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Indian Journal of Microbiology
The Official Publication of the
Association of Microbiologists of India
ISSN 0046-8991
Indian J Microbiol
DOI 10.1007/s12088-015-0534-8
Plausible Drug Targets in the
Streptococcus mutans Quorum Sensing
Pathways to Combat Dental Biofilms and
Associated Risks
Gurmeet Kaur, Shrinidhi Rajesh &
S.Adline Princy
1 23
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REVIEW ARTICLE
Plausible Drug Targets in the Streptococcus mutans Quorum
Sensing Pathways to Combat Dental Biofilms and Associated
Risks
Gurmeet Kaur
1
•Shrinidhi Rajesh
1
•S. Adline Princy
1
Received: 9 March 2015 / Accepted: 8 May 2015
ÓAssociation of Microbiologists of India 2015
Abstract Streptococcus mutans, a Gram positive facul-
tative anaerobe, is one among the approximately seven
hundred bacterial species to exist in human buccal cavity
and cause dental caries. Quorum sensing (QS) is a cell-
density dependent communication process that respond to
the inter/intra-species signals and elicit responses to show
behavioral changes in the bacteria to an aggressive forms.
In accordance to this phenomenon, the S. mutans also
harbors a Competing Stimulating Peptide (CSP)-mediated
quorum sensing, ComCDE (Two-component regulatory
system) to regulate several virulence-associated traits that
includes the formation of the oral biofilm (dental plaque),
genetic competence and acidogenicity. The QS-mediated
response of S. mutans adherence on tooth surface (dental
plaque) imparts antibiotic resistance to the bacterium and
further progresses to lead a chronic state, known as peri-
odontitis. In recent years, the oral streptococci, S. mutans
are not only recognized for its cariogenic potential but also
well known to worsen the infective endocarditis due to its
inherent ability to colonize and form biofilm on heart
valves. The review significantly appreciate the increasing
complexity of the CSP-mediated quorum-sensing pathway
with a special emphasis to identify the plausible drug tar-
gets within the system for the development of anti-quorum
drugs to control biofilm formation and associated risks.
Keywords Streptococcus mutans Dental biofilms
Quorum Sensing CSP ComA Multi-drug resistance
Quorum Sensing System: Deciphering ‘Dental
Talk’
Streptococcus mutans, a Gram positive coccus, is an in-
habitant of human buccal cavity and is also a primary
causal organism of dental caries. The tooth surface act as
an essential ecological niche for S. mutans as it is very
difficult to be identified until the teeth is erupted in the
buccal cavity and dissipate immediately after the teeth is
lost due to infection or old age [1–3]. S. mutans has ad-
vanced to rely upon a biofilm ‘‘way of life’’ to survive and
persist for longer durations in its characteristic biological
community [4–6], dental biofilm, commonly known as
plaque. S. mutans with its own or other populations tend to
form oral biofilms (dental plaque) via., inter/intra-species
communication known as quorum sensing system (QS)
[6–8].
QS Pathways in S. mutans
ComCDE QS Pathway
QS in gram-positive bacteria like S. mutans, generally
comprise three components: a signal peptide (CSP), a two-
component regulatory system (TCTS) with a membrane-
bound histidine kinase (HK) sensor and an intracellular
response regulator (RR) [9]. Recently, it has been identified
that the CSP-mediated QS system in S. mutans up-regulates
the genes, cslAB (comAB) and comCDE that exhibits the
phenotypic traits like genetic competence, bacteriocin
production and biofilm [10,11]. The genes comC,comD,
&S. Adline Princy
adlineprinzy@biotech.sastra.edu
1
Quorum Sensing Laboratory, Centre for Research on
Infectious Diseases (CRID), School of Chemical and
Biotechnology, SASTRA University,
Thanjavur 613 402, Tamil Nadu, India
123
Indian J Microbiol
DOI 10.1007/s12088-015-0534-8
Author's personal copy
and comE encodes a precursor of competence-stimulating
peptide (CSP), the HK sensor protein, and a cognate RR,
respectively [10,12,13]. The genes comC and comDE are
closely located on the same chromosome and the peptide
(CSP) is synthesized as a collective result of their gene
products [14,15]. The genes, cslA and cslB, are divergently
mapped on the same chromosome also encodes a CSP-
specific maturation and secretion complex factors with an
ATP-binding cassette (ABC) transporter (ComA) and an
accessory factor (ComB). As these factors, specifically
involved in the post-translational processing of the CSP to
further secrete it towards the outside of the cell, as a mature
signaling peptide [16]. The QS signalling operates opti-
mally when the cells acquire and depend solely and ac-
tively on the biofilm lifestyle as the CSP concentration
reaches a threshold value [17]. Mature CSP binds to the
conserved HK residue (ComD) present in the membrane,
resulting in its own phosphorylation and the subsequent
relay of this process to its cognate RR protein (ComE) [18].
Thus the cell-density dependent process elicits a cellular
response to activate the gene loci such as comA and comB
with a simultaneous feed forward circuit for comCDE.
Also, the same response was extended to the genes, comR
and sigX expression, for which the mechanism is still un-
known [2].
ComRS Pathway
Interestingly, S. mutans possess ComCDE as well as
ComRS quorum sensing pathways. The ComRS QS system
is activated on sensing the extracellular tryptophan signal
peptide pheromone, XIP and get internalized to the cells
through a membrane-bound oligopeptide ABC transport
system, Opp/Ami [2]. Further, XIP binds to a transcrip-
tional regulator, ComR, in turn regulates the sigX, an al-
ternative sigma factor SigX (ComX), to switch on the late
competence genes responsible for the genetic transforma-
tion. The ComCDE operon regulates the production of anti-
microbial peptides, toxins and adherence factors, whereas
the functional characterization of the ComRS quorum-
sensing circuit show a pre-dominant role in the genetic
competence via., the regulator, SigX [19,20].
LuxS Pathway
Previously, it has been reported that S. mutans interact with
the other oral flora of the dental plaque to mediate inter-
species communication. LuxS is reported to involve in
S-adenosylmethionine catabolism and converts ribose ho-
mocysteine into homocysteine and 4,5-dihydroxy-2,3-
pentanedione that act as a precursor of Autoinducer-2 (AI-
2) [21–23]. The LuxS-mediated QS are well characterised
to elicit interspecies communication and modulate multiple
traits crucial to establish S. mutans pathogenesis. So, the S.
mutans flourish in the buccal cavity via., activation of the
luxS gene that leads to the production of AI-2 which en-
sures it survival and virulence expression in multispecies
environment. The researchers has shown that the luxS-
deficient strains affects the expression of the virulence
determinant to a greater extent (
[50 %) and in parallel
upregulate their acid-adaptive behaviours to increase their
survival rate [24]. Also, the luxS gene is highly conserved
among the Gram positive and Gram negative bacteria and
may operate as a global regulator to be an essential factor
for a drug target [22,23].
S. mutans and Dental Caries
Clarke, in 1924, designated S. mutans, after he could iso-
late a bacterial species from the carious lesions and looked
like a mutant form of a coccus. The relationship of S.
mutans with dental caries was not largely perceived until
dental practitioners and researchers in the 1960s revived
interest in this organism. Since then, various studies con-
firmed the relationship of S. mutans with dental decay and
carious lesions and longitudinal studies followed the pre-
dominance of S. mutans on infected sites that ultimately
became cariogenic. An experimental study on the mono-
infected rats revealed the cariogenic potential of the var-
ious plaque species and notably, S. mutans was found to be
predominant among cariogens. The research has led to the
‘‘specific plaque hypothesis’’ and stated that the S. mutans,
were primarily responsible for the dental decay. With the
taxonomical advances, it has become apparent that the S.
mutans-like isolates actually accounts for several species
and was collectively known as the mutans streptococci
(MS).
Like many other diseases, dental caries, also have
multiple etiologies. However, the topmost variable for
caries occurrence has been sucrose rich diet. Frequent
consumption of sucrose has also been implicated in
building plaque ecological niche and provide a substrate
for MS to synthesize adhesive glucans to promote their
own colonization and accumulation on the tooth surface
[25]. The acid tolerance capability of the MS further en-
hances their adhesion and growth as well as the growth of
other acid-tolerant species such as the lactobacilli. Due to
highly acidogenic nature of these species [26], the fer-
mentable dietary carbohydrate results in a huge drop in
plaque pH than normal paving way for demineralization
and decalcification of the tooth surface. The scenario also
promoted researchers worldwide to focus on the adhesion,
acidogenicity, and acid-tolerant properties of the MS [27].
The focus of this review is to primarily describe the
quorum sensing–mediated cell–cell communication in S.
mutans and its various components. Since most of the
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virulence properties (Table 1) are shared among the var-
ious MS, the review will address on the S. mutans as a
paradigm for the virulence of dental caries under the
regulation of QS. Also the review discusses the structural
and functional aspects of various quorum molecules that
would provide an insight to exploit them as drug targets.
The application potential of this review would also provoke
the scientific community towards target based drug dis-
covery in synthetic biology to effectively control the bac-
terial biofilms and its associated risks.
Plausible Drug Targets: Hindering ‘Bacterial
Chat’
Signals
CSP
In S. mutans comC encodes a precursor signal peptide, a
21-amino acid Competence Stimulating Peptide (CSP)
processed and exported out of the cell to elicit its response
via., the ComCDE QS pathway. The formation of biofilm
and virulence traits in S. mutans such as acidogenicity,
bacteriocin production, genetic competence solely depends
on the signal, CSP-mediated QS [22]. S. mutans has
evolved various mechanisms to allow the survival and
persistence of the species in a broad range of adapting to
frequent environmental pressure such as acidic environ-
ment that act as a constant stress in the oral cavity to en-
hance the production of CSP [28]. The stress-inducible
activation of CSP induces the cell death of a sub-popula-
tion accounting for the altruistic nature of bacteria to be
beneficial for its own population. Similarly, the native CSP
of Steptococcus pneumonia was known to cause chronic
diseases in humans such as pneumococcal meningitis and
pneumonia [13]. Earlier reports shows that the S. mutans
JH1005 (deletion of three amino acid residues in the C-
terminal domain of the peptide, CSP) has overexpressed
bacteriocin and resulted in 600-fold reduction in the com-
petence and drive a greater significance of those residues in
QS signalling pathway [29]. The CSP has been reported to
exist as a factor to be structurally random coiled and highly
soluble nature to increase its diffusion rate in the aqueous
environment [10,30]. The randomly coiled CSP undergoes
conformational change and folding upon binding to the
membrane bound Histidine Kinase (HK) receptor resulting
in the formation of an amphipathic a-helix with a hy-
drophobic face. The change in the conformation enables
the CSP to show a stronger interface of its interaction with
the HK receptor binding pocket via., hydrophobic inter-
actions [29,31]. Till date, approximately 700 species has
been reported in the buccal cavity and a few cariogenic
pathogens, including S. mutans are well characterized to
mediate dental caries. So as to reduce or completely
eliminate dental caries, alternate strategies has to be pri-
oritized as a one-step forward to prevent the S. mutans
adherence on the tooth surface without affecting the normal
oral flora. In recent years, antimicrobial peptides (AMPs)
have received attention of the researchers worldwide, as a
novel class of antimicrobial agents because of their ability
to kill a wide range of pathogenic species, including bac-
teria, fungi, and viruses, through various mechanism of
action [9]. The AMPs are also reported to be effective
against resistant pathogens with their ability to disrupt the
functions of the cellular membranes and nucleic acids di-
rectly and moreover the rate to which AMP-resistant
strains appears, is very low or almost negligible [32].
Similarly, a new class of pathogen-specific antimicro-
bials, STAMPs (selectively targeted antimicrobial pep-
tides) were discovered against the dental pathogen, S.
mutans. The peptides were designed to have a targeted
domain (8-aminoacid region of the CSP) fused with an
antimicrobial peptide domain to show a robust species
Table 1 QS controlled genes and their phenotypic traits in S. mutans
QS components Regulatory gene Group-derived benefits (Virulence factors) References
CSP (ComC) comC/comD/comE Bacteriocins, Biofilm Formation [10,11,14,55]
XIP (ComS) comR Competence [10,11]
SigX (ComX) comR DNA transport, uptake and Recombination [12,36]
ComAB nlmAB (comAB) Mutacin IV (bacteriocins), Maturation and export of CSP
outside the cell
[16,49]
ComD comC/comD/comE (TCSTS) Class II bacteriocins and HK receptor for CSP signal [14,22]
immA and immB (VicRK) comC Self- immunity proteins for protection from bacteriocins,
acid tolerance
[55]
Lux S luxS Interspecies communication [39,40]
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specific activity and eliminate the S. mutans from multi-
species biofilm without affecting the normal (non-car-
iogenic) streptococci [33]. Syvitski et al. [29] reported that
the C-terminal truncated CSP peptides could competitively
affect the QS activity and the structural motif in the
C-terminal domain restores the activation of the QS signal
transduction pathway [34,35]. Further the mutational
studies (Point deletion) revealed the loss of function of
CSP in promoting genetic competence without affecting
the binding of peptide to the receptor [34–37].
XIP
XIP (Sig X Inducing Peptide) modulates the expression of
r
x
(ComX) via., ComRS signaling pathway. The comS
encodes the XIP precursor is processed to mature and in-
ternalize the cells through the transporter, Opp/Ami. The
mature signal in-turn interacts with the comR and activate
the gene comX to express the factor, r
x
[36,37]. Recent
data has shown a drastic reduction of XIP levels in the
comX (r
x
) deficient S. mutans strains, as it evidence the
positive feed-back role of ComX in ComRS/XIP expres-
sion where its binding sites were localized either within or
upstream of ComRS [38]. Earlier reports have also
demonstrated that the increase in the concentration
(2–4 lM) of CSP mediated cell-killing and growth arrest of
S. mutans [39,40]. Likewise, the S. mutans on exposure to
10 lM of synthetic XIP (sXIP) has shown the killing ef-
fect, naturally score the phenomena of exploiting the
pheromones (XIP) as a potent effector molecules to treat
dental caries [41].
Autoinducer-2 (AI-2)
The previous studies had established that in dental plaque,
the oral streptococci and other oral bacterial species in-
teract with each other and form an ideal system to mediate
interspecies signalling and biofilm formation in a particular
niche [42]. The AI-2 mediated interspecies QS response is
a well-characterized cross-talk strategy, a known co-op-
erative synergy exists in both gram-positive and gram-
negative pathogens [43]. As it is evidenced from the fact
that they all have a highly conserved luxS gene encoding
autoinducer-2 (AI-2) serve the function of optimization of
the virulence gene expression in a cell density dependent
manner in a particular biological niche. A study carried out
by Merritt et al. [44], Yoshida et al. [42] demonstrated that
the mutation in the luxS gene of S. mutans leads to an
altered biofilm structure and also affected the production of
the bacteriocin and mutacin I. In Escherichia coli and
Serratia marcescens the mutacin production was lexA and
recA dependent whereas in S.mutans, it was solely de-
pendent on a Lex-A like factor, IrvR [45]. Rickard et al.
[43] and McNab et al. [46] have also shown the involve-
ment of AI-2 multispecies biofilm communities and thus,
an approach leading to the suppression of luxS gene ex-
pression could lead an altered interspecies behavior in the
oral microbial community leading to hindrance in the QS
system.
Receptor and Response Regulator
ComDE
The two signal transduction system, ComDE are proven to
elicit CSP-mediated responses to the neighbouring cells
via., a phospho-relay mechanism and activate its target
genes, comCDE, comAB and comX to establish biofilm,
stress response and bacteriocin production. These virulent
traits show an anti-bacterial effect to the other commensal
species, so that their DNA released in the growth sur-
roundings, would provide an ecological advantage to S.-
mutans to compete the natural ecosystem i.e. multi-species
dental biofilms in oral cavity. Recent studies had revealed
the bifunctional nature of the ComED system showing a
dual response as an activator of nlmC and repressor of
comC. The researchers also reveal the puzzle behind the
dual opposing characteristics of ComED as nlmC share an
intergenic region (IGSA499) with the divergently tran-
scribed comC [47,48]. Further, experiments show that the
nlmC expression in comD mutant show [40-fold fold and
[fourfold reduction in comparison with the wild-type
under CSP-induced or un-induced state respectively [49].
Also, the ComE share higher sequence homology to the
DNA binding domains of AlgR, AlgA, LytR family
members and provide an insight to be exploited for de-
veloping broad-spectrum anti-biofilm compounds. Li et al.
suggest that those compound would also block the quorum
sensing dependent virulence properties and reduce the
cariogenic properties of S. mutans, regardless of the fact
that the organism is still active in oral biofilms [50,51].
ABC Transporters
ComA
The gene comAB encodes ComA, to act in the initial step of
the QS pathway of Streptococcus, consists of bacteriocin-
associated ATP-binding cassette (ABC) transporters, that
employs ComB as an accessory protein for specific pro-
cessing and maturation of the precursor (AMS family of
ABC transporters involved in maturation and secretion of
molecules) CSP [16]. The ComA has three domains (1)
N-terminal Peptidase domain comprising of 150-amino
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acid (2) a six membrane-spanning segments of transmem-
brane domain (3) an ATP-binding C-terminal motif local-
ized on the cytoplasmic face of the membrane. The
functional aspects of the peptidase domain was understood
specifically to cleave their cognate propeptides after the
consensus Gly–Gly motif [52].
The ATP-binding cassette (ABC) transporters are the
largest well characterized protein super-family from var-
ious organisms to participate in a wide array of phys-
iological functions, that includes virulence, antigen
presentation in pathogenic bacteria, multi-drug resistance
and regulation of ion channels across the bacterial mem-
branes [53]. The core structural organization of the ABC
transporter consists of a two transmembrane domains
(TMD) and two cytosolic nucleotide-binding domains
(NBD) [53]. The sequence variation in the several mem-
brane-spanning a- helices that form the transport pathway
for TMDs reflects its complement sequence variation in
their ligands for the ABC transporters and the highly
conserved NBDs provide energy on ATP hydrolysis for the
substrate transport [54].
In ABC exporters, a linker region with a 20 amino
acids adjoins the last a-helix of TMD and the first b-
strand of NBD. The highly conserved glycine residue is
located near the C-terminal end of the linker and 11
amino-acid residues proceeds the adenine stacking the
tyrosine residue of the NBDs [53,55,56]. To date, the
molecular characterization of the three-dimensional
structures of the four full-length ABC exporters are
known to be Sav1866 from Staphylococcus aureus,
MsbA from Salmonella typhimurium, and Thermotoga
maritima TM287/288 [5,54–57]. Whereas, the protein
sequence alignment of ComA with the ABC exporters
evidence that the residue Gly526 occupy a significant
position in the ABC exporters [53,58,59].
Since, ABC transporters have been reported to play a
vital role in the maturation and secretion of CSP, anti-
quorum compounds targeting the vital glycine residue and
blocking the ABC transporter (ComA) in S. mutans may
pave a way to novel drug design [52]. Moreover, the
family of the bacteriocin- associated ABC transporters has
so far been found only in prokaryotes, the PEP domains
of ComAs would be an ideal target for the development
of drugs that inhibit the biofilm formation of Strepto-
coccus [52] (Fig. 1). Mutations at the active site of PEP
domain has resulted in the complete loss of the catalytic
activity of PEP domain [16]. Based on the observation
and substrate specificity of peptidase domain of ComA,
we could hypothesize that the therapeutic inhibition of
PEP domain of ComA will halt the maturation and se-
cretion of CSP from S. mutans and thus hindering the
virulence expression associated with a quorum sensing
circuit of S. mutans.
ComX
ComX, an alternative sigma factor (r
x
) activates the late
competence genes primarily involved in the processing and
recombination of the foreign DNA to the chromosome of
Streptococci [12]. In S. pneumoniae, the up-regulation of
r
x
is induced in response to the activation of the compe-
tence cascade via., CSP-mediated ComCDE pathway.
Upon activation with the CSP, the membrane-bound HK
receptor integrates the signal to its cognate cytoplasmic
response regulator (ComD) to further activate the alternate
sigma factor, SigX (ComX) [60]. Also, its recognizing sites
are well characterized to be a set of non-canonical con-
sensus region (CIN box) that lie in the promoter regions
(TACGAATA) of various ComX dependent genes, ssbB,
dalA,ccl,celAB,cAAB, and cinA-recA [12,61]. Similarly,
in S. mutans, it is postulated that the CSP-mediated acti-
vation of r
x
, an alternate sigma factor up-regulates the
expression of several competence genes that includes
comX,comCDE (positive feedback loop) and other early
com genes, comW (r
x
Stabilizing factor). ComX (r
x
)
predominates to induce CSP-dependent com regulon ex-
pression and a sole key factor to resist stress responses in
gram-positive bacteria, could probably be chosen as an
ideal target for disarming the pathogen against its evolving
trend to resist antibacterial agents.
Conclusion
Streptococcus mutans mediated dental caries is a multi-
factorial disease are being ignored due to its non-life
threatening nature [62,63]. An unknown fact exists as the
inflammation process takes its pace slowly and gradually, it
would act as a ‘silent killer’ for the patients suffering from
dental caries. Therefore the untreated carious dentine can
lead to the development of systemic diseases such as in-
fective endocarditis [61–65]. Meta-analysis reports be-
tween 2003 and 2009 has shown a weak but statistically
significant correlation between cardiovascular disease and
dental diseases [66–68]. Based on the reports of meta-
analysis, a conclusion can be drawn, that an individual with
severe dental infections is at higher risk of either having or
developing cardiovascular disease and rheumatoid arthritis
[25,69]. Since the association of S. mutans with dental
caries was established, research was focused on recogniz-
ing its virulence properties along with their role in plaque
formation and the progress of dental diseases. The review
has highlighted the importance of QS pathway in S.mutans
and its role in formation of biofilm along with other viru-
lence properties. Quite a few efforts has been taken to
modulate QS to reduce the production of biofilm and as-
sociated virulence factors like Hentzer and Givskov,
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Ravichandran et al., Arya et al., Hema et al., and Wright
et al., have described the application of antagonists that act
as QS inhibitors to attain the inhibition of virulence genes
under QS circuit to prevent the various infections caused
by Pseudomonas aeruginosa,Vibrio cholerae and Sta-
phylococcus aureus [28,33,70–76]. The approach ensures
its success in the fact that, anti-quorum compounds may
control virulence traits of pathogenic microbes without
significant effects on viability of bacterial cells [77]. Bio-
film cells have been shown to be several fold more tolerant
to antibiotics than planktonic cells, and this makes it hard
to treat S. mutans with modern medicine [78,79]. Thus, the
importance of developing an anti-quorum compounds as
alternate therapy for multi-drug resistant bacteria, has
positive future perspectives with respect to medicine.
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