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.
VOL. 78, 2010CANDIDA ALBICANS-STREPTOCOCCUS INTERACTIONS 4645
Role of Als3p in early-stage biofilm formation. S. gordonii
forms dual-species biofilms with C. albicans in which strep-
tococcal cells clearly associate more avidly with hyphal fil-
aments than with yeast cells (1). Therefore, we hypothesized
that a hypha-specific component or components provided
receptors for binding by S. gordonii. The Als3p protein is
one of several hypha-specific glycoproteins involved in ad-
hesion to host tissues and tissue proteins and has been
shown to be required for mature-biofilm formation (39, 56).
Accordingly, we first investigated the role of Als3p in early-
stage biofilm formation in our system, and then the interac-
tion of Als3p with S. gordonii. Glass coverslips were coated
with human salivary proteins as described in Materials and
Methods and incubated with suspensions of C. albicans
NGY152 (CAI4/Clp10) (here referred to as WT) or the 1843
als3?/als3? mutant (here designated ?ALS3). Biofilms were
developed at 37°C in YPT-Glc medium with gentle agitation
and visualized by light microscopy at intervals over 6 h. In
the first hour, similar numbers of WT and ?ALS3 cells
attached to the surface, and hyphal formation was clearly
initiated in similar proportions of cells (Fig. 1A). At 3 h,
similar patterns of colonization were observed for the two
strains, with cells forming hyphal filaments up to 20 ?m in
length and relatively evenly distributed across the salivary-
pellicle surface (Fig. 1A). At 6 h, C. albicans WT formed a
dense biofilm consisting of networks of hyphal filaments,
pseudohyphae, and yeast cells. However, the ?ALS3 mutant
had not formed a biofilm, and only a few hyphae and yeast
cells remained attached to the pellicle-coated surface (Fig.
1A). These results suggested that although biofilm forma-
tion was initiated in the ?ALS3 mutant, most of the cells
developing hyphae then failed to remain attached to the
surface and became dispersed. Quantification of the biofilms
confirmed the visual observations showing little or no dif-
ference in biomass between the two strains after 1 h or 3 h
of growth. However, the biomass of the ?ALS3 mutant
biofilm was significantly reduced at 6 h compared to the WT
parent strain (P ? 0.05) (Fig. 1C). To confirm that the
?ALS3 mutant was not deficient in growth, biofilms were
produced in which C. albicans cells were allowed to adhere
to the saliva-coated coverslip for 1 h and then nonattached
cells were removed and replaced with fresh medium. The
OD600of planktonic-phase samples taken at intervals up to
6 h increased markedly for the ?ALS3 mutant compared to
the WT strain (Fig. 1D). These results suggested that WT
cells adhered to the substratum with only a small fraction of
cells becoming detached, whereas the ?ALS3 mutant hy-
phae became detached from the surface and continued to
undergo growth and hyphal development in the planktonic
Saliva-coated coverslips were then incubated with S. gordonii
DL1 cells, which readily form a confluent layer under these
conditions (1), and biofilms of C. albicans were allowed to
develop on this bacterial base layer. In these dual-species bio-
films, C. albicans WT hyphal filaments were more prolific than
those formed on the salivary-pellicle base, and dense networks
of hyphae were associated with the S. gordonii monolayer (Fig.
1B). As before, the ?ALS3 mutant showed levels of attach-
ment to similar those of the WT after 1 h or 3 h of incubation.
However, after 6 h there were virtually no hyphal filaments or
yeast cells of the ?ALS3 mutant attached to the streptococcal
cell monolayer (Fig. 1B). These experiments showed that the
?ALS3 mutant became detached not only from a pellicle-
coated surface, but also from a streptococcal monolayer. The
detachment occurred concomitantly with the elongation of
preexisting hyphal cells as opposed to the initiation of emerg-
ing germ tube filaments.
Als3p is necessary for S. gordonii binding to C. albicans
hyphae. To further characterize the physical interactions oc-
curring between C. albicans Als3p and S. gordonii DL1 cells, a
fluorescence-based coaggregation assay was developed. Fluo-
rescently labeled S. gordonii cells were incubated with C. albi-
cans cells that had been induced to form hyphal filaments for
3 h. Because of heterogeneity within the population of C.
albicans with respect to the degree of hyphal formation, and in
levels of bacterial attachment to hyphal filaments, we catego-
rized the hyphal-bacterial interactions into three groupings:
2?, streptococci forming dense accumulations around hyphae
(Fig. 2A and B); 1?, distinct patches of streptococci aligned
alongside regions of hyphae (Fig. 2C and D); 0, few or no
streptococcal cells associated with hyphae (Fig. 2E and F). In
Fig. 2G, summarizing these results, the bars above the baseline
represent percentages of hyphae with streptococci bound,
while the bars below the baseline show percentages of hyphae
scoring 0. On the basis of this assessment, it was estimated that
60% of wild-type hyphal filaments demonstrated 2? binding of
streptococci, 25% showed 1? binding, and 15% of hyphae had
no streptococci bound to them (Fig. 2G). For the C. albicans
?ALS3 strain, coaggregation was ablated, with 97% of hyphal
filaments having no streptococcal cells attached and thus scor-
ing 0 in the assay (Fig. 2G).
S. gordonii SspB interacts with C. albicans hyphae. Previous
studies have suggested that the S. gordonii antigen I/II proteins
SspA and SspB were involved in binding to C. albicans cells (1).
Coaggregation assays performed between C. albicans wild-type
hyphae and S. gordonii UB1360 ?(sspA sspB) showed only
small, and statistically insignificant, differences in mutant-cell
binding levels compared with the S. gordonii DL1 wild type
(data not shown). This is probably because other S. gordonii
surface components are able to interact with C. albicans (21).
We then tested the ability of a surrogate host bacterium, L.
lactis expressing SspB, to coaggregate with C. albicans hyphae.
Cell wall localization of SspB in L. lactis UB1586 had previ-
ously been demonstrated (27), although expression of the pro-
tein on the cell surface had not. Accordingly, we confirmed
surface expression of SspB by immunofluorescence experi-
ments (Fig. 3). Antibodies reactive with SspB bound only to L.
lactis UB1568 cells containing pUB1000-sspB and not to con-
trol cells (Fig. 3A to D).
In coaggregation assays of L. lactis MG1363 wild-type
cells with C. albicans WT, low levels of interaction were
observed, with 45% of hyphae showing no bound lactococci
and 52% with a 1? level of coaggregation (Fig. 4). However,
with L. lactis expressing SspB, ?50% of wild-type hyphae
showed 2? coaggregation levels, significantly more than
with L. lactis WT (P ? 0.05), and ?25% showed 1? binding
(Fig. 4), thus confirming a direct role for SspB in hyphal-
filament recognition. On the other hand, cells of L. lactis
4646SILVERMAN ET AL.INFECT. IMMUN.
expressing SspB were deficient in binding to hyphae formed
by the C. albicans ?ALS3 mutant. Less than 5% of ?ALS3
mutant hyphae avidly bound L. lactis expressing SspB, and
60% of hyphae showed no binding (P ? 0.05) (Fig. 4). There
were lower levels of coaggregation between C. albicans
?ALS3 and L. lactis WT than between C. albicans WT and
L. lactis WT (Fig. 4). This suggests that Als3p might also
weakly recognize an irrelevant lactococcal surface compo-
SspB interacts with Als3p. The previous results suggested
that S. gordonii SspB might interact directly with C. albicans
Als3p. Therefore, we further characterized the interactions
by utilizing S. cerevisiae expressing heterologous Als3p. Ex-
pression of Als3p in this host was confirmed following im-
munoblot analysis of proteins released from cell walls fol-
lowing lyticase treatment. Blots of cell wall protein extracts
were probed with monoclonal antibody to Als3p (9), and
this detected a band with an approximate molecular mass of
105 kDa (Fig. 3E). A band of ?120 kDa was present in
extracts from C. albicans wild-type hyphal cell walls, corre-
sponding well to the predicted mass of ?124 kDa (Fig. 3E).
No band was observed on immunoblots of cell wall extracts
FIG. 1. Als3p in mono- or dual-species biofilms. (A) C. albicans wild type or C. albicans als3?/als3? biofilms that formed on saliva-coated
glass at 1 h, 3 h, or 6 h stained with CV and visualized by microscopy. The images are representative of experiments performed in triplicate.
Scale bars, 40 ?m. (B) C. albicans wild type or C. albicans als3?/als3? biofilms that formed on S. gordonii DL1 cells at 1 h, 3 h, or 6 h stained
with crystal violet and visualized by microscopy. The images are representative of experiments performed in triplicate. Scale bars, 25 ?m.
(C) Biomasses of biofilms of C. albicans WT or C. albicans ?ALS3 assessed by CV release. (D) Cell densities of planktonic-phase samples
from developing C. albicans biofilms. The data are means and standard deviations (SD) from experiments performed in triplicate (?, P ?
VOL. 78, 2010 CANDIDA ALBICANS-STREPTOCOCCUS INTERACTIONS 4647
obtained from the C. albicans als3?/als3? mutant (Fig. 3E).
Als3p is predicted to be heavily glycosylated (23), and thus,
the differences in observed masses between S. cerevisiae
recombinant Als3p and C. albicans Als3p might result from
Recombinant S. cerevisiae cells expressing Als3p were
found to bind S. gordonii DL1 wild-type cells avidly, with
60% of S. cerevisiae cells binding S. gordonii compared with
?10% of S. cerevisiae WT cells (Fig. 5E). Deletion of the
sspA and sspB genes in S. gordonii UB1360 led to signifi-
cantly reduced numbers of S. gordonii cells attaching to S.
cerevisiae ALS3?(Fig. 5E). We then determined if L. lactis
expressing SspB interacted with S. cerevisiae. L. lactis cells
expressing SspB bound only weakly to wild-type S. cerevisiae
cells (Fig. 5A, B, and F). However, SspB-expressing cells of
L. lactis interacted strongly with ?85% of S. cerevisiae cells
expressing Als3p (Fig. 5C, D, and F). Wild-type L. lactis
interacted with only ?25% of S. cerevisiae cells expressing
Als3p (Fig. 5F). Taken collectively, these data show that S.
gordonii SspB interacts directly with C. albicans Als3p, thus
providing a molecular mechanism for mediating binding of
S. gordonii cells to C. albicans hyphal filaments.
Attachment of microorganisms to oral or dental surfaces,
and the subsequent formation of biofilms, may lead to per-
sistent oral colonization by C. albicans. These biofilms may
subsequently act as reservoirs of C. albicans cells that could
become dispersed and lead to systemic disease (51). Within
the oral cavity, attachment to a surface is important for
microorganisms to avoid being cleared by salivary flow (30,
43). It is essential to understand the complex physical and
chemical interactions occurring between C. albicans and the
host, and other microorganisms present in biofilm commu-
nities. Mature C. albicans biofilms comprise a mixture of
yeast cells, pseudohyphae, and hyphal filaments. The last
form a network of interlocked branches that is thought to
give the biofilm structure and rigidity. The current notion is
that C. albicans surface proteins, some of which are hypha
specific (7), are necessary for initiation of biofilm formation
and for interhyphal communication within biofilms (24).
Thus, when genes encoding the hyphal-wall protein Hwp1
and the related adhesins Hwp2 and Rbt1 were deleted,
biofilm formation was inhibited (14, 39, 40). The surface
protein adhesin Eap1, which is not hypha specific, also con-
FIG. 2. Coaggregation of C. albicans WT or ?ALS3 with S. gordonii. (A to F) Fluorescently (FITC) labeled S. gordonii DL1 cells were incubated
with C. albicans hypha-forming cells for 1 h. Coaggregated microorganisms were visualized by phase-contrast microscopy (A, C, and E) or by
fluorescence microscopy (B, D, and F). Scale bars, 25 ?m. Images A to F are representative of samples of S. gordonii DL1 and C. albicans WT
exhibiting population heterogeneity with respect to the ability of hyphae to bind bacteria. (G) Percentages of total C. albicans hyphae with attached
S. gordonii DL1 determined on the basis of the following binding levels (see Materials and Methods): 2?, hyphae completely surrounded by
streptococci (B); 1?, hyphae with areas where streptococcal cells are attached (D); 0, little or no bacterial binding (F). The data are means ? SD
from duplicate experiments.
4648SILVERMAN ET AL.INFECT. IMMUN.
tributes to biofilm formation (35). The hypha-specific sur-
face protein Als3p is involved in hyphal aggregation, and
als3?/als3? null mutants formed very weak biofilms after
48 h in a catheter model (56).
In this study, we have shown that als3?/als3? mutant cells
were able to attach to a saliva-coated surface in a manner
similar to that of C. albicans WT parent cells and formed
very early-stage biofilms up to 3 h. This result might have
been anticipated, because initial attachment of yeast cells
must involve adhesins other than Als3p, which is a hypha-
specific protein. At 3 h, short hyphal filaments of WT and
?ALS3 strains could be seen associated with the saliva-
coated surface, but by 6 h, the als3?/als3? mutant cells
forming hyphae had become detached from the surface.
This suggests that the initial adherence interactions were
not sufficient to sustain biofilm formation or that the medi-
ators of initial attachment were downregulated upon hyphal
formation and Als3p expression. Interestingly, hyphal devel-
opment by the ?ALS3 strain continued to occur in the
planktonic phase, confirmed by growth and development
measurements (Fig. 1D), but these hypha-producing cells
did not subsequently become attached to the substratum.
This suggests a critical role for Als3p in hyphal-filament
adherence to the substratum. Recent studies (40) have
shown that an als1?/als1? als3?/als3? double mutant, or an
hwp1?/hwp1? mutant, was unable to form biofilms individ-
ually. However, when the two mutants were coincubated,
biofilms were formed that were as thick as those formed by
the WT strain (40). These observations indicate that there
may be complementary adhesin functions between Hwp1p,
Als1p, and Als3p.
A majority of studies investigating C. albicans adherence and
biofilm formation have been concerned with monospecies cul-
tivation. However, within the natural environment of the oral
cavity, hundreds of different species of bacteria are present
that potentially influence colonization by C. albicans. For ex-
ample, S. gordonii enhances biofilm formation when it is in
association with C. albicans and promotes hyphal-filament pro-
duction (1). It is suggested that close proximity of the two
microorganisms may aid exchange and sensing of diffusible
signals. Since Als3p is reported to bind a range of molecules
(24), we investigated whether a complementary adhesin mech-
anism, as described above, might operate between the als3?/
als3? mutant cells and S. gordonii. However, in dual-species
biofilm formation with S. gordonii, the ?ALS3 strain forming
hyphae became detached as before (Fig. 1B), suggesting that
Als3p might be a protein receptor for binding of S. gordonii to
Previous studies investigating the physical interactions be-
tween S. gordonii and C. albicans have focused mainly upon the
streptococcal adhesins (21, 22). These studies have suggested
that the antigen I/II family polypeptides SspA and SspB and
the surface fibrillar polypeptide CshA (36) were involved in
adherence of C. albicans to immobilized streptococcal cells. In
coaggregation assays, S. gordonii wild-type cells demonstrated
a preference for binding hyphal filaments, suggesting that a
hypha-specific component may act as a receptor for S. gordonii.
Since there was little or no coaggregation between S. gordonii
and the ?ALS3 mutant, it seemed likely that a major receptor
was Als3p (Fig. 2G). An alternative possibility was that dele-
tion of ALS3 affected the expression or presentation of other
hyphal surface molecules. We therefore utilized an S. cerevisiae
strain expressing Als3p in coaggregation assays with S. gordonii
to avoid these potential issues and corroborate the current
findings. This clearly showed that S. gordonii interacted with
Als3p, complementing previous studies by Klotz et al. suggest-
ing that Als5p bound S. gordonii (32).
To identify the S. gordonii factor recognizing Als3p, we
utilized an L. lactis strain expressing SspB on the cell sur-
face, based on evidence from mutagenesis studies that the
SspB and SspA proteins were involved in C. albicans recog-
nition (22). The SspB polypeptide has been shown to inter-
act with P. gingivalis and to promote biofilm formation by
this oral anaerobic and pathogenic bacterium (11). The
SspB protein also mediates coaggregation of S. gordonii with
A. naeslundii to form early dental plaque biofilms (27). The
SspB-expressing strain of L. lactis, but not control L. lactis,
mediated strong coaggregation with S. cerevisiae expressing
Als3p. These results identify the first molecular mechanism
for binding of a Gram-positive bacterium to C. albicans. We
FIG. 3. Expression of heterologous proteins in L. lactis or S.
cerevisiae. Lactococcal cells were incubated with antibodies to SspB,
and antibody reactivity was detected with FITC-labeled anti-rabbit
secondary antibody. (A and C) Light microscopy. (B and D) Fluo-
rescence microscopy. (A and B) L. lactis MG1363 (control) cells
nonreactive with antibody. (C and D) L. lactis expressing SspB.
(E) Western blots of lyticase-extracted fungal cell wall proteins
reacted with monoclonal antibody to Als3p and antibody binding
detected with HRP-linked anti-mouse secondary antibody. Lane 1,
S. cerevisiae wild type; lane 2, S. cerevisiae expressing Als3p; lane 3,
C. albicans NGY152 wild type; lane 4, C. albicans als3?/als3?.
VOL. 78, 2010 CANDIDA ALBICANS-STREPTOCOCCUS INTERACTIONS4649
have not yet investigated the potential role of the CshA
fibrillar protein or SspA in interaction with Als3p. Since
deletion of the sspA and sspB genes in S. gordonii did not
ablate coaggregation with C. albicans wild-type hyphae or S.
cerevisiae expressing Als3p, it seems likely that other surface
components of S. gordonii may also interact with Als3p. The
possibility that alternative streptococcal adhesins are in-
volved in currently under investigation.
A feature of S. gordonii coaggregation with C. albicans
was that, in addition to adhering to hyphal filaments, the
bacteria adhered to each other. Since the streptococcal
strain utilized here does not self-aggregate, these observa-
tions suggest that upon attachment to hyphae, the bacteria
acquired the ability to recruit additional streptococcal cells.
One explanation might be that adherence of streptococci to
hyphae led to upregulation or unmasking of cell surface
factors that promoted streptococcal cell-cell aggregation.
Another possibility is that a diffusible signal from C. albicans
led to cell surface expression of streptococcal self-aggrega-
tion factors. These effects may be relevant to the develop-
ment of biofilm architecture, promoting the formation of
societies of streptococci that can be seen among the C.
albicans hyphae (1). Discrete groupings or societies of mi-
croorganisms are frequently observed in dual-species bio-
films (34), and these could be initiated and sustained by
In summary, we have investigated the physical interac-
tions occurring between C. albicans and S. gordonii and
demonstrated that the hypha-specific protein Als3p is a ma-
jor receptor for streptococcal attachment. In addition, we
provide evidence that the S. gordonii SspB protein interacts
directly with Als3p, though this should be confirmed by
further investigation utilizing protein-protein interaction
technologies. The region of Als3p that is recognized by S.
gordonii is not known but is currently under study. It has
recently been suggested that a tridecapeptide structure
present within the N termini of Als1p, Als3p, and Als5p has
amyloid-forming potential in catalyzing the formation of
parallel ?-sheets in Als protein-protein interactions (44).
This may, in part, account for the aggregative properties of
Als3p that presumably contribute to the frequently reported
clumping of hyphae. Interestingly, the SspB polypeptide se-
quence also contains three potential ?-aggregation se-
quences, predicted by the TANGO algorithm (15). One of
these (742 to 746 amino acids [aa]) is within the central V
region of the protein and, on the basis of the three-dimen-
sional (3D) crystal structure (4), is predicted to be at the
opening of the receptor-binding pocket (16). It remains to
FIG. 4. Coaggregation of L. lactis expressing SspB with C. albicans hyphae. (B to I) FITC-labeled L. lactis MG1363 (Ll control) or L. lactis
MG1363(pUB1000-sspB) (LlSspB) was incubated in suspension with hypha-forming cells of C. albicans NGY152 (CaWT) or the 1843 als3?/als3?
mutant (Ca?ALS3). Aggregates were visualized by light or fluorescence microscopy. (A) Percentages of hyphae with attached bacteria were
calculated on the basis of bacterial binding levels, as described in the legend to Fig. 2. The data are means ? standard errors (SE) from 4
independent experiments (?, P ? 0.05).
4650SILVERMAN ET AL.INFECT. IMMUN.
be determined if these self-propagating sequences are in-
volved in interactions of Als3p with SspB.
We thank Lois Hoyer for supplying monoclonal antibody and for
helpful advice. We also thank Scott Filler for the provision of S.
cerevisiae strains, Caroline Bamford and Angela Nobbs for helpful
discussions, and Jane Brittan and Lindsay Dutton for excellent tech-
This work was supported by NIH (NIDCR) grant R01-DE016690
awarded to H.F.J. and M.M.V.
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Editor: G. S. Deepe, Jr.
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