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Biofilm formation and virulence expression by Streptococcus mutans are altered
when grown in dual-species model
BMC Microbiology 2010, 10:111
Zezhang T Wen (email@example.com)
David Yates (firstname.lastname@example.org)
Sang-Joon Ahn (email@example.com)
Robert A Burne (firstname.lastname@example.org)
11 November 2009
14 April 2010
14 April 2010
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Biofilm formation and virulence expression by Streptococcus
mutans are altered when grown in dual-species model
Zezhang T. Wen'*, David Yates2, Sang-Joon Ahn2 and Robert A. Burne2
Addresses: 1Department of Oral and Craniofacial Biology and Department of
Microbiology, Immunology and Parasitology, Louisiana State University Health Sciences
Center, New Orleans, LA 70119, USA; 2Department of Oral Biology, College of
Dentistry, University of Florida, Box 100424, Gainesville, FL 32610, USA.
Email: ZT Wen*, email@example.com; D Yates, firstname.lastname@example.org; SJ Ahn,
email@example.com; RA Burne, firstname.lastname@example.org
Background: Microbial cell-cell interactions in the oral flora are believed to play an
integral role in the development of dental plaque and ultimately, its pathogenicity. The
effects of other species of oral bacteria on biofilm formation and virulence gene
expression by Streptococcus mutans, the primary etiologic agent of dental caries, were
evaluated using a dual-species biofilm model and RealTime-PCR analysis. Results: As
compared to mono-species biofilms, biofilm formation by S. mutans was significantly
decreased when grown with Streptococcus sanguinis, but was modestly increased when
co-cultivated with Lactobacillus casei. Co-cultivation with S. mutans significantly
enhanced biofilm formation by Streptococcus oralis and L. casei, as compared to the
respective mono-species biofilms. RealTime-PCR analysis showed that expression of
spaP (for multi-functional adhesin SpaP, a surface-associated protein that S. mutans uses
to bind to the tooth surface in the absence of sucrose), gtfB (for glucosyltransferase B that
synthesizes Ul,6-linked glucan polymers from sucrose and starch carbohydrates) and
gbpB (for surface-associated protein GbpB, which binds to the glucan polymers) was
decreased significantly when S. mutans were co-cultivated with L. casei. Similar results
were also found with expression of spaP and gbpB, but not gtfB, when S. mutans was
grown in biofilms with S. oralis. Compared to mono-species biofilms, the expression of
luxS in S. mutans co-cultivated with S. oralis or L. casei was also significantly decreased.
No significant differences were observed in expression of the selected genes when S.
mutans was co-cultivated with S. sanguinis. Conclusions: These results suggest that the
presence of specific oral bacteria differentially affects biofilm formation and virulence
gene expression by S. mutans.
Oral biofilms are compositionally and structurally complex bacterial communities.
To date, more than 750 different species or phylotypes of bacteria have been identified in
mature dental plaque . Microbial cell-cell interactions in the oral flora and their
impact on bacterial adherence and biofilm formation are beginning to be appreciated [1-
4]. Cross-feeding or metabolic cooperation is well-documented among certain bacterial
species in the oral flora. Veillonellae can utilize the lactic acid produced by streptococci
and Porphyromonas gingivalis benefits from succinate produced by T. denticola.
Similarly, isobutyrate secreted by P. ginivalis stimulates the growth of T. denticola [2, 3].
Adhesin-ligand mediated physical interactions such as those between Streptococcus
gordonii and P. gingivalis may be important for secondary colonizers like P. gingivalis to
establish and persist in the oral cavity . A recent study has also provided evidence that
a mutualistic effect in biofilm formation between Actinomyces naeslundii and
Streptococcus oralis is facilitated by autoinducer-2 (Al-2) . Intra- and inter-species
interactions are believed to play a crucial role in community dynamics, contributing to
the formation of plaque and, ultimately, the development of polymicrobial diseases,
including caries and periodontitis [2, 5]. Therefore, a better understanding of cell-cell
interactions between oral pathogens and commensal bacteria, and the impact of these
interactions on expression of virulence factors and pathogenicity, could lead to
development of novel preventive and therapeutic strategies against dental caries and
As the principal etiological agent of human dental caries, Streptococcus mutans has
developed multiple mechanisms to colonize the tooth surface and, under certain
conditions, to become a numerically significant species in cariogenic biofilms . The
multi-functional adhesin SpaP, also called P1 and PAc 1, is considered the primary factor
mediating early attachment of S. mutans to tooth enamel in the absence of sucrose . S.
mutans also produces at least three glucosyltransferases (GtfB, -C & -D), which
polymerize the glucosyl moiety from sucrose and starch carbohydrates into al,3- and
al,6-linked glucans [7, 9]. Binding to glucans by glucan binding proteins (GbpA, -B, -C
and -D) and by the Gtfs facilitates bacterial adherence to tooth surfaces, inter-bacterial
adhesion and accumulation of biofilms [9, 10]. GtfBC&D and GbpABC&D, together
with the adhesive extracellular glucans, constitute the sucrose-dependent pathway for S.
mutans to establish on the tooth surface and are of central importance in plaque formation
and development of caries [7, 9-14].
Multiple regulatory networks that integrate external signals, including the cell
density-dependent Com system and other two-component regulatory systems, including
CiaHR, LiaSR and VicRK, with CiaH, LiaS and VicK being the sensor kinases and CiaR,
LiaR and VicR the response regulators of two-component system, are required for
biofilm formation [15-20, 21]. S. mutans also possesses a LuxS-mediated signaling
pathway that affects biofilm formation and bacteriocin production [18, 22, 23]. LuxS is
the enzyme that catalyzes the reactions leading to the production of the AI-2 signal
molecule . In addition, a number of other gene products, such as BrpA (a cell
surface-associated biofilm regulatory protein), have also been shown to play critical roles
in environmental stress responses and biofilm development by S. mutans [25, 26]. While
much effort has been devoted to understanding the molecular mechanisms of adherence,
biofilm development and virulence gene expression by S. mutans in pure cultures, there
are large gaps in our knowledge of how this cariogenic bacterium behaves in response to
inter-generic interactions with bacteria commonly found in the supragingival plaque.
In this study, we developed a dual-species in vitro model to examine the impact of
co-cultivation of S. mutans with S. oralis or S. sanguinis, two primary colonizers and
members of the normal flora, or with Lactobacillus casei, a bacterium frequently isolated
from carious sites, on biofilm formation by these bacteria and expression of known
virulence factors of S. mutans. Data presented here suggest that growth in dual-species
impacts surface biomass accumulation by some of the bacterial species analyzed, as
compared to the respective mono-species biofilms and that the expression of known
virulence factors by S. mutans can be differentially modulated by growth with other
bacteria commonly found in dental plaque. Such interactions may influence the
formation, architecture and pathogenic potential of human dental plaque.
Bacterial strains and growth conditions. S. mutans UA159, S. oralis SK92 and S.
sanguinis SK150 were maintained in Brain Heart Infusion (BHI, Becton, Dickinson and
Company, MD), and L. casei 4646 was maintained in Lactobacillus MRS (Difco
Laboratories, MI). For biofilm formation, all cultures were grown on semi-defined
biofilm medium (BM)  with 18 mM glucose and 2 mM sucrose as the supplemental
carbohydrate sources (BMGS) [18, 26]. Biofilms were grown in a 5% CO2-aerobic
atmosphere at 370C. For growth studies using a Bioscreen C (Oy Growth Curves AB
Ltd, Finland), cultures were grown at 370C aerobically and the optical densities were
monitored every 30 minutes, with shaking for 10 seconds before measurement .
Growth of dual-species biofilms. Sterile glass slides were used as substratum and
biofilms were grown by following a protocol described previously [25, 26]. Briefly,
overnight broth cultures were transferred by 1:50 dilutions into fresh, pre-warmed, broth
medium (BHI for streptococci and MRS for lactobacillus) and were allowed to grow until
mid-exponential phase (OD600nm 0.5) before transfer to BMGS for biofilm formation.
For mono-species biofilms, 450 pl of the individual cultures was added to the culture
tube, and for two-species biofilms, 450 pl of each culture was used as inoculum. The
biofilms grown on the glass slides that were deposited in 50 ml Falcon tubes were
aseptically transferred daily to fresh BMGS. After four days, the biofilms were scratched
off with a sterile spatula and suspended in 7.5 ml of 10 mM potassium phosphate buffer,
pH 7.0. To de-chain and separate the cells, the biofilms were sonicated using a Sonic
Dismembrator (model 100, Fisher Scientific, Idaho) at energy level 3 for 25 seconds,
twice, with 2 minutes on ice between treatments. To determine the total number of viable
bacterial cells (colony-forming units, CFU), 100 p1 from dispersed, four-day biofilms
was serially diluted in potassium phosphate buffer, 10 mM, pH 7.0, and plated in
triplicate on BHI agar plates.
RNA extraction. Immediately after sampling for plating, bacterial cells were treated
with RNAProtect (Qiagen Inc., CA) as recommended by the supplier. The cells were
then pelleted by centrifugation and total RNA extractions were performed using a hot
phenol method [18, 26]. To remove all DNA, the purified RNAs were treated with
DNAse I (Ambion, Inc., TX) and RNA was retrieved with the Qiagen RNeasy
purification kit, including an additional on-column DNAse I treatment with RNase-free
RealTime-PCR. For RealTime-PCR, gene-specific primers were designed using the
DNA mfold program (http://www.bioinfo.rpi.edu/application/mfold/old/dna) and Beacon
Designer 3.0 (PREMIER Biosoft International, Palo Alto, CA) using the following
criteria: primer length 20-22 nucleotides, Tm > 60C with 50 mM NaCl and 3 mM MgC12,
and the expected length of PCR products 85-150 bp (Table 1). For RealTime-PCR,
cDNA was generated with gene-specific primers using SuperScript III First Strand
Synthesis Kit (InVitorgen, CA) by following the supplier's recommendations. For
validation assays, iScript Reverse Transcriptase was also used to generate cDNA
templates with random nanomers as primers (Bio-Rad laboratories, CA). RealTime-PCR
was conducted using the iCycler iQ real time PCR detection system (Bio-Rad
Laboratories), with controls and standards as described previously [20, 26].
Data analysis. The mRNA copy number of selected virulence factors was determined
per pg of total RNA. When grown in the dual-species model, the values were further
normalized to relative numbers of S. mutans by multiplying the copy number by the ratio
of S. mutans CFU to the total CFU in the mixed-species biofilms. The resulting data
were expressed as copy number per pg of S. mutans total RNA. Statistical analysis was
carried out using the non-parametric Kruskal-Wallis test and t-test.
Results and Discussion
Establishment of a suitable biofilm model for the reliable monitoring of gene
expression in S. mutans. Glass slides can be used very effectively to cultivate biofilms
of oral bacteria [26, 29]. As compared to tooth enamel model systems, e.g.
hydroxylapatite disks, glass slides are easier to handle, stable and non-reactive. By daily
transfer to fresh medium, bacteria on glass surfaces continue to accumulate and generate
sufficient biofilms after 3-4 days for multiple experiments , including whole genome
transcriptional profiling .
For measurement of the expression levels of selected virulence factors by S. mutans,
total RNA was extracted from mono- and dual-species biofilms and RealTime-PCR
reactions were carried out using gene-specific primers (Table 1). To confirm that no
genomic DNAs left in the RNA preps, cDNA synthesis reactions that received no reverse
transcriptase were used as controls and results of RealTime-PCR using gene-specific
primers (Table 1) showed that none of the RNA preps used in this study had any
significant genomic DNA contamination. To verify that the primers did not amplify non-
S. mutans genes under the conditions tested, total RNA of S. oralis, S. sanguinis and L.
casei, either alone or in mixtures with known quantities of S. mutans RNA, were used as
a template for reverse transcription and RealTime-PCR. No cDNA was detected when S.
oralis, S. sanguinis or L. casei total RNA alone was used as a template with primers for
spaP, gtfB, gbpB, luxS, and brpA, as well as the Idh gene encoding lactate
dehydrogenase) (data not shown). Melting curves consistently presented unique
amplification products for every amplicon tested. Results also demonstrated that the
presence of RNA from non-mutans streptococci or from lactobacilli had no significant
effect on the efficiency of amplification of S. mutans specific products. Specifically,
quantification of the respective genes in mixed RNA samples yielded results that were
proportional to the amount of S. mutans RNA used in the reactions (data not shown).
Similar results were also obtained with genomic DNA from the respective strains as
templates (data not shown).
The choice of appropriate controls for this study was carefully considered.
Ribosomal RNA is the most commonly used reference in single species transcriptional
analysis, and has often been used as a control in Northern analysis of S. mutans RNA [18,
30]. However, use of ribosomal RNAs could be misleading when it is used for analysis
of gene expression in mixed-species biofilms, especially when closely related species are
present in the consortium. Specifically, during calibration of the methods, cross-reactions
between rRNA of different bacterial sources were noted, as shown by multiple peaks in
the melting curves in the RealTime-PCR reactions (data not shown). Therefore, rather
than use rRNA total viable counts (CFU) were used to normalize the RealTime-PCR
data. Brief sonication was used to disperse the biofilms. When plated on BHI agar
plates, the distinctive colony morphology of S. mutans (flat, opaque, dry colonies with
rough surface) versus S. oralis (small, flat and smooth colonies), S. sanguinis and L. casei
(both forming small, wet, convex colonies with shiny and smooth surfaces) made it easy
to distinguish S. mutans from the other streptococci and L. casei. For S. mutans-L. casei
dual-species biofilms, an erythromycin resistant L. casei strain (Browngardt and Bume,
unpublished data) was also used in dual-species biofilms, and BHI agar plates containing
erythromycin (5 pg ml -1) were used for viable counts of L. casei. The results were
similar to those when BHI agar plates were used (data not shown).
The lactate dehydrogenase gene Idh of S. mutans has been reported to be
constitutively expressed  (Wen and Bume, unpublished data), so we also examined
whether this gene may serve as a suitable reference. No cross-reactions were detected
between primers of S. mutans Idh and genes of other bacteria (data not shown). As
expected, no significant difference in expression of Idh was observed between S. mutans
grown in mono-species biofilms and those in dual-species biofilms, following proper
normalization to CFU (Figure 1)._ Similar results were obtained when random primers
were used to generate cDNA template instead of Idh-specific primers. These results add
additional support to the finding that RealTime-PCR with normalization to CFU is a
reliable approach for assessment of gene regulation in S. mutans growing in this mixed-
species biofilm model.
S. mutans enhances biofilm formation by S. oralis and L. casei in dual-species model.
When grown on glass slides, S. mutans accumulated an average of 8.8x1010 CFU after 4
days (Figure 2). S. sanguinis also formed biofilms efficiently on glass surfaces,
averaging 8.2x1010 CFU after 4 days. When these two bacteria were grown in the dual-
species model, the level of S. mutans decreased by more than 8-fold (P<0.05), yielding
an average of 1.0x1010 CFU, while S. sanguinis accumulated to 5.1x1010 CFU. S. oralis
displayed a relatively poor capacity to form biofilms when grown alone, averaging
2.6x109 CFU after 4 days. When grown in dual-species with S. mutans, however, the
number of S. oralis in the biofilms increased to an average of 1.4x1010 CFU (P<0.01).
On the other hand, biofilm formation by S. mutans was decreased when grown together
with S. oralis, although the difference between mono-species and dual-species was not
statistically significant. L. casei alone formed biofilms poorly, accumulating only
2.9x107 CFU after 4 days. However, the capacity of L. casei to form biofilms was
enhanced significantly (P<0.001) when co-cultivated with S. mutans, resulting in an
increase of more than 60-fold to an average of 1.7x109 CFU after 4 days. Notably, when
S. mutans was cultivated in dual-species biofilms with L. casei, the organisms
accumulated in about 2-fold greater numbers than when S. mutans was grown alone,
averaging 1.8x1011 CFU.
Various bacterial cell-cell interactions may exist when growing in dual-species
biofilms, including competition for binding sites and nutrients available. In this study,
the same amount of inoculum was used in mono- and dual-species biofilms. For bacteria
that grow similarly well in mono-species model, slight decreases will be expected for
both constituents when grown in dual-species biofilms. The observed decreases in
population of both S. mutans and S. sanguinis when they were cultivated together (Figure
2), as compared to the respective mono-species biofilms, could be at least in part
attributed to competition for binding sites. Both S. sanguinis and S. oralis grew well in
BMGS broth, with a doubling time of 86.5 (2.7) and 80 (6.1) minutes, respectively,
whereas S. mutans took 134.7 (11.6) minutes to double its optical density. These results
suggest that S. sanguinis and S. oralis should possess advantages over S. mutans for
available nutrients when grown in a mixed-species consortium. Disadvantages in nutrient
competition could certainly affect the capacity of S. mutans to accumulate on the glass
surfaces, contributing to the observed decreases in biofilm formation when grown
together with S. sanguinis or S. oralis (Figure 2). S. sanguinis is also known to produce
hydrogen peroxide, which can inhibit the growth of S. mutans [4, 32], although such an
impact on S. mutans growth was shown to be limited when the organisms were
inoculated simultaneously , as they were in this study.
L. casei did not grow well in BMGS broth, yielding an average of 4.7x107 CFU ml-1
after 24 hours, as compared to 6.0xl08 CFU ml-1 for S. mutans. Poor growth could
certainly contribute to poor biofilm formation by this bacterium. As was observed with
dual-species biofilms, however, co-cultivation of L. casei and S. mutans planktonically in
BMGS broth also increased S. mutans CFU by more than 3-fold, with an average CFU of
2.3x109 ml-1, although the numbers of L. casei remained similar to those in mono-species
cultures (data not shown). The mixed-species broth cultures also had a slightly decreased
doubling time (121.48.8 minutes), as compared to S. mutans (134.711.8 minutes) and
L. casei (24024 minutes) in mono-species planktonic cultures. BHI, and especially
MRS, yielded much better growth of L. casei than BMGS, although no major differences
were observed in biofilm formation by L. casei when grown in BHI or MRS (data not
Oral lactobacilli, such as L. casei, are a group of acid tolerant bacteria that are
commonly isolated in relatively significant proportions from cariogenic dental plaque
[33-36]. However, the ability of lactobacilli to adhere to the tooth surface was known to
be poor . Results presented here also suggest that L. casei alone does not form
biofilms on glass surfaces very effectively, but biofilm formation by this bacterium can
be dramatically improved when mixed with S. mutans. S. mutans produces at least three
Gtf enzymes  that produce high molecular weight glucans that promote bacterial
adhesion and biofilm accumulation. Recent studies have shown that these enzymes,
especially GtfB, are capable of directly binding to L. casei and other oral bacteria .
Adsorption of Gtfs to the surface of non-mutans streptococci can promote adhesion of the
bacteria via glucan-mediated pathways . Thus, the Gtf enzymes of S. mutans and the
adhesive glucans likely contribute to the enhanced biofilm formation by L. casei, and
probably S. oralis, when grown in mixed-species biofilms with S. mutans. Notably,
enhanced biofilm formation by Lactobacillus plantarum and Lactobacillus rhamnosus
was noted in a mucin-based medium , so the presence of polysaccharides may have a
general ability to promote biofilm formation by lactobacilli. However, the actual
mechanism for the enhancement of L. casei levels in biofilms with S. mutans requires
While the close association of L. casei and S. mutans in carious sites is well
documented, little information is available concerning the interaction between these two
bacteria with respect to S. mutans biofilm formation and its cariogenicity. While co-
cultivation with S. mutans significantly enhanced biofilm formation by L. casei, the
sessile population of S. mutans was also found to be increased by more than 2-fold in
dual species model with L. casei (Figure 2), which is contrary to what was observed with
the other bacteria studied. While the exact nature and the underlying mechanism await
further investigation, the interaction observed between S. mutans and L. casei may partly
explain the prevalence and the close association of these two bacteria in cariogenic
Expression of genes critical to cariogenicity of S. mutans can be altered when grown
in mixed-species biofilms. RealTime-PCR was used to analyze the expression of several
genes that have critical roles in bacterial adherence and biofilm accumulation by S.
mutans [7-10], including spaP, gtfB and gbpB. As shown in Figure 3, slight decreases
were observed in expression of spaP, gtfB and gbpB by S. mutans when grown in dual-
species with S. sanguinis as compared to those in mono-species biofilms, although the
differences were not statistically significant. When grown in dual-species with L. casei,
however, expression of spaP, gbpB and gtfB by S. mutans was decreased by as much as
40-fold, at a significance level of P<0.05 for spaP and P<0.001 for gtfB and gbpB,
respectively, as compared to cells in mono-species biofilms. The expression of spaP
(P<0.05) and gbpB (P<0.001), but not gtfB, was also lower by more than 30-fold in S.
mutans when grown with S. oralis. As compared to mono-species biofilms, expression of
luxS was decreased by more than 7-fold in cells grown with L. casei (P<0.001) and by
more than 15-fold in cells with S. oralis (P<0.001), but again no significant differences
were observed when S. mutans was grown with S. sanguinis. Expression of brpA was
decreased by more than 3-fold (P<0.05) in cells grown with S. oralis, but no major
differences were observed when grown with S. sanguinis and L. casei. As a control, the
expression of the Idh gene, a constitutively expressed gene (Wen and Burne, unpublished
data) , was also analyzed and no significant differences were observed between S.
mutans grown in single-species and those grown in dual-species (Figure 1).
Glucosyltransferases and glucan-binding proteins of S. mutans are known to be
differentially expressed in response to environmental conditions, such as carbohydrate
source and availability, pH and growth of the bacteria on surfaces [9, 39-41]. Results
presented here further demonstrate that the level of expression of these known virulence
attributes can be altered when S. mutans is grown in dual-species biofilms and that the
effect varies as a function of the species of bacteria in the biofilms. Among the three
different bacterial species analyzed, the most significant effect on the expression of the
selected genes was seen with L. casei, followed by S. oralis. No significant effect was
observed with S. sanguinis in expression of either spaP, gtfB or gbpB. As described
above, nutrient availability, especially when grown together with faster growing
microorganisms, such as S. oralis, could have an impact on gene expression in S. mutans,
and consequently affect biofilm formation .
L. casei, as a frequent isolate from the cariogenic plaque, is also known for its high
capacity for acid production from carbohydrates. When grown on BMGS in mono-
species cultures, S. mutans overnight (24-hour) cultures had an average pH of 5.75
(0.28). As expected, the pH was decreased slightly when grown in dual species with L.
casei, averaging 5.63(0.20). Similar results were also observed when S. mutans was
grown together with S. oralis, with an average pH measured at 5.69 (0.08). In contrast,
however, the pH of overnight cultures of S. mutans co-cultured with S. sanguinis was
5.95(+0.03). Environmental pH has been shown to influence the expression of some of
the genes selected . Although not necessarily fully reflective of what occurs in
sessile populations, the decreases in culture pH suggest that S. mutans may endure a more
significant acid challenge when grown in dual-species with L. casei as well as S. oralis
and that such decreases could at least in part contribute to the down-regulation of the
selected genes in S. mutans grown in dual-species with L. casei and S. oralis.
Many bacteria produce autoinducer 2 through LuxS enzymes . Recent studies of
the oral flora have yielded evidence that AI-2 mediated signaling in the flora may play an
important role in inter-species communication, affecting plaque formation and,
ultimately, development of oral diseases [6, 23, 44, 45]. Neither S. oralis nor A.
naeslundii alone were found to form good biofilms, but growth in the two-species model
resulted in abundant mutualistic growth . AI-2 of S. oralis was recently found to be
critical for such a mutualistic interaction . Below and above the optimal
concentration, mutualistic biofilm growth was suppressed. In S. mutans, LuxS was
shown to be involved in biofilm formation and to affect the structure of biofilms [18, 22,
23], although its role in regulation of factors critical to bacterial adherence and biofilm
formation is somewhat controversial. As shown previously, LuxS-deficiency
significantly decreased brpA expression, but no major differences were seen between
wild-type and the LuxS-deficient mutants in expression of gtfBC, gbpB or spaP .
Similar results were also obtained by DNA microarray analysis in both planktonic 
(Wen et al., unpublished data) and sessile populations (Wen et al., unpublished data). In
a study using RealTime-PCR, however, Yoshida et al.  reported that transcription of
gtfB and gtfC, but not gtfD, was up-regulated in response to LuxS-deficiency. Like S.
mutans and S. oralis, both S. sanguinis (www.oralgen.lanl.gov) and L. case (Wen and
Burne, unpublished data) possess LuxS. It remains unclear, however, whether LuxS in
these bacteria is in fact involved in cell-cell communication. Nevertheless, down
regulation of luxS expression in S. mutans when grown in dual-species with L. casei and
S. oralis would likely affect the absolute concentration of AI-2 in the biofilms. Studies
are ongoing to determine whether AI-2 signaling is functional between these bacterial
species and whether alterations in luxS expression does in fact affect the expression of
known virulence factors by S. mutans in mixed-species biofilms.
It is well established that GtfB and GbpB are critical components of the sucrose-
dependent pathway in S. mutans biofilm formation and cariogenicity. In the presence of
sucrose, GtfB synthesizes copious cl,3-linked, water insoluble glucan polymers. Then,
surface-associated glucan-binding protein GbpB and others bind to these polymers,
facilitating intercellular adherence and biofilm accumulation by S. mutans. It would be
expected that down-regulation of GtfB and GbpB would result in less biofilm formation.
Surprisingly, our S. mutans-L. casei dual-species data showed that S. mutans accumulated
more than 2-fold more biofilms while the expression of gtfB and gbpB was decreased.
One possible explanation is that down regulation of GtfB and GbpB (and probably some
other members of the Gtfs and Gbps) when grown together with L. casei altered the
balance of glucans to glucan-binding proteins ratio or altered the glucan structure in a
way that altered biofilm architecture. In fact, similar observations have also been
reported recently by us and some other groups [11, 12, 48]. In particular, deficiency of
trigger factor (RopA) in S. mutans reduced production of GtfB and -D as revealed by
Western blotting, but the ropA-mutant formed more than 50% more biofilms than the
parental strain when sucrose was provided as the supplemental carbohydrate source .
During characterization of GbpA of S. mutans, the Banas group showed that strains
deficient in GbpA were more adherent in vitro and more cariogenic in vivo than the
parental strain [11, 12]. As compared to the biofilms by the parent strain, which were
composed of big cellular clusters with large gaps in between, the biofilms formed by the
gbpA- mutant were relatively small, but more compact and more evenly distributed.
Interestingly, GbpA-deficiency was later found to increase the frequency of
recombination between the tandemly arranged, highly homologous gtfB and gtfC genes,
resulting in a dramatic decrease in production of water-insoluble glucans. Additional
experiments that probe the basis for altered gtfand gbp expression, coupled with
measurements of Gtf and Gbp protein and activity and glucan structure will be needed to
shed light on the basis for the observations.
In vitro dual-species biofilm model and RealTime-PCR analysis showed that biofilm
formation and virulence expression by S. mutans could be altered in response to the
presence of other oral bacterial species. Effort is currently directed to further
investigation of the underlying mechanism of the altered expression of selected genes and
the impact of such alterations on biofilm formation by S. mutans. Considering the
frequent association of L. casei and S. mutans in carious sites and their role in caries
development, information yielded from these studies could be used to formulate novel
strategies against human dental caries.
List of abbreviations
AI-2, autoinducer 2; GtfB, -C & -D, glucosyltransferase B, C & D; GbpA, -B, -C, & -
D, glucan-binding protein A, B, C & D; CFU, colony-forming-unities; BHI, brain heart
infusion; MRS, lactobacilli MRS medium; SpaP, adhesin Pl; BrpA, biofilm regulatory
protein A; Idh, lactate dehydrogenase gene; BM, biofilm medium; BMG, BM plus
glucose; BMS, BM plus sucrose; BMGS, BM plus glucose and sucrose.
ZTW conceived the study, designed and implemented most of the experiments, and
drafted the manuscript; DY carried out most of the biofilm assays and RealTime-PCR
analysis; SJA was involved in parts of experimental design and data analysis; RAB
participated the experimental design and data analysis and revised critically the
manuscript. All authors have read and approved the manuscript.
This work is supported by NIDCR grants DE13239 and 12236 to R.A.B. and DE15501
and DE019452 to Z.T.W. We thank Mr. Christopher Browngardt for his kind help with
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Table 1. Primers used for RealTime-PCR
Primer DNA sequence (5' 3') Application Amplicon
Figure 1. RealTime-PCR analysis of Idh gene as an internal control.
Data presented here were generated from at least four separate sets of biofilm cultures
and RealTime-PCR was carried out in triplicate and was repeated at least once. These
data were normalized and further analyzed using a non-parametric Kruskal-Wallis Test
and student t-test. The bar graphs represent the average standardd deviation in error
bars) of normalized copy numbers x (pg S. mutans total RNA)-1. No significant
differences were observed between S. mutans grown in mono-species and those grown in
dual-species biofilms. Abbreviations: Sm, S. mutans; Ss, S. sanguinis; So, S. oralis; Lc,
L. case, with Sm-Ss, Sm-So and Sm-Lc indicating dual-species biofilm of the selected
Figure 2. Biofilm formation in mono- and dual-species model.
Data presented here were generated from more than ten independent sets of experiments
and were further analyzed using a non-parametric Kruskal-Wallis Test and student t-test.
This graph shows the average standardd deviation, in error bars) of CFU in biofilms
formed by S. mutans and the other oral bacteria tested when grown in the mono- and
dual-species models with S. mutans. A *, # and @ indicate significant difference at
P<0.05, 0.01 and 0.001, respectively, when compared to those grown in mono-species
biofilms. All abbreviations are the same as in Figure 1.
Figure 3. RealTime-PCR analysis of selected genes.
RealTime-PCR for specific genes was carried out in triplicate and repeated at least once.
Data presented here were generated from at least four independent sets of experiments.
These data were normalized and further analyzed using a non-parametric Kruskal-Wallis
Test and student t-test. The bar graphs represent the average (+standard deviation in error
bars) of copy numbers x (pg S. mutans total RNA) -1, with *, # and @ illustrating
statistical differences at P<0.05, 0.01, and 0.001, respectively, when compared to the
respective genes in mono-species biofilms.
S. mutans grown in mono- and dual-
species with So, Ss and Lc
0 sm-so E2Sm-Lc
Sm, Ss, So and Lc
Ss, So and Lc in
Sm in mono- and
dual-species with Ss,
So and Lc
. . . .
Figure 3 spaP
SS. mutans/S. sanguinis