ORAL COLONIZATION BY CANDIDA ALBICANSR.D. Cannonl*W.L. Chaffin2'Department of Oral Sciences and Orthodontics, Faculty of Dentistry, University of Otago, PO Box 647, Dunedin, New Zealand; 2Department of Microbiology and Immunology, Texas TechUniversity Health Sciences Center, Lubbock, Texas, USA; *corresponding author

ABSTRACT: Candida albicans is a commensal yeast normally present in small numbers in the oral flora of a large proportion ofhumans. Colonization of the oral cavity by C. albicans involves the acquisition and maintenance of a stable yeast population.Micro-organisms are continually being removed from the oral cavity by host clearance mechanisms, and so, in order to surviveand inhabit this eco-system, C. albicans cells have to adhere and replicate. The oral cavity presents many niches for C. albicanscolonization, and the yeast is able to adhere to a plethora of ligands. These include epithelial and bacterial cell-surface mole-cules, extracellular matrix proteins, and dental acrylic. In addition, saliva molecules, including basic proline-rich proteins,adsorbed to many oral surfaces promote C. albicans adherence. Several adhesins present in the C. albicans cell wall have nowbeen partially characterized. Adherence involves lectin, protein-protein, and hydrophobic interactions. As C. albicans cells evadehost defenses and colonize new environments by penetrating tissues, they are exposed to new adherence receptors andrespond by expressing alternative adhesins. The relatively small number of commensal Candida cells in the oral flora raises thepossibility that strategies can be devised to prevent oral colonization and infection. However, the variety of oral niches and thecomplex adherence mechanisms of the yeast mean that such a goal will remain elusive until more is known about the contri-bution of each mechanism to colonization.

Key words. Candida albicans, colonization, adherence, candidiasis.

(1) IntroductionThe presence of Candida albicans in the oral cavity is not

indicative of disease. In many individuals, C. albicans isa minor component of their oral flora, and they have noclinical symptoms. In certain sections of the population,however, oral candidiasis occurs frequently and necessi-tates antifungal therapy. Oral presentations of candidia-sis vary from the large white plaques of pseudomembra-neous candidiasis on the tongue and buccal mucosa tothe palatal erythematous lesions of chronic atrophic can-didiasis, and to angular cheilitis on the labial commis-sures (Samaranayake, 1990; Scully et al., 1994; Shay et al.,1997). The primary etiological agent of oral candidiasis isthe yeast C. albicans; however, other species that causedisease less commonly include C. tropicalis, C. glabrata, C.krusei, C parapsilosis, C. guilliermondii, and C. dubliniensis(Odds, 1988; Fridkin and Jarvis, 1996; Sullivan andColeman, 1998). Sequelae of mucosal colonization, par-ticularly of the gastrointestinal tract, may include pene-tration of the vascular system by Candida cells andhematogenous dissemination (Cole et al., 1996). Thesecells can then infect a variety of organs in immunocom-promised individuals and cause disseminated or sys-temic disease.

It is difficult to give a precise oral carriage rate for C.albicans, since this depends on the age and health of the

population studied. A compilation of data from a num-ber of reports showed that the mean carriage rate forhealthy individuals (no known underlying disease) was17.7% (range, 1.9-62.3%), whereas mean carriage in hos-pitalized individuals (without clinical candidiasis) was40.6% (range, 6.0-69.6%) (Odds, 1988). These data indi-cate that the health of an individual is a predisposingfactor for C. albicans colonization. A large number of sitesin the oral cavity can be colonized; in healthy individuals,C. albicans is most commonly isolated from the mid-lineof the middle and posterior thirds of the tongue, thecheek, or the palatal mucosa (Arendorf and Walker, 1979,1980; Borromeo et al., 1992).

It is of interest that only a proportion of the popula-tion is colonized by C. albicans, and only a subset of theseindividuals develops candidiasis. Few longitudinal stud-ies have been carried out on healthy individuals to see ifCandida colonization is continuous. However, daily sam-pling has shown that C. albicans carriage persisted in aproportion of healthy people and that colonizationrecurred in a majority of the remaining subjects (Gergelyand Uri, 1966; Williamson, 1972). In a study of 163neonates in an intensive care and surgical unit, 21 of theneonates initially carried C. albicans in their mouths, butonly five yielded 6 or more yeast-positive cultures overthe 17-week study period (Sharp et al., 1992). Theseneonates were colonized for periods of between 7 and 63

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(2) AcquisitionColonization Acquisition (a) Candida species inhabit a variety of

environments (Odds, 1988). C. albi-

b)cans has been isolated from primates,domesticated and other mammals,

GWt C. albicans Systemic marsupials, and birds. In contrast,\ (d) Disease other Candida species have been iso-\i' X lated from a much narrower range ofMucosl hosts (Odds, 1988). In humans, C.

Removal (c) disease Oral cavity Ialbicans preferentially colonizess Oa mucosal surfaces, and the intestinaltract is believed to be a major reser-voir for infection (Odds, 1988; Cole etFigure 1. A model showing the interrelationship of factors involved in colonization of the oral al., 1996). C. albicans can colonize

cavity by C. albicans: (a) acquisition, (b) growth, (c) removal, and (d) tissue damage and practically any site in the gastroin-penetration. testinal tract (Cole et al., 1996), from

the oral cavity to the rectum andperi-anal tissues, allowing anal-oralinoculation to occur (Soll et al., 1991).

days. The C. albicans strains were biotyped, and there was The vulvovaginal regions of approximately 40% ofunequivocal evidence for more than one infecting bio- healthy women are colonized by Candida species (Soll ettype in only 8.1% of colonized neonates. In immunocom- al., 1991), and the genito-urinary tract presents anotherpromised hosts, candidiasis is often caused by a resident reservoir for oral inoculation. C. albicans survives betterstrain (Powderly et al., 1993; Voss et al., 1994), and the on moist surfaces than dry inanimate objects, but if thesame strain can cause recurrent infections (Miyasaki et degree of contamination is high enough, viable cells willal., 1992). Some of the factors involved in the balance remain on dry surfaces for at least 24 hours (Rangel-among clearance of C. albicans, colonization, and the Frausto et al., 1994). Many studies of nosocomial can-development of candidiasis have been reviewed previ- didiasis in clinical settings have been carried out toously (Cannon et al., 1995a). The objective of this review determine how patients acquire infections (Hunter et al.,is to focus on the initial, critical, step of colonization, 1990; Vazquez et al., 1993, 1998; Fridkin and Jarvis, 1996).and to discuss the factors involved in colonization and It is evident that the most common means of transfer ishow current research might lead to therapeutic interven- contact with carriers, often the hands of hospital staff,tions that could prevent colonization and, thus, preclude although various Candida species can be cultured fromcandidiasis. inanimate objects (Hunter et al., 1990; Vazquez et al., 1993,

Colonization of the oral cavity by C. albicans can be 1998; Strausbaugh et al., 1994; Jarvis, 1996; Pfaller, 1996).defined as the acquisition and maintenance of a stable A worrying finding in one of these studies was that C.population of C. albicans cells which does not give rise to albicans could be cultured from the food given to twoclinical disease. A model based on this definition is patients in a bone marrow transplant unit (Vazquez et al.,shown in Fig. 1. Colonization depends on the rate of 1993). Indeed, yeasts, including Candida species, are rela-acquisition-that is, the rate at which yeast cells enter tively common contaminants of both processed andthe oral cavity-growth, and removal of cells from the unprocessed foods (Buck et al., 1977; Viljoen andmouth by swallowing and oral hygiene. In a simplified Greyling, 1995). In a dental setting, the internal surfacesmodel, if the rate of removal is greater than that of acqui- of dental unit water lines can become coated with bacte-sition and growth, clearance will take place. If the rate of ria-rich biofilms (Tippett et al., 1988; Peters and McGaw,removal is the same as that of acquisition and growth, 1996). Although there are no reports of yeasts being pres-then there will be colonization. If the rate is lower and ent in these biofilms, this may reflect the culturingthere is tissue damage, it will lead to candidiasis. The methods used. If C. albicans were a component of suchpresentation of candidiasis will depend on the tissue biofilms, contaminated water lines could constitute acolonized, the virulence factors expressed by the Candida significant risk of inoculating oral cavities with yeast. Incells, and the host response. So, colonization depends people whose mouths are colonized with C. albicans, theon several factors: the acquisition or entry of cells into yeast can be found in saliva at an average concentrationthe oral cavity, the attachment and growth of those cells, of 300 to 500 cells per mL (Arendorf and Walker, 1980).the penetration of tissues, and the removal of cells from This will allow for transfer during kissing and other directthe oral cavity. Each of these factors will be examined. saliva-saliva contact. There are ample opportunities,

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therefore, for the entry of Candidaspecies into the oral cavity by manualinoculation, saliva transfer, or contami-nated food and drink.

(3) Maintaining an OralCandida Population

The entry of Candida cells into the oralcavity is not sufficient for colonization;they must be stably maintained. Sincethe oral cavity is a continuous-flowenvironment, yeast cells will be washedout by saliva and swallowed unlessthey adhere and replicate. Growth con-ditions in the oral cavity are so poor(there is practically no growth in salivaunless it is supplemented with glucoselSamaranayake et al., 19861) that cellshave to adhere to be maintained.Adhesion is therefore of critical impor-tance in colonization. Adherence ismediated between moieties of theCandida cell wall and host surfaces, andso an understanding of colonizationrelies upon knowledge of these sur-faces.

(A) THE C. ALBICANS CELL WALLThe cell wall is essential both to the biology of C. albicansand to its interactions with the human host in health anddisease. Although frequently called a dimorphic fungus,the organism is, in fact, polymorphic and may adoptgrowth not only in yeast or hyphal modes but also aspseudohyphae and may produce chlamydospores in cer-tain growth conditions (Odds, 1988). The initial emer-gence of hyphae from yeast cells is often referred to asgerm tube formation. While both yeast and hyphae canbe found in lesions, and different adhesins are expressedon hyphae as discussed below, hyphal cells clump exten-sively and have been less-well-studied in adherenceassays than yeast. The cell wall is the structure responsi-ble for supplying the rigidity that maintains the uniqueshapes that characterize fungal growth. The surface ofthe organism is the site of the physical interactionsbetween the fungus and host proteins and tissues thatlead to adherence, and between the fungus and theimmune system that lead to clearance.

The cell wall is composed primarily of carbohydrate(80-90%), 1-glucan, chitin, and mannan (Fig. 2a; for moreextensive discussion, see reviews by Shepherd, 1987;Cassone, 1989; Fleet, 1991; Fukazawa and Kagaya, 1997;Chaffin et al., 1998). The components of the cell wallsfrom yeast and hyphal forms are similar, although thereis some quantitative variation. Chitin (an unbranched

(a)

(b)

Figure 2. Molecular interactions between the cell wall of C. albicans and oral surfaces. (a)Schematic representation of the architecture and composition of the C. albicans cell wall:41M chitin, 41i49 3(1,3)-glucan, "`..i 13(1,6)-glucan, .L, mannoprotein, (&phosphodiester linkage, and plasma membrane. (b) Interactions of C. albicans withmolecules and surfaces in the oral cavity that may contribute to colonization.

polymer of N-acetylglucosamine) is a minor constituentthat is variously reported to contribute from 1 to 10% ofthe cell wall's dry weight. The higher levels are associat-ed with hyphal cells which are reported to containapproximately three times more chitin than yeast cells.However, a recent study reports that chitin measure-ments depend greatly on the method used (Munro et al.,1998). (-glucan (a branched polymer containing 3-1,3and 3-1,6 linkages) is the main constituent, accountingfor 47 to 60% of the cell wall's dry weight. These twomicrofibrillar polysaccharides, while found throughoutthe cell wall, are more concentrated in the inner portionnear the plasma membrane and provide a rigid skeleton.The other main component is mannan, also sometimescalled phosphomannoprotein or phosphopeptidoman-nan complex, which accounts for about 40% of the cellwall. Mannans are composed of mannose polymers cova-lently linked to a protein moiety mostly by N-glycosidiclinkages through di-N-acetylchitobiose to asparagineresidues. The mannose component consists of a back-bone of (x-1,6-linked mannose molecules to which areattached oligosaccharide side-chains containing man-nose residues with ax-1,2, ox-1,3, 3-1,2, 13-1,6 linkages andsome a-1,6 branches. Some of these side-chains alsocontain a phosphodiester linkage to short 1-1,2 manno-oligosaccharides. The N-glycosyl moieties of high-molec-

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TABLE 1C. albicans Adhesins and BEC Ligands

Adhesin Ligand ReferenceCarbohydrate

Chitin Unknown Segal, 1996Factor 6 oligomannosaccharide Unknown Miyakawa et al., 1992

Protein66-kDa fimbrial protein Glycosphingolipid Yu et al., 1994aFibronectin binding protein

(multiple candidates) Fibronectin Klotz and Smith, 1992; Gozalbo et al., 1998;Yan et al., 1998b

iC3b binding protein(multiple candidates) iC3b Eigentler et al., 1989; Hostetter et al., 1990;

Alaei et al., 1993Fucose binding protein Fucose-containing oligosaccharide

(1 5-kDa fragment) (blood group antigen?) Cameron and Douglqs, 1996GIcNAc or glucosamine Host oligosaccharide-containing

binding protein (190 kDa) GIcNAc or glucosamine Enache et al., 1996SAP (secreted aspartyl Unknown (proteolytic modification

proteinase) of host or fungus?) Watts et al., 1 998ALS gene familyALSI Unknown Fu et al., 1998ALA1 Unknown Gaur and Klotz, 1997Other proteins (38-kDa, 54-kDa

candidate species) Unknown Imbert-Bernard et al., 1995

ular-weight yeast cell mannoproteins average more than components, 3-1,3-glucan, 3-1,6-glucan, chitin, and600 mannose residues and those from germ tubes more mannoprotein (Kollar et al., 1997). The analysis of thisthan 300 residues. In addition, single mannose residues material suggested that 3-1,6-glucan with some f-I,3-and short, unbranched manno-oligosaccharides may be glucan branches may be linked to the reducing end of0-linked to protein through serine and threonine. chitin. Covalent attachment of mannoprotein to 3-1,6-Mannoproteins are found throughout the cell wall and glucan is through a remnant of the mannoprotein GPIappear to be the dominant component at the cell sur- (glycosyl phosphatidyl inositol) anchor. However, eachface. Electron microscopic analysis shows a variable complex may not contain all four components, and thenumber of cell wall layers (from 3 to 8) that seems to be proportion of cell wall polysaccharide involved in thisrelated to the technique used, the strain, and growth type of structure is unclear.conditions of the fungus (Cassone et al., 1973; Rico et al.,1991). This layering appears to be the result of quantita- (B) C. ALBICANS ADHESINStive differences in the individual components in different Adhesins are the fungal surface moieties that mediateregions of the wall. Fimbriae, which may extend 110 to binding of C. albicans to other cells (host or microbial),300 nm, radiate from the surface (Fig. 2a; Yu et al., 1994a). inert polymers, or proteins. Different experimentalThe fimbrial subunit appears to be a highly glycosylated approaches and reagents have been used to identify C.glycoprotein with an apparent molecular mass of 66 kDa albicans adhesins (also called binding proteins or recep-(Yu et al., 1994a). tors) and host ligands (sometimes also called receptors).

The architecture of the yeast wall has been studied There is disagreement among some of these studies asmore extensively in Saccharomyces cerevisiae, and a number to the identity, number of candidal receptors for variousof observations suggest that the candidal cell wall will fit ligands, and the inhibitors of adherence (reviewedthe same model. In a recent study, material was isolated recently in Fukazawa and Kagaya, 1997; Sturtevant andfrom a cell wall digest that contained all of the major wall Calderone, 1997; Chaffin et al., 1998). Our incomplete

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understanding of the factors that influence the adher-ence interactions is a likely source of apparently conflict-ing observations. Following are examples of five factorswith the potential to affect observations:

(1) Some C. albicans strains are more adherent thanothers (Schmid et al., 1995b).

(2) Some strains possess adhesins with differentspecificities (Critchley and Douglas, 1987a,b).

(3) In vitro growth conditions of the fungus-such astemperature (Lee and King, 1983), medium composition(Alloush et al, 1996; Yan et al., 1998b), carbon source(McCourtie and Douglas, 1985; Gustafson et al., 1991), orthe presence of a specific inducer (Yan et al., 1998a)-may alter the expression of an adhesin.

(4) Fungal cell viability may affect the extent of bind-ing (Gorman et al., 1986).

(5) The binding capacity of exfoliated human cellsused in adherence assays may differ among donors andfrom the same donor on different days (Sandin et al.,1987b) and with hormonal status (Theaker et al., 1993).

Despite differences between some studies, the gen-eral conclusions are that C. albicans possesses multipleadhesins and that there may be more than one adhesinthat recognizes a host ligand or cell. Most adhesins iden-tified to date are mannoprotein, and, for individualadhesins, both the protein and/or carbohydrate portionshave been implicated in adherence.

(C) ADHERENCE TO SKINSkin is a site of normal colonization as well as infectionssuch as diaper rash and intertriginous candidiasis.Infections usually occur in individuals with some loss ofnormal skin defenses such as abrasion and maceration,and yeast growth is promoted by a warm, moist environ-ment (Samaranayake, 1990; Scully et al., 1994). Systemicconditions such as diabetes, obesity, and various medicaltreatments may also contribute to susceptibility to skinand other mucocutaneous infections (Odds, 1988;Samaranayake, 1990). C. albicans cells bind in vitro to cor-neocytes (keratinized cells of stratum corneum) fromindividuals in these susceptible groups at twice the fre-quency with which they bind to corneocytes from healthyindividuals (Srebrnik and Segal, 1990). Amino sugars,mannosamine, glucosamine, and galactosamine inhibit-ed binding of C. albicans to human corneocytes and tobuccal epithelial cells (BECs) (Collins-Lech et al., 1984). Achitin-soluble extract (CSE) also inhibited binding of C.albicans to human corneocytes (Kahana et al., 1988). C. albi-cans cells exposed to nikkomycin, a chitin synthetaseinhibitor, had decreased chitin content and showed a cor-responding decrease in adherence to BECs (Segal et al.,1997). The site of adherence between the yeast cells andepithelial cells labeled intensely with wheat germ agglu-tinin, a lectin-recognizing chitin. In a porcine stratum

corneum model, three isolates from oral infectionsadhered more than a commensal isolate (Law et al., 1997).Removal of lipid from the stratum corneum led to dou-bling of the number of adhered organisms. Specificepithelial lipids can modulate fungal adherence, sincebinding was inhibited by fatty acids, sterols, andceramides and was unaffected by squalene, steryl esters,cholesterol esters, and triglycerides. In a murine stratumcorneum model, yeast cells of C. albicans and C. stellatoideaadhered in greater numbers than those of C. tropicalis,while C. guilliermondii, C. krusei, and C. parapsilosis cellsshowed little or no adherence (Ray and Payne, 1988). Thishierarchy of adherence was similar to that observed withhuman epidermal corneocytes and BECs (Ray et al., 1984).In the murine stratum corneum model, the adherent cellsacquired fibrils and strands of an amorphous materialbetween the yeast and corneocyte cell surface, formedcavitations at the site, and produced hyphae that invadedcorneocytes distal to the yeast attachment (Ray andPayne, 1988). Depletion of lipids had no effect on adher-ence in this study, but pepstatin, an inhibitor of the fun-gal secreted aspartyl proteinase, inhibited the formationsof cavities around the adherent cells. Epidermolytic pro-teases, likely including the secreted aspartyl proteinase,have been isolated from strains recovered from patientswith cutaneous disease (El-Maghrabi et al., 1990).Pepstatin, bovine brain gangliosides, and convalescenthuman serum all reduced binding of yeast cells to cor-neocytes. Although there may be differences amongadhesins for corneocytes and BECs and vaginal epithelialcells (VECs), it is likely that there are at least some com-mon adhesins. It appears, therefore, that C. albicans chitinand proteinase may be important in skin colonization.

(4) Adherence to Oral SurfacesThe oral cavity presents a number of surfaces for C. albi-cans adhesion. These include BECs, the inert polymers ofdental prostheses, teeth, and other oral micro-orga-nisms. Adherence to each of these surfaces and the mod-ulating effect of saliva on adhesion will be discussed.

(A) ADHERENCE TO BECsExfoliated BECs are probably the best-investigatedhuman cell type in C. albicans adherence studies, and sev-eral adhesin/ligand interactions have been proposed(Table 1). In interpreting the results of BEC adhesionassays, one should note the following features: Fungalstrain and source of epithelial cells affect adherence(Sandin et al., 1987b), the number of C. albicans cells thatbind to individual BECs is variable (Sandin et al., 1987a),there are both binding and non-binding BECs (Gorman etal., 1986; Polacheck et al., 1995), and both viable and non-viable C. albicans cells bind to BECs, with the non-viablecells having a greater adherence (Gorman et al., 1986).

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The binding capacity of BECs from newborn full-terminfants for Candida is less than that of BECs from prema-ture infants, school-age children, and adults, but increas-es over the infants' first few days (Davidson et al., 1984;Cox, 1986; Polacheck et al., 1995). Adherence was greaterto BECs from children with oral infection or colonizationthan to BECs from uninfected controls but increasedduring a course of antibiotic therapy in previously unin-fected children (Cox, 1983). Menstrual cycle affectsepithelial binding capacity, since BECs collected on day5 showed a binding capacity higher than that of cells col-lected on day 15, 22, or 28 (Theaker et al., 1993). In onestudy, VECs from the first and fourth weeks had a bind-ing capacity higher than that of VECs from the second orthird week (Segal et al., 1984), while another study sug-gested that binding capacity peaked between the thirdand fourth weeks (Bibel et al., 1987). VECs collected frompregnant or diabetic women also bound more C. albicanscells than those from non-pregnant or non-diabetic con-trols, and 'infection isolates' adhered better than 'colo-nizing isolates' (Segal et al., 1984). However, there was nodifference in the binding capacity of VECs from womenwith recurrent vaginitis compared with healthy controls(Trumbore and Sobel, 1986). Palatal epithelial cells fromacrylic-denture-wearers with non-insulin-dependent dia-betes bound more fungal cells than did epithelial cells ofnon-diabetic individuals (Dorocka-Bobkowska et al.,1996). In another study, adherence to BECs from diabet-ic individuals was similar to adherence to BECs from nor-mal individuals (Polacheck et al., 1995). Thus, adherenceto BECs is affected by many host factors, and hormonaleffects on adherence could be mediated by altering theexpression of adhesins on C. albicans cells or ligands onhost cells.

There is also variability in binding to BECs from dif-ferent donors (Sandin et al., 1987b). Adherence differedwhen epithelial cells were collected on different dates,but gender was not a factor. C. albicans adhered in greaternumbers to BECs from AIDS patients than to BECs fromhealthy individuals or transplant patients (Schwab et al.,1997). C. albicans isolates from patients in the early stagesof AIDS adhered to BECs less well than did those fromhealthy individuals. However, adherence of isolatesincreased with the progression of AIDS until it exceededthat of control isolates (Pereiro et al., 1997). Isolates fromimmunocompetent patients with esophageal candidiasisadhered better than isolates from patients who wereheavily colonized but not symptomatic (Wellmer andBernhardt, 1997). Although there were differences amongstrains, isolates from candidiasis patients were moreadherent and formed germ tubes more rapidly than theother isolates. Analysis of these results is complicated bythe fact that the adherence of strains from AIDS patientshas mostly been measured in vitro and may not correlate

to adherence in vivo for patients who are on courses ofantifungal drugs.

The effect of treating BECs and/or C. albicans cellswith antimicrobial agents on subsequent adherence hasbeen studied extensively. The consensus of opinion isthat treatment of BECs or yeast cells with any of a varietyof agents-including chlorhexidine, hexetidine,dequalinium chloride, cetrimide, cetylpyridinium chlo-ride, octenidine, pirtenidine, taurolidine, propamidineisethionate, noxythiolin, and aqueous garlic extract-reduced adherence (Gorman et al., 1986, 1987a,b; Tobgi etal., 1987; Ghannoum, 1990; Ghannoum et al., 1990; Jonesand Fowler, 1994). Also, treatment of C. albicans withpropamidine isethionate, octenidine, pirtenidine, andaqueous garlic extract reduced germ tube formation(Jones and Fowler, 1994; Jones et al., 1997). Exposure toantifungal drugs may also reduce adherence to epithelialcells. Subinhibitory concentrations of amphotericin B,nystatin, miconazole nitrate, and 5-fluorocytosinereduced binding of C. albicans, C. tropicalis, and C. kefyr toBECs, and the effect of amphotericin B and 5-fluorocyto-sine combined was greater than that of either alone(Abu-el Teen et al., 1990). A one-week course of flucona-zole also reduced the adherence of C. albicans to BECs(Darwazeh et al., 1991). Drug treatment could be affectingcell-surface charge, or wall and membrane biosynthesisand structure.

Binding of C. albicans to exfoliated epithelial cells isaffected by growth conditions of the fungus and can beinhibited by several reagents. Although there are somedifferences among studies that may reflect the complex-ity of growth conditions and adhesin expression, there isprogress in characterizing the interactions and identify-ing the fungal adhesins and host receptors. Growth of C.albicans in media containing glucose, sucrose, galactose,xylitol, or maltose enhanced binding to BECs and HeLacells; maltose was the most effective and glucose theleast effective sugar (Samaranayake and MacFarlane,1982). Growth in the presence of glucocorticoids, dexa-methasone, or triamcinolone acetonide also increasedadherence to BECs (Ghannoum and Abu Elteen, 1987).In one study, organisms grown at 250C were more adher-ent than those grown at 37C (Lee and King, 1983). Cell-surface hydrophobicity, which is increased at the lowergrowth temperature, is suggested to contribute to, butnot be the predominant mechanism of, adherence toBECs (Hazen, 1989). A decrease in hydrophobicity maycontribute partially to the decrease in binding followingtreatment of C. albicans with cetylpyridium chloride, tau-rolidine, chlorhexidine acetate, or providone-iodine(Jones et al., 1991, 1995). Another study demonstratedthat an increase in temperature during growth promotedadherence, and, as with growth on different carbonsources, this may be due to increased expression of an

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adhesin for a high copy number receptor (Staddon et al.,1990).

Concanavalin A, a lectin-recognizing mannan, is aninhibitor of adherence to BECs (Sandin and Rogers,1982; Sandin, 1987; Macura and Tondyra, 1989). Glucose,galactose, sucrose, or mannose enhanced adherence toBECs, while xylose, ribose, fructose, maltose, lactose, orraffinose had no effect on adherence (Macura andTondyra, 1989). Another study found no effect of galac-tose, N-acetylglucosamine (GIcNAc), ribose, or xylose(Sandin, 1987). Thus, certain sugar residues may beinvolved in a lectin-like adherence interaction or maycross-bridge between adhesin and ligand. Lipids extract-ed from C. albicans or C. tropicalis inhibited binding toBECs and involved individual phospholipids, sterols,and steryl ester but not triacylglycerols or free fatty acids(Ghannoum et al., 1986).

Several C. albicans cell wall proteins have been iden-tified as adhesin candidates for epithelial cells. In onestudy, a yeast cell wall extract fractionated by con-canavalin A-affinity chromatography followed by ion-exchange chromatography yielded a fraction that sub-stantially inhibited yeast cell binding to BECs (Imbert-Bernard et al, 1995). This fraction contained four moi-eties, of which the 38- and 54-kDa proteins were sug-gested as adhesins. 0-linked mannoproteins may also beinvolved in C. albicans adherence to epithelial cells. The C.albicans CaMNTI gene encodes a mannosyl transferaseinvolved in 0-linked mannosylation, and a Camntl null-mutant showed reduced adherence to BECs (Buurman etal., 1998).

Fibronectin was one of the first molecules to be sug-gested as a ligand recognized by a C. albicans adhesin(Skerl et al, 1984). Both BECs and VECs stained with anti-fibronectin antibody, and yeast cells pre-treated withfibronectin showed reduced binding to BECs and VECscompared with untreated cells (Skerl et al., 1984; Kalo etal., 1988). The complement fragment iG3b has also beenimplicated as a ligand involved in epithelial andendothelial cell adherence (Gustafson et al., 1991; Bendeland Hostetter, 1993; Bendel et al., 1995; also see reviewsby Hostetter, 1994; Chaffin et al., 1998). Glucose-growncells express more iC3b receptor than glutamate-growncells and show increased binding to human umbilicalvein cells (HUVCs) (Gustafson et al., 1991). Antibody tothe human iC3b integrin receptor, iC3b, and several RGD(arginine-glycine-aspartic acid)-containing peptidesfrom iC3b reduced binding of C. albicans to HUVCs orHeLa cells (Bendel and Hostetter, 1993). After growth ofHeLa cells in serum-free medium, iC3b and fibronectinwere detected on the cell surface, and treatment withanti-C3 antibody, but not anti-fibronectin antibody,reduced adherence of C. albicans. although the reverseeffect was observed with C. tropicalis. The candidates for

fibronectin and iC3b adhesin(s) are described in moredetail below.

The secreted aspartyl proteinases (SAPs) also appearto contribute to C. albicans adherence to BECs and othersubstrates (Ghannoum and Abu Elteen, 1986; El-Maghrabi et al., 1990; Watts et at., 1998). The SAP genefamily consists of at least seven members encoding 42-to 45-kDa aspartyl proteinases (Hube et al., 1994; Monodet al., 1994). The expression of proteinase isozymesdepends on the strain, cellular morphology, and environ-mental factors (White and Agabian, 1995). Strains defi-cient in one or more of these genes have been con-structed. Deletions in SAPI, SAP2, or SAP3 reducedadherence of the organism to poly-L-lysine, an extracel-lular matrix (ECM) preparation, or (slightly) to BECs(Watts et al., 1998). However, a triple Asap 4-6 mutantshowed decreased adherence to the first two substratesbut increased adherence to BECs. Pepstatin inhibitedbinding of the parental strain to all three substrates. Inaddition to any direct effect on adhesion, proteinasesmay act on the yeast surface to modify adhesins or hostsurfaces to expose ligands.

BEC glycosphingolipid is also an adherence targetfor C. atbicans. Several pathogenic yeasts, including C.albicans, bind to lactosylceramide ltGal( 1 -4)3Glc(l - )Cerl(limenez-Lucho et al., 1990). C. albicans fimbriae bound toBECs and reduced the binding of C. albicans yeast cells toBECs (Yu et al., 1994b). Purified fimbriae bind to an asialo-GM, Igangliotetraosylceramide: 3Gal(1 -3)f3GalNAc( 1-4)3Gal( l-4)f3Glc( 1-I )Cerl immobilized on microtiterplates. The binding of fimbriae to BECs was inhibited upto 80% by asialo-GMP. Pseudomonas aeruginosa also bindsto this glycosphingolipid through pili, and the adhesinsfrom P. aeruginosa and C. albicans appear to share a com-mon binding domain (adhesintope) (Yu et al., 1994c,1996). Antibodies to this domain in the P. aeruginosa pilusprotein inhibit binding of both organisms to BECs, and apeptide derived from this region is also inhibitory.

The presence of candidal lectin-like epithelialadhesins that recognize L-fucose or GlcNAc has beenreported (Critchley and Douglas, 1987a,b). Fucoseinhibits binding of some strains to BECs, and glu-cosamine or GlcNAc inhibits the binding of other strains,suggesting strain-specific receptors. Synthesis of thelectin-like material increased when organisms weregrown on galactose (McCourtie and Douglas, 1985).Extracellular material recognizing L-fucose inhibitedbinding of the homologous strain. Fucose has beenshown to bind to yeast and hyphal cells with approxi-mately 2 x 107 binding sites per hyphal cell, mostly locat-ed adjacent to the hyphal tip (Vardar-Unlu et al., 1998). Afragment of an L-fucose-binding protein was purified byaffinity chromatography with the blood group H trisac-charide antigen that terminates in fucose, and it was sug-

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gested that blood group antigens may act as epithelialcell receptors for C. albicans (Cameron and Douglas,1996). The purified fragment inhibited binding to BECsby up to 80%. Binding to an esophageal cell line (Het-l)is partially mediated by a lectin-like interaction (Enacheet al., 1996). Yeast cells grown on galactose adhere betterthan those grown on glucose, and GlcNAc or glu-cosamine reduce binding by about 40%. A 190-kDa gly-coprotein detected in cell wall extracts of galactose-grown cells was postulated to be responsible for theincreased adherence (Enache et al., 1996).

The serotype A determinant, factor 6, of C. albicansmannan has also been implicated in epithelial adher-ence (Miyakawa et al., 1992). A mutant strain deficient inthe factor 6 determinant, or serotype B strains, showedreduced adherence to BECs compared with non-mutantserotype A strains. Mannan from the wild-type strain andanti-factor 6 antibody inhibited adherence to BECs.

A genetic approach has yielded two candidate C. albi-cans adhesins for epithelial cells. The fact that S. cerevisiaecells adhere to a variety of surfaces significantly less wellthan do C. albicans cells has been used by several groupsto screen C. albicans genomic libraries for sequences thatconfer adherence on the non-adherent yeast. Separatescreens have identified two members of a family of relat-ed genes. Members of the C. albicans ALS (agglutinin-likesequence) family are related to S. cerevisiae agglutiningenes that mediate cell-cell interactions during matingof haploid cells (Hoyer et al., 1995). Als proteins have acentral domain of a tandemly repeated motif that is richin serine, threonine, and proline. The sequence of ALSIcarries a signal for a GPI (glycosyl phosphatidyl inositol)anchor. Another member of the family, ALAl, was isolat-ed by the screening of a library for sequences that con-ferred adherence to ECM (Gaur and Klotz, 1997).Transformed yeast cells bound to fibronectin, laminin,and collagen IV. In addition, adherence to BECs wasincreased, suggesting that the adhesin may be multi-functional, recognizing multiple ligands, and mediatingadherence to different tissues. More recently, ALS1 hasagain been isolated in a screen of a C. albicans genomiclibrary for sequences conferring increased adherence toendothelial cells (Fu et at., 1998). Expression of ALS1 alsosubstantially increased binding of S. cerevisiae to the FaDuoropharyngeal epithelial cell line. More definitive evi-dence for the role of these proteins in candidal adher-ence awaits further analysis in that organism.Nonetheless, members of the Als protein family are cer-tainly candidates for adhesins that mediate adherence ofC. albicans in the oral cavity.

In addition to physical immobilization, adherence ofCandida cells to BECs may lead to alterations in fungalgene expression (Bailey et al., 1995). Analysis of proteinssynthesized by C. albicans three hours following adhesion

to BECs showed that proteins of 52-56 kDa differed in theextract of attached yeast cells compared with those fromunattached yeast or from BECs alone. Furthermore, anti-phosphotyrosine antibodies recognized 54-kDa and 60-kDa species from the attached cells but not from cells incontrol cultures. These results suggest that contact of C.albicans with a surface may activate signaling pathwaysthat result in the expression of adhesins. Some C. albicansstrains demonstrate the phenomenon of phenotypicswitching (Soll, 1997). It is postulated that a 'masterswitch' is responsible for turning off one set of genes andswitching on another set, some of which may be involvedin virulence. It is possible that contact with a particularsurface activates a set of genes involved in adherence to,and penetration of, that surface.

(B) ADHERENCE TO INERT POLYMERSC. albicans adheres to a variety of materials found in med-ical devices, such as catheters and oral prostheses. Thisadherence may promote colonization and infection. C.albicans is able to form biofilms on the surfaces of thesematerials (reviewed in Chaffin et al., 1998). In addition,colonization may contribute to the deterioration of thedevices (Marcuard et al., 1993; Gottlieb and Mobarhan,1994; Busscher et al., 1997; van Weissenbruch et al., 1997),and adherent organisms may be less susceptible to anti-fungal drugs (Kayla and Ahearn, 1995; Hawser, 1996).Most studies have focused on oral devices which maycontain multiple materials. Since these studies used dif-ferent fungal growth conditions, different adherenceassays, and different methods of analysis, the resultscannot be compared directly.

Hydrophobicity has been frequently, but not univer-sally, implicated as a major factor in the adherence ofCandida species to inert polymers. The more hydrophobicspecies C. tropicalis, C. glabrata, and C. krusei adhered moreto these polymers, including those found in dentureresin materials, than the less hydrophobic C. albicans, C.stellatoidea, and C. parapsilosis (Klotz et al., 1985; Minagi etal., 1985, 1986; Miyake et al., 1986). Isolates of C. krusei, anemerging pathogen, showed variable but greaterhydrophobicity than C. atbicans isolates, and there was acorrelation between hydrophobicity and adherence toHeLa cells but not to denture acrylic (Samaranayake etal., 1995). This suggests that factors other thanhydrophobicity might contribute to the hierarchy of viru-lence among Candida species. In an earlier study, isolatesof C. albicans showed greater adherence to acrylic thanisolates of other species (Segal et al., 1988). Adherence isincreased on rough acrylic and silicone rubber surfacescompared with smooth surfaces (Verran and Maryan,1997). The acrylic base for dentures supported lessadherence of C. albicans than tissue conditioners and asoft liner (Okita et al., 1991).

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As dental prostheses are exposed to saliva and oralbacteria, a complex biofilm develops to which C. albicanscells can adhere. The fungus is present in biofilms in var-ious morphological forms, and extracellular materialmay also be present (Hawser and Douglas, 1994). Theextent of biofilm formation is dependent on the nature ofthe inert material; the greatest biofilm formation wasfound on latex, which is frequently used in urinarycatheters, followed by silicone elastomer and polyvinylchloride, often found in central venous catheters.Formation of a biofilm was least on polyurethane and100% silicone. In vitro, gentle liquid flow increased theformation of the extracellular matrix material, in whichthe organism was embedded, compared with static con-ditions (Hawser et al., 1998).

As with adhesion to BECs, treatment of either dentalacrylic or C. albicans cells with antimicrobial agentsaffects adhesion. Incubating acrylic with chlorhexidinegluconate, amphotericin B, nystatin, but not a histidinepolypeptide, reduced binding to the polymer (McCourtieet al., 1985, 1986a,b; Spiechowicz et al., 1990). Exposure ofstationary-phase cells to chlorhexidine for a short peri-od, or growth of C. albicans in a sublethal concentration ofchlorhexidine, reduced the adherence of the cells com-pared with unexposed cells, and the treated cells weremore susceptible to the action of :3-glucanase. This sug-gests an effect of chlorhexidine on the fungal cell wall.When C. albicans was grown in subinhibitory concentra-tions of antifungals, exposure to azalomycin F andaculeacin A increased subsequent adherence to acrylic,while exposure to miconazole, ketoconazole, andamphotericin B did not alter adherence (Miyake et al.,1990). Exposure to drugs did not change cell-surfacehydrophobicity, while the negative charge of the cell sur-face decreased in the more adherent cells, suggestingthat a decrease in electric repulsive force enhanced bind-ing. Growth of C. albicans, C. krusei, C. kefyr, C. tropicalis, C.parapsilosis, and C. guilliermondii in subinhibitory concen-trations of sodium hypochlorite resulted in subsequentreduction in adherence of all C. albicans strains and mostother species to polystyrene and BECs (Webb et al.,1995). Growth in hypochlorite appeared to increase thenumbers and amounts of certain proteins in cell wallextracts from C. albicans and C. parapsilosis, again indicat-ing alterations in the cell wall composition.

Several surface mannoproteins, among themhydrophobic proteins, have been suggested as adhesincandidates for plastics (reviewed by Fukazawa andKagaya, 1997; Chaffin et al., 1998). Yeast cells grown ingalactose were more adherent to acrylic than thosegrown in medium containing glucose, sucrose, fructose,or maltose (McCourtie and Douglas, 1981). Materialfound in the growth medium, when used to pre-treatacrylic or BECs, promoted adherence of C. albicans cells to

acrylic but reduced adherence to BECs (McCourtie andDouglas, 1985). When germ tubes that adhered to poly-styrene were physically removed, several mannoproteinswere subsequently solubilized from the plastic (Tronchinet al., 1988). Two major constituents of 60 and 68 kDa andtwo minor constituents of high molecular mass (. 200kDa) were obtained. While the relationship of the small-er species to similar-sized proteins described below asrecognizing other ligands is unknown, the size similarityhas supported conjecture that there may be multi-func-tional adhesins recognizing a variety of ligands. A 58-kDaand a 37-kDa protein which bind fibrinogen and laminin,respectively, also bind to plastic and have been suggest-ed to possess hydrophobic domains (Lopez-Ribot et al.,1991, 1995). Among extracted cell wall proteins, there aremany that have hydrophobic domains. Analysis of pro-teins adsorbed to latex beads showed a spectrum of pro-teins in the 20- to 67-kDa range that may be more abun-dant in extracts from germ tubes (Lopez-Ribot et al.,1991). Hydrophobic interaction chromatography ofextracted proteins suggested that the hydrophobic pro-teins were usually smaller (< 50 kDa) than thehydrophilic proteins (> 90 kDa), perhaps reflecting theextent of glycosylation (Hazen and Hazen, 1992, 1993;Hazen and Glee, 1994). Hydrophilic cells exhibit a denselayer of fibrils not observed on hydrophobic cells, and ithas been proposed that this layer masks the hydropho-bic species. In keeping with this suggestion, the abun-dance of the acid-labile phosphodiester-linked manno-oligosaccharides was less in mannan from hydrophobiccells than in that from hydrophilic cells (Masuoka andHazen, 1997).

(C) ADHESION TO TEETHThe mouth is a unique part of the body in that it containsexposed mineralized tissues, in the form of teeth. Beadsof crystalline hydroxyapatite (HA) have been used inadhesion assays as a model for studying microbial adhe-sion to tooth surfaces (Clark et al., 1978). C. albicans cellsdo not bind well to hydrated HA beads, but adherence isstimulated greatly by pre-incubation of the beads witheither whole or parotid saliva ( Cannon et al., 1995b;O'Sullivan et al., 1997). Adherence to saliva-coatedhydroxyapatite (SHA) beads is strain-specific (O'Sullivanet al., 1997), and strains more frequently associated withcandidiasis adhere significantly better to SHA beadsthan do less pathogenic strains (Schmid et al., 1995b).

(D) CO-ADHERENCEC. albicans cells co-adhere with several species of oralbacteria, including Streptococcus gordonii, S. mutans, S. oralis,S. sanguis, S. salivarius, and Actinomyces species (Richardsand Russell, 1987; Branting et al., 1989; Jenkinson et al.,1990; Holmes et al., 1995b; Millsap et al., 1998). The

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growth conditions for the bacteria, however, can affectco-adherence (Richards and Russell, 1987; Millsap et al.,1998), and some assays do not take into account thekinetics associated with the larger size of yeast cells(Millsap et al., 1998). Colonizing acrylic with oral strepto-cocci in the presence, but not in the absence, of sucroseenhanced binding of C. albicans (Richards and Russell,1987; Branting et al., 1989). C. albicans bound in greaternumbers to acrylic pieces coated with S. sanguis, S.mutans, or S. sobrinus than to uncoated acrylic, but in thiscase, pre-incubation of the bacteria with sucrose toinduce synthesis of extracellular polymers did notincrease binding (Vasilas et al., 1992). Protein-protein andlectin interactions have been proposed for the adhesiveinteractions between Candida and bacteria, althoughhydrophobic and electrostatic interactions may also takepart (Millsap et al., 1998). Both carbohydrate (Holmes etal., 1995b) and protein molecules (Holmes et al., 1996)that act as C. albicans receptors have been identified onthe surface of S. gordonii. A carbohydrate containingrhamnose, glucose, GlcNAc, and galactose, isolated fromthe cell walls of S. gordonii cells, acted as a receptor for C.albicans adherence in an in vitro assay (Holmes et al.,1995b). Gene disruption experiments have shown that C.albicans adherence to bacteria is multifactorial, and inter-actions involving the S. gordonii cell-surface polypeptidesCshA, CshB, SspA, and SspB contribute to co-adherence(Holmes et al., 1996). The co-adherence of C. albicans withoral bacteria is species-specific. Pre-treating BECs or den-ture acrylic with S. salivarius, Escherichia coli, or Porphyromonasgingivalis reduced subsequent adherence of C. albicans cells(Nair and Samaranayake, 1996a,b). Also, in one report, abiofilm of S. gordonii reduced adherence of most C. albicansstrains and other species to polystyrene (Webb et al.,1995). This would suggest that C. albicans recognizes spe-cific receptors on certain oral bacteria which are expressedunder particular growth conditions.

(E) ADHERENCE TO SALIVA MOLECULESIn the oral cavity, proteins from saliva selectively adsorbto surfaces to form acquired pellicles. The acquiredenamel pellicle has been particularly well-studied, andafter two hours' formation it has been found to containimmunoglobulins, mucin, at-amylase, cystatins, proline-rich proteins, lysozyme, glucosyltransferases, albumin,fibrinogen, and serum components (Kraus et al., 1973;Rolla et al., 1983; Al-Hashimi and Levine, 1989; Jensen etal., 1992; Edgerton et al., 1996). The composition of thepellicle depends on the underlying surface (Edgerton etal., 1996) and the composition of the saliva (Jensen et al.,1992; Edgerton et al., 1996). The intra-oral composition ofsaliva varies (Sas and Dawes, 1997), and this affects thepellicles formed at different sites, and hence the patternof microbial colonization. Since all surfaces are coated

with a salivary pellicle, it is reasonable to suppose thatmicrobial adherence interactions involve adsorbed salivamolecules. Saliva pellicles increase the adherence of C.albicans cells to HA beads (Cannon et al., 1995b), poly-methylmethacrylate (Edgerton et al., 1993), and to S. gor-donii cells (Holmes et al., 1995a) (Fig. 2b). Adherence of C.albicans was greater to dental acrylic coated with wholesaliva than to uncoated acrylic, and a coating of parotidsaliva stimulated adherence more than a coating of sub-mandibular-sublingual saliva (Vasilas et al., 1992). In anearlier study, however, adhesion to acrylic was reducedby an 18-hour whole-saliva pellicle (Samaranayake et al.,1980). Also, coating of acrylic surfaces with whole salivareduced the contact angle and decreased the binding ofhydrophobic Candida strains, while the adherence ofmore hydrophilic C. albicans was unaffected (Miyake et al.,1986). Adherence to two experimental silicone soft-lin-ing materials was less than to a commercial product orthe acrylic base, varied with the strains, and was reducedwhen the materials were coated with saliva (Waters et al.,1997). In another study, however, coating soft liners withsaliva or serum increased adherence of C. albicans andbiofilm formation, although the effect varied with thematerial and protein source (Nikawa et al., 1997). Coatingalso increased firm colonization and hyphal invasion,although the plasticizer used affected cavitation.Incubating polymethylmethacrylate beads with sub-mandibular-sublingual saliva enhanced C. albicans bind-ing compared with coating them with parotid saliva(Edgerton et al., 1993). Binding was reduced by treatmentof yeast cells with protease or glycosidase or incubationwith mannose or galactose. Interestingly, C. albicans cellsdo not detectably bind proteins from saliva in the fluidphase, apart from small amounts of mucin MG1 and MG2(Edgerton et al., 1993; Newman et al., 1996), which wouldexplain why added saliva did not inhibit adherence of C.albicans to SHA beads (Cannon et al., 1995b). This indi-cates that C. albicans may have specific adhesins that rec-ognize cryptitopes on saliva molecules that are exposedwhen the molecules adsorb to surfaces (Fig. 2b). Suchadhesins would promote colonization and prevent sali-va-mediated aggregation and clearance from the oralcavity.

In order to identify the saliva proteins to which C.albicans cells adhere, investigators have developed blotoverlay assays (Newman et al., 1996; O'Sullivan et al.,1997), in which saliva proteins are separated by SDSpolyacrylamide gel electrophoresis, electroblotted ontonitrocellulose membranes, and incubated with eitherradiolabeled (O'Sullivan et al., 1997) or fluorescentlylabeled (Newman et al., 1996) yeast cells.Autoradiography or photography, respectively, revealsthe protein bands to which the cells bind. These studieshave identified basic proline-rich proteins, including IB-

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6 (O'Sullivan et al., 1997) and Psi (Newman et al., 1996),as receptors for C. albicans adhesion.

(5) GrowthIn order to maintain Candida populations in the oral cav-ity, cells must grow and multiply at a rate at least equalto that of clearance. The growth rate of C. albicans in sali-va is too low to be measured accurately, due to carbonsource limitation (Samaranayake et al., 1986), which ispresumably caused by the large number of bacteria insaliva. Therefore, any metabolic activity that helps C. albi-cans acquire carbon or nitrogen will aid its growth andsurvival in the oral cavity. C. albicans secretes aspartyl pro-teinases, which are believed to contribute to the orga-nism's virulence in several ways (Hoegl et al., 1996).Tissue destruction may aid fungal penetration, but thisprocess could also release peptides as a source of nitro-gen or carbon. The proteinase Sap2, for example,degrades gastrointestinal mucin, and mucin can act as asole nitrogen source for C. albicans (Colina et al., 1996). Inaddition, C. albicans secretes the hydrolytic enzyme N-acetylglucosaminidase (also called hexosaminidase),which cleaves chitobiose, the dimer of GIcNAc, into twomolecules of GIcNAc (Sullivan et al., 1984; Niimi et al.,1997a). C. albicans can use GlcNAc as either a carbon ornitrogen source. N-acetylglucosaminidase activity isshown by C. albicans and C. dubliniensis and to a lesserextent by C. tropicalis cells (Niimi and Cannon, unpub-lished observation), species found relatively frequently inthe oral cavity. It is tempting to speculate that a functionof this enzyme may be to cleave terminal GIcNAc residuesfrom host glycoproteins, and that this scavenging activitygives these Candida species a selective growth advantage.

Competition with other oral micro-organisms fornutrients, such as glucose, affects the growth rate ofCandida cells. It is recognized that antibiotic treatment,which reduces the number of oral bacteria, is a predis-posing factor for oral candidiasis (Samaranayake, 1990).Oral bacteria are present in most oral sites at concentra-tions much higher than C. albicans, and so the Candidacells must compete with them for adhesion sites andnutrients, and be exposed to bacterial toxins and by-products.

(6) Evading Host Clearance MechanismsA major factor influencing the balance among clearance,colonization, and candidiasis is the interaction betweenC albicans cells and the host defenses (Cannon et al.,1 995a). Immune system defects are a major risk factor forcandidiasis. Innate defenses include the epithelial barri-er and anti-candidal compounds in saliva such aslysozyme (Tobgi et al., 1988), histatins (Xu et al., 1991),lactoferrin (Nikawa et al., 1993), and calprotectin (Mulleret al., 1993; Challacombe, 1994). Acquired immunity

includes the production of immunoglobulins and, if tis-sues are penetrated, the involvement of macrophagesand polymorphonuclear leukocytes (Challacombe, 1994).The major immunoglobulin in saliva is secretory IgA(SIgA); serum immunoglobulins enter the saliva via thegingival crevicular fluid, but are present at low concen-trations. SIgA does not fix complement efficiently; itsmajor role is the agglutination of micro-organisms,which are then swallowed more easily. Anti-Candida SIgAcan be detected in saliva, and its concentration isincreased in whole or parotid saliva from HIV-positiveindividuals, but reduced in AIDS patients, suggestingthat a compensatory response is overcome with progres-sive immunodeficiency (Challacombe and Sweet, 1997).

C. albicans can be ingested by neutrophils andmononuclear phagocytic cells (for a review of interac-tions with macrophages, see Vazquez-Torres and Balish,1997). The interactions between these cells and C. albi-cans appear to involve both opsonic and non-opsonic fac-tors. Components from the classic and alternative com-plement pathways can also enhance phagocytosis bymacrophages and neutrophils (Solomkin et al., 1978;Marodi et al., 1991). C. albicans activates the alternatepathway of complement, and both iC3b and C3d frag-ments can bind to C. albicans (adhesins for these frag-ments are discussed below). A candidal-protective mech-anism has been proposed in which fungal binding ofiC3b blocks neutrophil CR3 recognition of iC3b andphagocytosis of iC3b-coated C. albicans is reduced. Yeastcells coated with an anti-human CR3 antibody, thatblocked the candidal binding protein, were phagocy-tosed by neutrophils to a greater extent than uncoatedcells (Gilmore et al., 1988). The clumping of C. albicanscells coated with C3 fragments has also been proposedas a candidal-protective effect, since these aggregatesare too large to be phagocytosed (Heidenreich andDierich, 1985). On the other hand, host protection ispostulated for the binding of serum vitronectin, sinceCandida cells coated with vitronectin show enhancedbinding to macrophages and phagocytosis (Limper andStanding, 1994).

Macrophage mannose receptors also mediate theadherence of C. albicans (reviewed by Vazquez-Torres andBalish, 1997). Binding of C. albicans to murine spleen andlymph node tissue is primarily to macrophages (Kanbe etal., 1993). A (31,2-linked mannotetraose in the acid-labileC. albicans mannan as well as an acid-stable structurewere identified as adhesins (Li and Cutler, 1993; Kanbeand Cutler, 1994). A monoclonal antibody to (1,2-linkedoligomannosaccharide, but not a monoclonal antibodyto the acid-stable mannan epitope, in the presence ofcomplement, enhanced phagocytosis of yeast cells byneutrophils (Caesar-TonThat and Cutler, 1997). Solublemannan can inhibit phagocytosis of complement-C3-

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coated C. albicans by macrophage (Kolotila et al., 1987).Thus, C. albicans phagocytosis may be mediated by eitherthe complement-dependent or mannose receptor path-ways. The effect of mannan on the complement-mediat-ed pathway, however, suggests that the pathways mayinteract physiologically or physically. C. albicans hasdeveloped other ways of evading the innate and theacquired immune system. The C. albicans cell wall manno-proteins proposed to be receptors for SIgA binding arereleased into the medium as yeast cells produce hyphae(Ponton et al., 1996). The shedding of these receptors mayenable the hyphae to escape clearance. In addition, theimmunoglobulins present in saliva, including SIgA, IgG,and IgM, are substrates of C. albicans secreted aspartylproteinase (Ruchel, 1986; Reinholdt et al., 1987).

(A) FINDING BETTER ENVIRONMENTSC. albicans will grow more quickly and reach higher cellconcentrations in environments with less effective clear-ance mechanisms and better nutritional supply. Candidacells have an array of mechanisms (virulence factors)which enable them to colonize new environments, aprocess often involving tissue penetration. Polarizedhyphal growth facilitates directional growth toward a dif-ferent environment, and thigmotropism or contact sen-sing (Sherwood et al., 1992) could allow the hyphae toinvade tissues. C. albicans cells also secrete a number ofhydrolytic enzymes which may play a role in tissuedestruction and penetration. Phospholipase or N-acetyl-glucosaminidase production by C. albicans strains, forexample, correlated with virulence in mouse infectionmodels (Jenkinson and Shepherd, 1987; Ibrahim et al.,1995). Many workers have investigated the role of secret-ed aspartyl proteinase isozymes in the pathogenesis ofcandidiasis, and demonstrated their expression in vivo(Borg and Ruchel, 1988; Ray and Payne, 1988; El-Maghrabi et al., 1990; De Bernardis et al., 1995; Hoegl et al.,1996; Borg-von Zepelin et al., 1998). In addition to playinga role in adherence, as discussed above, secreted pro-teinases may aid in tissue destruction and penetration.

Penetration of tissues brings C. albicans into intimatecontact with other cellular structures and host moleculeswhich could act as adhesion receptors. Tissue penetra-tion also often involves breaching endothelial barrierswith consequential endothelial cell injury (Filler et al.,1995). Depletion of endothelial cell iron reduces phago-cytosis of the fungus and results in less cell injury (Frattiet al., 1998). Phagocytosis and subsequent injury is alsoreduced by treatment of endothelial cells with gammainterferon (Fratti et al., 1996). Migration across anendothelial layer is facilitated by hyphal formation (Zinketal., 1996).

Several potential adhesins and ligands that mediatebinding of the fungus to endothelial cells have been

identified and may share identity with moleculesinvolved in epithelial adherence (reviewed by Hostetter,1994; Fukazawa and Kagaya, 1997; Chaffin et al., 1998).The iC3b binding protein(s) discussed below contributedto adherence to umbilical vein endothelium. Two mono-clonal antibodies that recognize human integrin subunittm partially inhibited adherence (Gustafson et al., 1991).Adherence to cultured human dermal microvascularendothelial cells was also reduced by treatment with ananti-human CR3 monoclonal antibody (Lee et al., 1997).Fibronectin has been observed, by indirect immunofluo-rescence, on endothelial cells as well as on epithelialcells (see above). Fibronectin was detected on rabbit aor-tic valves in a model of non-bacterial thrombotic endo-carditis (Scheld et al., 1985). C. albicans and C. tropicalis,which are often isolated from infections, bound signifi-cantly better to fibronectin in vitro than the infrequentlyisolated C. krusei. During the analysis of adherence toendothelial monolayers, it was noted that C. albicansadhered preferentially to subendothelial ECM (Klotz,1987; Klotz and Maca, 1988). The candidal binding pro-teins for iC3b and fibronectin are discussed below.

(B) ADHERENCE TO ECM AND SERUM PROTEINSC. albicans binds to several host proteins found in serumand in ECM (Table 2; reviewed by Hostetter, 1994;Fukazawa and Kagaya, 1997; Sturtevant and Calderone,1997; Chaffin et al., 1998). Serum proteins that bind to thefungus include serum albumin, transferrin, fibrinogen,and the complement C3 fragments, C3d and iC3b(Heidenreich and Dierich, 1985; Bouali et al., 1986; Pageand Odds, 1988). ECM components that bind to the fun-gus include fibronectin, laminin, entactin, collagen typeI and type IV, and vitronectin (Skerl et al., 1984; Boucharaet al., 1990; Klotz, 1990; Jakab et al., 1993; Lopez-Ribot andChaffin, 1994). While the host ligands have been identi-fied, less progress has been made with the identificationof the fungal adhesins, many of which appear to beexpressed more abundantly on the surfaces of germtubes than on yeast cells (Heidenreich and Dierich, 1985;Bouali et al., 1986; Bouchara et al., 1990; Lopez-Ribot andChaffin, 1994). Some adhesins may recognize multipleligands, since similar-sized proteins have been identifiedas potential adhesins for several ligands. Differences inadhesin identification among various studies furthercomplicate the issue.

The interactions between human serum albumin andtransferrin and C. albicans have not been studied exten-sively. The two proteins were observed by indirectimmunofluorescence to bind preferentially to germtubes (Page and Odds, 1988). More recently, binding of C.albicans cells to bovine serum albumin, immobilized onmicrotiter plates, was reported, and binding was affectedby pre-incubation of the yeast with alanine, proline, and

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TABLE 2Oral Ligands for C. albicans Adhesion, and Possible Interventions to Prevent Colonization

Adhesion Ligand Type of Interaction Reference Possible Interventionsa

BEC Protein-protein, lectin, hydrophobic, Hazen, 1989; Macura CHOb (e.g., CSEC), peptidefimbrial glycoprotein-BEC and Tondyra, 1989;glycosphingolipid Jimenez-Lucho et al., 1990;

Yu et al., 1 994a,b,c; Imbert-Bernardet al., 1995; Cameron andDouglas, 1996

Dental acrylic Hydrophobic, glycoprotein-acrylic Klotz et al., 1985; McCourtie Surface modificationand Douglas, 1985;Minagi et al., 1985

Oral bacteria Lectin, protein-protein Holmes et al., 1995b, 1996; ?CHOMillsap et al., 1998

Adsorbed salivary proteins Lectin, ?protein-protein Newman et al., 1996; ?CHOO'Sullivan et al., 1997

iC3b Protein-protein Hosteffer et al., 1990; Alaei Peptideet al., 1993

ECM proteins: Fibronectin Protein-protein, ?lectin Skerl et al., 1984; Klotz et al., Peptide1993; Gozalbo et al., 1998;Yan et al., 1998a

Laminin Protein-protein Bouchara et al., 1990; Gozalbo Peptideet al., 1998; Yan et al., 1 998a

Entactin Protein-protein L6pez-Ribot and Chaffin, 1994 PeptideCollagen Protein-protein Klotz et al., 1993; Chaffin et al., Peptide

1998Vitronectin Protein-protein, lectin Limper and Standing, 1994; ?CHO

Olson et al., 1996a Non-specific interventions and those involving multiple interactions include antimicrobial mouthwashes.b Carbohydrate (monosaccharides or oligosaccharides).c Chitin-soluble extract.

leucine but not with other amino acids (Hawser and (Bouali et al., 1987; Casanova et al., 1992; Martinez et al.,Islam, 1998). Human fibrinogen binds extensively to 1994). Binding of C. albicans to platelets appears to begerm tubes, while binding to yeast cells appears to mediated via the interaction with fibrinogen (Robert et al.,depend on growth conditions (Bouali et al., 1987; Page 1996). Since fibrinogen can be a component of theand Odds, 1988). The major protein that interacted with enamel pellicle (Kraus et al., 1973), it may also be a recep-fibrinogen had a molecular mass of 68 kDa and also tor for C. albicans adherence in the oral cavity.bound to plastic, laminin, and C3d (Tronchin et al., 1988; C. albicans germ tubes have the ability to rosette anti-Annaix et al., 1990; Bouchara et al., 1990). A 58-kDa pro- body-sensitized erythrocytes coated with C3d or iC3btein, encoded by FBP1, is also a fibrinogen-binding pro- (Heidenreich and Dierich, 1985). Functional and anti-tein, but it does not appear to recognize the other lig- genic similarities between the fungal and mammalianands (Casanova et al., 1992; Lopez-Ribot et al., 1997). The proteins that bind these moieties have led to frequent58-kDa fibrinogen-binding protein is modified by cova- use of terminology adopted from mammalian systems.lent attachment of the ubiquitin polypeptide (Seputlveda The fungal binding proteins are sometimes referred to aset al., 1996), contains epitopes or sequences from a type an integrin analog or CR3 (iC3b binding proteins) andIV collagen molecule (Seputlveda et al., 1995), and is CR2 (C3d binding protein). The receptor for iC3b mayexpressed in vivo (L6pez-Ribot et al., 1996). The fibrino- play a role in adherence to epithelial and endothelialgen-binding species are distributed heterogeneously on cells, as discussed above. Antibodies that recognize sub-the cell surface, as determined by transmission, scan- units of human integrins which bind iC3b react with thening electron, and confocal fluorescence microscopy fungal cell surface. Using an anti- aXm antibody and fluo-

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rescence flow cytometry, Gilmore et al. (1988) observed alow level of receptor expression on yeast cells that wasaffected by the concentration of glucose in the growthmedium and was strain-specific. There is no consensuson the identity of the iC3b binding protein(s), since vari-ous studies implicate different components, including165-, 130-, 66-, 55-, and 42-kDa proteins (Eigentler et al.,1989; Hostetter et al., 1990; Alaei et al., 1993).Oligonucleotide probes based on the postulated similar-ity to integrins were used to isolate a candidal integrinhomologue Intlp (Gale et al., 1996). Although it has lim-ited similarity to integrins, the deduced Intlp sequencecontains a membrane spanning region and divalentcation-binding motifs. Expression of the gene in S. cere-visiae resulted in aberrant cell morphology with formationof germ-tube-like structures (Gale et al., 1998). A mutantstrain deficient in expression of this gene exhibitedreduced binding to HeLa cells, reduced hyphal formationunder some conditions, and reduced virulence in mice.Several hypotheses on the role of iC3b- and C3d-bindingin the pathogenesis of candidiasis have been proposed."Bystander" deposition of complement fragments onerythrocytes following activation of the alternate com-plement pathway by the fungus may promote fungalrosetting of coated erythrocytes. This could provide fun-gal access to erythrocyte iron through candidal surfacehemolysin-mediated erythrocyte lysis (Manns et al.,1994). Also, binding iC3b may mask the cells and preventphagocytosis, as discussed above.

Two major C3d-binding components have been iden-tified, a 60-kDa moiety expressed on the surfaces of germtubes and a 50-kDa moiety in the yeast plasma mem-brane (Calderone et al., 1988; Linehan et al., 1988). Inother studies, antibodies to the major C3d-binding pro-tein (60-kDa species) reacted with several components.These molecules included a major 50- to 60-kDa moietyand minor species of 94, 67-68, 60, 50, 40, 31, and 20 kDa(Kanbe et al., 1991; Franzke et al., 1993; Lopez-Ribot et al.,1995). The C3d receptor is expressed, in vivo, on orga-nisms recovered from peritoneal lavage and in kidneysfrom infected mice (Kanbe et al., 1991). A role for C3d-binding in pathogenesis is mostly speculative.Hypotheses include: that aggregation of opsonized andunopsonized cells could protect the fungus from phago-cytosis; a role in iron acquisition, as suggested above foriC3b binding; or binding to any host cell on which C3dwas deposited.

The ability of C. albicans to bind to the ECM ligandsfibronectin, laminin, entactin (nidogen), types I and IVcollagen, and vitronectin has been the focus of numer-ous studies in the last decade (for recent reviews, seeFukazawa and Kagaya, 1997; Sturtevant and Calderone,1997; Chaffin et al., 1998). Some of these ECM compo-nents are able to form complexes among themselves,

and, thus, ECM presents a host target with multiplebinding sites for the fungus. The identity of the adhesinsthat recognize ECM components is unresolved and thesubject of some disagreement between studies.Fibronectin is found in both plasma and ECM and wasone of the first host proteins identified as a ligand pro-moting C. albicans adherence (Skerl et al., 1984). Species of62 kDa and 72 kDa have been isolated by affinity chro-matography as adhesin candidates for fibronectin andcollagen (Klotz et al., 1993). The expression of fibronectin-binding capacity is regulated in part by environmentalconditions. Growth medium and temperature can alterthe extent of binding (Jakab et al., 1993; Negre et al., 1994).Recently, growth in the presence of hemoglobin has beenreported to induce enhanced expression of a 55-kDapromiscuous adhesin that recognized fibronectin,laminin, and fibrinogen (Yan et aci., 1998a).Glyceraldehyde-3-phosphate dehydrogenase, a 33-kDaprotein present predominantly on the yeast cell surface,is also a fibronectin- and laminin-binding protein(Gozalbo et al., 1998). Laminin adhesins of 68 kDa and 62kDa have been identified by ligand affinity blotting(Bouchara et al., 1990). These receptors may be the sameas the proteins binding to plastic (Tronchin et al., 1988)and fibrinogen (Annaix et al., 1990) and, indeed, lamininbinding was reduced competitively by fibrinogen(Bouchara et al., 1990). A 37-kDa receptor for lamininthat appeared not to recognize other ligands was firstdetected with an antibody produced to a human high-affinity laminin receptor (Lopez-Ribot et al., 1994). Threeproteins (65 kDa, 44 kDa, 25 kDa) were detected, by lig-and affinity blotting, as candidates for entactin bindingproteins (Lopez-Ribot and Chaffin, 1994). Binding of cellwall protein to immobilized entactin was reduced byfibronectin and laminin. Vitronectin appears to bindboth to cell wall protein (30-kDa moiety; Limper andStanding, 1994) and to 13-glucan (Olson et al., 1996).Fibronectin inhibited binding of vitronectin to the fungus(Jakab et al., 1993).

Several studies have reported that peptides with theRGD (arginine-glycine-aspartic acid) or related motifwere inhibitors of binding between fungal adhesins andECM components (Klotz et al., 1992; lakab et al., 1993;Lopez-Ribot and Chaffin, 1994), while other studies havesuggested that the RGD motif is not a contributor to theinteractions (Negre et al., 1994; Yan et al., 1998b). Parallelsbetween fungal ECM binding proteins and mammalianintegrins that recognize ECM ligands and the RGD motifraise the possibility of structural and antigenic related-ness. Antibody to the a531 mammalian integrin andmonoclonal antibodies to each subunit reacted with C.albicans, and the reactivity increased as yeast cells formedgerm tubes (Santoni et al., 1994). Both high- and low-affinity receptors have been detected, and the number of

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binding sites per C. albicans cell ranges from 5000 to98,000 (Bouchara et al., 1990; Limper and Standing, 1994;Negre et al., 1994). In vivo evidence for a role of adhesionto fibronectin or any other ECM ligand in pathogenesis isscant. In a rabbit model of infection, administration of anRGD-containing peptide reduced fungal counts in sever-al organs (Klotz et al., 1992). The promiscuity of adhesins,the involvement of multiple adhesins in adherence, andthe presence of some host ligands, such as fibronectin,in both superficial and deep sites suggest that there isnot a strict separation between the adhesins and mech-anisms the fungus uses to colonize cutaneous andmucocutaneous surfaces and that those that areinvolved in deep tissue invasion.

(7) Clinical Significance of Colonizationand Its Prevention

(A) INCIDENCE OF CANDIDIASISThe balance between colonization and mucosal candidia-sis (Fig. 1) depends on the effectiveness of the hostdefenses. A corollary of this is that candidiasis is often anindication of an underlying immune deficiency. Oral can-didiasis affects a large proportion of HIV-positive individ-uals and those with AIDS (Greenspan and Greenspan,1996; Darouiche, 1998), and approximately 90% of AIDSpatients have suffered from oropharyngeal or esophagealcandidiasis at some stage of their illness (Alexander andPerfect, 1997) Oropharyngeal candidiasis is also oftenseen in the elderly, and merits investigation of potentialpre-disposing factors (Shay et al., 1997). A comprehensiveanalysis of the literature concerning the treatment ofimmunocompromised patients with oropharyngeal oresophageal candidiasis indicates that optimal results areachieved with the triazoles fluconazole and itraconazole(Darouiche, 1998). They offer clinical efficacy at least com-parable with that of the polyenes and imidazoles, togeth-er with a highly favorable mycological cure rate.

During the 1980s and 1990s, the frequency of nosoco-mial candidiasis has increased dramatically (Beck-Sagueand Jarvis, 1993; Pfaller, 1995, 1996). In a study of datafrom the USA National Nosocomial InfectionsSurveillance System, C. albicans was the most frequentlyisolated fungal pathogen (59.7%) in hospital environ-ments (Beck-Sague and Jarvis, 1993). Transfer of Candidabetween individuals often occurs via the hands of healthcare workers (Strausbaugh et al., 1994), and nosocomialtransmission can occur without candidiasis outbreaks(Schmid et al., 1995a). This reinforces the need for appro-priate cross-infection control in the dental surgery.

(B) RECURRENCEA common feature of candidiasis is recurrence. Factorswhich favor recurrence include: re-inoculation from colo-

nized individuals and the environment; underlyingimmune suppression; and endogenous reservoirs forreinfection. We have discussed the ease with which C.albicans can enter the oral cavity and the central impor-tance of immune suppression in the development of can-didiasis. There can also be reservoirs for re-infectionwithin the oral cavity. C. albicans can be isolated fromplaque (Arendorf and Walker, 1980), oral biofilms (Kaylaand Ahearn, 1995; Hawser, 1996), and occluded acrylicdenture surfaces, where it will be relatively well-protect-ed from topical antifungal agents but could be releasedinadvertently by routine dental hygiene procedures andlead to colonization of other oral sites.

(C) CANDIDA SPECIES AND DRUG RESISTANCEAlthough C. albicans remains the most common cause ofnosocomial candidiasis, infections due to non-albicansspecies are increasing (Pfaller, 1996). Some of thesespecies, namely, C. glabrata and C. krusei, are intrinsicallyless sensitive to azole drugs. Another interesting trend isthe increased association of a novel Candida species, C.dubliniensis, with oral infection in AIDS patients (Coleman etal., 1997; Sullivan and Coleman, 1998). C. dubliniensis isclosely related to C. albicans (Coleman et al., 1997), and aswith C. albicans, stable fluconazole resistance can beinduced by exposure to the drug (Albertson et al., 1996;Moran et al., 1997). Treatment of AIDS patients with pro-longed courses of azole antifungal agents appears to haveselected for the development of azole-resistant C. albicansstrains (White, 1997a; Darouiche, 1998). Resistance can bedue to mutations in the drug target (White, 1997b;Sanglard et al., 1998) or over-expression of drug effluxpumps (Sanglard et al., 1995; Albertson et al., 1996). Over-expression of drug pumps from the ATP binding cassette(ABC) family of efflux pump can lead to cross-resistance toseveral azole antifungals (Albertson et al., 1996; Niimi et al.,1997b). Most azole-resistant strains, however, retain sen-sitivity to amphotericin B (Albertson et al., 1996).

(D) PROSPECTS FOR STRATEGIESTO PREVENT COLONIZATION

An attractive alternative to treating patients with can-didiasis, which is often recurrent, is to prevent the infec-tion from occurring. This could be achieved by increasingthe concentration of natural anti-candidal compounds inthe mouth or by preventing adherence. Denture resin hasbeen modified to enhance adsorption of the anti-candi-dal salivary protein histatin 5 (Edgerton et al., 1995).Although adsorbed histatin 5 did not have an anti-candi-dal effect, desorbed histatin did, and modified dentureacrylic loaded with histatin 5 could provide a localizedcontrolled release of the protein. There is also theprospect of using gene therapy to over-express histatinsin saliva (O'Connell et al., 1996).

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Another approach is the prevention of colonizationby inhibiting C. albicans adherence. This could beachieved by immunizing the host or by physical interfer-ence with adherence mechanisms (Table 2).Theoretically, the application of soluble receptors, lig-ands, or the domains of these molecules involved inadherence could be used to prevent microbial coloniza-tion (Mandel, 1996). There is evidence that chitin isinvolved in C. albicans adherence, and a chitin-solubleextract (CSE) has been used to inhibit yeast adhesion toa variety of cells (Segal, 1996). Although most adhesioninhibition experiments have been carried out in vitro, ini-tial in vivo results appear to be promising (Segal, 1996).The oral environment is rich in proteinases, and any useof peptides to inhibit adhesion may necessitate frequentapplication. Other approaches to prevent microbial colo-nization include immunization (lenkinson and Lamont,1997). Passive immunization with recombinant plantmonoclonal secretory antibodies to an S. mutans adhesinhas been used to inhibit specific microbial colonizationin humans (Ma et al., 1998). As significant adhesins in C.albicans are identified, this approach could also be usedto preclude candidiasis. Salivary IgA antibodies havebeen shown to reduce the adherence of C. albicans cells toBECs (Epstein et al., 1982; Challacombe, 1994), and thestimulation of a mucosal immune response with a C. albi-cans adhesin, possibly expressed by another resident oralmicrobe, may prevent colonization.

The inhibition of S. mutans colonization is being con-sidered for the prevention of caries (Ma et al., 1998), aprocess which will make a significant, and possibly dele-terious, change to the oral flora. C. albicans cells, howev-er, are present in low numbers, and so removal is notlikely to have adverse effects on the remaining flora.From this review, it is evident that C. albicans can utilize anumber of adherence mechanisms, and it is far from cer-tain that inhibiting any one will prevent colonization. Inaddition, C. albicans embedded in plaque may be protect-ed from anti-adhesive compounds. However, the prophy-lactic treatment of susceptible individuals with thesecompounds after aggressive dental hygiene might pre-vent the incorporation of C. albicans into plaque and thedevelopment of a protected reservoir.

(8) ConclusionC. albicans is truly an opportunistic organism. It is themost frequent cause of candidiasis because it is the mostsuccessful yeast at colonizing the oral cavity and so isoften in a position to take advantage of immune sup-pression in the host. C. albicans is the most adherentCandida species, which is probably due to its ability toadhere to many different ligands (Fig. 2b, Table 2). It pos-sesses other virulence factors, such as the secretion ofhydrolytic enzymes and the ability to evade the immune

system, which give it a growth advantage over otheryeast. Subsets of these virulence factors may be activat-ed by changes in the environment or contact with sur-faces. In colonized individuals, however, C. albicans is usu-ally present in the oral cavity only in relatively low num-bers. This indicates that there is a fine balance betweencolonization and clearance, and there is the prospect,with further analysis of major adherence interactions, fortipping this balance in favor of clearance.

AcknowledgmentsRDC acknowledges financial support from the New Zealand LotteriesBoard and the University of Otago. WLC acknowledges support fromUS Public Health Service grants A123416 and A140675. We aregrateful to Dr. Ann Holmes for helpful discussions.

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