Interaction of Candida albicans cell wall Als3 protein with Streptococcus gordonii SspB adhesin promotes development of mixed-species communities.
ABSTRACT 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, als3Δ/als3Δ (ΔALS3) 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 ΔALS3 mutant. Saccharomyces cerevisiae S150-2B cells expressing Als3p, but not control cells, supported binding of S. gordonii DL1. However, S. gordonii Δ(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.
- SourceAvailable from: Ernesto Cota[Show abstract] [Hide abstract]
ABSTRACT: C. albicans binds various bacteria, including the oral commensal Streptococcus gordonii. Published reports documented the role of C. albicans Als3 and S. gordonii SspB in this interaction, and the importance of the Als N-terminal domain (NT-Als) in C. albicans adhesion. Here, we demonstrate that Als1 also binds S. gordonii. We also describe use of the NT-Als crystal structure to design mutations that precisely disrupt peptide-binding cavity (PBC) or amyloid-forming region (AFR) function in Als3. C. albicans displaying Als3 PBC mutant proteins showed significantly reduced binding to S. gordonii; mutation of the AFR did not affect the interaction. These observations present an enigma: the Als PBC binds free C termini of ligands, but the SspB C terminus is covalently linked to peptidoglycan and thus unavailable as a ligand. These observations and the predicted SspB elongated structure suggest that partial proteolysis of streptococcal cell wall proteins is necessary for recognition by Als adhesins.Frontiers in Microbiology 11/2014; 5:564. · 3.94 Impact Factor
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ABSTRACT: In polymicrobial biofilms a high level of interspecies interactions occur with often detrimental effect to the host. Many chronic infections are attributed to polymicrobial biofilms which tend to exhibit increased resistance to antimicrobial therapy. Yet despite the gravity of such infections, areas of study in polymicrobial diseases are in their infancy. Thus, much work is needed to promote a better understanding of emerging concepts in the biofilm development process such as interspecies communication and host immune response to microbial biofilms. The key challenges are to design effective therapeutic strategies to impede microbial colonization and prevent development of polymicrobial infections. Therefore, future research directions should focus on designing animal model systems to study in vivo-grown polymicrobial biofilms and infections. This review summarizes our limited knowledge about the nature of these complex communities and examines their role in disease, highlighting the challenges and novel approaches that are being pursued to combat polymicrobial biofilms and infections.The Open Mycology Journal 06/2011; 5(1).
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ABSTRACT: The human microbiome contains diverse microorganisms, which share and compete for the same environmental niches [1, 2]. A major microbial growth form in the human body is the biofilm state, where tightly packed bacterial, archaeal, and fungal cells must cooperate and/or compete for resources in order to survive [3-6]. We examined mixed biofilms composed of the major fungal species of the gut microbiome, Candida albicans, and each of five prevalent bacterial gastrointestinal inhabitants: Bacteroides fragilis, Clostridium perfringens, Escherichia coli, Klebsiella pneumoniae, and Enterococcus faecalis [7-10]. We observed that biofilms formed by C. albicans provide a hypoxic microenvironment that supports the growth of two anaerobic bacteria, even when cultured in ambient oxic conditions that are normally toxic to the bacteria. We also found that coculture with bacteria in biofilms induces massive gene expression changes in C. albicans, including upregulation of WOR1, which encodes a transcription regulator that controls a phenotypic switch in C. albicans, from the "white" cell type to the "opaque" cell type. Finally, we observed that in suspension cultures, C. perfringens induces aggregation of C. albicans into "mini-biofilms," which allow C. perfringens cells to survive in a normally toxic environment. This work indicates that bacteria and C. albicans interactions modulate the local chemistry of their environment in multiple ways to create niches favorable to their growth and survival.Current biology : CB. 10/2014;
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, 2010 CANDIDA 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 ?
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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.