Archives of
Microbiology
Arch Microbiol (1984) 139: 221 - 224
9 Springer-Verlag1984
Cytochemical and ultrastructural studies of Candida albicans III. Evidence for modifications of the cell wall coat during adherence to human buccal epithelial cells G. Tronchin, D. Poulain, and A. Vernes INSERM U 42, Unit~ de Biologic et de Biochimie Parasitaires et Fongiques, Domaine du Certia, 369, rue Jules Guesde, F-59650 Villeneuve d'Ascq, France Abstract. Ultrastructural modifications of the cell wall coat of Candida albicans during adherence to host cells were investigated using various cytochemical techniques. Attachment of the fungus to buccal epithelial cells appeared to involve spatial rearrangement of their cell wall surface. In particular adhering yeast developed a fibrogranular surface layer visualized by the periodic acid - thiocarbohydrazidc - silver proteinate technique (a polysaccharide detection technique); Concanavalin A binding sites detected on their cell wall coat were highly increased. Attachment of yeasts to epithelial cells appeared mediated by fibrillar structures or polysaccharidic granules distributed on the cell wall coat. But free extra-cell wall material containing mannoproteins released from the yeast surface suggested additional mechanisms. Key words: Adherence albicans - Cell wall coat
Buccal
cells
-
Candida
Adherence of Candida albieans to human epithelial cells has received considerable attention the last few years as an important event of fnngal infection. Contrary to numerous microorganisms, adherence of C. albicans to mucosal surfaces is neither necessary for its survival nor for its growth. It represents an initial step of a more complex phenomenon such as colonization of tissue surfaces for which effective mechanisms of cellular attachment have been developed. Among previous reports on this subject, McCourtie and Douglas (1981), and Douglas et al. (1981) demonstrated that adherence of C. albicans to human epithelial cells or acrylic surfaces was proportional to the concentration of sugar i n the growth medium. This enhanced adherence appeared to be related with the production of an additional wall surface layer. Recently, Samaranayake and Mac Farlane (1981) suggested that this phenomenon could also imply an extracellular metabolic product of the yeast, probably a glycoprotein (Maisch and Calderone 1981). We have previously described a cell wall coat containing mannoproteins with e-linked D-mannosyl residues using concanavalin A on Offprint requests to: G. Tronchin Abbreviations: Con A: Concanavalin A; Man-fer: mannosyl ferritin; PATAg: Periodic acid-thiocarbohydrazide-silver proteinate
cryostat sections of C. albicans blastospores (Tronchin et al. 1981 b). Since the initial yeast-substratum interactions are surface phenomena, we initiated an ultrastructural and cytochemical study of the surface of adhering C. albicans blastospores.
Material and methods Yeasts. Candida albicans VW32 originally isolated from a case of human candidosis was used throughout. Cultures were routinely maintained by subculture on Sabouraud dextrose agar twice a month. Blastospores grown on this medium after culture for 24 h at 37~ were harvested by centrifugation and washed three times in phosphatebuffered saline (PBS, pH 7.2). The cells were enumerated by hemacytometer counts and were then resuspended at 5 x 106 cells/ml of PBS. Epithelial cells. Buccal cells were collected by gently rubbing the mucosal surface of the cheeks of human volunteers with sterile swabs. Epithelial cells were washed three times in PBS and standardized to 1 x 105 cells/rot in PBS. Adherence assay. The adherence method of Gibbons and Van Houte (1975) was used with some modifications. One ml each of epithelial cells and yeast suspension was mixed in tubes and incubated on a shaker at 37~ for 60 min. Control tubes contained epithelial cells and yeasts alone. Epithelial cells and adhering yeasts were collected on polycarbonate filters (10 ~tm pore size; Nucleopore c o r p . , Pleasanton, California, USA), and washed with 50 ml of PBS to remove unattached yeast. The membranes filters were gently pressed against glass slides previously coated with albumin. Cells on the slide were fixed in 10% formalin 10 min, and P.A.S.-tohiidine blue stained. The number of fungi adhering to 200 buccal cells was determined by light microscopy. Double blind conditions were used in all studies. Enzyme treatment of yeast. The cells were treated for 60 min at 37 ~C with 250 gg/ml pronase and 0.2 M mercaptoethanol in 0.05 M Tris-HC1 buffer, ph 7.4, (Tronchin et al. 1981 b) and washed with buffer before adherence test. Incubation of buccal cells with a culture supernatant of yeast. Yeast incubated alone for 2 h at 37~ were removed by centrifugation. The supernatant was collected and used
222
Figs. 1 - 4 . Ultrastructural aspects of the cell wall coat (arrows) of blastospores adhering to epithelial cells after 30 min of incubation (Figs. 1 and 3), compared to blastospores harvested from Sabouraud medium after culture for 24 h (Figs. 2 and 4); Figs. 1 and 2: PATAg technique; Figs. 3 and 4: Con A and Man-fer Figs. 5--7. Modifications of the cell wall coat during adherence assay (Con A and Man-fer) Figs. 5 and 7. In some cases, detachment of numerous isolated fragments (arrows) only labelled on their external face, left behind a few or unreactive cell wall surface (arrowheads) Fig. 6. Detachment of an outer sublayer (arrows) of cell wall coat components Fig. 8. Yeast-epithelial cell interactions; uranyl acetate and lead citrate. Attachment of blastospores via external granules (arrow) lodged into the bilayer membrane of a buccal cell; bars represent 0.5 I.tm
for incubation o f buccal cells for 1 h at 37~ ence assay.
before adher-
Electron microscopy. Washed epithelial cells and yeasts were prepared for electron microscopy as previously described (Tronchin et al. 1981 b). Thin sections were treated by one of the following techniques: concanavalin A (Con A) 100 gg/ml in 0.1 M sodium cacodylate buffer (pH 7.4) and mannosyl ferritin (Man-fer) 100 gg/ml (Industrie biologique frangaise); Periodic acid - thiocarbohydrazide - silver
proteinate technique (PATAg) according to Thiery (1967); Lead citrate and uranyl acetate by the method of Reynolds (1963). Thin sections were examined with a Sopelem 75 or Hitachi HU-11E electron microscope.
Results Electron microscopy of thin sections of adhering yeasts stained by the PATAg technique clearly demonstrated the
223 Table 1. Effect ofpronase and culture supernatant on adherence of C. albicans to buccal cells Pre-treatment
Mean number Relative of adherent adherence yeasts/t00 epithelial cells + SD a
t-test
PBS control Yeast treated with pronase PBS control Epithelial cells exposed to the culture supernatant of yeasts
212 + 21
1.0
94 + 11 159 + 17
0.44 1.0
b
101 + 13
0.63
c
" Standard deviation of the mean b Significant difference at the 99% level Significant difference at the 95% level
presence of a fibrillar cell wall coat composed of thin filaments arranged perpendicularly to the cell surface (Fig. 1). Numerous small electron opaque polysaccharidic granules appeared scattered on these filaments. With Con A and Man-fer, the cell wall coat showed an intensive binding of ferritin particles (Fig. 3). The increased proliferation of this external layer during the adherence phenomenon, and its reorganization, is evident by comparing the cell wall of adhering yeast with that of cells taken directly from the culture medium (Figs. 2 and 4). An abundant extracellular material with numerous binding sites for Con A and Manfer appeared to be released from the yeast surface. Detachment of numerous fragments of the cell wall coat (Fig. 5), or of an outer sublayer (Fig. 6) occurred during agitation. Only the external face of these fragments were labelled with Con A and Man-fer, leaving behind unreactive surfaces (Fig. 7). Yeasts epithelial cell interactions involved a direct epithelial-yeast cell attachment through fibrogranular components of the cell wall coat which bound to the bilayer membrane of the epithelial cell (Fig. 8). Treatment of the yeast cells with pronase and 2 mercaptoethanol before mixing with buccal cells inhibited adherence significantly, and supernatants of cultures of C. albicans reduced attachment of yeasts to epithelial cells after one hour of incubation (Table 1).
Discussion
The relationship between the yeast and its environment is largely conditioned by the external layers of its cell wall. In this sense yeasts must mediate adherence via their cell wall surface. The results reported herein indicate that adherence of C. albicans to host cells requires an ability of the yeast to actively rearrange substructural aspects of their cell wall coat in response to external stimuli. This point is supported by two observations: (a)The adhering blastospores produced a fibrogranular cell wall coat which was much less developed or not detected in non-adhering forms. McCourtie and Douglas (1981) also detected an additional, outermost fibrillar, floccular layer in blastospores harvested from medium containing high concentrations of certain
sugars. It was suggested that this layer may be responsible for an enhanced adherence of yeast to acrylic surfaces; (b) the cell wall coat of adhering yeasts was intensively labelled with Con A and Man-fer. A spatial reorganization of the glycoproteins of the cell wall is probably of primary importance in the attachment of C. albicans to host cells. Whether adherence of C. albieans reflects a difference in the number of specific surface structures involved in attachment, or a difference in their organization and/or in their presentation remains to be determined. Nevertheless, there is evidence to suggest that ready accessibility of the external cell wall fibrogranular components to the cell receptors is necessary for attachment of yeasts. It seems reasonable to suggest that yeast adherence may involve glycoproteinic structures of their cell wall surface. This suggestion is evidenced by reduction of adherence obtained with pronase treatment which is known to removed mannoproteins of the cell wall coat (Tronchin et al. I981 b). Sandin et al. (1982) demonstrated that mannose-containing moieties on the surface of C. albicans and buccal cells could mediate in vitro adherence. Maisch and Calderone (1981) also point out a role for yeast-surface mannan in the adherence of C. albicans to fibrin-platetet clots. Thus it seems that cell wall coat glycoproteins (notably mannoproteins) play an important role in yeast adherence. Our preliminary observations further suggest additional mechanisms during this penomenon. Indeed material released from the yeast cell wall coat into the medium could constitute an excess of components able to partially modify the adherence proper by binding to specific receptors on host cells (Sobel et al. 1981 ; Lehrer et al. 1983), and so could act as a competitive inhibitor. Moreover loss of material from the yeast cell wall surface could unrnask underlying ligands (i.e., N-acetyl-D-glucosamine residues) present under the external cell wall coat (Tronchin et al. 1981a). These hypothesis will require further biochemical studies to clarify the molecular basis of the substructural component of the cell wall coat. Acknowledgements. The authors are grateful to Dr R. G. Garrison
(Kansas City, Missouri) for the reviewing of the manuscript. They also wish to thank Mrs Masurelle for help in the preparation of the manuscript.
References
Douglas LJ, Houston JG, Mc Courtie J (1981) Adherence of Candida albicans to human buccal epithelial cells after growth on different carbon sources. FEMS Microbiol Lett 12:241243 Gibbons RP, Van Houte J (1975) Bacterial adherence in oral microbial ecology. Annu Rev Microbio129:19-44 Lehrer N, Segal E, Barr-Nea L (1983) In vitro and in vivo adherence of Candida albicans to mucosal surfaces. Ann Microbiol (Inst Pasteur) 134 B: 293 - 306 Maisch AP, Calderone RA (1981) Role of surface mannan in the adherence of Candida albicans to fibrin-platelet clots formed in vitro. Infect Immun 32 :9 2 - 97 Mc Courtie J, Douglas J (1981) Relationship between cell surface composition of Candida albicans and adherence to acrylic after growth on different carbon sources. Infect Immun 32:12341241 Reynolds E (1963) The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J Cell Biol 17:208 -212
224 Samaranayake LP, Mac Farlane TW (1981) The adhesion of the yeast Candida albicans to epithelial cells of human origin in vitro. Arch Oral Biol 26: 815 - 820 Sandin RL, Rogers AL, Patterson RJ, Beneke ES (1982) Evidence for mannose-mediated adherence of Candida albicans to human buecal cells in vitro. Infect Immun 35:79-85 Sobel JD, Hyers PG, Kaye D. Levison ME (1981) Adherence of Candida albieans to human vaginal and buccal epithelial cells. J Infect Dis 143:76-82 Thiery JP (1967) Mise en 6vidence des polysaccharides sur coupes fines en microscopic 6lectronique. J Micros 6:978-1018
Tronchin G, Poulain D, Herbaut J, Biguet J (1981 a) Localization of chitin in the cell wall of Candida albicans by means of wheat germ agglutinin. Fluorescence and ultrastructural studies. Eur J Cell Biol 26:121 - 128 Tronchin G, Poulain D, Herbaut J, Biguet J (1981 b) Cytochemical and ultrastructural studies of Candida albicans. II. Evidence for a cell wall coat using concanavalin A. J Ultruct Res 75 : 5 0 - 59
Received February 28, 1984/Accepted April 22, 1984