Vol. 24, No. 3
INFECTION AND IMMUNITY, June 1979, p. 912-919
0019-9567/79/06-0912/08$02.00/0
Distribution of Anionic Groups at the Cell Surface of Different Sporothrix schenckii Cell Types MARLENE BENCHIMOL, W. DE SOUZA, AND L. R. TRAVASSOSt* Instituto de Biofisica and Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 20,000, Brazil Received for publication 18 January 1979
The distribution of anionic groups at the cell surface of yeastlike forms, hyphae, and conidia of Sporothrix schenckii was studied by staining with colloidal iron hydroxide and cationized ferritin. By using colloidal iron hydroxide it was shown that the external cell wall layer of one strain (strain 1099.18) could be resolved into two reactive sublayers and that these layers were present in many but not all cells of the same population. In contrast, most cells of another strain (strain 1099.12) were stained by colloidal iron hydroxide, but only one reactive layer was seen. Acidic layers of the yeastlike forms of the two strains were much thicker than those of conidia and hyphae. By the cationized ferritin staining procedure it was observed that the acidic layers of yeast forms sloughed off of cells, probably due to cell-cell or cell-medium attrition in shaken submerged cultures or to a process by which the outer layers detach from cells as they are replaced by newly synthesized ones. The colloidal iron hydroxide- and cationized ferritin-reactive cell surface layers of S. schenckii correspond to the previously described (L. R. Travassos et al., Exp. Mycol. 1:293-305, 1977) concanavalin A-reactive peptidorhamnomannan complexes, and their reactivity is probably due to the presence of acidic amino acids of low pK values rather than to glucuronic acid units.
Previous studies (17) have shown that different cell types of the human pathogen Sporothrix schenckii form cell walls containing external layers of concanavalin A-reactive peptido-rhamnomannans. These layers are loosely bound to the inner cell wall structures and are often detached into the suspending medium of shaken cultures. Microtubules or vesicles consisting of aggregates of a corresponding cell wall material also formed in supernatants of cultures of Ceratocystis ulmi (14), an ascomycete which, like other species of Ceratocystis, synthesizes rhamnomannans (6). Peptido-rhamnomannans from S. schenckii cell walls carry antigenic determinants (9, 11), and the polysaccharide moieties have fine structures which are characteristic of the yeastlike or conidial and mycelial cell types (12, 15, 16). Garrison et al. (2, 3) have observed a microfibrillar material at the surface of S. schenckii cells which showed enhanced electron density in the yeastlike forms or yeast primordia on hyphae as compared with the hyphal cell wall. This microfibrillar material could be stained by acidified dialyzed iron and probably corresponds to the concanavalin A-reacting outer layer of S. t Present address: Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
schenckii cell walls (17). Depending on the S. schenckii strain and the morphological phase, this layer was very prominent in some but not all cells of the same culture. In fact, a proportion of the yeast population had a much thinner layer reacting with the lectin and in some cases an unreactive external layer. A variation in the response of different cells to antisera specifically recognizing certain structures in the rhamnomannans was attributed in part to differences in antigen concentration on the cell surface (10). In the present work we studied the distribution of cell surface anionic groups in different S. schenckii cell types in an attempt to further define the nature of the cell wall outer layer. By using colloidal iron hydroxide (CIH) and cationized ferritin (CF), we attempted to localize acidic groups carried by aspartic and glutamic acid residues, which are present in S. schenckii peptido-rhamnomannans (9, 17), and by glucuronic acid units, which are present in alkali-extractable acidic rhamnomannans (5, 16).
MATERIALS AND METHODS Microorganisms. S. schenckii strains 1099.12 and 1099.18 were obtained originally from the Mycology Laboratory, Department of Dermatology, Columbia
University, New York. Stock cultures were maintained
912
CELL SURFACE OF S. SCHENCKII
VOL. 24, 1979 at 40C on Sabouraud agar covered with a layer of mineral oil. Transfers were made at 6-month intervals. Culture media. Yeastlike forms and mycelial forms were obtained by growing S. schenckii in brain heart infusion broth (Difco Laboratories) at 370C and in liquid Sabouraud medium at 250C, respectively. Conidia were separated from the mycelial cultures by filtration through gauze. For the cytochemical study, cells were harvested by centrifugation and washed twice in 0.9% NaCl. Electron micro8copy. Cells were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4 for 2 to 12 h at room temperature. After a thorough rinse in cacodylate buffer, cells were postfixed in 1% OS04 in 0.1 M cacodylate buffer for 1 h at 40C, dehydrated in acetone, and embedded in Epon. Ultrathin sections were cut with an LKB Ultratome III ultramicrotome displaying light-gold to silver interference colors. The sections were then collected on copper grids and examined, after staining with uranyl acetate and lead citrate, in an AEI EM6-B electron microscope with a 50-ttm objective aperture and operating at 60 kV. Cytochemistry. Two methods were used for labeling the anionic groups on the cell surface. (i) CIH method. After fixation in glutaraldehyde, the cells were kept for 60 min at room temperature in a nondialyzed suspension of CIH at pH 1.8 (4) and washed twice in 12% acetic acid and once in distilled water before postfixation in 1% OS04. (ii) CF method. After fixation in glutaraldehyde, the cells were kept for 60 min at room temperature in a suspension of CF (1 mg/ml; Miles-Yeda Ltd., Rehovoth, Israel) in phosphate buffer, pH 7.2, washed in buffer, and postfixed in OSO4 (1). In some experiments the cells were treated overnight with 0.05 M NH4Cl and incubated with CF in the presence of NH4IC to control nonspecific binding of CF to free aldehyde groups (7).
RESULTS A comparison between an unstained and a CIH-stained yeast form of S. schenckii 1099.18 can be made from Fig. 1 and 3. The reactivity of different cells of the same culture is shown in Fig. 2. Binding of colloidal iron by yeast forms of strain 1099.18 was very irregular. Some cells reacted strongly with CIH, and some were completely negative. Intermediate reactivities were also observed. Reaction with CIH could involve one or two layers of cell surface components (Fig. 2). Details of the bilayer reactivity of yeast forms of strain 1099.18 with CIH are shown in Fig. 3 and 4. Both reactive layers of the bilayer are 50 to 70 nm wide, and between these layers there is an apparently empty area of approximately 50 nm, except where the outer layer seems to detach from the cell wall structural complex. In S. schenckii strain 1099.12 the yeast forms reacted more uniformly with CIH than did the yeast forms of strain 1099.18, but unlike
913
the latter they did not form a visible bilayer of reactive components (Fig. 5). Similarly, S. schenckii hyphae had only a single CIH-reactive layer (Fig. 6 and 7), which was thinner than the bilayer complex of the yeast forms. The reactivity of conidia of strain 1099.18 was as irregular as that of the yeast forms, with a greater proportion of cells showing a weak reaction with CIH (Fig. 8). Reaction of S. schenckii 1099.18 with CF usually stained the outer layer of the bilayer much more intensely than the inner layer (Fig. 9 and 11), so that a clear resolution of the bilayer was not possible by this method except at sites where there occurred detachment of the outer layer after a strong reaction with CF (Fig. 12). Although in some cases detachment of the outer layer left behind a CF-reactive inner layer, in other cases there was a single reactive layer which was detached, and the resulting cell surface was not stained by CF (Fig. 12). After cell division the CF-reactive material seemed to be immediately synthesized along the septum (Fig. 10). The reactivity of hyphae with ferritin was similar to that observed with CIH (Fig. 13). However, in some cases the CF label was bound to aggregates of reactive materials along the cell wall rather than staining a continuous monolayer (Fig. 14). In several micrographs of S. schenckii there were primarily two cell wall layers which did not react with either CIH or CF: one was electron transparent and the other had increased electron density. The chemical nature of these layers was not investigated in the present work.
DISCUSSION The external layer(s) of S. schenckii cell walls react with concanavalin A and mannose-sensitive bacterial fimbriae (17), and the present results show that these structures also contain anionic groups which bind colloidal iron or ferritin. Binding of CIH is electrostatic and thus depends on the pH of the reaction medium and the pK values of the anionic groups present on the cell surface. At pH values below 2.5, CIH binds to sulfate groups of acid carbohydrates and carboxyl groups from mucopolysaccharides or from acidic amino acids or from both (8, 13). The polycationic derivative of ferritin described by Danon et al. (1), on the other hand, is used to stain anionic groups at neutral pH. In S. schenckii the, use of both methods gave comparable results, but the resolution of the reactive outer layer into two sublayers was clearly seen only with the CIH method. This is probably due to
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4.
FIG. 3 and 4. Details of cell wall bilayer reactivity with CIH in yeast forms of S. schenckii 1099.18. Figure 3, x30,000; Fig. 4, x90,000. FIG. 5. Staining of the anionic groups in yeast forms of S. schenckii 1099.12. A more uniform reactivity of different cells is observed. x9,000. 915
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FIG. 9 and 11. Labeling of S. schenckii 1099.18 yeast forms with ferritin. Note that the inner portion of the outer layer is poorly labeled. Figure 9, x30,000; Fig. 11, x30,00O. FIG. 10. Ferritin-labeled material being synthesized after septum formation and cell division of a yeast form of strain 1099.12. x30,000. FIG. 12. Detachment of the outer sublayer of cell wall components reacting with ferritin in yeast forms of S. schenckii 1099.18 (arrows). In one instance detachment of a single ferritin-reactive layer (curved arrow) left behind an unreactive cell surface. x30,000. 917
918
BENCHIMOL, DE SOUZA, AND TRAVASSOS
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FIG. 13 and 14. Reaction of hyphae of S schenckii 1099.18 and 1099.12 with ferritin. In Fig. 14 (strain 1099.12) a discontinuous reaction was observed (arrows). Figure 13, x15,000; Fig. 14, x45,000.
the size of the ferritin particles, which do not penetrate the external sublayer as well as CIH, thus staining the internally located charged groups less efficiently. The fact that different S. schenckii yeast cells contained either one or two layers of CIH-reactive components, and in some instances no reactive layer at all, suggests that these components are synthesized constantly and that detachment usually occurs after the bilayer is
formed. Detachment from the cell wall of the outer sublayer was a frequent observation; this
exposes the inner sublayer and allows further growth of this structure. Variations of this model are common; for example, in several cases a single CF-reactive layer was detached from the cell wall, leaving behind an unreactive surface. We cannot assess with the present evidence whether the cells bearing one or two CIH-reactive layers are in different stages of the cell cycle,
VOL. 24, 1979
CELL SURFACE OF S. SCHENCKII
919
ACKNOWLEDGMENTS because detachment of these structures can be This work was supported by Financiadora de Estudos e due to the normal replacement of these acidic Projetos, the Brazilian National Research Council (CNPq), layers by newly synthesized ones or to increased and the Research Council of Federal University of Rio de cell-cell or cell-medium attrition in shaken sub- Janeiro, Brazil. In either circumstance it seems merged cultures. LITERATURE CIMD clear that layers stained by colloidal iron, fer-'D., L. Goldstein, Y. Marikovsky, and E. Skutin, or concanavalin A are linked to the inner 1. Danon, telsky. 1972. Use of cationized ferritin as a label of layers of the cell wall by very weak bonds and negative charges on cell surfaces. J. Ultrastruct. Res. that their removal apparently does not hamper 38:500-510. cell viability. These layers are thicker and often 2. Garrison, R. G., K. S. Boyd, and F. Mariat. 1975. Ultrastructural studies of the mycelium-to-yeast transduplicated in yeastlike forms as compared with formation of Sporothrix schenckii. J. Bacteriol. 124: the thin monolayers of conidia and hyphae. In 959-968. hyphae, staining with ferritin showed in some 3. Garrison, R. G., K. S. Boyd, and F. Mariat. 1976. Etude sur l'ultrastructure de la transformation mycelium-leinstances the presence of acidic components in vure du Sporothrix schenckii et du Ceratocystis stenoa discontinuous arrangement at the cell surface ceras. Bull. Soc. Mycol. Med. 5:69-74. rather than in a monolayer. 4. Gasic, G. J., L. Berwick, and M. Sorrentino. 1968. The main components of the external layer of Positive and negative iron as cell surface electron stain. Lab. Invest. 18:63-71. S. schenckii cell wall probably include the pepL R. Travassos, and tido-rhamnomannans and a few other polymers, 5. Gorin, P. A. J., R. H. Haskins, L Mendonea-Previato. 1977. Further studies on the such as neutral galactose-containing polysaccharhamnomannans and acidic rhamnomannans of Sporides, which are responsible for the reaction of rothrix schenckii and Ceratocystis stenoceras. Carboyeast forms with anti-Hormodendrum antisehydr. Res. 55:21-33. rum (K. 0. Lloyd, unpublished data) and are 6. Gorin, P. A. J., and J. F. T. Spencer. 1970. Structures of the L-rhamno-D-mannan from Ceratocystis ulmi and possibly similar to the polysaccharide isolated the D-gluco-D-mannan from Ceratocystis brunnea. Carfrom Ceratocystis stenoceras (5), and a low probohydr. Res. 13:339-349. portion of starch (Previato et al., submitted for 7. Grinnell, F., M. Q. Tobleman, and C. R. Hackenbrock. 1975. The distribution and mobility of anionic publication). It is likely, then, that the acidic sites on the surfaces of baby hamster kidney cells. J. groups are located in the peptido-rhamnomanCell Biol. 66:470-479. nan complexes. In the peptido-rhamnomannan 8. Gros, D., and C. E. Challice. 1975. The coating of mouse from the yeast forms of one strain of S. schenckii myocardial cells. A cytochemical electron microscopical study. J. Histochem. Cytochem. 23:727-744. (9), the proportions of glutamic and aspartic K. O., and M. A. Bitoon. 1971. Isolation and acids in the peptide were 9 and 9.6%, respec- 9. Lloyd, purification of a peptido-rhamnomannan from the yeast tively. In the yeast forms of strains 1099.12 and form of Sporothrix schenckii. Structural and immuno1099.18 studied in the present work, the proporchemical studies. J. Immunol. 107:663-671. tions of glutamic acid were 7.6 and 6.7%, respec- 10. Lloyd, K. O., L. Mendonca-Previato, and L. R. Travassos. 1978. Distribution of antigenic polysaccharides tively, and those of aspartic acid were 7.6 and in different cell types of Sporothrix schenckii as studied 7.3%, respectively (17). These proportions by immunofluorescent staining with rabbit antisera. should provide enough acidic groups for a strong Exp. Mycol. 2:130-137. reaction with CIH or CF, particularly if several 11. Lloyd, K. O., and L. R. Travassos. 1975. Immunochemical studies on L-rhamno-D-mannans of Sporothrix peptide complexes aggregate to form continuous schenckii and related fungi by use of rabbit and human and frequently superimposed layers at the cell antisera. Carbohydr. Res. 40:89-97. surface. The low pK values of the acidic amino 12. Mendonga, L, P. A. J. Gorin, K. O. Lloyd, and L R. Travassos. 1976. Polymorphism of Sporothrix schenacids provide the basis for the reactivity with ckii surface polysaccharides as a function of morphoCIH. The presence of glucuronic acid units is, differentiation. Biochemistry 15:2423-2431. logical not presumably, essential for the reactivity with 13. Rambourg, A. 1971. Morphological and histochemical CIH and CF observed in the present study beaspects of glycoproteins at the surface of animal cells. cause only the rhamnomannan from S. schenckii Int. Rev. Cytol. 31:57-114. strain 1099.12 grown in the mycelial form may 14. Takai, S. 1974. Pathogenicity and ceratoulmin production in Ceratocystis ulni. Nature (London) 252:124-126. contain these acidic units (16). Alkali-extracta- 15. Travassos, L. R., and L. Mendonca-Previato. 1978. ble rhamnomannans from strain 1099.18 are not Synthesis of monorhamnosyl L-rhamno-D-mannans by acidic, and yet the cell wall of this strain is conidia of Sporothrix schenckii. Infect. Immun. 19:1-4. strongly reactive with CIH and CF. Moreover, 16. Travassos, L. R., L. Mendonia-Previato, and P. A. J. Gorin. 1978. Heterogeneity of the rhamnomannans glucuronic acid units recognizable by 13C nuclear from one strain of the human pathogen Sporothrix magnetic resonance spectroscopy (5, 16) are a schenckii determined by 13C nuclear magnetic resocharacteristic of mycelial polysaccharides and nance spectroscopy. Infect. Immun. 19:1107-1109. were not detected in the yeast rhamnomannans. 17. Travassos, L. R., W. Souza. L. Mendonga-Previato, and K. 0. Lloyd. 1977. Location and biochemical naAs shown in the present study, yeast forms of ture of surface components reacting with concanavalin both S. schenckii strains are the most reactive A in different cell types of Sporothrix schenckii. Exp. cells with CIH or CF. Mycol. 1:293-305.
INFECTION AND IMMUNITY, June 1979, p. 912-919
0019-9567/79/06-0912/08$02.00/0
Distribution of Anionic Groups at the Cell Surface of Different Sporothrix schenckii Cell Types MARLENE BENCHIMOL, W. DE SOUZA, AND L. R. TRAVASSOSt* Instituto de Biofisica and Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 20,000, Brazil Received for publication 18 January 1979
The distribution of anionic groups at the cell surface of yeastlike forms, hyphae, and conidia of Sporothrix schenckii was studied by staining with colloidal iron hydroxide and cationized ferritin. By using colloidal iron hydroxide it was shown that the external cell wall layer of one strain (strain 1099.18) could be resolved into two reactive sublayers and that these layers were present in many but not all cells of the same population. In contrast, most cells of another strain (strain 1099.12) were stained by colloidal iron hydroxide, but only one reactive layer was seen. Acidic layers of the yeastlike forms of the two strains were much thicker than those of conidia and hyphae. By the cationized ferritin staining procedure it was observed that the acidic layers of yeast forms sloughed off of cells, probably due to cell-cell or cell-medium attrition in shaken submerged cultures or to a process by which the outer layers detach from cells as they are replaced by newly synthesized ones. The colloidal iron hydroxide- and cationized ferritin-reactive cell surface layers of S. schenckii correspond to the previously described (L. R. Travassos et al., Exp. Mycol. 1:293-305, 1977) concanavalin A-reactive peptidorhamnomannan complexes, and their reactivity is probably due to the presence of acidic amino acids of low pK values rather than to glucuronic acid units.
Previous studies (17) have shown that different cell types of the human pathogen Sporothrix schenckii form cell walls containing external layers of concanavalin A-reactive peptido-rhamnomannans. These layers are loosely bound to the inner cell wall structures and are often detached into the suspending medium of shaken cultures. Microtubules or vesicles consisting of aggregates of a corresponding cell wall material also formed in supernatants of cultures of Ceratocystis ulmi (14), an ascomycete which, like other species of Ceratocystis, synthesizes rhamnomannans (6). Peptido-rhamnomannans from S. schenckii cell walls carry antigenic determinants (9, 11), and the polysaccharide moieties have fine structures which are characteristic of the yeastlike or conidial and mycelial cell types (12, 15, 16). Garrison et al. (2, 3) have observed a microfibrillar material at the surface of S. schenckii cells which showed enhanced electron density in the yeastlike forms or yeast primordia on hyphae as compared with the hyphal cell wall. This microfibrillar material could be stained by acidified dialyzed iron and probably corresponds to the concanavalin A-reacting outer layer of S. t Present address: Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
schenckii cell walls (17). Depending on the S. schenckii strain and the morphological phase, this layer was very prominent in some but not all cells of the same culture. In fact, a proportion of the yeast population had a much thinner layer reacting with the lectin and in some cases an unreactive external layer. A variation in the response of different cells to antisera specifically recognizing certain structures in the rhamnomannans was attributed in part to differences in antigen concentration on the cell surface (10). In the present work we studied the distribution of cell surface anionic groups in different S. schenckii cell types in an attempt to further define the nature of the cell wall outer layer. By using colloidal iron hydroxide (CIH) and cationized ferritin (CF), we attempted to localize acidic groups carried by aspartic and glutamic acid residues, which are present in S. schenckii peptido-rhamnomannans (9, 17), and by glucuronic acid units, which are present in alkali-extractable acidic rhamnomannans (5, 16).
MATERIALS AND METHODS Microorganisms. S. schenckii strains 1099.12 and 1099.18 were obtained originally from the Mycology Laboratory, Department of Dermatology, Columbia
University, New York. Stock cultures were maintained
912
CELL SURFACE OF S. SCHENCKII
VOL. 24, 1979 at 40C on Sabouraud agar covered with a layer of mineral oil. Transfers were made at 6-month intervals. Culture media. Yeastlike forms and mycelial forms were obtained by growing S. schenckii in brain heart infusion broth (Difco Laboratories) at 370C and in liquid Sabouraud medium at 250C, respectively. Conidia were separated from the mycelial cultures by filtration through gauze. For the cytochemical study, cells were harvested by centrifugation and washed twice in 0.9% NaCl. Electron micro8copy. Cells were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4 for 2 to 12 h at room temperature. After a thorough rinse in cacodylate buffer, cells were postfixed in 1% OS04 in 0.1 M cacodylate buffer for 1 h at 40C, dehydrated in acetone, and embedded in Epon. Ultrathin sections were cut with an LKB Ultratome III ultramicrotome displaying light-gold to silver interference colors. The sections were then collected on copper grids and examined, after staining with uranyl acetate and lead citrate, in an AEI EM6-B electron microscope with a 50-ttm objective aperture and operating at 60 kV. Cytochemistry. Two methods were used for labeling the anionic groups on the cell surface. (i) CIH method. After fixation in glutaraldehyde, the cells were kept for 60 min at room temperature in a nondialyzed suspension of CIH at pH 1.8 (4) and washed twice in 12% acetic acid and once in distilled water before postfixation in 1% OS04. (ii) CF method. After fixation in glutaraldehyde, the cells were kept for 60 min at room temperature in a suspension of CF (1 mg/ml; Miles-Yeda Ltd., Rehovoth, Israel) in phosphate buffer, pH 7.2, washed in buffer, and postfixed in OSO4 (1). In some experiments the cells were treated overnight with 0.05 M NH4Cl and incubated with CF in the presence of NH4IC to control nonspecific binding of CF to free aldehyde groups (7).
RESULTS A comparison between an unstained and a CIH-stained yeast form of S. schenckii 1099.18 can be made from Fig. 1 and 3. The reactivity of different cells of the same culture is shown in Fig. 2. Binding of colloidal iron by yeast forms of strain 1099.18 was very irregular. Some cells reacted strongly with CIH, and some were completely negative. Intermediate reactivities were also observed. Reaction with CIH could involve one or two layers of cell surface components (Fig. 2). Details of the bilayer reactivity of yeast forms of strain 1099.18 with CIH are shown in Fig. 3 and 4. Both reactive layers of the bilayer are 50 to 70 nm wide, and between these layers there is an apparently empty area of approximately 50 nm, except where the outer layer seems to detach from the cell wall structural complex. In S. schenckii strain 1099.12 the yeast forms reacted more uniformly with CIH than did the yeast forms of strain 1099.18, but unlike
913
the latter they did not form a visible bilayer of reactive components (Fig. 5). Similarly, S. schenckii hyphae had only a single CIH-reactive layer (Fig. 6 and 7), which was thinner than the bilayer complex of the yeast forms. The reactivity of conidia of strain 1099.18 was as irregular as that of the yeast forms, with a greater proportion of cells showing a weak reaction with CIH (Fig. 8). Reaction of S. schenckii 1099.18 with CF usually stained the outer layer of the bilayer much more intensely than the inner layer (Fig. 9 and 11), so that a clear resolution of the bilayer was not possible by this method except at sites where there occurred detachment of the outer layer after a strong reaction with CF (Fig. 12). Although in some cases detachment of the outer layer left behind a CF-reactive inner layer, in other cases there was a single reactive layer which was detached, and the resulting cell surface was not stained by CF (Fig. 12). After cell division the CF-reactive material seemed to be immediately synthesized along the septum (Fig. 10). The reactivity of hyphae with ferritin was similar to that observed with CIH (Fig. 13). However, in some cases the CF label was bound to aggregates of reactive materials along the cell wall rather than staining a continuous monolayer (Fig. 14). In several micrographs of S. schenckii there were primarily two cell wall layers which did not react with either CIH or CF: one was electron transparent and the other had increased electron density. The chemical nature of these layers was not investigated in the present work.
DISCUSSION The external layer(s) of S. schenckii cell walls react with concanavalin A and mannose-sensitive bacterial fimbriae (17), and the present results show that these structures also contain anionic groups which bind colloidal iron or ferritin. Binding of CIH is electrostatic and thus depends on the pH of the reaction medium and the pK values of the anionic groups present on the cell surface. At pH values below 2.5, CIH binds to sulfate groups of acid carbohydrates and carboxyl groups from mucopolysaccharides or from acidic amino acids or from both (8, 13). The polycationic derivative of ferritin described by Danon et al. (1), on the other hand, is used to stain anionic groups at neutral pH. In S. schenckii the, use of both methods gave comparable results, but the resolution of the reactive outer layer into two sublayers was clearly seen only with the CIH method. This is probably due to
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2 FIG. 1. S. schenckii 1099.18 yeast form unlabeled for anionic groups. x45,000. FIG. 2. Heterogeneous CIH labeling of anionic groups in different yeastlike forms of strain 1099.18 from the same culture. Some cells have a bilayer of CIH-reactive components, whereas others are poorly labeled or completely unreactive. x9,000.
4.
FIG. 3 and 4. Details of cell wall bilayer reactivity with CIH in yeast forms of S. schenckii 1099.18. Figure 3, x30,000; Fig. 4, x90,000. FIG. 5. Staining of the anionic groups in yeast forms of S. schenckii 1099.12. A more uniform reactivity of different cells is observed. x9,000. 915
916
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1
X
SS
4.~~~~~~~~~~~~~~~~
is
H
FIG. 9 and 11. Labeling of S. schenckii 1099.18 yeast forms with ferritin. Note that the inner portion of the outer layer is poorly labeled. Figure 9, x30,000; Fig. 11, x30,00O. FIG. 10. Ferritin-labeled material being synthesized after septum formation and cell division of a yeast form of strain 1099.12. x30,000. FIG. 12. Detachment of the outer sublayer of cell wall components reacting with ferritin in yeast forms of S. schenckii 1099.18 (arrows). In one instance detachment of a single ferritin-reactive layer (curved arrow) left behind an unreactive cell surface. x30,000. 917
918
BENCHIMOL, DE SOUZA, AND TRAVASSOS
INFECT. IMMUN.
13 'CX
X
Oft
t; ^ XA
I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~l r
a'~~~~ifr4'SS'~~~~&flrZ'PZ~~.#OP..
FIG. 13 and 14. Reaction of hyphae of S schenckii 1099.18 and 1099.12 with ferritin. In Fig. 14 (strain 1099.12) a discontinuous reaction was observed (arrows). Figure 13, x15,000; Fig. 14, x45,000.
the size of the ferritin particles, which do not penetrate the external sublayer as well as CIH, thus staining the internally located charged groups less efficiently. The fact that different S. schenckii yeast cells contained either one or two layers of CIH-reactive components, and in some instances no reactive layer at all, suggests that these components are synthesized constantly and that detachment usually occurs after the bilayer is
formed. Detachment from the cell wall of the outer sublayer was a frequent observation; this
exposes the inner sublayer and allows further growth of this structure. Variations of this model are common; for example, in several cases a single CF-reactive layer was detached from the cell wall, leaving behind an unreactive surface. We cannot assess with the present evidence whether the cells bearing one or two CIH-reactive layers are in different stages of the cell cycle,
VOL. 24, 1979
CELL SURFACE OF S. SCHENCKII
919
ACKNOWLEDGMENTS because detachment of these structures can be This work was supported by Financiadora de Estudos e due to the normal replacement of these acidic Projetos, the Brazilian National Research Council (CNPq), layers by newly synthesized ones or to increased and the Research Council of Federal University of Rio de cell-cell or cell-medium attrition in shaken sub- Janeiro, Brazil. In either circumstance it seems merged cultures. LITERATURE CIMD clear that layers stained by colloidal iron, fer-'D., L. Goldstein, Y. Marikovsky, and E. Skutin, or concanavalin A are linked to the inner 1. Danon, telsky. 1972. Use of cationized ferritin as a label of layers of the cell wall by very weak bonds and negative charges on cell surfaces. J. Ultrastruct. Res. that their removal apparently does not hamper 38:500-510. cell viability. These layers are thicker and often 2. Garrison, R. G., K. S. Boyd, and F. Mariat. 1975. Ultrastructural studies of the mycelium-to-yeast transduplicated in yeastlike forms as compared with formation of Sporothrix schenckii. J. Bacteriol. 124: the thin monolayers of conidia and hyphae. In 959-968. hyphae, staining with ferritin showed in some 3. Garrison, R. G., K. S. Boyd, and F. Mariat. 1976. Etude sur l'ultrastructure de la transformation mycelium-leinstances the presence of acidic components in vure du Sporothrix schenckii et du Ceratocystis stenoa discontinuous arrangement at the cell surface ceras. Bull. Soc. Mycol. Med. 5:69-74. rather than in a monolayer. 4. Gasic, G. J., L. Berwick, and M. Sorrentino. 1968. The main components of the external layer of Positive and negative iron as cell surface electron stain. Lab. Invest. 18:63-71. S. schenckii cell wall probably include the pepL R. Travassos, and tido-rhamnomannans and a few other polymers, 5. Gorin, P. A. J., R. H. Haskins, L Mendonea-Previato. 1977. Further studies on the such as neutral galactose-containing polysaccharhamnomannans and acidic rhamnomannans of Sporides, which are responsible for the reaction of rothrix schenckii and Ceratocystis stenoceras. Carboyeast forms with anti-Hormodendrum antisehydr. Res. 55:21-33. rum (K. 0. Lloyd, unpublished data) and are 6. Gorin, P. A. J., and J. F. T. Spencer. 1970. Structures of the L-rhamno-D-mannan from Ceratocystis ulmi and possibly similar to the polysaccharide isolated the D-gluco-D-mannan from Ceratocystis brunnea. Carfrom Ceratocystis stenoceras (5), and a low probohydr. Res. 13:339-349. portion of starch (Previato et al., submitted for 7. Grinnell, F., M. Q. Tobleman, and C. R. Hackenbrock. 1975. The distribution and mobility of anionic publication). It is likely, then, that the acidic sites on the surfaces of baby hamster kidney cells. J. groups are located in the peptido-rhamnomanCell Biol. 66:470-479. nan complexes. In the peptido-rhamnomannan 8. Gros, D., and C. E. Challice. 1975. The coating of mouse from the yeast forms of one strain of S. schenckii myocardial cells. A cytochemical electron microscopical study. J. Histochem. Cytochem. 23:727-744. (9), the proportions of glutamic and aspartic K. O., and M. A. Bitoon. 1971. Isolation and acids in the peptide were 9 and 9.6%, respec- 9. Lloyd, purification of a peptido-rhamnomannan from the yeast tively. In the yeast forms of strains 1099.12 and form of Sporothrix schenckii. Structural and immuno1099.18 studied in the present work, the proporchemical studies. J. Immunol. 107:663-671. tions of glutamic acid were 7.6 and 6.7%, respec- 10. Lloyd, K. O., L. Mendonca-Previato, and L. R. Travassos. 1978. Distribution of antigenic polysaccharides tively, and those of aspartic acid were 7.6 and in different cell types of Sporothrix schenckii as studied 7.3%, respectively (17). These proportions by immunofluorescent staining with rabbit antisera. should provide enough acidic groups for a strong Exp. Mycol. 2:130-137. reaction with CIH or CF, particularly if several 11. Lloyd, K. O., and L. R. Travassos. 1975. Immunochemical studies on L-rhamno-D-mannans of Sporothrix peptide complexes aggregate to form continuous schenckii and related fungi by use of rabbit and human and frequently superimposed layers at the cell antisera. Carbohydr. Res. 40:89-97. surface. The low pK values of the acidic amino 12. Mendonga, L, P. A. J. Gorin, K. O. Lloyd, and L R. Travassos. 1976. Polymorphism of Sporothrix schenacids provide the basis for the reactivity with ckii surface polysaccharides as a function of morphoCIH. The presence of glucuronic acid units is, differentiation. Biochemistry 15:2423-2431. logical not presumably, essential for the reactivity with 13. Rambourg, A. 1971. Morphological and histochemical CIH and CF observed in the present study beaspects of glycoproteins at the surface of animal cells. cause only the rhamnomannan from S. schenckii Int. Rev. Cytol. 31:57-114. strain 1099.12 grown in the mycelial form may 14. Takai, S. 1974. Pathogenicity and ceratoulmin production in Ceratocystis ulni. Nature (London) 252:124-126. contain these acidic units (16). Alkali-extracta- 15. Travassos, L. R., and L. Mendonca-Previato. 1978. ble rhamnomannans from strain 1099.18 are not Synthesis of monorhamnosyl L-rhamno-D-mannans by acidic, and yet the cell wall of this strain is conidia of Sporothrix schenckii. Infect. Immun. 19:1-4. strongly reactive with CIH and CF. Moreover, 16. Travassos, L. R., L. Mendonia-Previato, and P. A. J. Gorin. 1978. Heterogeneity of the rhamnomannans glucuronic acid units recognizable by 13C nuclear from one strain of the human pathogen Sporothrix magnetic resonance spectroscopy (5, 16) are a schenckii determined by 13C nuclear magnetic resocharacteristic of mycelial polysaccharides and nance spectroscopy. Infect. Immun. 19:1107-1109. were not detected in the yeast rhamnomannans. 17. Travassos, L. R., W. Souza. L. Mendonga-Previato, and K. 0. Lloyd. 1977. Location and biochemical naAs shown in the present study, yeast forms of ture of surface components reacting with concanavalin both S. schenckii strains are the most reactive A in different cell types of Sporothrix schenckii. Exp. cells with CIH or CF. Mycol. 1:293-305.
