INFECTION AND IMMUNITY, Nov. 2010, p. 4644–4652
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 78, No. 11
Interaction of Candida albicans Cell Wall Als3 Protein with
Streptococcus gordonii SspB Adhesin Promotes Development
of Mixed-Species Communities?
Richard J. Silverman,1Angela H. Nobbs,1M. Margaret Vickerman,2
Michele E. Barbour,1and Howard F. Jenkinson1*
School of Oral and Dental Sciences, University of Bristol, Lower Maudlin Street, Bristol BS1 2LY, United Kingdom,1and
Department of Oral Biology, Foster Hall, State University of New York at Buffalo, Buffalo, New York 142142
Received 25 June 2010/Returned for modification 16 July 2010/Accepted 22 August 2010
Candida albicans colonizes human mucosa and prosthetic surfaces associated with artificial joints,
catheters, and dentures. In the oral cavity, C. albicans coexists with numerous bacterial species, and
evidence suggests that bacteria may modulate fungal growth and biofilm formation. Streptococcus gordonii
is found on most oral cavity surfaces and interacts with C. albicans to promote hyphal and biofilm
formation. In this study, we investigated the role of the hyphal-wall protein Als3p in interactions of C.
albicans with S. gordonii. Utilizing an ALS3 deletion mutant strain, it was shown that cells were not affected
in initial adherence to the salivary pellicle or in hyphal formation in the planktonic phase. However, the
Als3?mutant was unable to form biofilms on the salivary pellicle or deposited S. gordonii DL1 wild-type
cells, and after initial adherence, als3?/als3? (?ALS3) cells became detached concomitant with hyphal
formation. In coaggregation assays, S. gordonii cells attached to, and accumulated around, hyphae formed
by C. albicans wild-type cells. However, streptococci failed to attach to hyphae produced by the ?ALS3
mutant. Saccharomyces cerevisiae S150-2B cells expressing Als3p, but not control cells, supported binding
of S. gordonii DL1. However, S. gordonii ?(sspA sspB) cells deficient in production of the surface protein
adhesins SspA and SspB showed >50% reduced levels of binding to S. cerevisiae expressing Als3p.
Lactococcus lactis cells expressing SspB bound avidly to S. cerevisiae expressing Als3p, but not to S150-2B
wild-type cells. These results show that recognition of C. albicans by S. gordonii involves Als3 protein-SspB
protein interaction, defining a novel mechanism in fungal-bacterial communication.
Candida species are the fourth most common causative
agents of nosocomial bloodstream infections (2, 47, 54). Crude
mortality rates for Candida infections exceed 50% (10, 52), and
attributable mortality rates vary between 5 and 48% (3, 10, 13).
Candida albicans accounts for 62% of invasive candidiasis in-
fections (46, 47) and is commonly isolated from the oral cavity,
gastrointestinal tract, and vagina. The oral carriage rate of C.
albicans in healthy subjects ranges from 25 to 60% (28, 42, 48).
In the oral cavity, there are estimated to be approximately 700
different species of microorganisms present (45). C. albicans is
able to interact physically, by coaggregation, or chemically,
through small-molecule signaling, with some of these other
microorganisms (1, 18, 20, 29, 33). Interactions of C. albicans
with bacteria may be antagonistic, e.g., with Pseudomonas
aeruginosa (20), or synergistic, e.g., with Streptococcus gordonii
(1), resulting in the formation of diverse polymicrobial com-
Streptococcus gordonii is a primary colonizer of the oral
cavity and may be isolated from mucosal or hard surfaces
present there (17, 41). It has previously been shown that S.
gordonii, and other viridans streptococci, can coaggregate
with C. albicans cells both in vitro and in vivo (21, 29, 57).
The interactions between oral streptococci and C. albicans
are recognized as contributing to formation of enhanced
biofilms (1), which may occur on dentures, leading to den-
ture stomatitis (42). Oral streptococci express a range of cell
surface polypeptides, many of which act as adhesins to pro-
mote colonization (31, 38). The antigen (Ag) I/II family of
polypeptides are cell wall-anchored proteins produced by
most indigenous species of oral streptococci (4). These ad-
hesins have been shown to bind a wide range of host cell
proteins, including fibronectin (49) and salivary agglutinin
gp-340 (5, 12, 27). In addition, the Ag I/II family polypep-
tide SspB from S. gordonii has been shown to interact di-
rectly with other microorganisms, including Actinomyces
naeslundii (27), Porphyromonas gingivalis (11), and C. albi-
cans (1, 22). It is thus proposed that oral streptococci may
promote colonization by these other microorganisms by pro-
viding alternative surfaces to adhere to (30) and possibly
metabolic benefits (25).
Candida albicans is a pleomorphic fungus, with the two most
commonly identified morphologies being yeast cells and hy-
phae. Hyphal-filament formation may be induced by many
factors, including pH, serum, temperature, nutrient availabil-
ity, and diffusible cell signaling molecules (53). In a mixed-
species biofilm model, S. gordonii enhances hyphal formation,
and there is evidence that this may be mediated, at least in
part, by soluble factors released by streptococci (1). Within
mixed-species biofilms of S. gordonii and C. albicans, strepto-
* Corresponding author. Mailing address: Oral Microbiology,
School of Oral and Dental Sciences, University of Bristol, Lower
Maudlin Street, Bristol BS1 2LY, United Kingdom. Phone: 44-117-
342-4424. Fax: 44-117-342-4313. E-mail: howard.jenkinson@bristol
?Published ahead of print on 30 August 2010.
cocci were found associated with yeast cells, pseudohyphae,
and hyphae, but preferentially with hyphal filaments (1).
The hyphal cell wall comprises a mixture of chitin, ?-1,3
glucans, and ?-1,6 glucans, as well as a vast array of proteins
(7). One of the major families of C. albicans adhesins is the
ALS (agglutinin-like sequence) group of cell wall glycopro-
teins (24). The family comprises 8 members, several of
which have adhesive functions involved in host-pathogen
interactions (24). One of these adhesins, Als3p, is a hypha-
specific protein (9, 23) and has been shown to be required
for mature-biofilm formation, binding extracellular matrix,
adhesion to host cells, and internalization of C. albicans by
endothelial cells (24, 50, 56). There is also evidence that the
Als5 protein is involved in recognition of S. gordonii by C.
In this study, we investigated the role of hypha-specific Als3p
in early-stage biofilm formation and in intergeneric interac-
tions of C. albicans with S. gordonii. The results suggest that
Als3p interacts directly with SspB on the surface of S. gordonii,
a binding event that may then enable additional concerted
adhesin-receptor interactions to become established.
MATERIALS AND METHODS
Strains and growth conditions. The bacterial strains used in this study were
S. gordonii DL1 (Challis) wild type (WT), S. gordonii UB1360 ?(sspA sspB)
(19), Lactococcus lactis MG1363, and L. lactis UB1586(pUB1000-sspB), a
strain constitutively expressing heterologous SspB (27). Streptococci were
routinely maintained on BHY agar (37 g/liter brain heart Infusion [Difco], 5
g/liter yeast extract, and 1.5% agar). Liquid cultures were grown statically in
BHY broth in capped bottles at 37°C. S. gordonii UB1360 cultures were
supplemented with spectinomycin (100 ?g/ml). Lactococci were cultivated on
M17 medium (Difco) containing 0.5% glucose and 2% agar. Liquid cultures
were grown statically in M17-glucose at 30°C in capped tubes. Strain UB1586
containing plasmid pUB1000-sspB was grown in the presence of erythromycin
(5 ?g/ml). The yeast strains used in this study were C. albicans strain NGY152
(CAI-4/CIp10) (6, 37) or 1843 als3?/als3? (55) and Saccharomyces cerevisiae
S150-2B containing plasmid pADH or pADH-ALS3, constitutively expressing
heterologous ALS3 under the alcohol dehydrogenase (ADH) promoter (50).
C. albicans NGY152 expresses URA3 in a CAI-4 (Ura-negative) background
and was used as a control strain for comparison with the als3?/als3? (?ALS3)
mutant. C. albicans strains were maintained aerobically on Sabouraud dex-
trose agar (Difco) at 37°C, and broth cultures were grown in yeast extract-
peptone-dextrose (YPD) medium (10 g/liter yeast extract, 20 g/liter peptone,
20 g/liter dextrose) at 37°C with orbital shaking at 200 rpm. S. cerevisiae
containing pADH or pADH-ALS3 was maintained on synthetic medium
lacking uracil (CSM-glu) (0.077% CSM-ura [Formedium], 0.67% yeast nitro-
gen base [Difco], 2% glucose, and 2.5% agar). Liquid cultures were grown
aerobically at 30°C with shaking. C. albicans biofilm formation and hyphal
induction were performed at 37°C in YPT-Glc medium (yeast nitrogen base
in 10 mM NaH2PO4-Na2HPO4buffer, pH 7.0, containing 0.05% Bacto tryp-
tone and 0.5% glucose) (1).
Saliva preparation. Unstimulated whole human saliva was collected from at
least 5 healthy volunteers with Institutional Review Board approval. Saliva was
pooled and mixed with dithiothreitol (2.5 mM) before clarification by centrifu-
gation (10,000 ? g; 10 min). The supernatant was diluted to 25% in distilled
water (dH2O) and filter sterilized through a 0.22-?m nitrocellulose membrane.
Aliquots of prepared saliva were stored at ?20°C.
Biofilm formation. Sterile glass coverslips (13-mm diameter) were incubated
with filter-sterilized 10% saliva in distilled water for 16 h at 4°C and washed twice
with dH2O. For monospecies biofilms, late-stationary-phase cells of S. gordonii in
BHY medium were harvested by centrifugation (5,000 ? g; 5 min) and sus-
pended in YPT-Glc at an optical density at 600 nm (OD600) of 0.1 (?5 ? 107
CFU/ml). Portions (0.5 ml) were added to wells of 24-well polystyrene tissue
culture plates containing saliva-coated coverslips, and the plates were incubated
at 37°C with gentle shaking at 50 rpm. After 1 h of incubation, nonattached
bacteria were aspirated and the coverslips were gently washed twice with YPT-
Glc. Fresh YPT-Glc medium was added to the wells, and the biofilms were grown
for up to 6 h, with the coverslips recovered at intervals for microscopic or
biomass analysis. C. albicans cells were grown for 16 h in YPD medium at 37°C
and harvested by centrifugation (5,000 ? g; 5 min). The cells were suspended in
YPT-Glc at an OD600of 0.1 (3 ? 106CFU/ml), and portions (0.5 ml) were added
to wells containing saliva-coated coverslips. The plates were then incubated for
various times at 37°C with gentle movement (50 rpm) to promote biofilm for-
To produce mixed-species biofilms, C. albicans suspensions prepared as de-
scribed above were added to coverslips that had previously been incubated with
S. gordonii cells for 1 h and washed. After inoculation with C. albicans, the plates
were incubated at 37°C for up to 6 h. At intervals, duplicate coverslips were
removed, washed with PBS, and air dried. The biofilms were then stained with
crystal violet (CV) and visualized by light microscopy. Quantification of biofilm
formation was achieved by releasing crystal violet with 10% acetic acid and
measuring the absorbance at 595 nm.
Coaggregation assays. Streptococci or lactococci were cultured for 16 h
under their respective growth conditions. Bacteria were harvested by centrif-
ugation (5,000 ? g; 5 min), suspended in 1.5 mM fluorescein isothiocyanate
(FITC) solution in 0.05 M Na2CO3containing 0.1 M NaCl (pH 7.5), and
incubated at 20°C for 30 min. The fluorescently labeled cells were washed
thoroughly by alternate centrifugation and suspension in TNMC buffer (1
mM Tris-HCl, pH 8.0, containing 0.15 M NaCl, 0.1 mM MgCl2, and 0.1 mM
CaCl2) (8) to remove excess FITC, and suspended in TNMC at an OD600of
0.5. C. albicans filamentation was induced by incubation of cells in YPT-Glc
for 3 h at 37°C. S. cerevisiae was grown in CSM-ura medium for 16 h at 30°C
with vigorous aeration. Yeast or hyphal cells were collected by centrifugation,
washed with TNMC buffer, and suspended in TNMC at an OD600of 1.0. For
coaggregation assays, FITC-labeled bacterial suspension (1 ml) was mixed
with yeast cell suspension (1 ml) and incubated for 1 h at 37°C (C. albicans)
or at 30°C (S. cerevisiae) with shaking. Portions of the suspensions were then
deposited onto microscope slides and visualized by light or fluorescence
microscopy. Degrees of coaggregation of S. gordonii or L. lactis with C.
albicans were assigned to one of three categories: 2?, extensive attachment
of bacteria to hyphae or yeast cells with bacterial-cell clumping; 1?, align-
ment of bacterial cells along hyphae in distinct patches; 0, sparse or no
interactions between bacteria and hyphae. The numbers of hyphae with
degrees of bacterial binding, expressed as percentages of the total number of
hyphae counted, were determined from 2 independent experiments. We
found that the C. albicans strains NGY152 (CAI4-4/CIp10) and SC5314 (wild
type) behaved identically in all assays. For S. cerevisiae coaggregations, the
yeast cells binding bacteria were counted and expressed as the proportion of
the total number of yeast cells visualized from two independent experiments.
For aggregation assays, between 50 and 100 hyphal cells and between 300 and
500 S. cerevisiae cells were counted for each coaggregation pairing.
Yeast cell wall extracts. Late-stationary-phase S. cerevisiae cells or C. albicans
cells, induced to form hyphal filaments, were harvested by centrifugation and
washed in Tris-buffered saline (TBS) (10 mM Tris-HCl containing 0.15 M NaCl,
pH 8.0). The cell pellets were suspended in 1 M sorbitol containing 40 mM
2-mercaptoethanol and 125 U/ml lyticase (Sigma) (1 ml), and incubated for 4 h
at 37°C with shaking. Crude cell wall fractions were separated from cell debris by
centrifugation (3,000 ? g; 10 min; 4°C). The supernatant containing cell wall
proteins was then centrifuged (5,000 ? g; 5 min), and portions of the supernatant
were mixed with sample buffer (50 mM Tris-HCl, pH 6.8, containing 1% SDS)
and subjected to SDS-PAGE. Proteins were stained with Coomassie blue R250
or electroblotted onto a nitrocellulose membrane (Hybond). The blots were
probed with monoclonal antibody to Als3p (3-A5) (9) diluted 1:1,000. Antibody
binding was detected with an appropriate horseradish peroxidase (HRP)-conju-
gated secondary antibody (Dako), and the blots were developed with 4-chloro-
L. lactis expression of SspB. Lactococcal cells were harvested from late-expo-
nential-phase cultures and suspended in TNMC buffer at an OD600of 0.5.
Anti-Ag I/II antibody (26) diluted 1:500 or irrelevant rabbit antiserum (1:500)
was mixed with 0.5 ml cells and incubated for 1 h at 37°C. Bacteria were
harvested, washed three times with TNMC buffer to remove nonspecific anti-
body, and then incubated with FITC-conjugated anti-rabbit antibody (Dako)
(diluted 1:1,000) for 30 min. Cells were collected by centrifugation and washed
three times, and portions were applied to glass slides and visualized by light or
fluorescence microscopy. Western immunoblot analysis of cell surface protein
extracts of L. lactis expressing SspB reactive with Ag I/II antibody has been
previously described (26).
Statistics. Data were processed using PRISM software (Graph Pad). An un-
paired Student’s t test was used to perform statistical analyses at a confidence
level with a P value of ?0.05.
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Editor: G. S. Deepe, Jr.
4652 SILVERMAN ET AL.INFECT. IMMUN.