Synthetic analogues of beta-1,2 oligomannosides prevent intestinal colonization by the pathogenic yeast Candida albicans.

Article abstract of DOI:10.1128/AAC.46.12.3869-3876.2002

The pathogenic yeast Candida albicans displays at its cell surface beta-1,2 oligomannosides (beta-1,2-Mans). In contrast to the ubiquitous alpha-Mans, beta-1,2-Mans bind to galectin-3, a major endogenous lectin expressed on epithelial cells. The specific

Full text of DOI:10.1128/AAC.46.12.3869-3876.2002

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 2002, p. 3869–3876
0066-4804/02/$04.00ϩ0 DOI: 10.1128/AAC.46.12.3869–3876.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 46, No. 12
Synthetic Analogues of ␤-1,2 Oligomannosides Prevent Intestinal
Colonization by the Pathogenic Yeast Candida albicans
Fran¸coise Dromer,1 Reynald Chevalier,2 Boualem Sendid,3 Luce Improvisi,1 Thierry Jouault,3
Raymond Robert,4 Jean Maurice Mallet,2 and Daniel Poulain3*
Unit´e de Mycologie Mol´eculaire, Institut Pasteur, 75015 Paris,1 D´epartement de Chimie, Ecole Normale Sup´erieure,
75005 Paris,2 Equipe Inserm 9915, Facult´e de M´edecine, Poˆle Recherche, Centre Hospitalier-Universitaire,
59045 Lille,3 and Laboratoire de Parasitologie, Facult´e de Pharmacie,
Boulevard Daviers, 49000 Angers,4 France
Received 1 April 2002/Returned for modification 13 May 2002/Accepted 15 July 2002
The pathogenic yeast Candida albicans displays at its cell surface ␤-1,2 oligomannosides (␤-1,2-Mans). In
contrast to the ubiquitous ␣-Mans, ␤-1,2-Mans bind to galectin-3, a major endogenous lectin expressed on
epithelial cells. The specific role of ␤-1,2-Mans in colonization of the gut by C. albicans was assessed in a mouse
model. A selected virulent strain of C. albicans (expressing more ␤-1,2-Man epitopes) induced more intense and
sustained colonization than an avirulent strain (expressing less ␤-1,2-Man epitopes). Synthetic (⌺) ␤-and
␣-linked tetramannosides with antigenicities that mimicked the antigenicities of C. albicans-derived oligom-
annosides were then constructed. Oral administration of ⌺␤-1,2-Man (30 mg/kg of body weight) prior to
inoculation with the virulent strain resulted in almost complete eradication of yeasts from stool samples,
whereas administration of ⌺␣-Man at the same dose did not. As most cases of human systemic candidiasis are
endogenous in origin, this first demonstration that a synthetic analogue of a yeast adhesin can prevent yeast
colonization in the gut opens the possibility of new prophylactic strategies.
Since the 1980s the incidence of systemic Candida infections
in hospitalized patients has increased dramatically, and yeasts
of the genus Candida are now the fourth most important cause
of nosocomial infection (1a, 35). Patients at risk of developing
systemic candidosis are immunosuppressed as a result of their
primary illness and/or the medicosurgical procedures designed
to control or cure it. These patients are usually hospitalized on
highly specialized wards (oncology wards, hematology wards,
intensive care, neonatal units, and surgical wards, including
organ transplantation units). The medical and economic prob-
lems associated with these opportunistic infections result from
the difficulties in establishing a rapid and specific diagnosis and
have led to considerable investment in both basic academic
research and antifungal drug development.
Candida albicans, the most pathogenic Candida species, is
responsible for 60 to 80% of infections and is a harmless
commensal of the digestive tract of at least 50% of healthy
subjects (31). The percentage of colonized subjects and the
intensity of gut colonization both increase as a result of per-
turbation of host homeostasis during hospitalization (31). In
both intensive care units (33) and clinical hematology units,
colonization by C. albicans has been shown to be an indepen-
dent risk factor for the development of systemic candidosis
(26). In addition, genetic similarities have been found between
strains colonizing patients and those recovered from blood
cultures (29, 36, 50). Thus, the prevention or reduction of yeast
adherence in the gut could have prophylactic effects. Several
studies have established the role of ligand-receptor interac-
tions in colonization of different segments of the host digestive
tract by C. albicans. Although mannose residues make up 40%
of the cell wall (25) and/or play a part in the relationship
between the cell surface of C. albicans and its environment (6),
few studies have investigated the role of mannose residues in
this interaction. Among them, a role for C. albicans antigen 6,
specific for serotype A, has been demonstrated in the adher-
ence to buccal epithelial cells (30). In nonpathogenic yeasts
such as Saccharomyces cerevisiae and in mannoconjugates
formed by enzymes encoded by viruses, bacteria, and parasites,
most mannose sequences correspond to mannose residues
linked through ␣-1,6, ␣-1,2, or ␣-1,3 bonds. C. albicans con-
tains enzymes capable of constructing unique sequences of
mannose residues linked through an unusual anomer type of
linkage, ␤-1,2 oligomannosides (␤-1,2-Mans) (42). These res-
idues are expressed in large quantities at the cell wall surface
in association with different molecules, either mannan (42),
mannoproteins (47, 48), or glycolipids (49). ␤-1,2-Mans act as
adhesins (14, 28) and trigger macrophages producing different
mediators modulating the host response (23, 24), while ␤-1,2-
Mans at the nonreducing end of ␣-linked chains have been
shown to act as adhesins for buccal epithelial cells (30). ␤-1,2-
Mans also induce specific antibodies, which, in contrast to
antibodies directed against ␣-linked mannose residues, protect
animals from either systemic candidosis (18) or vaginal can-
didosis (19). Furthermore, a previous study in our laboratory
demonstrated that recognition of ␤-1,2-Mans by endogenous
lectins of mammals occurred through galectin-3 and did not
involve C-lectins, which bind to mannose residues with an
␣-anomer type of linkage (15). Galectin-3 is a major lectin with
pleiotropic effects expressed on a large variety of cells includ-
* Corresponding author. Mailing address: Inserm E9915, Facult´e de
M´edecine, Poˆle 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@univ-lille2.fr.
3869

3870
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ANTIMICROB. AGENTS CHEMOTHER.
FIG. 1. Preparation of ␤-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, Ϫ78°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, Ϫ78°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-A molecular sieves, CH2Cl2, Ϫ20°C, 1 h, 70% ␤/␣ ϭ 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%. Abbrevi-
ations: Ph, phenyl; Bn, benzyl; TBDMS, tertbutyldimethylsilyl.
MATERIALS AND METHODS
ing intestinal epithelial cells (9). This suggests that ␤-1,2-Mans
could have a specific role in the interaction between C. albicans
and its natural ecological niche.
C. albicans strains. The C. albicans strains used in this study were kindly
provided by A. Schmidt (Bayer AG, Wuppertal, Germany), who has previously
shown that these strains are reproducibly distributed into three groups according
to their virulence in mouse and rat models of C. albicans systemic infection.
From this panel of strains, we selected only the seven serotype A strains. They
are classified as 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 suspensions at Ϫ80°C in
40% glycerol. The yeasts were always used after culture on Sabouraud’s dextrose
agar at 37°C and three washes in sterile saline.
Polyclonal antibodies and MAbs against C. albicans oligomannose residues.
Monospecific rabbit antisera (Candida check; Iatron Laboratories Inc., Tokyo,
Japan) were used. Specifically, for the present study, serum factor 1, which
recognizes ␣-1,2 mannose sequences [Man (␣-1,2-Man)n (␣-1,2-Mans with n Ն
0)], and serum factor 5, which reacts with a ␤-1,2 oligomannose sequence [Man
(␤-1,2-Man)n (␤-1,2-Mans with n Ն 1)] (45), were used.
An interdisciplinary approach was therefore designed to de-
termine the role of ␤-1,2-Mans in gut colonization in compar-
ison to those of the ␣-linked mannose residues expressed by
other commensal organisms or pathogens of the gut. In a
preliminary study, the virulence of a number of C. albicans
strains was demonstrated to correlate closely with the level of
␤-1,2-Man epitope surface expression. Synthetic ␤- and ␣-1,2
mannotetraoses were then constructed, and their antigenicities
were shown to mimic the antigenicities of native C. albicans
homologues by using polyclonal and monoclonal antibodies
(MAbs) specific for the native epitopes. By using the highly
standardized infant mouse model developed by Cole et al. (7),
administration of synthetic (⌺) ␤-1,2-Mans (⌺␤-Mans) prior to
inoculation with C. albicans was shown to prevent gut coloni-
zation by a virulent strain, whereas synthetic ␣-Mans (⌺␣-
Mans) had no effect.
MAb AF1 from Cassone et al. (5) and MAb DF9-3 from Borg-Von Zepelin
and Gruness (2) were also used. These MAbs react specifically with ␤-1,2-Mans
(48, 49). MAb EBCA1 was used as a control for ␣-linked mannose (22). This
antibody is used in a commercially available test (Platelia Candida; Bio-Rad,
Marnes-la-Coquette, France) for the detection of mannanemia (41).

VOL. 46, 2002
␤-Man SYNTHETIC ANALOGUES AGAINST C. ALBICANS
3871
FIG. 2. Preparation of ␣-mannosides, reagents, and conditions (numbers in boldface refer to the various compounds). (a) Glycosylation by
˚
activation of the trichloroacetimidate: trimethylsilyl triflate, 4-A molecular sieves, CH2Cl2, Ϫ10°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-A-mesh-size molecular sieves, CH2Cl2, Ϫ10°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-A molecular sieves, CH2Cl2, Ϫ20°C, 1 h, 72% ␤/␣ ϭ 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.
Chemical synthesis of C. albicans oligomannose analogues. The chemical
reactions used to synthesize the C. albicans oligomannose analogues are shown
schematically in Fig. 1 for ⌺␤-Mans and in Fig. 2 for ⌺␣-Mans. The ⌺␤-Mans
D-Manp ␤(132)4 (compound 8) and D-Manp ␤(132)4 O-(CH2)8-COOH (com-
pound 9) were synthesized from compound 1 (Fig. 1), prepared from D-mannose
(D-Man) by previously published methods (32, 46) by using the sulfoxide strategy
recently applied to the construction of ␤-manno linkages by Crich and Sun (10).
This glycosylation method is compatible with the presence of a thiophenyl group
in the acceptor and thus allows convergent blockwise synthesis of the tetrasac-
charide (compound 7). This pivotal compound was either 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.
The synthesis of the ␣-mannosides (46) D-Manp ␣(132)4 (compound 16; Fig.
2) and D-Manp ␣(132)4 O-(CH2)8-COOH (compound 17) was carried 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 of compound 14 being stable under trichloroacetimidate (13)
activation conditions. The tetramannoside (compound 15) was converted into
compounds16 and 17 in a manner analogous to that described for glycosylation
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 reducing terminal ends. The
connecting residue was then covalently linked to the wells of a microtiter plate
(CovaLink NH2; Nunc, Roskilde, Denmark) by using carbodiimide (43) to pro-
vide serial concentrations. The plates were rinsed three times with phosphate-
buffered saline (PBS; 200 ␮l/well) and blocked with PBS plus 5% nonfat dry milk
overnight at 4°C (200 ␮l/well). The plates were washed five times with 200 ␮l 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 ␤-1,2-Man surface expression by latex agglutination. Carboxyl-
modified microspheres (Estapore K1-0.8) were obtained from Prolabo (Fon-
tenay-sous-Bois, France) and coated with purified MAbs AF1 and DF9-3 as
described previously (39) by a procedure used in the Bichro-latex test developed
by one of us. Sensitized red particles were then suspended in a green buffer
designed to prevent direct yeast agglutination. Before use, the reagent was
homogenized to form a dark brown suspension.
A suspension of 106 yeasts/ml was prepared from fresh cultures for each
experiment. The coagglutination test was performed by mixing 15 ␮l of yeast
suspension (106 yeasts/ml) with 15 ␮l of sensitized particles, followed by rotation
of the mixture. Preliminary experiments showed that a scoring procedure en-
sured the reproducibility of the results. Thus, the absence of reactivity producing
a homogeneous dark brown spot due to nonagglutinated red particles evenly
suspended in the green buffer was assigned a score of 0, while positive results
were graded 0.5 to 2 as follows: reactions in which all the agglutinated beads
formed a thick, red edge around a clear, green central area were given a score of
2; reactions with weaker agglutination, a thinner colored edge, and some red
agglutinates remaining in the central part of the spot in contrast to the green
background were given a score of score 1; very weak reactions that gave only
small red aggregates without any visible edge were given a score of score 0.5.
Agglutination scores were recorded after 1, 2, and 3 min; their sums gave the
score for each MAb; and these scores were added to give a final score, which thus
took into account the speed and intensity of the agglutination reaction. The tests
were performed blindly three times with each of the strains provided by A.
Schmidt, and the results were subsequently deciphered.
Experimental model of gastrointestinal colonization. The experimental model
of gastrointestinal colonization was based on that described by Pope et al. (34).

3872
DROMER ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
Suckling Swiss mice (age, 3 to 4 days; CD1, Crl:CD1, BR mice; ICR) were
obtained from Charles River Laboratories (St. Aubin les Elbeuf, France) as
litters of 10 to 15 animals. Six-day-old infant mice weighing approximately 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 determination
of differences in their levels of surface ␤-1,2-Man expression (see Results). The
yeast suspensions were adjusted to 2 ϫ 109/ml in sterile water, and viability was
determined by counting the numbers of CFU in duplicate. The suspension was
administered by gavage in a 50-␮l volume with a 1-ml syringe equipped with the
Teflon tubing of a sterile intravenous catheter (Insyte 24 GA, 0.7 by 19 mm;
Becton Dickinson and Co., Paramus, N.J.).
The course of the infection was monitored daily by counting the number of
surviving infant mice in each cage. The degree of gut colonization was assessed
by measuring the numbers of CFU in the fecal pellets (8). Fresh fecal pellets
were collected from each mouse and immediately homogenized in 100 ␮l of
sterile distilled water, and the suspension obtained was plated onto Sabouraud’s
dextrose agar containing 50 ␮g of chloramphenicol per ml. Since the small size
of the fecal pellets prevented accurate weighing, the first dropping was always
used for the experiments. After incubation at 30°C for 24 h, the number of CFU
was counted. When the CFU counts were Ͼ300 to 400, a quick evaluation was
performed on a small portion of the plate and an arbitrary number was used to
allow comparison between groups: 1,000 for colonies that were still distinct,
10,000 for nonconfluent growth, and 100,000 for confluent growth. It must be
noted that for the noninfected animals the first fecal pellets obtained from infant
mice (on day 13 after birth) and those from adults were consistently negative
(data not shown).
The synthetic oligosaccharides (⌺␤-Mans and ⌺␣-Mans) were dissolved in
sterile distilled water to a concentration of 3 or 1 mg/ml. The solution was
administered by gavage in a 50-␮l volume 1 h prior to oral inoculation. The
effects of pretreatment with water (litter 1), ⌺␤-Mans at 50 ␮g/infant mouse (10
mg/kg of body weight; litter 2), ⌺␤-Mans at 150 ␮g/infant mouse (30 mg/kg; litter
3), or ⌺␣-Mans at 150 ␮g/infant mouse (litter 4) on gut colonization were
evaluated.
Statistical analysis. The percent mortality and the percentage of animals with
positive feces were compared between litters infected with strains ATCC 10231
and ATCC 10261 by the Fisher exact test. Nonparametric tests (the Mann-
Whitney or the Kruskal-Wallis test, depending on the number of groups) were
used for comparison of gut colonization and agglutination scores.
FIG. 3. Binding of polyclonal factor sera to synthetic ␣- and ␤-man-
notetraoses as measured by EIA. Serum factor 5 (reactive with ␤-1,2
mannose sequence; closed bars) and serum factor 1 (reactive with
␣-1,2 mannose sequence; open bars) were allowed to react with plates
coated with increasing concentrations of synthetic ␣-1,2-linked man-
notetraose (A) and ␤-1,2-linked mannotetraose (B).
RESULTS
Synthetic oligomannose residues with antigenicities that
mimic the antigenicities of natural components of the C. albi-
cans cell wall can be constructed. Figure 3 shows the reactiv-
ities of serum factors 1 and 5, specific for ␣-1,2 oligomannose
and ␤-1,2 oligomannose sequences, respectively, with ⌺␣-
Mans and ⌺␤-Mans coupled to EIA plates. Increasing concen-
trations of both ⌺␤-Mans and ⌺␣-Mans were associated with a
dose-dependent binding of serum factors specific for each
epitope, whereas only weak reactivity was observed with the
irrelevant epitope. Figure 4 shows the reactivity of MAb AF1
(specific for ␤-linked oligosaccharides) and MAb CA1 (specific
for ␣-linked oligomannosides) with ⌺␤-Mans. Poor binding of
MAb CA1 was observed, whereas the binding of MAb AF1
increased in parallel with the amount of ⌺␤-Mans coated onto
the plates. Thus, the antigenicities of the synthetic oligoman-
nosides appeared to mimic closely the antigenicities of native
C. albicans molecules.
Surface expression of ␤-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 pre-
viously shown (40) to exhibit reproducible differences in viru-
lence in rat and mouse models of systemic infection were
tested blindly for their abilities to react with MAbs specific for
␤-1,2-Mans. The level of epitope expression was measured by
determination of both the sizes of the agglutinates and the
times required for the reaction to develop, with the highest
agglutination score relating to the highest level of surface ex-
pression of ␤-1,2-Mans.
As shown in Table 1, the agglutination scores were signifi-
cantly lower for strains with low levels of virulence than for
those with high or intermediate levels of virulence (medians,
5.5 and 7.7, respectively [P ϭ 0.034]), suggesting a correlation
between the surface expression of ␤-1,2-Mans and virulence.
Virulence of two C. albicans strains in the infant mouse
model of gut colonization correlates with virulence in systemic
models and surface expression of ␤-1,2-Mans. One virulent
strain (ATCC 10261) with a high level of ␤-1,2-Man surface
expression and one avirulent strain (ATCC 10231) with a low
level of ␤-1,2-Man surface expression were selected. Their
virulences were compared in six independent experiments.
Preliminary analysis showed that the degree of gut colonization
evaluated according to the number of CFU per fecal pellet was
reproducible, allowing the data for two litters infected with the
same strain to be pooled (data not shown). Results from rep-
resentative experiments are shown in Table 2. The results are
for three litters inoculated with each strain, with the results for
a total of 31 animals infected with strain ATCC 10261, 30
animals infected with strain ATCC 10231, and 217 stool cul-
tures provided.

VOL. 46, 2002
␤-Man SYNTHETIC ANALOGUES AGAINST C. ALBICANS
3873
FIG. 4. Binding of anti-C. albicans MAbs to synthetic ␤-1,2-linked mannotetraose measured by EIA. MAb AF1, which is specific for C. albicans
␤-1,2-Mans (closed bars), and MAb CA1, which reacts with C. albicans ␣-linked mannose residues (open bars), were incubated with increasing
concentrations of synthetic ␤-1,2-linked mannotetraose.
From day 7 to day 33 after inoculation (day 7 was the first
day on which it was possible to assess colonization), coloniza-
tion was variable from mouse to mouse but was always signif-
icantly higher in mice colonized with strain ATCC 10261 than
in those colonized with strain ATCC 10231. Thus, colonization
was more protracted in animals infected with ATCC 10261
than in those infected with ATCC 10231 (at day 33 postinocu-
lation, 11 of 11 animals inoculated with ATCC 10261 were still
colonized, whereas 5 of 11 animals infected with ATCC 10231
were still colonized [P ϭ 0.006]). The rate of mortality for
animals without subsequent induced immunosuppression was
low. However, the rate of cumulative acute mortality from the
six independent experiments was higher among infant mice
inoculated with ATCC 10261 than among infant mice inocu-
lated with ATCC 10231, although the difference did not reach
statistical significance (6 of 63 versus 1 of 62 mice [P ϭ 0.059]).
Synthetic ␤-1,2 tetramannosides but not ␣-1,2 tetramanno-
sides inhibit gut colonization by a virulent C. albicans strain.
The last set of experiments was designed to test the prophy-
lactic effects of ␤-1,2-Mans. Synthetic tetramannosides were
administered as a single dose prior to C. albicans inoculation.
In a preliminary experiment, the effect of the administration of
50 ␮g of ⌺␤-Mans prior to inoculation of C. albicans strains
ATCC 10261 and ATCC 10231 was evaluated at day 7 posti-
noculation. Pretreatment had no effect when the mice were
inoculated with the strain expressing less ␤-1,2-Mans at the cell
surface (ATCC 10231), while there was a reduction (although
it was not significant) in the numbers of CFU from mice inoc-
ulated with the strain expressing more ␤-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 to use the more virulent strain expressing
more ␤-1,2-Mans, larger numbers of animals, different doses of
⌺␤-Mans, and ⌺␣-Mans as a control (Fig. 5). At day 7 after
inoculation, administration of ⌺␣-Mans had no effect on gut
colonization compared to the effects of distilled water (control
treatment) (median counts, 196 CFU [range, 11 to 1,000 CFU]
versus 258 CFU [range, 31 to 1,000 CFU] in the ⌺␣-Man-
treated group versus the controls [P Ͼ 0.05]). By contrast,
administration of 50 ␮g of ⌺␤-Mans led to a decrease in the
number of CFU per fecal pellet, which was significant. The
effect of ⌺␤-Mans was dose dependent, since the effect became
highly significant when 150 ␮g was administered. The efficacy
of this treatment was further demonstrated by the complete
disappearance after treatment with ⌺␤-Mans of the variability
in the level of gut colonization observed in all experiments
among untreated infant mice and in this experiment among
untreated and ⌺␣-Man-treated animals (Fig. 5 and Table 2).
We also sampled the animals on day 10 after inoculation and
observed that the level of colonization had dramatically in-
creased in all mice, but it had not increased as much in mice
treated with 150 ␮g of ⌺␤-Mans than in the other groups (P ϭ
0.0220).
TABLE 1. Agglutination scores for C. albicans strains reacted with
Bichro-latex particles sensitized with MAbs DF9-3 and AF1 specific
for ␤-1,2-Man epitopes compared with virulence determined from
systemic Candida infection of rats and micea
Cumulated score obtained with
Virulence observed
in vivo
beads sensitized with MAb:
Strain
DF9-3
AF1
Final score
ATCC 44505
ATCC 62342
ATCC 10261
ATCC 32354
ATCC 44831
ATCC 18804
ATCC 10231
4
2.5
4
4.5
2.5
3
2.5
2
6.5
8.5
9
Virulent
4.5
4.5
4.5
2.5
3
Virulent
Virulent
7
Intermediate
Avirulent
Avirulent
Avirulent
5.5
5.5
5.5
3.5
a The agglutination scores (obtained at 1, 2, and 3 min with the same suspen-
sion) were determined, and the final score (addition of the scores obtained with
MAbs DF9-3 and AF1) was used for statistical comparison (results of a typical
experiment). The virulences of the strains were determined as described previ-
ously (40).

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ANTIMICROB. AGENTS CHEMOTHER.
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 ␤-1,2-Mans, respectively, at their cell surfaces
Median (range) no. of CFU/fecal pelleta (no. of expts)
Day after
inoculation
Significanceb
ATCC 10231
ATCC 10261
Expt A
7
8
9
0.5 (0–31) (n ϭ 10)
2 (0–40) (n ϭ 11)
64.5 (0–475) (n ϭ 10)
510 (8–1,000) (n ϭ 10)
64.5 (0–1,000) (n ϭ 12)
1,000 (35–1,000) (n ϭ 13)
256 (160–10,000) (n ϭ 13)
1,000 (300–10,000) (n ϭ 12)
0.0011
0.0001
0.0001
0.0001
10
Expt Bc
12
107 (8–500) (n ϭ 19)
135 (25–1,000) (n ϭ 12)
17 (4–167) (n ϭ 11)
7.5 (0–82) (n ϭ 11)
0 (0–9) (n ϭ 11)
548 (144–1,000) (n ϭ 18)
10,000 (500–10,000) (n ϭ 11)
1,000 (196–1,000) (n ϭ 11)
398 (28–1,000) (n ϭ 11)
Ͻ0.0001
0.0003
Ͻ0.0001
0.0006
Ͻ0.0001
15
19
26
33
484 (7–10,000) (n ϭ 11)
a CFU were enumerated, and an arbitrary number (see the text) was used for counts Ͼ300 to allow statistical comparison by nonparametric tests.
b P value for comparison of the number of CFU per fecal pellet in ATCC 10231-infected mice versus the number in Mann-Whitney test. ATCC 10261-infected mice
on the same day. Significance was determined by the Mann-Whitney test.
c Results for 2 litters were pooled after the reproducibilities of the results between litters infected with the same isolate were checked (data not shown). The groups
were each composed of 5 to 10 animals, depending on the day of study.
DISCUSSION
adhesins (28), stimulators of immune response mediator se-
cretion (23, 24), and inducers of protective antibodies (18, 19).
The recent identification of galectin-3 (previously, the Mac 2
antigen) as a ␤-1,2-Man receptor on host cells provides further
evidence for the specificity of these interactions (15). It indeed
appears that C. albicans has a unique biological trait related to
its ability to express ␤-1,2-Mans at its cell surface and that this
biological trait has its counterpart in two major host recogni-
tion systems, namely, antibodies and endogenous lectins. It
therefore seemed worthwhile to explore the role of this system
Although C. albicans is not a true pathogen, it has some
biological characteristics, termed virulence factors (3), which
help it to invade debilitated hosts. Among these factors are cell
wall molecules that act as adhesins (44). C. albicans has been
shown to adhere to a wide variety of host cells and molecules
through protein-protein (17), yeast lectin-host carbohydrate (4,
52), or yeast carbohydrate-host lectin interactions. A growing
body of evidence suggests that ␤-1,2-Mans have an important
role in pathogenesis since they have been shown to act as
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 ␤-1,2 mannose at its cell surface. Infant mice received either distilled water (control) or solutions providing 50 ␮g of synthetic ␤-1,2
tetramannose (␤-Man), 150 ␮g of ␤-1,2 tetramannose, or 150 ␮g of synthetic ␣-1,2 tetramannose (␣-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%. ء, P Ͻ 0.02 versus 50 ␮g of ␤-1,2 tetramannose; ءء, P Ͻ 0.0001 versus 150 ␮g of ␤-1,2
tetramannose; †, not significant versus 150 ␮g of ␣-1,2 tetramannose (P values were determined by the Kruskal-Wallis test).

VOL. 46, 2002
␤-Man SYNTHETIC ANALOGUES AGAINST C. ALBICANS
3875
in the process of colonization of the gut, where galectin-3 is
widely expressed (9).
indigenous flora by providing a local competitor. When infant
mice were inoculated with two different Candida species, the
number of mice colonized with Candida glabrata increased as
the number of mice colonized with C. albicans decreased (13).
The C. albicans competitor used in human studies is the non-
pathogenic yeast Saccharomyces boulardii (21), but conflicting
results and rare cases of fungemia due to Saccharomyces spe-
cies may discourage use of this approach (38).
To address this question of the role of ␤-1,2-Mans in colo-
nization, a multiple-step study was designed. Previous data
suggested a relationship between the expression of ␤-1,2-Mans
at the yeast cell surface and the virulence of strains (16). By
testing several unrelated strains of C. albicans that have been
shown to exhibit various degrees of virulence in animal models
of systemic infection (40), a clear correlation was found be-
tween these two parameters; namely, the weaker the expres-
sion of ␤-1,2-Mans is, the lower the reported level of virulence
is, and vice versa. This is the first set of experiments suggesting
a correlation between surface expression of ␤-1,2-Mans and C.
albicans strain pathogenicity in animal models. The same trend
was observed when more than 200 C. albicans clinical isolates
were tested in a previous study (16). The isolates recovered
from colonized patients were less agglutinated by an MAb
designated 5B2 than those isolated from infected patients.
Interestingly, subsequent immunochemical experiments
showed that MAb 5B2 is specific for ␤-1,2-Man epitopes (48).
In the present study the strain with the higher levels of expres-
sion of ␤-1,2-Man epitopes was shown to induce higher levels
of colonization and more protracted colonization of the gut of
infected mice than the strain with lower levels of expression of
␤-1,2-Man epitopes.
To further assess the role of ␤-1,2-Mans in intestinal colo-
nization, synthetic oligosaccharides were administered as po-
tential competitors prior to inoculation with the most virulent
strain. Preparation of large quantities of mannan-derived oli-
gomannosides is based on a long and tedious procedure and
requires nuclear magnetic resonance verification of the struc-
tures obtained (12). As previous studies with macrophages
demonstrated that synthetic molecules exhibiting a minimum
polymerization of four residues inhibit ␤-1,2-Mans binding and
stimulation of mammalian cells (14, 24), it was decided to
chemically synthesize ␤-1,2 mannotetraoses. Appropriate con-
trols, consisting of ␣-1,2-Man tetramannoses, were also syn-
thesized. EIA demonstrated that the antigenicities of both
⌺␣-Mans and ⌺␤-Mans mimicked the antigenicities of C. al-
bicans-derived homologues. When animals were given ⌺␤-
Mans (30 mg/kg) prior to inoculation with C. albicans, a dra-
matic decrease in the number of yeasts recovered from stools
was observed; however, this decrease was not seen following
administration of the same dose of ⌺␣-Mans. To us, this effect
is of real biological significance because it was evidenced 7 days
and even 10 days after administration. Furthermore, it was
dose and structure dependent.
Even if the efficacy of ⌺␤-Mans is transient (since we chose
to administer only a single dose), our data suggest that this
innovative approach may be useful for the prevention of sys-
temic Candida infection in at-risk patients. This strategy has
several potential advantages over existing prophylactic regi-
mens: (i) the chemical procedure can be easily adapted to the
production of large quantities of ␤-Mans; (ii) ⌺␤-Mans are
resistant to gastric pH and should also be well tolerated, as
␤-1,2-Mans (from C. albicans) are already present in the hu-
man gut; (iii) ⌺␤-Mans should 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 models are in progress to determine the influence of
the administration regimen (timing, doses) on the therapeutic
efficacies of ⌺␤-Mans for the long-lasting prevention of gut
colonization and to determine whether it extends to a reduc-
tion in the level of existing colonization or the treatment of
existing colonization. In vitro studies are also in progress since,
besides their therapeutic applications, the clear-cut results ob-
tained here are of basic pathophysiological and phylogenetic
interest for the understanding of host-endogenous microbe
interactions.
ACKNOWLEDGMENTS
This work was supported by the Programme de Recherche Fonda-
mentale en Microbiologie, Maladies Infectieuses et Parasitaires, and
collaborative research was developed through “Re´seau Infections
Fongiques,” supported by the same program.
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