Synthetic Analogues of {beta}-1,2 Oligomannosides Prevent Intestinal Colonization by the Pathogenic Yeast Candida albicans

The pathogenic yeast Candida albicans displays at its cell surfaceß-1,2 oligomannosides (ß-1,2-Mans). In contrastto the ubiquitous ${alpha}$ -Mans, ß-1,2-Mans bind to galectin-3,a major endogenous lectin expressed on epithelial cells. Thespecific role of ß-1,2-Mans in colonization of thegut by C. albicans was assessed in a mouse model. A selectedvirulent strain of C. albicans (expressing more ß-1,2-Manepitopes) induced more intense and sustained colonization thanan avirulent strain (expressing less ß-1,2-Man epitopes).Synthetic ( ${Sigma}$ ) ß-and ${alpha}$ -linked tetramannosides with antigenicitiesthat mimicked the antigenicities of C. albicans-derived oligomannosideswere then constructed. Oral administration of ${Sigma}$ ß-1,2-Man(30 mg/kg of body weight) prior to inoculation with the virulentstrain resulted in almost complete eradication of yeasts fromstool samples, whereas administration of ${Sigma}$ ${alpha}$ -Man at the same dosedid not. As most cases of human systemic candidiasis are endogenousin origin, this first demonstration that a synthetic analogueof a yeast adhesin can prevent yeast colonization in the gutopens the possibility of new prophylactic strategies.

INTRODUCTION

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Introduction

Since the 1980s the incidence of systemic Candida infectionsin hospitalized patients has increased dramatically, and yeastsof the genus Candida are now the fourth most important causeof nosocomial infection (1a, 35). Patients at risk of developingsystemic candidosis are immunosuppressed as a result of theirprimary illness and/or the medicosurgical procedures designedto control or cure it. These patients are usually hospitalizedon highly specialized wards (oncology wards, hematology wards,intensive care, neonatal units, and surgical wards, includingorgan transplantation units). The medical and economic problemsassociated with these opportunistic infections result from thedifficulties in establishing a rapid and specific diagnosisand have led to considerable investment in both basic academicresearch and antifungal drug development.

Candida albicans, the most pathogenic Candida species, is responsiblefor 60 to 80% of infections and is a harmless commensal of thedigestive tract of at least 50% of healthy subjects (31). Thepercentage of colonized subjects and the intensity of gut colonizationboth increase as a result of perturbation of host homeostasisduring hospitalization (31). In both intensive care units (33)and clinical hematology units, colonization by C. albicans hasbeen shown to be an independent risk factor for the developmentof systemic candidosis (26). In addition, genetic similaritieshave been found between strains colonizing patients and thoserecovered from blood cultures (29, 36, 50). Thus, the preventionor reduction of yeast adherence in the gut could have prophylacticeffects. Several studies have established the role of ligand-receptorinteractions in colonization of different segments of the hostdigestive tract by C. albicans. Although mannose residues makeup 40% of the cell wall (25) and/or play a part in the relationshipbetween the cell surface of C. albicans and its environment(6), few studies have investigated the role of mannose residuesin this interaction. Among them, a role for C. albicans antigen6, specific for serotype A, has been demonstrated in the adherenceto buccal epithelial cells (30). In nonpathogenic yeasts suchas Saccharomyces cerevisiae and in mannoconjugates formed byenzymes encoded by viruses, bacteria, and parasites, most mannosesequences correspond to mannose residues linked through ${alpha}$ -1,6, ${alpha}$ -1,2, or ${alpha}$ -1,3 bonds. C. albicans contains enzymes capable ofconstructing unique sequences of mannose residues linked throughan unusual anomer type of linkage, ß-1,2 oligomannosides(ß-1,2-Mans) (42). These residues are expressed inlarge quantities at the cell wall surface in association withdifferent molecules, either mannan (42), mannoproteins (47,48), or glycolipids (49). ß-1,2-Mans act as adhesins(14, 28) and trigger macrophages producing different mediatorsmodulating the host response (23, 24), while ß-1,2-Mansat the nonreducing end of ${alpha}$ -linked chains have been shown toact as adhesins for buccal epithelial cells (30). ß-1,2-Mansalso induce specific antibodies, which, in contrast to antibodiesdirected against ${alpha}$ -linked mannose residues, protect animals fromeither systemic candidosis (18) or vaginal candidosis (19).Furthermore, a previous study in our laboratory demonstratedthat recognition of ß-1,2-Mans by endogenous lectinsof mammals occurred through galectin-3 and did not involve C-lectins,which bind to mannose residues with an ${alpha}$ -anomer type of linkage(15). Galectin-3 is a major lectin with pleiotropic effectsexpressed on a large variety of cells including intestinal epithelialcells (9). This suggests that ß-1,2-Mans could havea specific role in the interaction between C. albicans and itsnatural ecological niche.

An interdisciplinary approach was therefore designed to determinethe role of ß-1,2-Mans in gut colonization in comparisonto those of the ${alpha}$ -linked mannose residues expressed by othercommensal organisms or pathogens of the gut. In a preliminarystudy, the virulence of a number of C. albicans strains wasdemonstrated to correlate closely with the level of ß-1,2-Manepitope surface expression. Synthetic ß- and ${alpha}$ -1,2mannotetraoses were then constructed, and their antigenicitieswere shown to mimic the antigenicities of native C. albicanshomologues by using polyclonal and monoclonal antibodies (MAbs)specific for the native epitopes. By using the highly standardizedinfant mouse model developed by Cole et al. (7), administrationof synthetic ( ${Sigma}$ ) ß-1,2-Mans ( ${Sigma}$ ß-Mans) priorto inoculation with C. albicans was shown to prevent gut colonizationby a virulent strain, whereas synthetic ${alpha}$ -Mans ( ${Sigma}$ ${alpha}$ -Mans) had noeffect.

MATERIALS AND METHODS

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Materials and Methods

C. albicans strains. The C. albicans strains used in this study were kindly providedby A. Schmidt (Bayer AG, Wuppertal, Germany), who has previouslyshown that these strains are reproducibly distributed into threegroups according to their virulence in mouse and rat modelsof C. albicans systemic infection. From this panel of strains,we selected only the seven serotype A strains. They are classifiedas avirulent (strains ATCC 44831, ATCC 18804, and ATCC 10231),virulent (ATCC 44505, ATCC 62342, and ATCC 10261), and intermediate(ATCC 32354). The strains were maintained as stock suspensionsat -80°C in 40% glycerol. The yeasts were always used afterculture on Sabouraud's dextrose agar at 37°C and three washesin sterile saline.

Polyclonal antibodies and MAbs against C. albicans oligomannose residues. Monospecific rabbit antisera (Candida check; Iatron LaboratoriesInc., Tokyo, Japan) were used. Specifically, for the presentstudy, serum factor 1, which recognizes ${alpha}$ -1,2 mannose sequences[Man ( ${alpha}$ -1,2-Man)_n ( ${alpha}$ -1,2-Mans with n $>=$ 0)], and serum factor 5,which reacts with a ß-1,2 oligomannose sequence [Man(ß-1,2-Man)_n (ß-1,2-Mans with n $>=$ 1)] (45),were used.

MAb AF1 from Cassone et al. (5) and MAb DF9-3 from Borg-VonZepelin and Gruness (2) were also used. These MAbs react specificallywith ß-1,2-Mans (48, 49). MAb EBCA1 was used as acontrol for ${alpha}$ -linked mannose (22). This antibody is used in acommercially available test (Platelia Candida; Bio-Rad, Marnes-la-Coquette,France) for the detection of mannanemia (41).

Chemical synthesis of C. albicans oligomannose analogues. The chemical reactions used to synthesize the C. albicans oligomannoseanalogues are shown schematically in Fig. 1 for ${Sigma}$ ß-Mansand in Fig. 2 for ${Sigma}$ ${alpha}$ -Mans. The ${Sigma}$ ß-Mans D-Manp ß(1 $->$ 2)₄(compound 8) and D-Manp ß(1 $->$ 2)₄ O-(CH₂)₈-COOH (compound9) were synthesized from compound 1 (Fig. 1), prepared fromD-mannose (D-Man) by previously published methods (32, 46) byusing the sulfoxide strategy recently applied to the constructionof ß-manno linkages by Crich and Sun (10). This glycosylationmethod is compatible with the presence of a thiophenyl groupin the acceptor and thus allows convergent blockwise synthesisof the tetrasaccharide (compound 7). This pivotal compound waseither deprotected to give compound 8 or coupled with 8-methoxycarbonyloctanol,prepared from azelaic acid by the method of Lemieux et al. (27),to give compound 9 after deprotection.

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FIG. 1. Preparation of ß-mannosides, reagents, and conditions (numbers in boldface refer to the various compounds). (a) Protection of OH-2 by a tertbutyldimethylsilyl group: tertbutyldimethylsilyl triflate, triethylamine, CH₂Cl₂, 20°C, 15 h, 90%. (b) Oxidation of the phenylsulfide into a phenylsulfoxide: meta-chloroperbenzoic acid, CH₂Cl₂, 79%. (c) Glycosylation by activation of the anomeric phenylsulfoxide: triflic anhydride, 2,6-ditertbutyl-4-methylpyridine, -78°C, CH₂Cl₂, 72%. (d) Oxidation of the phenylsulfide into a phenylsulfoxide: meta-chloroperbenzoic acid, CH₂Cl₂, 91%. (e) Removal of the tertbutyldimethylsilyl group at OH-2: N(n-C₄H₉)₄F, aqueous tetrahydrofuran, 20°C, 1 h, 90%. (f) Glycosylation by activation of the anomeric phenylsulfoxide: triflic anhydride, 2,6-ditertbutyl-4-methylpyridine, CH₂Cl₂, -78°C, 55%. (g) Deprotection (three steps): first step (removal of the tertbutyldimethylsilyl group at OH-2), N(n-C₄H₉)₄F, aqueous tetrahydrofuran, 60°C, 12 h, 92%; second step (hydrolysis of the thiophenyl group), N-bromosuccinimide, acetone-H₂O, 0°C, 30 min, 84%; third step (removal of the benzyl ethers and benzylidene groups), H₂, Pd-C, methanol, 85%. (h) Glycosylation of the linker and deprotection (four steps): first step (introduction of the linker), 8-methoxycarbonyloctanol, N-bromosuccinimide, triflic acid, 4-Å molecular sieves, CH₂Cl₂, -20°C, 1 h, 70% ß/ ${alpha}$ = 6/1; second step (removal of the tertbutyldimethylsilyl group at OH-2), N(n-C₄H₉)₄F, aqueous tetrahydrofuran, 60°C, 12 h, 80%; third step (saponification of the ester group), NaOH, aqueous tetrahydrofuran, 82%; fourth step (removal of the benzyl ethers and benzylidene groups), H₂, Pd-C, methanol, 83%. Abbreviations: Ph, phenyl; Bn, benzyl; TBDMS, tertbutyldimethylsilyl.

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FIG. 2. Preparation of ${alpha}$ -mannosides, reagents, and conditions (numbers in boldface refer to the various compounds). (a) Glycosylation by activation of the trichloroacetimidate: trimethylsilyl triflate, 4-Å molecular sieves, CH₂Cl₂, -10°C, 30 min, 72%. (b) Preparation of the disaccharide donor (two steps): first step (hydrolysis of the anomeric phenyl sulfide), N-bromosuccinimide, acetone-H₂O, 20°C, 15 min, 87%; second step (formation of the anomeric trichloroacetimidate), CCl₃CN, 1,8-diazabicyclo(5,4,0)undec-7-ene, CH₂Cl₂, 0°C, 20 min, 81%. (c) Preparation of the disaccharidic acceptor: Na, methanol, toluene, 20°C, 15 min, 95%. (d) Glycosylation by activation of the trichloroacetimidate: BF₃(C₂H₅)2O, 4-Å-mesh-size molecular sieves, CH₂Cl₂, -10°C, 40 min, 60%, and then removal of the acetate group: Na, toluene, methanol, 20°C, 15 min, 71%. (e) Deprotection (two steps): first step (hydrolysis of the anomeric phenyl sulfide), N-bromosuccinimide, acetone-H₂O, 0°C, 30 min, 84%; second step (removal of the benzyl ethers), H₂, Pd-C, methanol, 85%. (f) Glycosylation of the linker and deprotection (three steps): first step (introduction of the linker), 8-methoxycarbonyloctanol, N-bromosuccinimide, triflic acid, 4-Å molecular sieves, CH₂Cl₂, -20°C, 1 h, 72% ß/ ${alpha}$ = 1/1; second step (saponification of the ester group), NaOH, aqueous tetrahydrofuran, 78%; third step (removal of the benzyl ethers), H₂, Pd-C, methanol, 95%. Abbreviations: Ph, phenyl; Bn, benzyl.

The synthesis of the ${alpha}$ -mannosides (46) D-Manp ${alpha}$ (1 $->$ 2)₄ (compound16; Fig. 2) and D-Manp ${alpha}$ (1 $->$ 2)₄ O-(CH₂)₈-COOH (compound 17) wascarried out starting from compounds 10 and 11, respectively,prepared from D-Man by previously published methods (51, 53).A blockwise synthesis was also used, the thiophenyl group ofcompound 14 being stable under trichloroacetimidate (13) activationconditions. The tetramannoside (compound 15) was converted intocompounds16 and 17 in a manner analogous to that described forglycosylation and deprotection of compound 7.

Assessment of synthetic oligomannose antigenicities against MAbs by EIA. The oligosaccharides 9 and 17 were first coupled to CH₂-(CH₂)₇-COOH(linker developed by Lemieux et al. [27]) through their reducingterminal ends. The connecting residue was then covalently linkedto the wells of a microtiter plate (CovaLink NH₂; Nunc, Roskilde,Denmark) by using carbodiimide (43) to provide serial concentrations.The plates were rinsed three times with phosphate-buffered saline(PBS; 200 µl/well) and blocked with PBS plus 5% nonfatdry milk overnight at 4°C (200 µl/well). The plateswere washed five times with 200 µl of TNT (50 mM Tris-HCl,150 mM NaCl, 0.05% Tween 20 [pH 7.5]) per well. Enzyme immunoassay(EIA) was then performed as described previously (12, 41).

Evaluation of ß-1,2-Man surface expression by latex agglutination. Carboxyl-modified microspheres (Estapore K1-0.8) were obtainedfrom Prolabo (Fontenay-sous-Bois, France) and coated with purifiedMAbs AF1 and DF9-3 as described previously (39) by a procedureused in the Bichro-latex test developed by one of us. Sensitizedred particles were then suspended in a green buffer designedto prevent direct yeast agglutination. Before use, the reagentwas homogenized to form a dark brown suspension.

A suspension of 10⁶ yeasts/ml was prepared from fresh culturesfor each experiment. The coagglutination test was performedby mixing 15 µl of yeast suspension (10⁶ yeasts/ml) with15 µl of sensitized particles, followed by rotation ofthe mixture. Preliminary experiments showed that a scoring procedureensured the reproducibility of the results. Thus, the absenceof reactivity producing a homogeneous dark brown spot due tononagglutinated red particles evenly suspended in the greenbuffer was assigned a score of 0, while positive results weregraded 0.5 to 2 as follows: reactions in which all the agglutinatedbeads formed a thick, red edge around a clear, green centralarea were given a score of 2; reactions with weaker agglutination,a thinner colored edge, and some red agglutinates remainingin the central part of the spot in contrast to the green backgroundwere given a score of score 1; very weak reactions that gaveonly small red aggregates without any visible edge were givena score of score 0.5. Agglutination scores were recorded after1, 2, and 3 min; their sums gave the score for each MAb; andthese scores were added to give a final score, which thus tookinto account the speed and intensity of the agglutination reaction.The tests were performed blindly three times with each of thestrains provided by A. Schmidt, and the results were subsequentlydeciphered.

Experimental model of gastrointestinal colonization. The experimental model of gastrointestinal colonization wasbased on that described by Pope et al. (34). Suckling Swissmice (age, 3 to 4 days; CD1, Crl:CD1, BR mice; ICR) were obtainedfrom Charles River Laboratories (St. Aubin les Elbeuf, France)as litters of 10 to 15 animals. Six-day-old infant mice weighingapproximately 5 g were removed from their mothers for 3 h, inoculated,and placed back with their dams 1 h after gavage.

Two strains (ATCC 10241 and ATCC 10261) were selected for determinationof differences in their levels of surface ß-1,2-Manexpression (see Results). The yeast suspensions were adjustedto 2 x 10⁹/ml in sterile water, and viability was determinedby counting the numbers of CFU in duplicate. The suspensionwas administered by gavage in a 50-µl volume with a 1-mlsyringe equipped with the Teflon tubing of a sterile intravenouscatheter (Insyte 24 GA, 0.7 by 19 mm; Becton Dickinson and Co.,Paramus, N.J.).

The course of the infection was monitored daily by countingthe number of surviving infant mice in each cage. The degreeof gut colonization was assessed by measuring the numbers ofCFU in the fecal pellets (8). Fresh fecal pellets were collectedfrom each mouse and immediately homogenized in 100 µlof sterile distilled water, and the suspension obtained wasplated onto Sabouraud's dextrose agar containing 50 µgof chloramphenicol per ml. Since the small size of the fecalpellets prevented accurate weighing, the first dropping wasalways used for the experiments. After incubation at 30°Cfor 24 h, the number of CFU was counted. When the CFU countswere >300 to 400, a quick evaluation was performed on a smallportion of the plate and an arbitrary number was used to allowcomparison between groups: 1,000 for colonies that were stilldistinct, 10,000 for nonconfluent growth, and 100,000 for confluentgrowth. It must be noted that for the noninfected animals thefirst fecal pellets obtained from infant mice (on day 13 afterbirth) and those from adults were consistently negative (datanot shown).

The synthetic oligosaccharides ( ${Sigma}$ ß-Mans and ${Sigma}$ ${alpha}$ -Mans)were dissolved in sterile distilled water to a concentrationof 3 or 1 mg/ml. The solution was administered by gavage ina 50-µl volume 1 h prior to oral inoculation. The effectsof pretreatment with water (litter 1), ${Sigma}$ ß-Mans at 50µg/infant mouse (10 mg/kg of body weight; litter 2), ${Sigma}$ ß-Mansat 150 µg/infant mouse (30 mg/kg; litter 3), or ${Sigma}$ ${alpha}$ -Mansat 150 µg/infant mouse (litter 4) on gut colonizationwere evaluated.

Statistical analysis. The percent mortality and the percentage of animals with positivefeces were compared between litters infected with strains ATCC10231 and ATCC 10261 by the Fisher exact test. Nonparametrictests (the Mann-Whitney or the Kruskal-Wallis test, dependingon the number of groups) were used for comparison of gut colonizationand agglutination scores.

RESULTS

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Results

Synthetic oligomannose residues with antigenicities that mimic the antigenicities of natural components of the C. albicans cell wall can be constructed. Figure 3 shows the reactivities of serum factors 1 and 5, specificfor ${alpha}$ -1,2 oligomannose and ß-1,2 oligomannose sequences,respectively, with ${Sigma}$ ${alpha}$ -Mans and ${Sigma}$ ß-Mans coupled to EIAplates. Increasing concentrations of both ${Sigma}$ ß-Mans and ${Sigma}$ ${alpha}$ -Mans were associated with a dose-dependent binding of serumfactors specific for each epitope, whereas only weak reactivitywas observed with the irrelevant epitope. Figure 4 shows thereactivity of MAb AF1 (specific for ß-linked oligosaccharides)and MAb CA1 (specific for ${alpha}$ -linked oligomannosides) with ${Sigma}$ ß-Mans.Poor binding of MAb CA1 was observed, whereas the binding ofMAb AF1 increased in parallel with the amount of ${Sigma}$ ß-Manscoated onto the plates. Thus, the antigenicities of the syntheticoligomannosides appeared to mimic closely the antigenicitiesof native C. albicans molecules.

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FIG. 3. Binding of polyclonal factor sera to synthetic ${alpha}$ - and ß-mannotetraoses as measured by EIA. Serum factor 5 (reactive with ß-1,2 mannose sequence; closed bars) and serum factor 1 (reactive with ${alpha}$ -1,2 mannose sequence; open bars) were allowed to react with plates coated with increasing concentrations of synthetic ${alpha}$ -1,2-linked mannotetraose (A) and ß-1,2-linked mannotetraose (B).

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FIG. 4. Binding of anti-C. albicans MAbs to synthetic ß-1,2-linked mannotetraose measured by EIA. MAb AF1, which is specific for C. albicans ß-1,2-Mans (closed bars), and MAb CA1, which reacts with C. albicans ${alpha}$ -linked mannose residues (open bars), were incubated with increasing concentrations of synthetic ß-1,2-linked mannotetraose.

Surface expression of ß-1,2-Man epitopes correlates with virulence of C. albicans serotype A strains in systemic models of candidosis in rats and mice. Seven nonisogenic strains previously shown (40) to exhibit reproducibledifferences in virulence in rat and mouse models of systemicinfection were tested blindly for their abilities to react withMAbs specific for ß-1,2-Mans. The level of epitopeexpression was measured by determination of both the sizes ofthe agglutinates and the times required for the reaction todevelop, with the highest agglutination score relating to thehighest level of surface expression of ß-1,2-Mans.

As shown in Table 1, the agglutination scores were significantlylower for strains with low levels of virulence than for thosewith high or intermediate levels of virulence (medians, 5.5and 7.7, respectively [P = 0.034]), suggesting a correlationbetween the surface expression of ß-1,2-Mans and virulence.

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TABLE 1. Agglutination scores for C. albicans strains reacted with Bichro-latex particles sensitized with MAbs DF9-3 and AF1 specific for ß-1,2-Man epitopes compared with virulence determined from systemic Candida infection of rats and mice^a

Virulence of two C. albicans strains in the infant mouse model of gut colonization correlates with virulence in systemic models and surface expression of ß-1,2-Mans. One virulent strain (ATCC 10261) with a high level of ß-1,2-Mansurface expression and one avirulent strain (ATCC 10231) witha low level of ß-1,2-Man surface expression were selected.Their virulences were compared in six independent experiments.Preliminary analysis showed that the degree of gut colonizationevaluated according to the number of CFU per fecal pellet wasreproducible, allowing the data for two litters infected withthe same strain to be pooled (data not shown). Results fromrepresentative experiments are shown in Table 2. The resultsare for three litters inoculated with each strain, with theresults for a total of 31 animals infected with strain ATCC10261, 30 animals infected with strain ATCC 10231, and 217 stoolcultures provided.

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TABLE 2. Kinetics of gut colonization in infant mice after oral inoculation with C. albicans strains ATCC 10231 and ATCC 10261 expressing low and high levels of ß-1,2-Mans, respectively, at their cell surfaces

From day 7 to day 33 after inoculation (day 7 was the firstday on which it was possible to assess colonization), colonizationwas variable from mouse to mouse but was always significantlyhigher in mice colonized with strain ATCC 10261 than in thosecolonized with strain ATCC 10231. Thus, colonization was moreprotracted in animals infected with ATCC 10261 than in thoseinfected with ATCC 10231 (at day 33 postinoculation, 11 of 11animals inoculated with ATCC 10261 were still colonized, whereas5 of 11 animals infected with ATCC 10231 were still colonized[P = 0.006]). The rate of mortality for animals without subsequentinduced immunosuppression was low. However, the rate of cumulativeacute mortality from the six independent experiments was higheramong infant mice inoculated with ATCC 10261 than among infantmice inoculated with ATCC 10231, although the difference didnot reach statistical significance (6 of 63 versus 1 of 62 mice[P = 0.059]).

Synthetic ß-1,2 tetramannosides but not ${alpha}$ -1,2 tetramannosides inhibit gut colonization by a virulent C. albicans strain. The last set of experiments was designed to test the prophylacticeffects of ß-1,2-Mans. Synthetic tetramannosides wereadministered as a single dose prior to C. albicans inoculation.In a preliminary experiment, the effect of the administrationof 50 µg of ${Sigma}$ ß-Mans prior to inoculation of C.albicans strains ATCC 10261 and ATCC 10231 was evaluated atday 7 postinoculation. Pretreatment had no effect when the micewere inoculated with the strain expressing less ß-1,2-Mansat the cell surface (ATCC 10231), while there was a reduction(although it was not significant) in the numbers of CFU frommice inoculated with the strain expressing more ß-1,2-Mans(median counts, 0 CFU [range, 0 to 36 CFU] versus 1 CFU [range,0 to 9 CFU] and 71 CFU [range, 4 to 482 CFU] versus 14 CFU [range,0 to 171 CFU] for strains ATCC 10231 and ATCC 10261, respectively).To further study the specificity of the effect, we decided touse the more virulent strain expressing more ß-1,2-Mans,larger numbers of animals, different doses of ${Sigma}$ ß-Mans,and ${Sigma}$ ${alpha}$ -Mans as a control (Fig. 5). At day 7 after inoculation,administration of ${Sigma}$ ${alpha}$ -Mans had no effect on gut colonization comparedto the effects of distilled water (control treatment) (mediancounts, 196 CFU [range, 11 to 1,000 CFU] versus 258 CFU [range,31 to 1,000 CFU] in the ${Sigma}$ ${alpha}$ -Man-treated group versus the controls[P > 0.05]). By contrast, administration of 50 µg of ${Sigma}$ ß-Mans led to a decrease in the number of CFU perfecal pellet, which was significant. The effect of ${Sigma}$ ß-Manswas dose dependent, since the effect became highly significantwhen 150 µg was administered. The efficacy of this treatmentwas further demonstrated by the complete disappearance aftertreatment with ${Sigma}$ ß-Mans of the variability in the levelof gut colonization observed in all experiments among untreatedinfant mice and in this experiment among untreated and ${Sigma}$ ${alpha}$ -Man-treatedanimals (Fig. 5 and Table 2). We also sampled the animals onday 10 after inoculation and observed that the level of colonizationhad dramatically increased in all mice, but it had not increasedas much in mice treated with 150 µg of ${Sigma}$ ß-Mansthan in the other groups (P = 0.0220).

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FIG. 5. Prophylactic effects of synthetic mannotetraoses on gut colonization of infant mice by C. albicans ATCC 10261, a strain that expresses high levels of ß-1,2 mannose at its cell surface. Infant mice received either distilled water (control) or solutions providing 50 µg of synthetic ß-1,2 tetramannose (ß-Man), 150 µg of ß-1,2 tetramannose, or 150 µg of synthetic ${alpha}$ -1,2 tetramannose ( ${alpha}$ -Man) per mouse 1 h before being inoculated with ATCC 10261. The degree of gut colonization was evaluated at day 7 after inoculation by counting the number of CFU per fecal pellet (9 to 11 mice per group). Data are shown as boxes, in which the internal horizontal lines indicate medians; the tops and bottoms of the boxes represent the 25th and 75th percentiles, respectively; the upper and lower bars represent the 10th and 90th percentiles, respectively; and open circles represent values exceeding the range of 10 to 90%. _*, P < 0.02 versus 50 µg of ß-1,2 tetramannose; _*_*, P < 0.0001 versus 150 µg of ß-1,2 tetramannose; ${dagger}$ , not significant versus 150 µg of ${alpha}$ -1,2 tetramannose (P values were determined by the Kruskal-Wallis test).

DISCUSSION

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Discussion

Although C. albicans is not a true pathogen, it has some biologicalcharacteristics, termed virulence factors (3), which help itto invade debilitated hosts. Among these factors are cell wallmolecules that act as adhesins (44). C. albicans has been shownto adhere to a wide variety of host cells and molecules throughprotein-protein (17), yeast lectin-host carbohydrate (4, 52),or yeast carbohydrate-host lectin interactions. A growing bodyof evidence suggests that ß-1,2-Mans have an importantrole in pathogenesis since they have been shown to act as adhesins(28), stimulators of immune response mediator secretion (23,24), and inducers of protective antibodies (18, 19). The recentidentification of galectin-3 (previously, the Mac 2 antigen)as a ß-1,2-Man receptor on host cells provides furtherevidence for the specificity of these interactions (15). Itindeed appears that C. albicans has a unique biological traitrelated to its ability to express ß-1,2-Mans at itscell surface and that this biological trait has its counterpartin two major host recognition systems, namely, antibodies andendogenous lectins. It therefore seemed worthwhile to explorethe role of this system in the process of colonization of thegut, where galectin-3 is widely expressed (9).

To address this question of the role of ß-1,2-Mansin colonization, a multiple-step study was designed. Previousdata suggested a relationship between the expression of ß-1,2-Mansat the yeast cell surface and the virulence of strains (16).By testing several unrelated strains of C. albicans that havebeen shown to exhibit various degrees of virulence in animalmodels of systemic infection (40), a clear correlation was foundbetween these two parameters; namely, the weaker the expressionof ß-1,2-Mans is, the lower the reported level ofvirulence is, and vice versa. This is the first set of experimentssuggesting a correlation between surface expression of ß-1,2-Mansand C. albicans strain pathogenicity in animal models. The sametrend was observed when more than 200 C. albicans clinical isolateswere tested in a previous study (16). The isolates recoveredfrom colonized patients were less agglutinated by an MAb designated5B2 than those isolated from infected patients. Interestingly,subsequent immunochemical experiments showed that MAb 5B2 isspecific for ß-1,2-Man epitopes (48). In the presentstudy the strain with the higher levels of expression of ß-1,2-Manepitopes was shown to induce higher levels of colonization andmore protracted colonization of the gut of infected mice thanthe strain with lower levels of expression of ß-1,2-Manepitopes.

To further assess the role of ß-1,2-Mans in intestinalcolonization, synthetic oligosaccharides were administered aspotential competitors prior to inoculation with the most virulentstrain. Preparation of large quantities of mannan-derived oligomannosidesis based on a long and tedious procedure and requires nuclearmagnetic resonance verification of the structures obtained (12).As previous studies with macrophages demonstrated that syntheticmolecules exhibiting a minimum polymerization of four residuesinhibit ß-1,2-Mans binding and stimulation of mammaliancells (14, 24), it was decided to chemically synthesize ß-1,2mannotetraoses. Appropriate controls, consisting of ${alpha}$ -1,2-Mantetramannoses, were also synthesized. EIA demonstrated thatthe antigenicities of both ${Sigma}$ ${alpha}$ -Mans and ${Sigma}$ ß-Mans mimickedthe antigenicities of C. albicans-derived homologues. When animalswere given ${Sigma}$ ß-Mans (30 mg/kg) prior to inoculationwith C. albicans, a dramatic decrease in the number of yeastsrecovered from stools was observed; however, this decrease wasnot seen following administration of the same dose of ${Sigma}$ ${alpha}$ -Mans.To us, this effect is of real biological significance becauseit was evidenced 7 days and even 10 days after administration.Furthermore, it was dose and structure dependent.

Together these data strongly suggest that expression of ß-1,2-Mansat the cell surface of C. albicans has an important role ingut colonization. In hospitalized immunocompromised patients,it is now agreed that sustained gastrointestinal colonizationusually precedes disseminated infection (33). Prophylaxis withnonabsorbable oral antifungal agents has been advocated to reducegastrointestinal colonization, but the efficacy of this approachfor decontamination of the digestive tract has not been demonstratedconclusively (11, 20). Azole antifungal agents have been moresuccessful at reducing colonization and infection, but theirintensive and/or inappropriate use may select for resistantstrains or species (11, 37). Another means of controlling theproliferation of one species is to change the indigenous floraby providing a local competitor. When infant mice were inoculatedwith two different Candida species, the number of mice colonizedwith Candida glabrata increased as the number of mice colonizedwith C. albicans decreased (13). The C. albicans competitorused in human studies is the nonpathogenic yeast Saccharomycesboulardii (21), but conflicting results and rare cases of fungemiadue to Saccharomyces species may discourage use of this approach(38).

Even if the efficacy of ${Sigma}$ ß-Mans is transient (sincewe chose to administer only a single dose), our data suggestthat this innovative approach may be useful for the preventionof systemic Candida infection in at-risk patients. This strategyhas several potential advantages over existing prophylacticregimens: (i) the chemical procedure can be easily adapted tothe production of large quantities of ß-Mans; (ii) ${Sigma}$ ß-Mans are resistant to gastric pH and should alsobe well tolerated, as ß-1,2-Mans (from C. albicans)are already present in the human gut; (iii) ${Sigma}$ ß-Mansshould not generate resistance; and (iv) the effective dose(30 mg/kg) that leads to a lasting effect is relatively low,at least in mice. Further studies with different animal modelsare in progress to determine the influence of the administrationregimen (timing, doses) on the therapeutic efficacies of ${Sigma}$ ß-Mansfor the long-lasting prevention of gut colonization and to determinewhether it extends to a reduction in the level of existing colonizationor the treatment of existing colonization. In vitro studiesare also in progress since, besides their therapeutic applications,the clear-cut results obtained here are of basic pathophysiologicaland phylogenetic interest for the understanding of host-endogenousmicrobe interactions.

ACKNOWLEDGMENTS

This work was supported by the Programme de Recherche Fondamentaleen Microbiologie, Maladies Infectieuses et Parasitaires, andcollaborative research was developed through "RéseauInfections Fongiques," supported by the same program.

FOOTNOTES

* Corresponding author. Mailing address: Inserm E9915, Faculté de Médecine, Pôle Recherche, CHRU, Place de Verdun, 59045 Lille Cedex, France. Phone: 33 3 20 62 34 20. Fax: 33 3 20 62 34 16. E-mail: dpoulain{at}univ-lille2.fr.

REFERENCES

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