Additive attenuation of virulence and cariogenic potential of Streptococcus mutans by simultaneous inactivation of the ComCDE quorum-sensing system and HK/RR11 two-component regulatory system.
ABSTRACT The genome of Streptococcus mutans harbours 13 two-component signal transduction systems (TCSTSs). Of these, a peptide-mediated quorum-sensing system, ComCDE, and the HK/RR11 two-component system are well known to regulate several virulence-associated traits in in vitro experiments, including genetic competence, bacteriocin production, biofilm formation and stress responses. In this study, we investigated the hypothesis that inactivation of ComCDE, HK/RR11 or both systems would attenuate the virulence and cariogenicity of S. mutans. The results showed that simultaneous inactivation of both signal transduction systems additively attenuated S. mutans virulence and cariogenicity, since inactivation of either of these systems alone did not result in the same degree of effect. The double deletion mutant SMcde-hk11 was defective in genetic competence, had a reduced acid production, was unable to grow at pH 5.0 and formed an abnormal biofilm with reduced biomass. Animal studies showed that this mutant had reduced capabilities for oral colonization, succession and initiation of dental caries. A competitive index (CI) analysis using a mixed-infection animal model revealed that all the mutants, particularly SMcde-hk11, had reduced fitness in their ecological niches and were unable to compete with the wild-type strain for persistence in dental biofilms. The evidence from this study suggests that the ComCDE and HK/RR11 signal transduction systems can be considered to be novel targets for the development of strategies in the prevention and treatment of S. mutans infections.
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ABSTRACT: Objectives Glycemic index (GI) refers to the effect of food on glycemia and insulinemia. It is widely used in nutrition and diabetology to advise patients on adequate diets to maintain constant glucose and insulin blood levels. As GI is mainly related to the metabolic availability of glucides, it is believed it might influence the activity of oral bacteria. The aim of this work was to assess the relationships between the consumption of food with high GI and the Decayed, Missing, Filled Teeth (DMFT) index.Prevenzione & Assistenza Dentale 09/2009; 35(4):135-141.
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ABSTRACT: Many bacteria are known to regulate their cooperative activities and physiological processes through a mechanism called quorum sensing (QS), in which bacterial cells communicate with each other by releasing, sensing and responding to small diffusible signal molecules. The ability of bacteria to communicate and behave as a group for social interactions like a multi-cellular organism has provided significant benefits to bacteria in host colonization, formation of biofilms, defense against competitors, and adaptation to changing environments. Importantly, many QS-controlled activities have been involved in the virulence and pathogenic potential of bacteria. Therefore, understanding the molecular details of quorum sensing mechanisms and their controlled social activities may open a new avenue for controlling bacterial infections.Sensors 12/2012; 12(3):2519-38. · 2.05 Impact Factor
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ABSTRACT: High coverage, whole genome shotgun (WGS) sequencing of 57 geographically- and genetically-diverse isolates of Streptococcus mutans from individuals of known dental caries status was recently completed. Of the 57 sequenced strains, fifteen isolates, were selected based primarily on differences in gene content and phenotypic characteristics known to affect virulence and compared with the reference strain UA159. A high degree of variability in these properties was observed between strains, with a broad spectrum of sensitivities to low pH, oxidative stress (air and paraquat) and exposure to competence stimulating peptide (CSP). Significant differences in autolytic behavior and in biofilm development in glucose or sucrose were also observed. Natural genetic competence varied among isolates, and this was correlated to the presence or absence of competence genes, comCDE and comX, and to bacteriocins. In general strains that lacked the ability to become competent possessed fewer genes for bacteriocins and immunity proteins or contained polymorphic variants of these genes. WGS sequence analysis of the pan-genome revealed, for the first time, components of a Type VII secretion system in several S. mutans strains, as well as two putative ORFs that encode possible collagen binding proteins located upstream of the cnm gene, which is associated with host cell invasiveness. The virulence of these particular strains was assessed in a wax-worm model. This is the first study to combine a comprehensive analysis of key virulence-related phenotypes with extensive genomic analysis of a pathogen that evolved closely with humans. Our analysis highlights the phenotypic diversity of S. mutans isolates and indicates that the species has evolved a variety of adaptive strategies to persist in the human oral cavity and, when conditions are favorable, to initiate disease.PLoS ONE 11/2013; 8(4):e61358. · 3.53 Impact Factor
Additive attenuation of virulence and cariogenic
potential of Streptococcus mutans by simultaneous
inactivation of the ComCDE quorum-sensing
system and HK/RR11 two-component regulatory
Yung-Hua Li,1,2Xiao-Lin Tian,1Gillian Layton,1Chris Norgaard2
and Gary Sisson2
1Department of Applied Oral Sciences, Dalhousie University, Halifax, NS, Canada
2Department of Microbiology and Immunology, Dalhousie University, Halifax, NS, Canada
Received 11 April 2008
Revised31 July 2008
Accepted 5 August 2008
The genome of Streptococcus mutans harbours 13 two-component signal transduction systems
(TCSTSs). Of these, a peptide-mediated quorum-sensing system, ComCDE, and the HK/RR11
two-component system are well known to regulate several virulence-associated traits in in vitro
experiments, including genetic competence, bacteriocin production, biofilm formation and stress
responses. In this study, we investigated the hypothesis that inactivation of ComCDE, HK/RR11
or both systems would attenuate the virulence and cariogenicity of S. mutans. The results showed
that simultaneous inactivation of both signal transduction systems additively attenuated S. mutans
virulence and cariogenicity, since inactivation of either of these systems alone did not result in the
same degree of effect. The double deletion mutant SMcde-hk11 was defective in genetic
competence, had a reduced acid production, was unable to grow at pH 5.0 and formed an
abnormal biofilm with reduced biomass. Animal studies showed that this mutant had reduced
capabilities for oral colonization, succession and initiation of dental caries. A competitive index
(CI) analysis using a mixed-infection animal model revealed that all the mutants, particularly
SMcde-hk11, had reduced fitness in their ecological niches and were unable to compete with the
wild-type strain for persistence in dental biofilms. The evidence from this study suggests that the
ComCDE and HK/RR11 signal transduction systems can be considered to be novel targets for
the development of strategies in the prevention and treatment of S. mutans infections.
Streptococcus mutans is a Gram-positive bacterium that
depends on a ‘biofilm life-style’ for survival and persistence
in its natural ecosystem, dental plaque (Ajdic et al., 2002;
Burne, 1998; Cvitkovitch et al., 2003). Under appropriate
environmental conditions, this bacterium can rapidly
produce acids from fermentable dietary carbohydrates
and initiate demineralization of the tooth surface. S.
mutans is therefore considered to be an important
aetiological agent of dental caries (Kuramitsu, 2003). The
ability of S. mutans to initiate dental caries depends on
several significant virulence traits, including: (i) initiation
of biofilm formation by adherence and accumulation on
the tooth surface that is promoted by its synthesis of
insoluble, extracellular polysaccharides; (ii) production of
numerous bacteriocins that kill other species, favouring its
competition in dental biofilms; (iii) high efficiency in
catabolizing carbohydrates and producing acids; and (iv)
the ability to tolerate low pH (Belli & Marquis, 1991; Li &
Burne, 2001; Kuramitsu, 2003; Scheie & Petersen, 2004).
These virulence-associated traits provide S. mutans with
overwhelming ecological advantages in competition and
succession in dental biofilms that cause caries (Marsh,
2000; Kuramitsu, 2003). S. mutans also causes corrosion of
dental materials, leading to secondary caries around
restorations (Marsh, 2000). Moreover, it can be a cause
of subacute infective endocarditis (Ajdic et al., 2002;
To survive and initiate infections, bacteria must sense, and
respond and adapt to their environments, a process that
requires signal transduction across biological membranes
Abbreviations: CI, competitive index; CSP, competence-stimulating
peptide; RR, response regulator; TCSTS, two-component signal
A supplementary figure showing the PCR-ligation mutagenesis strategy
used for construction and confirmation of the comCDE deletion is
available with the online version of this paper.
Microbiology (2008), 154, 3256–3265
32562008/019455G2008 SGMPrinted in Great Britain
(Barrett & Hoch, 1998). A major mechanism of signal
transduction, widespread in bacteria, is represented by the
so-called two-component signal transduction systems
(TCSTSs), which enable bacteria to regulate their gene
expression and coordinate activities in response to
environmental stimuli (Barrett & Hoch, 1998; Beier &
Gross, 2006; Hoch, 2000). A typical TCSTS consists of a
membrane-associated histidine kinase (HK) protein, which
senses a specific stimulus, and a cytoplasmic response
regulator (RR) protein, which enables the cells to respond
to the stimulus via regulation of gene expression (Hoch,
2000). Upon stimulation, the histidine kinase sensor
protein interacts with a specific signal and activates
autophosphorylation at a conserved histidine residue.
The phosphoryl group is then transferred to the cognate
response regulator, which in turn activates or represses the
transcription of its target genes (Barrett & Hoch, 1998;
Hoch, 2000). Many TCSTSs have been found to function as
global regulators by initiating signalling cascades, in which
large sets of genes are switched on and/or off. These
systems provide the major means by which bacteria
communicate with each other and the outside world.
TCSTSs have been known to regulate diverse metabolic
processes, the bacterial cell cycle, cell–cell communication
and virulence factors in a wide range of bacterial species
(Hoch, 2000). Because of their importance in the
regulation of cellular physiology, adaptation to environ-
ments and virulence expression, TCSTSs have been
considered to be important targets for the development
of antimicrobial agents (Barrett & Hoch, 1998; Beier &
Gross, 2006; Hoch, 2000).
There are 13 two-component systems that have been
identified in the S. mutans genome (Ajdic et al., 2002).
Several TCSTSs have been characterized and recognized to
regulate physiological activities and virulence-associated
traits in S. mutans (Levesque et al., 2007; Biswas et al.,
2008). A signal peptide-mediated quorum-sensing system
encoded by comCDE has been found to play a central role
in regulation of genetic competence, bacteriocin produc-
tion, biofilm formation and stress response (Li et al., 2001a,
b, 2002a; van der Ploeg, 2005). Another system, called HK/
RR11, is involved in S. mutans survival at acidic pH (Li
et al., 2002b). Since inactivation of either hk11 (SMu.486)
or rr11 (SMu.487) results in an abnormal biofilm
phenotype, which is similar to that formed by the comC
mutant, HK11 is suspected to be the second receptor to the
competence-stimulating peptide (CSP) (Li et al., 2002a). In
S. mutans, the CiaHR system has also been characterized
and found to play a coordinate role with the ComCDE
quorum-sensing system in regulating genetic competence
and stress response (Ahn et al., 2006). The VicRK system,
which shares a high similarity to the CovSR of Streptococcus
pyogenes, has been found to regulate sucrose-dependent
biofilm formation in S. mutans (Lee et al., 2004;
Senadheera et al., 2005). The ScnRK system in S. mutans
has been found to regulate hydrogen resistance and
macrophage killing (Chen et al., 2008). In addition, an
orphan response regulator in the S. mutans genome has
been found to play a role in sucrose-dependent adherence
and cariogenicity (Idone et al., 2003). These studies have
shown that the expression of virulence traits by S. mutans
requires multiple signal transduction pathways and com-
plex regulatory networks. The TCSTSs can be therefore
considered to be an essential prerequisite for the virulence
and cariogenicity of S. mutans (Levesque et al., 2007;
Biswas et al., 2008). However, most of these studies are
based on investigations of S. mutans in in vitro experi-
ments. Little is known of how these systems play roles in
the virulence and pathogenesis of S. mutans in vivo. In this
study, we used a specific-pathogen-free rat model to assess
the effects of inactivation of the ComCDE, HK/RR11 or
both signal transduction pathways on oral colonization,
ecological fitness and the cariogenic potential of S. mutans.
Here, we report the results of the experiments in which we
address these questions.
Table 1. Bacterial strains and plasmids used in this study
Strain, amplicon or
Relevant characteristics Source or reference
S. mutans strains
Wild-type, Erms, Specs, Kans
NG8 harbouring pDL276 to confer Kanrmarker
NG8 Dhk11::PcEm, Ermr
NG8 DcomCDE::Em, Ermr, Kans
NG8 Dhk11::Em; DcomCDE::Spec, Ermr, Specs
NG8::pComE-KO; ComE, Emr
Li et al. (2001a)
Li et al. (2002b)
Li et al. (2002a)
Li et al. (2001a)
Amplicon of ermAM cassette (860 bp)
Amplicon of spec cassette (1145 bp) from pDL278
Streptococcus–E. coli shuttle vector, Kanr
Streptococcus–E. coli shuttle vector, Specr
Streptococcus–E. coli shuttle vector, Kanr
Dunny et al. (1991)
Dunny et al. (1991)
Li et al. (2001a)
Additive attenuation of S. mutans virulence
Bacterial strains, media and growth conditions. Bacterial strains
and their characteristics are listed in Table 1. The S. mutans wild-type
strain was routinely grown on Todd–Hewitt medium plus 0.3% yeast
extract (THYE), whereas the mutants were maintained on THYE plus
an appropriate antibiotic. Selective media, Mitis–Salivarius agar plus
0.2 U bacitracin ml21(MSB) or THYE agar supplemented with
appropriate antibiotics, were used to distinguish between the S.
mutans strains and other bacteria in oral swab samples or dental
plaques from animals. Blood agar plates were used to grow bacteria
for enumeration of total viable cell counts of the oral samples.
Construction of the ComCDE-HK11 double deletion mutant. To
determine the effect of simultaneous inactivation of both ComCDE
and HK/RR11 on the virulence and cariogenic potential of S. mutans,
we used an existing hk11 deletion mutant SMhk11 (Li et al., 2002b) to
construct a double deletion mutant by deleting the internal region of
the comCDE locus using a PCR-ligation mutagenesis strategy (Lau
et al., 2002; Supplementary Fig. S1). Briefly, a 992 bp fragment
(ComCDE-up) from the internal region of the comC start codon was
amplified from S. mutans NG8 genomic DNA using primers CDE-P1
(59-CAAAAGGAAAAAAACGGCAG-39) and CDE-P2 (59-GGCGCG-
Another fragment, ComCDE-dw (1408 bp), was amplified from an
internal region of comED using primers CDE-P3 (59-GGCCGGCC-
CGCAATGGTGGTTTCAAGACG-39, FseI site underlined) and CDE-
P4 (59-CGGGTTTCAGGAACAGAAGC-39). A spectinomycin-resist-
ance marker (1145 bp) from the Spec cassette of Streptococcus–
Escherichia coli shuttle vector pDL278 (Dunny et al., 1991) was
amplified using primers Spec-P1 (59-GGCGCGCCACTAATAACGT-
AACGTGACTGGC-39, AscI site underlined) and Spec-P2 (59-
underlined). These amplicons were digested, cleaned and ligated to
produce a ComCDE-up::Spec::ComCDE-dw fragment. The liga-
tion product was then transformed into the SMhk11 mutant (Emr)
derived from S. mutans NG8. Following double-crossover homolog-
ous recombination, the internal region of the comCDE locus was
completely replacedby thespectinomycin
mentary Fig. S1). Transformants that grew on THYE agar plates
supplemented with both spectinomycin (500 mg ml21) and erythro-
mycin (10 mg ml21) were selected for PCR confirmation. The PCR
fragments of CDE-P1-Spec-P2 and Spec-P1-CDE-P4 from the mutant
were sequenced to confirm the location and orientation of the
Growth rate and competence assays. Growth curves of all the
strains were assayed by growing cells in 10 ml THYE broth in glass
tubes for 20 h and OD600 readings were obtained using a
spectrophotometer. The mutant was also assayed for genetic
competence to confirm the effect of gene deletion on genetic
transformability using a protocol described previously (Syvitski et
al., 2007). A Streptococcus–E. coli shuttle vector pDL289 conferring
kanamycin resistance was used as transforming DNA. The cultures
were spread on THYE plates plus kanamycin (800 ng ml21), while an
aliquot of the cell suspension was spread on THYE plates to
determine the total numbers of recipient cells. The transformation
frequency was calculated from the number of transformants divided
by the total number of viable recipient cells (per millilitre cell
suspension) and was expressed as a percentage.
Glycolytic pH drop assay. To determine acid production by the
mutants, a glycolytic pH drop assay was performed using a method
described elsewhere (Belli & Marquis, 1991). Briefly, stationary-phase
cells (overnight culture) were harvested and resuspended in a salt
solution (50 mM KCl and 1 mM MgCl2) to make a final cell density
of OD600 1.0. The cell suspensions were adjusted to pH 7.4 and
glucose was then added to a final concentration of 56 mM. Changes
of the pH dropping profile were recorded for 2 h using a digital pH
meter (Fisher) at room temperature.
Acid resistance assay. The mutant strains were assayed for the
effect of acidic pH on their growth by growing bacterial cells on
THYE plates at pH 7.0 and 5.0 using a protocol described previously
(Li et al., 2002b). An aliquot (20 ml) of cell suspension diluted from
overnight culture was inoculated onto THYE plates (pH 7.0 or 5.0)
with additional buffer (10 mM potassium phosphate). The plates
were then incubated at 37 uC for 48 h before assessment of their
growth at low pH.
Biofilm formation assay. The mutants were also assayed for biofilm
formation on a polystyrene surface by a method described previously
(Li et al., 2002a). The growth of biofilms was initiated by inoculating
5 ml cell suspension into 300 ml 46 diluted THYE broth in a 96-well
microtitre plate or 25 ml into 2 ml broth in a 24-well microtitre plate
(some wells contained coverslips). The plates were incubated at 37 uC
for 16 h before removing planktonic cells. The 96-well microtitre
plate was then stained by 0.1% (w/v) safranin for 10 min, rinsed with
water and air-dried for 3 h. Biofilms were quantified by reading
OD490of stained biofilms using a multi-detection microplate reader
(Synergy BioKet). Biofilms formed on cover slides were carefully
removed for examination and photography by a phase-contract
microscope after staining with 0.1% crystal violet for 5 min.
Rat model of oral colonization and cariogenic potential. To
determine the effects of inactivation of comCDE, hk11 or both on oral
colonization, fitness and cariogenic potential, a total of 64 Sprague–
Dawley female rats (19 days old) were purchased from the Charles
River Breeding Laboratory. Upon arrival, the animals were divided
into eight groups (n58 per group). All the animals were fed with
erythromycin water (100 mg ml21) and a regular diet for 3 days to
lower the microbial load, and were tested to confirm the absence of S.
mutans by swabbing and plating the samples on MSB. The animals
then received a sucrose-containing diet (D12450B; Research Diet)
throughout the entire experiment. On day 4, the animals were
inoculated by pasting a bacteria–starch mix (108cells per millilitre of
cooked starch) onto the surfaces of the animal’s molars once a day for
five consecutive days to allow oral colonization. Swab samples were
taken from the surfaces of animal molars on the first day and at the
first, third, sixth, eighth and tenth weeks post-inoculation. The
samples from each group were pooled in 2 ml 10 mM potassium
phosphate buffer and sonicated for 30 s at a setting of 2 using a Fisher
Sonic Dismembrator (Model 100). The samples were serially diluted
and plated on MSB or THYE plates containing appropriate antibiotics
and on blood agar plates for total cell counts. Samples from the wild-
type group NG8 [pDL276] were plated on MSB plus kanamycin
(500 mg ml21). The plates were incubated at 37 uC for 2 days before
enumeration of colonies of S. mutans and total viable counts. The
percentages of the S. mutans cells were calculated to determine oral
colonization and succession profiles of individual strains in animals.
At the end of the tenth week, all the animals were sacrificed in order
to obtain dental plaque samples by a scaling and washing procedure.
Samples from the same group were pooled, sonicated, serially diluted
and inoculated on appropriate plates. The plates were incubated at
37 uC for 2 days before enumeration of colonies. Both jaws of the
animals were then removed and suspended in 3.7% formaldehyde
until caries scoring. All molars of the animals were examined under a
dissecting microscope (Fig. 6) and carious lesions were scored by a
modification of the Keyes method (Keyes, 1958; Lee & Boran, 2003;
Michalek et al., 1975; Yamashita et al., 1993). The results were
analysed by Student’s t test, with P,0.05 considered statistically
Y.-H. Li and others
Competitive index (CI) analysis. To determine the ecological
fitness of the mutants in the animals, we inoculated a 1:1 ratio of a
mutant to the wild-type strain NG8 [pDL276] to establish mixed
infections in the animals. A CI analysis was used to assess the
ecological fitness of the mutants in terms of their ability to compete
with the wild-type strain for oral colonization and succession. The CI
was defined as the ratio of the mutant to the wild-type strain in the
output samples (numbers of the organisms post-infection) divided by
the ratio of the two strains in the input inoculum (Auerbuch et al.,
2001). The CI values were determined by viable cell counts of the
strains recovered from the selective media, while the resident flora
was estimated from total viable counts on blood agar plates. The
mean CI values were calculated from eight animals in each group. The
data were analysed by Student’s t test, with P,0.05 considered
Characteristics of the double deletion mutant
Previous studies have shown that the comCDE knockout
mutant SMcde-4 is significantly defective in its genetic
transformability (Li et al., 2001a). Introduction of a second
genetic construct into this mutant through transformation
was, if not impossible, very difficult. However, deletion of
hk11 resulted in inactivation of the entire signal transduc-
tion pathway, but did not affect the natural transform-
ability of S. mutans (Li et al., 2002b). We took advantage of
this by using an existing hk11 deletion mutant, SMhk11, to
construct a double deletion mutant by transforming a
SMhk11. The internal region (2192 bp) of the comCDE
locus was completely replaced by a spectinomycin cassette
through allelic exchange during double-crossover recomb-
ination. The transformants that grew on THYE plates plus
both spectinomycin (500 mg ml21) and erythromycin
(10 mg ml21) were selected for PCR and sequencing
confirmation. The result confirmed that the Spec cassette
was inserted into the correct location and completely
replaced the internal region of the comCDE locus
(Supplementary Fig. S1). The newly constructed mutant
was named SMcde-hk11 (DcomCDE::Spec, Dhk11::Em,
Specr, Ermr). The double mutant had a slower growth rate
[doubling time (Td)51.72 h] than the parent (Td51.28 h)
when grown in THYE broth, and was significantly defective
in genetic competence (data not shown). In addition, the
mutant cells grown in THYE broth appeared to auto-
aggregate and deposit on the bottom of test tubes.
Interestingly, mutant SMcde-4 had the highest cell density
(OD6001.45) compared with the parent (OD6001.20) when
grown in THYE broth.
Effects on glycolytic pH drop
To determine the effects of inactivation of these systems on
acid production, all the mutants along with their parent
strain NG8 were assayed for glycolytic pH reduction. As
shown in Fig. 1, strain NG8 could rapidly generate acids
from glycolysis, reducing the pH to 4.75 in just 10 min.
The lowest pH value for NG8 to carry out glycolysis was
about pH 3.7. If the acid was neutralized glycolysis took
place again, suggesting that glycolytic activity ceased due to
inhibition by the lower pH but not by glucose depletion. In
comparison with the parental strain, the double deletion
mutant SMcde-hk11 had a slower acid production rate,
since the mutant took about 20 min to reach pH 4.76. This
was almost two times slower than the parental strain in
glycolytic pH drop. In addition, the lowest pH value that
allowed SMcde-hk11 to carry out glycolysis was pH 4.5,
which was 0.8 units higher than the value for the parent
strain (pH 3.7). Clearly, the double deletion mutant was
less tolerant of lower pH. Interestingly, it was observed that
similar pH values stopped the glycolysis of single deletion
mutants SMcde-4 and SMhk11, suggesting that both
mutants were less tolerant of acidic pH. Nevertheless,
these mutants appeared to have similar glycolytic pH
values in the first 10 min, when pH was not a major factor
in inhibiting their growth.
Effects on growth at low pH
To further determine how these mutants tolerated low pH,
we examined the ability of all three mutants, SMcde-4,
SMhk11 and SMcde-hk11, to grow at pH 5.0. As shown in
Fig. 2, inactivation of comCDE (SMcde-4) alone did not
appear to affect the growth of this mutant at pH 5.0, since
the numbers and size of the colonies of this mutant were
similar to those of the parental strain (Fig. 2a). At the same
pH, however, the growth of the SMhk11 mutant was
diminished and the double deletion mutant SMcde-hk11
completely stopped growing (Fig. 2b). We checked the
viability of the overnight culture of these strains and found
Fig. 1. Glycolytic pH drop assay of the S. mutans mutants. ($)
NG8 [wild-type (wt)]; (m) SMcde-4; (e) SMhk11; (#) SMcde-
hk11. The arrow indicates the difference in pH reduction between
SMcde-hk11 and other strains.
Additive attenuation of S. mutans virulence
no significant viability loss in the overnight culture
(pH 4.4). This was also supported by the fact that the
same strain from the same overnight culture grew
reasonably well on control plates (pH 7.0), although the
size of colonies was smaller than that of the parental strain.
The data suggest that simultaneous inactivation of both
signal transduction systems additively attenuated the
ability of S. mutans to grow at acidic pH.
Effects on biofilm formation
To determine the effect of inactivation of these systems on
biofilm formation, the double deletion mutant SMcde-
hk11 was assayed for biofilm formation on a polystyrene
surface. The results showed that SMcde-hk11 formed a
biofilm with reduced biomass and exhibited a sponge-like
architecture (Fig. 3a). Phase-contrast microscopy revealed
that the biofilm formed by SMcde-hk11 was composed of
cells in very long chains that appeared to favour cell
aggregation (Fig. 3b). We also found that the biofilm of
this mutant was loosely attached to the surface and could
be easily removed with a gentle wash or by a mechanical
Effects on oral colonization and succession
A specific-pathogen-free rat model with mono-infection
was used to determine the effects of inactivation of
ComCDE, HK11 or both on oral colonization and
succession of S. mutans. At the first week post-inoculation
all the strains successfully colonized the animals (Fig. 4),
although the numbers of colonized cells varied with the
strains. The swab samples taken from the animals during
the first week predominantly contained the target organ-
isms, accounting for 40–68% of the total viable cells.
Among all the strains, SMcde-hk11 had the lowest
percentage of recovered cells post-inoculation, suggesting
that this mutant was less efficient in establishing initial
colonization. However, SMcde-4 was very similar to the
parental strain in the percentage of recovered cells. We also
Fig. 2. Effects of pH on the growth of S. mutans strains. (a)
SMcde-4 and SMhk11 (single deletion mutants) versus the
parental NG8 strain. (b) SMcde-hk11 (double deletion mutant),
SMhk11 and the parental strain.
Fig. 3. Biofilm formation by the mutants and parental strain on the
surface of a polystyrene microtitre plate. (a) The biofilm formed by
SMcde-hk11 shows reduced biomass with a sponge-like architec-
ture. (b)Phase-contrastmicroscopyshows the SMcde-hk11biofilm
to be composed of cells in extremely long chains. Bars, 20 mm.
Y.-H. Li and others
3260 Microbiology 154
took swab samples at different time points throughout the
experiment to monitor succession of these strains in the
animals. Although the numbers of recovered cells varied
considerably, all the target organisms could be identified
from the viable cell counts at any given time, suggesting
that all the strains succeeded and persisted in the mono-
infection animals. However, the percentages of the target
organisms decreased, suggesting that the resident organ-
isms slowly extended their populations. At the end of the
10-week experiment, the percentages of target organisms in
samples taken from dental plaque were 29% for NG8
[pDL276], 27% for SMcde-4, 18% for SMhk11 and 9% for
the double deletion mutant SMcde-hk11 (Fig. 4).
CI analysis for ecological fitness
To determine the ecological fitness of the mutants in the
animals, we used a mixed-infection model for CI analysis
by inoculating a 1:1 ratio of a mutant to the wild-type
strain NG8 [pDL276]. CI analysis is considered to be a
sensitive measure of the virulence attenuation of bacterial
pathogens. It can distinguish between mutant strains
whose attenuation is too subtle to be detected in mono-
infections. By testing the virulence level of a mutant
versus the parental strain within the same animal, sample
variations from time to time or from animal to animal are
usually decreased. Our results revealed that at the first
week post-inoculation all the strains successfully colo-
nized and initiated a mixed infection in the animals. The
mean CI values at the first week post-inoculation were
0.69±0.32 for SMcde-4/NG8 [pDL276], 0.54±0.29 for
SMhk11/NG8 [pDL276] and 0.28±0.12 for SMcde-hk11/
NG8 [pDL276]. Interestingly, the percentages of recovered
S. mutans cells from all the groups were similar to that of
NG8-kan in the mono-infections (data not shown),
suggesting that the inoculated S. mutans strains domi-
nated in the flora during the first week post-inoculation.
However, the succession of the individual mutants in the
mixed infections differed dramatically from that in the
mono-infections in that the CI values in all the groups
were greatly decreased at the tenth weeks post-inoculation
(Fig. 5). The CI values of the groups of SMcde-4/NG8
[pDL276] and SMhk11/NG8 [pDL276] were about 1.5–2
orders of magnitude lower than those at the first week.
The CI value of group SMcde-hk11/NG8 [pDL276] was
almost four orders of magnitude lower than that at the
first week. Interestingly, the recovered S. mutans cells
from the mixed-infection animals at week 10 were not
significantly different from the values for NG8 [pDL276]
from the mono-infected animals. The percentages of the
recovered cells were 21% for SMcde-4/NG8 [pDL276],
17% for SMhk11/NG8 [pDL276] and 19% for SMcde-
hk11/ NG8 [pDL276]. Clearly, SMcde-hk11 was almost
completely overtaken by the population of NG8 [pDL276]
by the end of the experiment, indicating that this mutant
was much less competitive in dental biofilms than the
parental strain. The results suggest that simultaneous
inactivation of both signal transduction pathways sig-
nificantly reduces the ecological fitness of S. mutans in
Fig. 4. Oral colonization by S. mutans NG8 [pDL276] and
mutants SMcde-4, SMhk11 and SMcde-hk11 in mono-infected
animals during the first week and the last (tenth) week post-
Table 2. Caries scores for S. mutans NG8 [pDL276] and its
mutants in a rat caries model
of caries on
*n58 animals per group.
dA comparison with the wild-type group and significant by Student’s
t test (P,0.05).
ofsmooth surface caries/totalnumberofcaries
Additive attenuation of S. mutans virulence
Effects on cariogenic potential
The effects of inactivation of ComCDE, HK11 or both on the
cariogenic potential of S. mutans were evaluated in the rat
caries models (Fig. 6). All the animals were fed with a
sucrose-containing diet throughout the entire experiment.
The results revealed that all the mutants were compromised
in their ability to initiate dental caries, resulting in lower
caries scores than for the parental strain (Table 2).
Interestingly, the total caries score in the group of the
double deletion mutant was about twofold lower than that
for the wild-type NG8 [pDL276] (P,0.01), while those for
foldlowerthanthe score forNG8 [pDL276] (P,0.05). Again,
the results suggest that inactivation of both signal transduc-
tion systems additively attenuated the cariogenic potential of
S. mutans. We also examined the incidence of caries in
animalswitha mixedinfectionofa1:1ratioofa mutantand
NG8 [pDL276]. The results showed very similar numbers of
caries lesions in all the groups to that of control group NG8
[pDL276], suggesting that the wild-type strain played the
major role in caries development in these animals.
The human oral cavity is a highly dynamic environment
that undergoes rapid and often substantial changes in
nutrient availability, nutrient type, pH, oxygen tension and
inter-bacterial interactions involving either competition or
cooperation (Burne, 1998; Marsh, 2000). Bacteria such as
S. mutans living in dental biofilms are frequently exposed
to cycles of such environmental challenges (Marsh, 2000).
To colonize and succeed in dental biofilms, S. mutans has
evolved multiple signal transduction systems to cope with
rapid and unexpected environmental fluctuations. Two-
component signal transduction is one of the most common
mechanisms by which S. mutans regulates large sets of
genes in response to changing environments (Levesque
et al., 2007; Biswas et al., 2008). In this study, we have
Fig. 5. CI analysis of the mutants versus strain NG8 [pDL276]
(1:1 ratio) in mixed infections. The mixed infections include three
[pDL276] and SMcde-hk11/NG8 [pDL276].
Fig. 6. Photographs showing examples of caries in rats inoculated with different strains. (a) Normal teeth in negative control; (b)
example of dissected teeth; (c) caries in group NG8 [pDL276]; (d) caries in group SMcde-4; (e) caries in group SMhk11; (f)
relatively low numbers of caries in group SMcde-hk11. Arrows indicate caries lesions.
Y.-H. Li and others
investigated the effects of the inactivation of the signal
transduction systems ComCDE, HK/RR11 or both on oral
colonization, ecological fitness and cariogenic potential of
S. mutans in a specific-pathogen-free rat caries model. One
of the interesting findings from this study was that
simultaneous inactivation of both signal transduction
pathways additively attenuated the virulence and cario-
genic potential of S. mutans, since inactivation of either of
these systems alone did not result in effects of the same
degree or extent. The double deletion mutant was much
less able than the parent to succeed and persist in dental
biofilms. Clearly, inactivation of both signal transduction
systems greatly reduced the ecological fitness of S. mutans
in its natural ecosystem. The fact that inactivation of both
systems additively attenuated the virulence and cariogenic
potential of S. mutans suggests that the ComCDE and HK/
RR11 signal transduction systems function independently
to regulate the physiological activities, ecological fitness
and virulence of S. mutans. However, which gene products
are directly responsible for these traits and how these signal
transduction systems regulate these gene products remain
to be studied. Genome-wide analysis of individual path-
ways using microarray technology may provide clues to
answer these questions.
Another interesting finding from this study was that
inactivation of the ComCDE quorum-sensing system alone
did not affect oral colonization and succession of S. mutans
in mono-infected animals. However, the caries score in this
group was significantly lower than that of the parental
strain (P,0.05), indicating that inactivation of the
ComCDE quorum-sensing system still attenuated the
cariogenic potential of S. mutans. The results suggest that
colonization of S. mutans in dental biofilms may not be
sufficient to explain its virulence and cariogenic activity,
although it is a prerequisite for infection. The mechanism
behind this is not very clear. One possibility is that this
mutant is less tolerant the lower pH, so that it has less
potential to initiate caries. Another possibility is that
SMcde-4 in mono-infected animals had less inter-species
competition because of reduced numbers of the resident
flora due to the use of antibiotic water. In these animals,
SMcde-4 dominated until the sixth week post-inoculation,
and the mean viable cell count of this mutant at the tenth
week remained the same as that of the parental strain
(Fig. 4). Recent studies have shown that the same quorum-
sensing system ComCDE that regulates genetic competence
in S. mutans also controls the production of several
bacteriocins and bacteriocin immunity proteins (Kreth
et al., 2005a, b; van der Ploeg, 2005; Matsumoto-Nakano &
Kuramitsu, 2006). These compounds can kill other related
species and favour S. mutans for competition in multi-
species dental biofilms (Kreth et al., 2005a, b). These
quorum sensing-controlled compounds and activity are
believed to act as a ‘two-edged sword’ to kill other species
and release DNA, which can be used by S. mutans for
genetic exchange (Kreth et al., 2005b). In contrast, many
other species of streptococci, including Streptococcus
pneumoniae, need two independent quorum-sensing sys-
tems, the ComCDE and BlpRH systems, to regulate these
phenotypes (Martin et al., 2006). Thus, the ComCDE
quorum-sensing system in S. mutans forms a unique
regulatory mechanism that may provide S. mutans with an
ecological advantage to cope with competing species in its
The attenuation of the cariogenic potential of S. mutans by
inactivating the ComCDE quorum-sensing mechanism and
the activities that it controls may hold promise for
developing anti-quorum-sensing compounds. These com-
pounds could function as inhibitors to block the quorum
sensing-controlled activities and reduce the cariogenic
potential of S. mutans, even if this organism is still present
in dental biofilms. Several recent studies have described
such a strategy and the application of quorum-sensing
antagonists to achieve the inhibition of quorum sensing-
controlled activities and to prevent opportunistic infec-
tions caused by Pseudomonas aeruginosa and Staphylococcus
aureus (Hentzer & Givskov, 2003; Wright et al., 2005). Our
recent study has also identified several signalling peptide
antagonists that show some degree of inhibition of the
quorum-sensing activity in S. mutans (Syvitski et al., 2007).
One of the major advantages of using this strategy is that
such anti-quorum-sensing compounds that specifically
block or override bacterial signalling pathways may control
unwanted pathogenic activities without significant effects
on bacterial viability (Hentzer & Givskov, 2003; Wright et
al., 2005). As bacterial viability is not affected, there is
much less selection pressure to create resistant microbes
with the use of these novel anti-microbial compounds.
Unlike the ComCDE mutant, the HK11 deletion mutant
consistently showed noticeable levels of attenuation in oral
colonization, succession and cariogenic potential in both
single- and dual-infection models, although the degree of
attenuation of these phenotypes was less severe than that
for SMcde-hk11. In the in vitro experiments, we also found
that SMcde-hk11 exhibited some phenotypes that looked
similar to mutant SMhk11 in terms of their growth in
broth, acid-resistance profile (Fig. 2) and formation of
biofilms (Fig. 3). The HK/RR11 two-component system
has been previously suspected to act as the second pathway
to CSP (Li et al., 2002a), since deletion of either hk11 or
rr11 results in a mutant that forms a biofilm with a sponge-
like architecture composed of cells in very long chains, a
feature that was also observed with the biofilm formed by
the comC mutant (Li et al., 2002b). More interestingly, the
HK11 histidine kinase protein has been found to cross-talk
with an unknown response regulator in response to an
acidic pH shift (Li et al., 2001b). However, there is no
additional evidence so far to determine whether or not
HK11 is the second receptor to CSP and how HK11 cross-
talks with a response regulator. Although the evidence
from this study is insufficient to answer these questions,
the additive effects on physiological activities, virulence
and the cariogenic potential of S. mutans of simultaneous
inactivation of both signal transduction systems appear to
Additive attenuation of S. mutans virulence
favour the possibility that the two signal transduction
systems function independently to regulate different sets of
genes, although the resulting phenotypes are similar.
Clearly, further study is necessary to answer these
In summary, this study has shown that simultaneous
inactivation of the ComCDE quorum-sensing system and
HK/RR11 two-component regulatory system additively
attenuates the virulence and cariogenic potential of S.
mutans. The evidence from this study suggests that the
ComCDE and HK/RR11 signal transduction systems can
be considered as novel targets for the development of
strategies for the prevention and treatment of S. mutans
We thank David Fillmore of the InVivo Research Laboratory, IWK
Children’s Hospital at Dalhousie University, for his excellent service
and animal care. We thank Drs Song Lee and Gordon Hall of the
Faculty of Dentistry at Dalhousie University for their advice and
assistance in gross dissection of animal jaws and teeth. We thank Dr
Dennis Cvitkovitch of the University of Toronto for bacterial strains.
We also thank Dr George Bowden of the University of Manitoba for
critical reading of the manuscript. This work was supported in part by
Grant MOP-74487 from the Canadian Institutes for Health Research
and by Discovery Grant RGPIN 311682-07 from the Natural Sciences
and Engineering Research of Canada. Y.-H.L. is a recipient of the
Nova Scotia-CIHR Regional Partnership New Investigator Award.
The authors declare that they have no competing financial interest.
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Additive attenuation of S. mutans virulence