Lectins as a tool for the study of yeast cell walls

EXPERIMENTAL I~I¥COLOG¥ 2, i 1 0 - 1 1 3

(1978)

BRIEF NOTES Lectins as a Tool for the Study of Yeast Cell Walls I{IVKA BARKAI-GOLANz AND NATHAN SHARON

Department of Biophysics, The Weizmann Institute of Science, Rehovoth, Israel Received August 3, 1977; revised November 29, 1977 BARKAI-GOLAN, R., AND SHARON, N. 1978. Lectins as a tool for the study of yeast cell walls. Experimental Mycology 2, 110-113. Fluorescein isothiocyanate (FITC)-conjugated lectins with different sugar specificities were found to be a useful aid for the study of the composition of yeast cell walls and of the organization of the polysaccharides in these walls. Using FITC-concanavalin A, mannan was shown to be located on the outer surface of Saccharomyces cerevisiae, S. bayanus, Candida mycoderma, and C. rugosa, as well as on Sporobolomyces roseus, but seemed to be overlayed by other components in the other yeasts examined. Schizosaccharomyces pombe, C. rugosa, and S. roseus reacted with both FITCsoybean agglutinin and FITC-peanut agglutinin, indicating the presence of galactosecontaining polysaccharides on their surface. Rhodotorula glutlnis readily combined with FITC-wheat germ agglutinin, indicating that chitin is present on its outer surface. In Spofobolomyces, however, a strong reaction with FITC-wheat germ agglutinin was observed only after the cells had been treated with KOH, indicating that the chitin is normally present in the interior. This was in contrast to the more homogeneous distribution of mannan and gal'actose in the walls of S. roseus.

INDEX DESCRIPTORS:coneanavalin A; soybean agglutinin; peanut agglutinin; wheat germ aggluUnin; species of Saccharomyces, Schizosaccharomyces, Candida, Cryptococcus, Rhodotorula, and Sporobolomyces. I n the majority of yeasts, the cell wall is comprised mainly of glucans, mannans, and chitin (Phaff, 1963). T h e relative proportions of these polysaccharides m a y differ widely a m o n g different genera (BartnickiGarcia and McMurrough, 1971). Thus, in the ascomycetous yeasts such as Saccharomyces spp., and the ascomycetous forms of Candida, glucan and m a n n a n are the principal cell wall constituents. In contrast, the basidiomycetous yeasts Sporobolomyces spp. and the related forms tlhodotorula

spp. contain mainly chitin and m a n n a n (Bartnicki-Garcia and McMurrough, 1971). During recent years it has b e e n demonstrated that cell wall composition correlates closely with the taxonomic position of the yeasts (Phaff, 1971), which also seems to apply to the entire spectrum of fungal species (Bartnicki-Garcia, 1968). Little is known, however, about the organization of the polysaccharides in the yeast cell wall. Tkacz et al. (1971) have shown that fluorescent concanavalin A (Con A), 2 a lee-

1 Permanent address: Department of Fruit and Vegetable Storage Research, The Volcani Center, Agricultural Research Organization, Bet-Dagan, Israel. To whom all correspondence should be sent.

2 Abbreviations used: Con A, coneanavalin A; SBA, soybean agglutinin; PNA peanut agglutinin; WGA, wheat germ agglutinin; FITC, fluorescein isothiocyanate. 110

0147-5975/78/00gl-011050g.00/0 Copyright O 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

BRIEF NOTES

111

TABLE 1 Binding of FITC Lectins to Yeasts Yeast species and taxonomic

group

Chemical category of cell walls~

Lectin and main specificityb Con A a-D-Man, a-D-Gle

PNA D-Gal

SBA GalNAc, n-Gat

WGA (GIcNAe)~

Ascomycetes yeasts

Naccharomyces cerevisiae t t a n s e n S. bayanus Sacc. Schizosaccharomyces pombe Lindner

Mannan-fl

glucan --

+

+

M a n n a n ~ gluean

-1÷

-+

-±

--

M a n n a n ~ glucan M annan-chitin

--

---

---

4-c +

Mannan-chitin

+

+

+

=t=d

Asporogenous yeasts

Candida mycoderma (t~eess) Lodder et Kreger-van Rij

C. rugosa (Anderson) Diddens et Lodder Cryptococcus albidus var. di~uens (Zaeh) Phaff et Fell

Rhodotorula glutinis (Fres.) Harrison Basidiomyectes yeasts

Sporobolomyces roseus iKluyver et van Niel

As proposed b y ]3artnicki-Garcia (1968), + , very strong to strong b i n d i n g ; =t=, weak b i n d i n g ; --, no binding." c Weak b i n d i n g was observed to b u d d i n g cells. a Weak b i n d i n g was observed to 10-30% of the cells (mainly b u d d i n g cells).

tin specific for a-linked D-glucose and Dmannose, binds to Saccharomyces cerevisiae and concluded that this binding is the result of specific interaction between Con A and the ~-mannan present on the surface of this yeast. Two other yeast species-Schizosaccharomyces pombe and Rhodotorula glutinis--did not bind the lectin, and it was suggested (Tkacz et al., 1971) that this in an indication that a-linked mannans are absent in their walls. In this report we present results on the binding of four lectins to yeasts of different taxanomic groups: Con A; soybean agglutinin (SBA), specific for N-acetyl-D-galactosamine and D-galactose; peanut agglutinin (PNA), specific for o-galaetose; and wheat germ agglutinin (WGA), specific for chitin oligosaccharides (Lis and Sharon, 1977). The yeasts used (Table 1) were of plant tissue origin, except for R. glutinis, which was of human origin. The fluorescein isothiocyanate ( F I T C ) conjugated leetins were prepared as described (Barkai-Golan et al., 1978). Labeling of cells by the FITC lectins (0.5-9. mg/ml) in buffered saline, pH 7.4, was

done according to the procedure of Mirelman et al. (1975). Control preparations were treated by FITC-eonjugated lectins preincubated for 30 min at 25°C with their specific inhibitors [0.4 ~ 9-galactose for SBA and PNA, 0.4 ~ methyl a-u-glucoside for Con A, and 2 mM (GlcNAe)s for WGA]. The preparations were examined under a standard tlA Zeiss fluorescence microscope using a BG-12 exciter filter and a No. 53 barrier filter. The binding ability of the FITC lectins to yeasts of different taxonomic groups and cell wall categories is given in Table 1. The pronounced labeling of S. cerevisiae by FITC-Con A is in agreement with the results of Tkacz et al. (1971). The binding of this lectin to Saccharomyces bayanus and to the two species of Candida tested, which belong to the same cell wall category as Saccharomgces spp., strongly suggests that the mannan present in their walls is located, at least in part, on the outermost surface of the cell wall. Binding of FITC-Con A to Candida albicans was recently reported (Stoddart and Herbertson, 1977). In contrast to Saccharomuces and Cam

112

BRIEF NOTES

dida species, no binding to FITC-Con A was found with the species of Schizosaccharomyces, Cryptococcus, and BhodotoruIa tested. These yeasts, which in early studies were reported to be devoid of mannan, were later found to contain some amounts of this polysaecharide (Crook and Johnston, 1962; Deshusses et al., 1969; Rippon, 1974). The lack of Con A binding to these yeasts suggests that the presumed mannans may be of a fl-linked type, as was found for the capsular mannan in tl. glutinis (Gorin et al., 1965), which reacts poorly, if at all, with Con A (Sharon and Lis, 1972). Another reason for the lack of Con A binding may be due to the location of the mannans in internal regions of the yeast walls, where they are overlayered by other components such as fi-glucans or chitin. On the other hand, Corin and Spencer (19'68) showed that various species of Schizosaccharomyces, including S. pombe, contain galaetomannan--a type of mannan which would probably not react with Con A. The lack of binding of Con A to Cryptococcus albidus var. diffiuens is in agreement with the lack of its binding to the human pathogen Crgptococcus neoformans (Stoddart and Herbertson, 1977) since, although the capsular polysaeeharide of Crgptococcus species contains a small proportion of mannose, it was shown to be an aeidic heterosaecharide (Phaff, 1971). The results obtained with FITC-PNA and FITC-SBA show that galaetose residues are of limited occurrence in yeast surfaces, as was also shown for mycelial fungi (Barkai-Golan et al., 1978). Our results show, however, that exposed galactose is present in the cell wall of S. pombe, Candida rugosa, and Sporobolomyces roseus (Table 1). This is in agreement with the report on the presence of small amounts of a galaetose in S. pombe (Deshusses et al., 1969) and in S. roseus (Crook and Johnston, 1962). In contrast to the cell walls of myeelial

TABLE 2 Labeling of Untreated and KOH-Treated Cells of Sporobolomyces roseus after Incubation with FITC Leetins Leetin

Degree of labeling~ Untreated

Con A PNA SBA WGA

+ + + + ~=

KOH

treated

+ + + + + + +

+ +, very strong labeling; +, strong labeling; :t:, weak labeling. fungi, which are generally rich in chitin (Bartnicki-Garcia, 1968) and bind F I T C WCA readily (Mh'elman et al., 1975; Barkai-Golan et al., 1978), the presence of this polysaeeharide on the outer surfaee of yeast cell walls is much less common. Of the yeasts tested, marked FITC-WGA binding was shown only to /l. glutinis. This is in agreement with the chitin content of this yeast's cell walls (Kreger, 1954). On the other hand, with S. roseus, whieh was also reported to eontain chitin in its wall (Kreger, 1954) and like tlhodoturuIa belongs to the mannan-chitin cell wall category, only weak labeling by FITC-WGA of part of the cell population (10-30%) was observed. Interestingly, binding of FITC-WGA was mainly to the young, newly formed buds, indicating that their chitin is exposed, as is the case in the chitinous hyphal fungi (MireIman et aI., 1975; Barkai-Golan et al., 1978), in the actively synthesized growing regions of the cells. The affinity of F I T C - W G A to yeast buds was also found in cell populations of Cryptococcus. Treatment of Sporobolymyces cells with 4.5% KOH [as described by Kleinsehuster and Baker (1974) for revealing leetin reeeptors of fungal spores] resulted in a marked increase in labeling by FITC-WGA as well as some increase by SBA, while no change occurred in the binding of Con A

BRIEF NOTES

113

1965. An exocellular mannan, alternately linked 1,3-/~ and 1,4-fl from Rhodotorula glutinis. Canad. 1. Chem. 43: 950--954. GomN, P. A. J., AND SPENCER, J. F. T. 1968. Galactomannans of Trichospora fermentans and other yeasts; proton magnetie resonance and chemical studies. Canad. 1. Chem. 46: 23002304, KLEINSCrrUSTER, S. M., ANn BAKER, R. 1974. Lectin-detectable difference in carbohydrate-containing surface moieties of maeroconidia of Fusarium roseum "Avenaceum" and Fusarium solani. Phytopathology 64: 394-399. KREGER, D. R. 1954. Observations on cell walls of yeasts, and some other fungi by X-ray diffracACKNOWLEDGMENT tion and solubility tests. Biochim. Biophys. Aeta 13: 1-9. We wish to thank Mrs. Ilana Harari for the Lis, H., AND SHARON, N. 1977. Leetins: Their preparation of the FITC-conjugated lectins. chemistry and application to immunology. In The Antigens (M. Sela, Ed.), Vol. 4, pp. 429529. Academic Press, London/New York. REFERENCES MIRELMAN, D., GALUN, E., SHARON, N., AND BARKAI-GOLAN, R., MIRELMAN, D., AND SHARON, LOTA~, R. 1975. Inhibition of fungal growth by N. 1978. Studies on growth inhibition by lections wheat germ agglutinin. Nature (London) 256: of penicillia and aspergilli. Arch. Microbiol., in 414-416. PILAFF, H. J. 1963. Cell wall of yeasts. Annu. Bey. press. BARTNICKI-GARclA, S. 1968. Cell wall chemistry, Microbiol. 17: 15--30. morphogenesis, and taxonomy of fungi. Annu. PILAFF, H. J. 1971. Structure and biosynthesis of the ),east cell envelope. In The Yeasts (A. H. Bee. Microbiol. 22: 87-108. BARTNICKI-GARcIA,S., AND McMmaaoucH, I. 1971. Rose and J. S. Harrison, Eds.), Vol. 2, pp. 135Biochemistry of morphogenesis in yeasts. In The 210. Academic Press, London/New York. Yeasts (A. H. Rose and J. S. Harrison, Eds.), RIPPON, J. W. 1974. Medical Mycology, p. 220. Vol. 2, pp. 441-491. Academic Press, London/ W. B. Saunders, Philadelphia/London/Toronto. New York. SHARON, N., AND LIS, I-I. 1972. Lectins: CellCRoo~:, E. M., ANn JOnNSTO~r, I. R. 1962. The agglutinating and sugar-specific proteins. Science qualitative analysis of the cell walls of selected 177: 949-959. species of fungi. Bioehem. ]. 83: 325-331. STODDAtlT, R. W., AND HEt/BERTSON, B. M. 1977. DESItUSSES, J., BEltTHOUD, S., AND POSTEt/NAK, TIL. The use of lectins in the detection and identifica1969. Reeherches biochimiques sur Schizotion of human fungal pathogens. Biochem. Soc. sacharomyces pombe en fonction de conditions Trans. 566th Meeting %3-235. de culture et de faction d'inhibiteurs. II. Com- TKACZ, J. S., CVZULSKA,E. B., AND LAMPEN, J. O. position de parois cellularies. Biochim, Biophys. 1971. Specific staining of wall mannan in yeast Act 176: 803-812. cells with fluorescein-conjugated concanavalin A. GORIN, P. A. J., HomTsu, K., AND SPENCER, J. F. T. ]. Bacteriol. 105: 1-5.

and PNA (Table 2). This finding indicates that fungal chitin may not be accessible to WGA, whereas KOH probably removes masking substances that are coating the target polymer. Our results demonstrate that lectins with different sugar specificities are a useful aid for the study of the composition of yeast cell wails and for obtaining an insight into the organization of the polysaccharides in these walls.