bra.si1ien.si.s 23!

FIG. 1. Thin sections of the Y cell wall. (a) Mother cell wall with a 2-layered bud cell wall. The young bud shows a heavily stained outer layer. Note the channels in the outer layer (arrowheads). Va, vacuole; Mi, mitochondrion. Uranyl acetate-lead citrate ~75,000. (b,c) Enlargements of part of the cell wall and plasma membrane region. Note the presence of giycogen-like material (GL) between the cytoplasmic membrane (pm) and the cell wall (CW) and Golgi-like cisternae (G). ER. endoplasmic reticulum. PATAg stain. (b): x 61,000 (c): x 74,500.

232 PARIS ET AL.

TABLE 1 Chemical Analysis of the Cell Walls of P. brusiliensis

(strain MTC)

Yeast Mycelium

Total neutral carbohydrate” 48% 31

Glucoseb 87 61 Mannoseb 8 23 Galactoseb 5 16

Amino sugaFjC 49% 43% Protein0 1% 1% % of cell wall hydrolysed

by laminarinase 7 4

Note. All values are averages of three independant experiments (in duplicate).

a Expressed as percentage of the organic material of the wall (dry wt without ashes).

b Neutral sugars released after methanolysis and determined as trifluoroacetylated methyl glucosides by gas chromatography. Values, expressed as free glucose, refer to the percentage of the total carbohydrate.

c Expressed as free glucosamine.

laminarinase (Table 1) and of snail gut enzymes (mostly p(1 --f 3) and p(1 -+ 6) glucanases: under our conditions snail gut enzymes degraded 50% of laminarin, 36% of pustulan, and 4% of mannan, data not shown). Walls of CFW or primulin-treated Y mother cells were brightly fluorescent. FITC-PNA and FITC-WGA which binds to galactosyl and N-acetylglucosaminyl residues, respectively, did not bind to Y cells. FITC-Con A gave a dim fluorescence (Figs. 2c-e).

2. Yeast-like Bud Cell Wall

The bud cell wall yielded different staining reactions from those described above for the wall of the mature Y mother cell. Conventional heavy metal stains of the thin sections of Y cells revealed that the initiation of the budding process was characterized by changes in electron opac- ity and in texture of the wall located at the site of bud emergence (Fig. la). Two well- separated layers appeared.

The outer layer of the newly forming bud was shown to be thicker and more electron opaque on conventional heavy metal staining than that of the outermost layer of

yeast-like mother cells. Substructural components of the bud cell wall outer layer be- came microfibrillar in texture and often showed electron translucent channels in the surrounding matrix (Fig. la). Figure 3 shows portions of thin sections of a PATAg- stained Y mother-daughter cell complex. The newly formed outer wall of the bud initial arose from a PATAg-reactive middle layer of the parental wall and was strongly PATAg reactive (Fig. 3a). The abscission areas of the cell walls of mature bud and mother cells were strongly PATAg reactive (Fig. 3b). Figures 3c and d illustrate portions of thin sections of budding yeast-like cells that were treated with protease prior to the application of the PATAg stain. The conspicuous PATAg staining of the cell wall of the bud initial (Fig. 3c) as well as that of the abscission regions between dividing cells (Fig. 3d) was markedly reduced. On staining with FITC-Con A, bud cell walls displayed a bright fluorescence that was re- stricted to the immediate sites of bud emergence (Figs. 2c, e) and to the yeast-like mother-daughter cell juncture (Fig. 2d). FITC-WGA and FITC-PNA were not bound. The localization of the positive staining of bud initials by the FITC-Con A reagent (bud and abscission areas) corre- lates with PATAg-reactive substances at these sites. The inner wall layer of buds was electron translucent (Fig. la), PATAg negative (Fig. 3b), and laminated after re- moval of the protease-labile outer layer like the mature Y inner layer (Figs. 3c, d). Bud cell wall structure was also unaffected by action of snail enzyme. This suggests a similar chemical composition as in mature Y cell wall.

3. Mycelial Bud Cell Wall

Figure 4 is an assembly of electron micrographs of thin sections illustrating PATAg-staining characteristics of mycelial buds.

The thin outer wall of the mycelial bud was strongly PATAg reactive; its origin was

CYTOCHEMISTRY OF Paracoccidioides brasiliensis 233

FIG. 2. Fluorescence micrographs of budding yeast-like cells stained with calcofluor (a,b) or with concanavalin A (c-e) x 3300. Bar represents 10 km. Note: (1) the bright fluorescence of mother cell and the slight fluorescence of the abscission area in (a) and (b); (2) the slight fluorescence of young buds (c,e) or of the abscission area; (3) the bud scar on mother cell in (d).

a b

FIG. 3. Thin sections of the budding yeast-like form. (a) Tangential section of a young bud. Note the numerous vesicular structures (V) and the heavily stained outer layer of the bud cell wall. B, bud; Y, yeast-like mother cell. PATAg stain. x 38,000. (b) Mature bud cell showing an intense staining reactivity at the mother bud junction (arrow). M, microbody; N, nucleus. PATAg stain. x 10,000 (cd) After treatment with protease, the heavily stained outer layer of young buds (c) or that of the abscis- sional region (d) has disappeared. Note the layered configuration (c, arrowheads). PATAg stain. (c) x 46,000; (d) x 10,000.

234

CYTOCHEMISTRY OF Paracoccidioides bmsilicnsis 235

from a PATAg-positive inner wall layer of the parental yeast-like cell (Figs. 4a, b). Sometimes (about 20% of the mycelial buds) there was a rupture of the PATAg- reactive outer wall layer of the parental yeast-like cell (Fig. 4~). The PATAg reactivity of the outer wall layer of the mycelial bud was labile to treatment with protease (Fig. 4d). FITC-Con A, -WGA, and -PNA did not bind to mycelial buds (not shown).

The inner layer was PATAg unreactive (Figs. 4b-d) and CFW did not bind to my- Celia1 buds (Fig. 5).

4. Hyphal Cell Wall

Transmission electron microscopy of medial longitudinal thin sections of conventional preparations of hyphal cells revealed the main wall to be composed of a thin, electron opaque outer layer and a thick- ened, electron translucent inner layer (Fig. 6c).

Thin sections of hyphal cells treated with the PATAg stain indicated that the outer wall layer was strongly PATAg reactive (Figs. 6d, e). The PATAg reactivity of the outer layer was sensitive to the lytic action of protease (Fig. 6f). Mycelial cells treated with FITC-WGA or FITC-Con A were unstained (not shown), but stained with FITC-PNA (Fig. 6b). The inner layer of the wall remained unstained by a PATAg treatment. Figure &a illustrates a typical staining reaction of the mycelium when treated with either CFW or primulin. The main walls of the mycelium were only slightly stained by these reagents but septa were brightly fluorescent. Prior treatment of hyphal cells with snail enzyme resulted in marked degenerative changes of the en- tirety of the cell wall proper (Fig. 6g). However, the wall was unaffected by an exop(l -+ 3) glucanase (Laminarinase) indicating that this glucan is branched.

DISCUSSION

1. Yeast-like Cell

The cytochemical and chemical results

presented in this study are in agreement with previous work demonstrating that I inner layer was mainly composed of CY(~ 3) glucan (insensitive to a p(1 -+ 3) glucanase treatment and PATAg negative) a fibrillar chitin (CFW positive) (Carbon& aZ., 1970; Grimaldi et al., 1979. Manetsuna et al., 1972). The yeast-like is different from the true yeast Sa omyces cerevisiae where chitin (ca. 1%) is concen- trated in the primary septum and localiz at the mother-bud cell junction (Cabib al., 1982; Sloat and Pringle, 197

The polysaccharide nature of the positive PATAg middle layer is unknown.

The absence of binding of PNA and WGA to the surface of U ce aies that determinants containing t CL-D- galactopyranosyl or M-acetylglucosaminyl units are not present in the outermost layer. The slight PATAg and Con A reactivity suggested the presence of terminal unsubstituted or 2-O substituted a-D-man- nopyranose.

Glycogen filled vesicles between t wall and the plasma membrane in mature Y cells are similar to those observed in regen- erating protoplasts of Candida a~~~ca~s (Tronchin et al., 1982) where these structures were shown to be involved in cell wall thickening. Some Golgi-like cisternae were observed in yeast but not in mycelial ccl These structures are different from t Golgi apparatus described by Campo- Aasen et al. (1982) that consists of large vacuoles, grains, flattened sacs, arrays of lamellar structures, and clusters of vesicles. The structures observed by these authors appeared to be more lyric strerctures than biosynthetic structures. However their cells were der cultures @to 15

) and were vacuo- lized. A role for Golgi-like cisternae and vesicular structures of the adjacent cyto- plasm in the mechanisms of cell wall synthesis merits further investigation.

2. Bud Cell

Our electron microscopic analyses p out significant substructural differences

236 PARIS ET AL.

CYTOCHEMISTRY OF Paracoccidioides bmsiiiemis 237

FIG. 5. Calcofluor staining of mycelial buds (arrows). x 4,000. Bar represents 10 Frn.

tween these wails and the wall of the parental Y mother cell.

The simultaneous appearence of a struc- turally different layer and of vesicles (Fig. 3a) suggests that these vesicles may be syn- thetic vesicles like the small membrane vesicles of 5;. cerevisiae (Hartwell, 1974).

The cytochemical characteristics of the outermost layer (sensitivity to protease, Con A and PATAg reactive) suggest that it is composed of proteins and polysaccharide with o~(l -+ 2) linked glucose or mannose residues. The presence of glucans is not probable because glucans in 19. brasi- Eiensis should be poorly PATAg reactive; Kanetsuna et al, (1972) showed that glucans of strain Pb9 does not contain ol(l -+ 2) linkages (PATAg+, Con A+) but mainly 1x(1 + 3) linkages (PATAg-, Con A-) and small amounts of a(1 -+ 6), a(1 +

4) linkages (PATAg+, Con A-). Since we found substantial amounts of mannose (8% of the total hexoses, Table 1) this electron opaque and PATAg-reactive outermost layer of strain MTC should be mai~iy composed of protein and mannose units. The slight PATAg reactivity and weak flmores- cence of Con A-treated mature Y cells suggest that the thin outermost layer of the Y cell wall and of the bud cell wall is of a similar chemical composition; the polymers being scattered on the surface of the mature Y cells.

The homogenous PATAg-nonreactive inner layer appeared finely laminated? after exposure to protease, in a manner previously described in thin sections of per- manganate-fixed cells (Garrison and 1975). The absence of fl~~ro~b~~~e binding to inner walls of very young bud

FIG. 4. Electron micrographs of mycelial buds. (a,b) Myce1ia.t bud (MB) not originating’from a yeast-like bud. (a) The MB cell wall is thinner than the Y cell wall. Note the highly vacuolated mother Y cell and the absence of glycogen and lipid bodies in the Y and MB. PATAg stain. x 7700. (b) High magnification of the base of the MB shown in (a). The MB wall originates from the inner layer of the Y cell wall. PATAg stain x 32,000. (c) Emergence of a mycelial bud from a Y bud. Note the rupture of the Y bud outer cell wall. PATAg stain. x 39,000. (d) After treatment with protease the outer layer of the mycelial bud has disappeared. PATAg stain. x 39.000.

238 PARIS ET AL.

CYTOCHEMISTRY OF Paracoccidioides brnsiliemis 239

initials provides two possible explanations. The wall may be composed of ol-glucans as structural polymers (o-polymers do not bind calcofluor, Maeda and Ishida, 1967). Alternatively, the structural polysaccharides may be chitin or P-glucans which exist in configurations not permitting the binding of fluorochrome. Binding of CFW would depend upon a microfibrillar configuration present only in mature cells.

3. kfyceliul bud

The cell wall of the mycelial buds (intermediate form of Y -+ M) was found to arise from the innermost layer as previously shown by Carbonell (1969) and Garrison and Boyd (1975). Mycelial buds emanated mostly (80%) from random points on mature Y cells (as did buds at 36°C) and sometimes (20%) from small buds. These results are similar to those of San-Blas et al. (1980). It seemed that 80% of the mycelial buds originated from undifferentiated sites; however, the presence of a common origin (e.g., the inner region of the wall) for the mycelial and yeast buds suggests that mycelial buds arise from an early stage of bud development.

The cytochemical staining affinities of the mycelial bud cell wall were different from both Y and M forms. The outer layer had the lectin reactivity (Con A- PNA-) of mature yeast-like cells but its PATAg reactivity and protease sensitivity were similar to that of the bud or hyphal outer layer. This finding suggests an intermediate composition between the two forms. The absence of CFW binding would indicate that

the inner layer more likely resem mycelial counterpart.

Our results show that if t of the mycelial bud was similar to the schematic representation of San-B and San- Blas (1985), the structure of mycelial bud cell wall should then be different from the structure of the yeast cell wall.

4. Mycelium

Conventional heavy metal and PATAg staining confirm that the hyphal wall could be divided in two layers as shown by Car- bone11 and Gil (1982) and is not composed of only one layer as proposed by Kanet- suna (1981) and San-Blas and San- (1985). The positive reaction to PATAg of the outermost layer an tivity to protease suggested tha is mainly composed of proteins and poly- sacharide (glucan or mannan PATAg’) with terminal galactopyranose directed to the exterior. Mycelium of strain Pb9 has a ga- lactomarman with a linear backbone of ii + 6) linked mannose residues (Con A- ,, PATAg+) with branches of one (1 -+ 2) galactofuranose residue (PNA-) (Azuma et aE., 1974; San-Bias and San-Has, 1985), The presence of an outermost layer coin posed of protein and gala~tomanna~ with an cr(1 -+ 6) mannan backbone and branches ‘of (1 --+ 2) galactose resid-ues would be in agreement with our cytochem-. ical results if the galactose were in the py- ranose form as it is observed in the exocellular yeast. galactomannan of strain B339 (Puccia et al., 1986). Azuma et nb. (19743 also found that gala~toma~~a~ was a

-

FIG. 6. Hyphal cells. (a) Mycelium stained with CFW: only septa are brightly fluorescent. x 2200. Bar represents 10 pm. (b) Mycelium stained with FITC-PNA. x 2200, (c) Transversal and longitudinal sections of hyphae stained with many1 acetate and lead cirate. x 15,000. (d,e) Longitudinal thin sections of hyphae showing the unstained inner layer (IL) and the heavily stained otter layer. The septum (S) and woronin bodies (WB) were negatively stained. Note the staining of the mesosomal-like membrane system (MS). PATAg stain. ~35,000. (f) Treatment of the mycelium with protease: the heavily stained outer layer has disappeared. PATAg stain. ~29,000. (g) Treatment of the mycelium with snail enzyme: the substructural integrity of the cell wall proper has been markedly altered. PATAg stain. x 14,000.

240 PARIS ET AL.

YEAST -k I

OL

PATAg(a' +

con A(') +

PNA") -

WGA(d' -

I PATAg t I

con A i I I

PNA I WGA - I

Protease + Protease + I I I

branched manndn 11nearu (l-3) glucan , + + I

p?Otel" manna" \ I

I

--MMYCELIAL BUD-

PAiAg +

con A

PNA

WGA -

Protease +

11near mannan +

protein

I

(l-3)'glucan +

chltln

r

calcofluor

I -1 MYCELIUM

I PATAg + I I

con A

I PNA + I I

WGA -

I Protease +

galactomannan

I t: (l-3) glucan

+ \ chlt,n chltln

I \ , \ PATAg PATAg

calcOfl"Or calcnfluor +

snail enzyme + sens1t,v,ty

FIG. 7. Schematic representation of cytochemical results and their interpretation. Strong (+), weak (k) and negative reaction (-). (a) PATAg reactivity: d$(l + 4) and o$(l + 6) glucans, ol(l -+ 4), (l-+ 6) mannans (Thierry, 1967). (b) Con A specificity: glucans or mannans with numerous branches of cu(1 -+ 2) or o(1 --;r 3) linked residues (Goldstein and Hayes, 1978). (c) PNA specificity: o-galactopyranosyl residues in external position (Lotan et nl., 1975). (d) WGA specificity: chitin (N-acetylglucoseamine polymer) (e) Calcofluor: 6 polymers (chitin, p glucans . ) (Maeda and Ishida, 1967). (I) Snail enzyme: l3 glucanases and traces of mannanase and chitinase (Peberdy, 1985, and text).

common antigen for Histoplasma, Para- coccidioides, and Blastomyces. These results led us to suppose that the galactomannan seems to be a quite stable structure in pathogenic dimorphic fungi. However, Azuma et al. (1974) found galactofuranose in the mycelium and Puccia et al. (1986) galactopyranose in the yeast phase. Permeth- ylation analysis should be done to know the form of the galactose in Y and M of our strain and to explain the difference of PNA sensitivity of yeast and mycelium. As hyphal walls of P. brasiliensis contain high amounts of chitin (47%) and glucans (20%), it was assumed that the inner layer was mainly composed of chitin and glucans (mainly p( 1 + 3) glucans, PATAg- and sensitive to snail enzyme). However, these

polymers did not react with CFW which bound only to septa indicating that the CFW results should be considered with caution. A positive result would indicate the presence of a fibrillar polysaccharide but a wall containing chitin and glucan may also give negative results with CFW.

Septal plates, as previously described by Garrison and Boyd (1975), were almost de- void of periodic acid-sensitive polysaccharides. They were brightly fluorescent with calcofluor and insensitive to snail enzyme. We conclude that septal areas contain a high concentration of unmasked calcofluor-binding chitin microfibrils.

5. Cell Walls in Relation to Dimorphism

Figure 7 gives a schematic representa-

tion of our results and interpretations of the needed for the visualization of wail sub- chemical composition of the cell wall layers structure as it undergoes alterations during during the Y to M transformation. This the critical stages of phase transition. figure permits us to review the models pro- Moreover chemical characterization by posed by Kanetsuna (1972, 1981) and San- degradation of cell wall using specific en- Blas and San-Blas (1985). zymes and permethylation of isolated poly-

The models proposed by Kanetsuna saccharides should be done. (1972) and San-Blas and San-Blas (1985) involved glucans as the most important poly- ACKNOWLEDGMENTS saccharide: a-linked in Y and p-linked in M. Wowever, some strains and one mutant

We thank F. Mar&t for his advice and encourage-

(Pb140) are able to produce Y forms ment and M. Coqttis for his photographic assistance.

without containing cr(1 + 3)-glucans in their walls but P-glucans (San-Blas, 1982; REFERENCES

San-Blas et al., 1983). In the Pb140 mutant, AWMA, I., KANETSUNA, F., TANAKA, Y., YAMA-

the cy(1 -+ 3)-mannan seems to fulfill the MURA, Y.,A,NDCARBONELL,L. M. 1974. Chemical

role played by the cx(1 -+ 3)-glucan. In and immunological properties of ga!actomannans obtained from Histoplasma duboisii, Hissopiasmu

strain MTC the mannan seems to be in- capsulatum. Pavacoccidioides brasiliensis and volved in some manner with dimorphism as Blastomyces dermatitidis. Mycopathologia ii4:

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cytochemical study of the yeast and mycelial cell walls ofparacoccidioides brasiliensis

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