Biotechnology Advances 29 (2011) 726–731

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Research review paper

Characteristics of yeast lectins and their role in cell–cell interactions

R.S. Singh ⁎, Ranjeeta Bhari, Hemant Preet KaurCarbohydrate and Protein Biotechnology Laboratory, Department of Biotechnology, Punjabi University, Patiala 147 002 (Pb.), India

⁎ Corresponding author. Tel.: +91 175 304 6262; faxE-mail addresses: rssingh11@lycos.com, rssbt@pbi.a

0734-9750/$ – see front matter © 2011 Elsevier Inc. Aldoi:10.1016/j.biotechadv.2011.06.002

a b s t r a c t

a r t i c l e i n f o

Article history:Received 22 June 2010Received in revised form 6 June 2011Accepted 6 June 2011Available online 14 June 2011

Keywords:AdhesinAgglutininCharacteristicsFlocculationFlocculation inhibitionPathogenicityYeast

Lectins are ubiquitous proteins with the ability to induce cell agglutination and, mediate cellular andmolecular recognition processes in a variety of biological interactions. Fungi display exquisite specificity fortarget tissues and attach to host glycoconjugates via these sugar-binding proteins. Although only few reportsare available on lectin activity of yeasts, these sugar binding proteins have been embraced for their role in cellflocculation, a commercially beneficial property, that simplifies downstream recovery operations in yeastfermentations. The lectins bind to cell wall mannans of the neighboring cells via hydrogen bonds leading tothe formation of cell aggregates which get interrupted in the presence of specific sugars. Attachment ofpathogenic yeasts to host cell surface is also a consequence of lectin-mediated recognition process. Thisreview provides a brief overview of yeast lectins, with an insight to lectin-mediated cellular recognitionphenomenon in yeasts.

: +91 175 2283073.c.in (R.S. Singh).

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© 2011 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7262. Localization of yeast lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7273. Purification of yeast lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7284. Characterization of yeast lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7295. Role of yeast lectins in flocculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7296. Lectin–receptor interactions in yeast pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7297. Conclusions and future focus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 730References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 731

1. Introduction

Lectins are mono- or multivalent proteins or glycoproteins of non-immunogenic origin, which recognize and bind diverse sugar structuresin a non-catalytic manner with a high degree of stereospecificity(Goldstein et al., 1980; Sharon and Lis, 1989). Owing to this propertythey are capable of binding with glyco-components on the cell surface.Carbohydrate binding sites of lectins recognize and adjust to thecarbohydrate ligand through lock and key complementarity involvingcomplex networks of hydrogen bonds. The formation of carbohydrate–protein complex involves the displacement of water moleculesassociated with polar groups, establishment of new hydrogen bondsand Van derWaals forces that determine the binding stability (Quiocho,

1986). There are numerous evidences that lectins mediate cellular andmolecular recognition in awide range of biological interactions (Sharonand Lis, 1989). Lectins from animals and plants have been extensivelyexploited as biochemical tools in biotechnological and biomedicalresearch. Thesearepresently beingused in the clinical laboratory to typeblood cells, as carriers of chemotherapeutic agents, as mitogens, forfractionation of animal cells and for investigations of cellular surfaces(Singh et al., 1999). Lectins can also be used as epidemiologic andtaxonomic markers of specific microorganisms and potential cost-effective diagnostic reagents (Slifkin and Doyle, 1990).

Though initially discovered in plants, lectins are distributedubiquitously in nature and have been isolated from diverse sources,ranging from viruses and bacteria to human beings (Drickamer andTaylor, 1993; Goldstein and Poretz, 1986; Singh et al., 1999; Singh et al.,2010,2011). Microbial lectins are represented by agglutinins, adhesins,precipitins, toxins and enzymes (Lakhtin, 1994). The co-functioning ofenzymes and lectins at different domains within the same molecule is

727R.S. Singh et al. / Biotechnology Advances 29 (2011) 726–731

well established by now and the applications of such association havebeen recently reviewed by Lakhtin et al. (2011). Fungal lectins aregaining greater interest from researchers as their wider biologicalroles are well established. Biomedical applications of lectins fromhigher fungi (Singh et al., 2011) and possible role of lectins in lowerfungi (Singh et al., 2010) have been recently reviewed by our group.Mushrooms constitute amajority (82%) of fungal lectins andhavegainedsignificant attention due to their immune enhancing and healthpromising benefits (Borchers et al., 2004). Molds comprise 15% of thetotal fungal lectinswhile yeast lectins occupy a small segment of merely3%. Fungal lectins are known to play an important biological role in hostrecognition and/or adhesion (Wimmerova et al., 2003). Lectin-mediatedhost cell attachment and colonization has been demonstrated innematode-trapping fungi Arthrobotrys oligospora (Nordbring-Hertz andMattiasson, 1979), Dactylaria candida (Nordbring-Hertz et al., 1981),Arthrobotrys conoides, Monacrosporium eudermatum, Monacrosporiumrutgeriensis (Rosenzweig and Ackroyd, 1983) and Arthrobotrys ellipsos-pora (Yamanaka et al., 1988). Lectins are involved in molecularrecognition processes during the early stage of mycorrhization e.g.,Lactaria mushroom/tree association (Giollant et al., 1993). These havealso been identified in succession, growth and morphogenesis of fungi(Singh et al., 2010,2011).

Yeast lectins are known to be involved in cell flocculation, whichshows their important commercial role in the brewing industry (Mikiet al., 1982a,b). Lectin synthesis and activation has been demonstratedin various brewing strains of Saccharomyces cerevisiae (Ngondi-Ekomeet al., 2003; Shankar and Umesh-Kumar, 1994; Stratford and Carter,1993). Yeast lectins are also involved in themating process as suggestedby properties of sexual agglutinins from Saccharomyces kluyveri 17 cells,

Table 1Localization and carbohydrate specificity of yeast lectins.

Yeast Lectin localization Inhibitory carbohydrate(s)

Candida albicans E D-Mannose, L-Fucose, N-Acetyl-D-glucoα-D-Mannosides

Candida glabrata CW D-Galactose, D-Lactose, N-Acetyl-D-lactoHansenula wingeia CW –

Histoplasma capsulatum CW D-Galactose, D-Galactosyl residuesSialic acid residues

Kluyveromyces bulgaricus E D-Fucose, D-Galactose, Lactose, MelibiosGal-β-1,4-Man, p-Nitrophenyl-α-D-galap-Nitrophenyl-β-D-galactoside, o-Nitropo-Nitrophenyl-β-D-galactoside, p-Nitropp-Nitrophenyl-β-D-lactoside

CW D-Galactose, D-Galactose-6-phosphate abearing terminal galactose at non-reduD-Fucose, D-Galactose, Lactose, Melibiosp-Nitrophenyl-α-D-galactoside, p-NitropMethyl-β-D-galactose

Kluyveromyces lactis CW bD-Galactose, Methyl-α-D-galactoside, pp-Nitrophenyl-β-D-galactoside, m-NitroThiodigalactoside, Lactose, Melibiose, Dcp-Nitrophenyl-α-D-galactoside, p-Nitrom-Nitrophenyl-β-D-galactoside, Thiodig

Paracoccidioides brasiliensis CW N-Acetyl-D-glucosamine, D-Glucose, LamPichia amethioninaa CW –

Saccharomycescarlsbergensis

CW D-Mannose, Maltose, Glucose, Phenyl-αMethyl mannose, p-Nitrophenyl-α-D-m

Saccharomyces cerevisiae CW D-Mannose-6-phosphate, p-α-NitrophenD-Mannosamine

Saccharomyces kluyveria CW –

Saccharomycodes ludwigii CW L-Arabinose, D-Fucose, D-Galactose, Lac

Saccharomyces uvarum CW D-Mannose, D-Mannose-6-phosphate, Oterminal mannose at non-reducing end

E: extracellular; CW: cell wall-associated.a Although these agglutinins bind cell wall mannoproteins, no glycosides have been repob Fully flocculating cells were taken in Helm's buffer.c Fully flocculating cells were taken in culture medium.

which bind to glycopeptides of S. kluyveri 16 cells through their sugarmoiety (Al-Mahmood et al., 1988),Hansenula wingei type 5 cells, whichspecifically agglutinate type 21 cells (Yen and Ballou, 1974) and Pichiaamethionina alpha cells (Mendonça-Previato et al., 1982). The role ofyeast lectins in parasitism has been proposed for pathogens Candidaalbicans (Brassart et al., 1991), Candida glabrata (Cormack et al., 1999),Histoplasma capsulatum (Taylor et al., 2000) and Paracoccidioidesbrasiliensis (Coltri et al., 2006). In the current review, an attempt hasbeen made to collate the available information on yeast lectins, with afocus on their role in flocculation and host cell adhesion.

2. Localization of yeast lectins

Within the yeast cells, lectins aremostly localized on the cell surface(Coltri et al., 2006; El-Behhari et al., 1998; Mendonça-Previato et al.,1982; Stratford and Pearson, 1992) with the exception of C. albicans(Critchley and Douglas, 1987b) and Kluyveromyces bulgaricus(Al-Mahmood et al., 1988), where lectins are secreted into thesurrounding medium. Table 1 documents the location of lectins indifferent yeasts. K. bulgaricus yeast cells are known to flocculatespontaneously upon growth. The aggregates are not dissociated bysuspension at pH 4.5 in Helm's buffer but deflocculated by suspensionin buffer containing D-galactose. These cell flocculation propertieshave been reported to be mediated by lectins recovered from culturebroth of spontaneously flocculating cells or supernatant of defloccu-lated cells suspended in galactose solution (Al-Mahmood et al., 1988).Three years later, these workers characterized another cell wallassociated lectin exhibiting twomolecular forms from the same yeastthat recognize cell wall phosphopeptidomannans (Al-Mahmood et

Reference(s)

samine, Critchley and Douglas (1987a,b);Cameron and Douglas (1996)

samine Cormack et al. (1999)Yen and Ballou (1974)Taylor et al. (1998)Mendes-Giannini et al. (2000);McMahon et al. (1995)

e,ctoside,henyl-α-D-galactoside,henyl-α-D-mannoside,

Al-Mahmood et al. (1988);Al-Mahmood et al. (1991)

nd oligosaccharidescing end

Hussain et al. (1986); Viard et al. (1993)

e, Raffinose,henyl-β-D-galactoside,

Al-Mahmood et al. (1991)

-Nitrophenyl α-D-galactoside,phenyl-β-D-galactoside,-fucose

El-Behhari et al. (1998)

phenyl-β-D-galactoside,alactoside, Melibiose

El-Behhari et al. (1998)

inin Coltri et al. (2006)Mendonça-Previato et al. (1982)

-D-mannoside,annoside

Dengis et al. (1995); Touhami et al. (2003)

yl mannopyranoside, Miki et al. (1982a); Terrance and Lipke (1981);Stratford and Carter (1993); Javadekar et al. (2000);Ngondi-Ekome et al. (2003)Weinstock and Ballou (1986)

tose, Lactulose, Melibiose Stratford and Pearson (1992)

ligosaccharides bearing Hussain et al. (1986)

rted to inhibit agglutination in any of these yeasts (Lipke and Kurjan, 1992).

728 R.S. Singh et al. / Biotechnology Advances 29 (2011) 726–731

al., 1991). Self-flocculant strains of Kluyveromyces lactis have alsobeen reported to possess a galactose-specific lectin. Extraction of theagglutinating factor with ion chelators or surfactants, suggests itsincidence in the cell envelope (El-Behhari et al., 1998).

Aggregation of Saccharomycodes ludwigii (Stratford and Pearson,1992) and Saccharomyces uvarum (Hussain et al., 1986) has beenreported to involve cell surface lectins and Ca2+ ions. C. albicans cells instationary phase express a lectin that preferentially binds to Galβ1,3-GalNAcβ1,4Galβ1,4Galβ1-1Ceramide (Yu et al., 1994). Another lectinrecognizingHblood group antigen Fucα1,2Galβ1,4GlcNAchas also beenreported from stationary cultures of C. albicans (Cameron and Douglas,1996).

3. Purification of yeast lectins

An initial step in lectin purification involves the preparation ofextracts in a buffer solution. Several methods have been used toisolate yeast agglutinins, employing limited proteolysis of the wall(Crandall and Brock, 1968), cell homogenization (Terrance and Lipke,1981) or digestion of cell wall with glucanase (Taylor, 1964) orzymolyase (Burke et al., 1980; Mendonça-Previato et al., 1982; Pierceand Ballou, 1983; Weinstock and Ballou, 1986). Phosphate buffer (pH7.0) containing EDTA has been used to extract lectins fromK. bulgaricus (Al-Mahmood et al., 1991). Straver and co-workers(1994) extracted an agglutinin from S. cerevisiae by heating the cellwalls at 60 °C in the presence of mannose. Non-ionic detergents havebeen used to isolate surface lectins from cell walls of flocculentS. cerevisiae strains (Javadekar et al., 2000; Ngondi-Ekome et al.,2003). Majority of yeast lectins have been purified using affinitychromatographic approaches (Al-Mahmood et al., 1988, 1991; Toshand Douglas, 1992; Yen and Ballou, 1974), but few reports document

Table 2Purification of yeast lectins.

Yeast Purification technique(s)

Candida albicans (fimbrial adhesin) HPLC on Protein-PAK 300 column anHPLC on Aquapore C4 column

Candida albicans (cell wall adhesin) Affinity adsorption on Synsorb H-2 adesalting on Sephadex G-25Affinity chromatography on ConA-Seion exchange chromatography on DE

Hansenula wingei Affinity chromatography on 21-H celgel filtration chromatography on Bio-

Kluyveromyces bulgaricus(extracellular GlcNAc specific lectin)

Affinity chromatography on Ultrogelwith β-D-GlcNAc

K. bulgaricus (extracellular Gal specific lectin) Affinity chromatography on Ultrogelwith α-D-Gal

K. bulgaricus(GlcNAc specific lectin from deflocculated cells)

Affinity chromatography on Ultrogelethanol precipitation and affinity chrsubstituted with p-aminophenyl-α-D

K. bulgaricus(Gal specific lectin from deflocculated cells)

Ethanol precipitation, affinity chromaAcA22 substituted with D-Gal

K. bulgaricus (cell wall associated Gal specificlectins Kb-CWL I and Kb-CWL II)

Affinity chromatography using yeastpolyacrylamide gel or using Sepharos

Paracoccidioides brasiliensis Affinity chromatography on GlcNAc-SSaccharomyces cerevisiae Desalting on polyacrylamide Biogel P

chromatography on mannose-SepharIon exchange chromatography on DEinteraction chromatography on phenchromatography on Sephacryl S-300Ammonium sulfate saturation (80%),chromatography on DEAE-Sepharosechromatography on phenyl-Sepharoson ConA-Sepharose column

Saccharomyces kluyveri (60 kDa lectin) Acid precipitation, ion exchange chroand affinity chromatography on Ultro

S. kluyveri (135 kDa lectin) Acid precipitation, ion exchange chroaffinity chromatography on Ultrogelchromatography on Sephacryl S-200

the use of conventional techniques involving ion exchange and gelfiltration chromatographies (Javadekar et al., 2000; Weinstock andBallou, 1986). Table 2 summarizes the techniques used by variousworkers for purification of yeast lectins.

Affinity chromatography is a highly reliable technique for lectinpurification based on lectin's ability to bind specifically and reversiblywith carbohydrates. Different affinity matrices can be selected basedon their carbohydrate specificity, which can be defined throughhapten inhibition assays using simple monosaccharides or complexcarbohydrates (Lis and Sharon, 1981). This approach has beenemployed for the purification of K. bulgaricus lectins. Precipitatesafter ethanol precipitation of supernatant from deflocculated cellswere loaded to Ultrogel AcA 22 column substituted with respectivespecific inhibitory sugar for the purification of extracellular GlcNAcand Gal specific lectins, eluted with 0.1 M acetic acid and 0.05 M HCl,respectively (Al-Mahmood et al., 1988). Cell wall associated lectinsfrom the same yeast have been purified using affinity chromatographyon yeast cells immobilized on non-denaturing polyacrylamide gel orSepharose 4B. The lectin was desorbed using 10 mM EDTA or 0.2 Mgalactose (Al-Mahmood et al., 1991). Paracoccin from P. brasiliensishas been purified by GlcNAc-Sepharose (Coltri et al., 2006).

Sexual agglutination reaction in H. wingei has been mostthoroughly investigated and complementary macromolecules havebeen isolated from cells of opposite mating types 5 and 21. Sexualagglutinin from H. wingei 21 cells has been purified by adsorption toan affinity column containing 21-H cells immobilized on cellulose andsuccessfully desorbed with buffer of pH 1.8 (Yen and Ballou, 1974).Mannoprotein adhesin from C. albicans has been isolated bysequential treatment with N-glucanase, papain, alkali and finallyrecovered by affinity adsorption on Synsorb H-2 matrix and, desaltedon Sephadex G-25 (Tosh and Douglas, 1992). Different approaches

Reference(s)

d Reversed phase Yu et al. (1994)

ffinity matrix and Tosh and Douglas (1992); Cameron andDouglas (1996)

pharose column,AE-Cellulose column

Critchley and Douglas (1987b)

ls immobilized to cellulose,Gel A column

Yen and Ballou (1974)

AcA 22 substituted Al-Mahmood et al. (1988)

AcA 22 substituted

AcA 22 substituted with β-D-GlcNAc,omatography on Ultrogel AcA 22-Galtography on Ultrogel

cells on non-denaturinge 4B

Al-Mahmood et al. (1991)

epharose column Coltri et al. (2006)2 column and affinityose column

Ngondi-Ekome et al. (2003)

AE-cellulose, hydrophobicyl-Sepharose and gel filtration

Javadekar et al. (2000)

dialysis, ion exchangecolumn, hydrophobice column, affinity chromatography

Shankar and Umesh-Kumar (1994)

matography on DEAE-Sephacelgel AcA 44 column

Weinstock and Ballou (1986)

matography on DEAE-Sephacel column,AcA 44 column, gel filtration

729R.S. Singh et al. / Biotechnology Advances 29 (2011) 726–731

involving ion exchange, hydrophobic and affinity chromatographyhave been used by various workers to isolate S. cerevisiae lectins(Javadekar et al., 2000; Ngondi-Ekome et al., 2003; Shankar andUmesh-Kumar, 1994).

4. Characterization of yeast lectins

Some reports are available on the detailed physicochemicalproperties of yeast lectins. Monomeric 61 kDa GlcNAc- and 65 kDagalactose-specific lectins from culture supernatant of K. bulgaricus havebeen demonstrated to exhibit pH dependent haemagglutination.GlcNAc-specific lectin agglutinates sheep erythrocytes at pH 7.4 withno agglutination of untreated rabbit erythrocytes or glutaraldehyde-fixed rabbit or sheep erythrocytes. However, the lectin agglutinatesglutaraldehyde-fixed sheep erythrocytes at pH 4.5. Galactose-specificlectin agglutinates both untreated and glutaraldehyde-fixed rabbiterythrocytes at pH 7.4 with no agglutination of untreated or glutaral-dehyde-fixed sheep erythrocytes. At pH 4.5, the lectin agglutinates bothglutaraldehyde-fixed rabbit and sheep erythrocytes (Al-Mahmood et al.,1988). Another 18.9 kDa galactose-specific lectin from K. bulgaricuscapable of readily agglutinating human type O erythrocytes has beenreported to be a glycoprotein in nature with protein part rich in lysine,glutamine and glycine, whereas the carbohydrate moiety consisting ofglucose, mannose and arabinose, constitutes only 1% of the entiremolecule. The calcium-dependent and strontium-inhibitable activity ofthese lectins has been reported to be optimal at pH4–6 (Al-Mahmood etal., 1991). The cell wall associated lectin KbCWL I from K. bulgaricus hasbeen reported to possess antifungal activity against Kluyveromyces,Saccharomyces, Pichia, Candida, Rhodotorula and Schizosaccharomyces.Blocking of D-galactose-binding site does not abolish the anti-yeastactivity of the lectin. Viard and co-workers (1993) proposed that thelectin behaves as a plurifunctional molecule which has at least twoindependent functionally active sites. The galactose-specific lectin ofK. lactis has been reported to agglutinate trypsinized human type Aerythrocytes (El-Behhari et al., 1998).

Molecular mass of cell surface mannoprotein lectin in brewingstrains of S. cerevisiae has been reported to be 13 kDa (Shankar andUmesh-Kumar, 1994), while Javadekar et al. (2000) isolated 40 kDalectin from S. cerevisiae. The latter is an acidic protein containing 44%hydrophobic amino acids with an isoelectric point at 4.0.

The 17-cell sexual agglutinin of S. kluyveri has been found topossess molecular massN200 kDa and high content of serine andglycine residues (Pierce and Ballou, 1983). The sexual agglutinationfactor of H. wingei has been reported to be phosphomannoprotein,rich in serine and threonine (Yen and Ballou, 1974). Activity of factor21 fromH. wingei (Yen and Ballou, 1974), α-factor from P. amethionina(Mendonça-Previato et al., 1982) and factor 17 from S. kluyveri (Pierceand Ballou, 1983) does not require carbohydrate moiety. The 66 kDapurified fimbrial subunit of C. albicans has been shown to contain 80–85% mannose and 10–15% protein, mainly composed of hydrophobicamino acid residues (Yu et al., 1994). Sialic acid-specific lectin hasbeen isolated from H. capsulatum (Mendes-Giannini et al., 2000). Thebudding regions of P. brasiliensis bears lectin, paracoccin which isreadily inhibited by GlcNAc, followed by D-glucose and D-mannose,but not by D-galactose, GalNAc or L-fucose. This 70 kDa lectin has alsobeen shown to bind strongly to laminin (Coltri et al., 2006).

5. Role of yeast lectins in flocculation

Flocculation in yeasts is an asexual aggregation of single cells, aproperty that has a commercially important role in the brewingindustry. Ideal brewing strains as single cells are expected to rapidlyconvert sugar to alcohol and flocculate after depletion of wort sugarleaving clear beer and a yeast crop suitable for repitching intosubsequent brews (Stratford and Carter, 1993). Commercial interestin immobilized yeast fermentations and economical separation of

cells from fermented broth to ease product recovery has led manyresearchers to investigate the mechanism underlying flocculation inyeasts (Fisher, 1975; Jansen, 1958; Lyons and Hough, 1971; Mill,1964; Stewart and Goring, 1976; Stratford, 1992; Taylor and Orton,1973). Calcium ion dependence of cell flocculation and its inhibitionby specific sugars prompted the lectin theory of flocculation (Fig. 1) byTaylor and Orton (1978), which was well elaborated by Miki et al.(1981, 1982a,b). The theory proposes that specific bonding occursbetween surface proteins (lectins) on thewalls of flocculated cells andsugar residues intrinsic to cell wall mannans of neighboring cells.Although lectins are present only on flocculent cells, lectin receptorsoccur on all yeasts with mannan-containing cell walls. Calcium ionshave been proposed tomaintain lectins in their active conformation tobind to receptors. The structure of these receptors was shown to benon-reducing termini of α-(1–3)-linked mannan side branches, twoor three mannopyranose residues in length (Stratford and Assinder,1991).

Miki et al. (1982a) proposed that gene FLO1 in S. cerevisiae maygovern the expression of a proteinaceous lectin-like activity, firmlyassociated with the cell walls of flocculent cells, which bind to the α-mannan carbohydrates of the adjoining cells. They further reportedthat the phenotypic expression of FLO1gene could be reproduciblymanipulated by aeration and factors are expressed only duringanaerobic growth in strain S646-1B of S. cerevisiae. Thus it mayprovide a model system for studies on the genetic control of cellsurface recognition mechanisms (Miki et al., 1982b). Later studiesindicating the inhibition of S. cerevisiae flocculation by sugars otherthan mannose pointed out the possibility of another phenotypeNewFlo, where flocculation was inhibited by gluco- and manno-pyranoses (Stratford and Assinder, 1991). Flocculation of Schizosac-charomyces pombe is known to involve galactose/fucose-specific lectin(Johnson et al., 1988). Wide array of sugars capable of inhibitingflocculation of different yeasts are collated in Table 1.

Stratford and Carter (1993) investigated the role of surface lectins inthe onset of flocculation in brewing strains of S. cerevisiaewith NewFlophenotype. They speculated that lectin synthesis and activation are twoseparateprocesses. Lectins are synthesizedearlyduringgrowth, secretedfrom the cell and exported to cell wall where they are held in a non-functional state, until activated at the onset of flocculation. Differentyeasts flocculate at different phases of growth. S. cerevisiae cells becomeflocculent in the stationary phase (Stratford and Carter, 1993), whileS. uvarum cells flocculate very early in the beginning of exponentialgrowth phase (Hussain et al., 1986). S. ludwigii (Stratford and Pearson,1992) andS. cerevisiae cellswith Flo1phenotype (Stratford andAssinder,1991) aggregate constitutively throughout the growth phase.

Flocculation of S. uvarum, K. bulgaricus and bottom fermentingyeasts Saccharomyces carlsbergensis has been demonstrated toinvolve lectinic mechanism, induced in the presence of Ca2+ ionsand reversed by specific sugars (Dengis et al., 1995; Hussain et al.,1986; Touhami et al., 2003). S. ludwigii, an occasional cider spoilageorganism, forms flaky deposits due to the presence of galactose-specific surface lectin. The strain has been reported to co-flocculatepoorly with mannan-containing S. cerevisiae cells but more stronglywith S. pombe containing galactan in the cell wall (Stratford andPearson, 1992). Galactose-specific lectins have been implicated inaggregation phenomenon of K. bulgaricus (Al-Mahmood et al.,1991) and K. lactis (El-Behhari et al., 1998). These galactose-specificlectins have been demonstrated to induce co-flocculation of flocculentK. bulgaricus and K. lactis 5c with non-flocculent yeast S. pombe, whereKluyveromyces sp. lectins bind to cell wall galactomannans of S. pombe(El-Behhari et al., 2000).

6. Lectin–receptor interactions in yeast pathogenesis

The essential role of microbial adhesion in pathogenesis of anumber of infectious diseases is well established. Pathogen entry is

Sugar Residues

Cell Wall

Lectin-like Proteins

Flocculent Cell

Non Flocculent

Cell

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Activated Flocculent

Cell

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Ca

Flocculated yeast cells (Lines showing the binding of lectin molecules to surface sugar residues of neighbouring cells)

Fig. 1. The lectin theory of yeast flocculation (Miki et al., 1981, 1982a,b; Taylor and Orton, 1978). Sugar residues are present on cell walls of yeast cells. Flocculent cells bear lectin-likesurface proteins, activated in the presence of calcium ions. These interact with sugar receptors on the cell walls of neighboring cells and form flocs.

730 R.S. Singh et al. / Biotechnology Advances 29 (2011) 726–731

initiated by adherence of yeast cells to the surface of host tissue thatgenerates an uptake signal which may induce its cytoplasmicinternalization (Mendes-Giannini et al., 2000). Mounting evidencessuggest the involvement of surface carbohydrate-binding proteins(lectins) in specific recognition between parasite and their target hostcells. The ability of C. albicans to adhere to host mucosal surface,involving adhesin–receptor interactions, facilitating colonization isthe first step in pathogenesis of C. albicans infection. Adhesion of C.albicans to buccal or vaginal epithelium has been demonstrated toinvolve lectin-like interactions between the protein component ofyeast mannoprotein adhesin capable of binding to GlcNAc, D-mannoseand L-fucose, and glycoside receptor on host cells (Critchley andDouglas, 1987a,b; Tosh and Douglas, 1992). Cormack et al. (1999)cloned a gene (EPA1) encoding a Ca+2-dependent surface lectin ofC. glabrata, which mediates its adherence to human epithelial cells.The lectin specifically recognizes asialo-lactosyl-containing carbohy-drates on the host cells. Deletion of EPA1 gene has been demonstratedto reduce adherence of yeast cells by 95%.

The virulence ofH. capsulatum is also known to involve interaction oflectinic component on yeast cell surfacewith complementary galactosylreceptors on membrane glycoproteins of murine macrophages as wellas on the surface ofmammalian erythrocytes, though sialic acid has alsobeen found as another specific carbohydrate associated with thisinteraction (Taylor et al., 1998, 2000). GlcNAc-binding surface lectin(paracoccin) from P. brasiliensis is shown to be directly related topathogenesis of the yeast strain. It binds to extracellular matrix

component laminin implicated in P. brasiliensis pathogenicity duringthe initial phase of infection and also in invasion and establishment ofinfection. Paracoccin also stimulates macrophages to release TNF-α andnitric oxide, which are the key mediators in paracoccidioidomycosis(Coltri et al., 2006).

7. Conclusions and future focus

Yeasts have many important applications in food and beverageindustry, most of which are based on specific interactions of surfaceproteins (lectins) with cell surface carbohydrates of adjoining cells.These lectins are also involved in interactions with host tissue leadingto parasitism. Over 1000 species are known to date but the presence oflectins has been investigated only in a few species. The occurrence oflectins in other known species should be explored and the detailedmechanism underlying lectin–receptor interactions in different phe-nomenon should be elucidated. This could be helpful in designinganti-adhesion therapies for biocontrol of disease causedby these strains.Lectins can also be helpful as biomarkers for species or even strainidentification as already demonstrated for several bacteria and molds.The authors realize theneed for detailedbiochemical characterization ofknown yeast lectins so as to identify homology if any, among theselectins or with known lectins from other fungi. This would be helpful toestablish new families or extend already known families within thefungal lectins.

731R.S. Singh et al. / Biotechnology Advances 29 (2011) 726–731

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