Progress of Antimicrobial Discovery Against

Curr. Issues Mol. Biol. (2019) 32: 601-644. DOI: https://dx.doi.org/10.21775/cimb.032.
601
Progress of Antimicrobial Discovery Against

the Major Cariogenic Pathogen Streptococcus
mutans
Tao Cui1,2*, Wenfu Luo1, Letong Xu1, Baoqiang Yang1, Wen Zhao1, Huaixing
Cang2*
1
Research & Development Institute of Northwestern Polytechnical University in
Shenzhen, Shenzhen 518057, Guangdong, P. R. China.

2
School of Life Sciences, Northwestern Polytechnical University, Xi’an 710072,
Shaanxi, P. R. China.
*Corresponding authors: [email protected], [email protected]
Abstract
Dental caries, also known as tooth decay, or cavities, remains a global public
health issue. Streptococcus mutans is considered the most significant contributor
to dental caries. Its cariogenicity typically depends on its unique metabolic
activity and lifestyle, including acid production, acid tolerance and biofilm
formation. Currently used anti-caries therapies, such as fluoride and
chlorhexidine, are characterized by side-effects and drug resistance. Therefore,
the development of alternative inhibitors against S. mutans growth is urgently
needed. In the last decade, a larger number of natural products and their
derivatives from plants, marine organisms and microorganisms were studied to
evaluate their antibacterial activity against S. mutans. In addition,
caister.com/cimb 601 Curr. Issues Mol. Biol. (2019) Vol. 32

Antimicrobial Discovery Cui et al
drug-repositioning base screening and target based high-throughput screening
were employed, resulting in inspiring progresses in recent years. In this review,
we summarized the available evidences regarding the inhibition of S. mutans
growth. We focus on the sources, structures and potential mechanism of action
of these inhibitors. Beside small molecular compounds, we also considered
antibacterial peptides and protein inhibitors developed in this field.
Introduction
Tooth decay, also known as dental caries or cavities, is a significant public health
problem worldwide (Pitts et al., 2017). Approximately 2.43 billion people (36% of
the population) have dental caries. Tooth decay is a result of demineralization of
the tooth by acids made by oral microorganisms (Figure 1). Food debris and
sugars on the surface of the tooth are metabolized by microorganisms, which
produce acids and lead to tooth decay. The oral cavity is inhabited by over 600
different bacterial species (Dewhirst et al., 2010; Kilian et al., 2016), among
which S. mutans is the most prosperous colonizer causing dental caries. During
carbohydrate metabolism, S. mutans produce lactic acid, leading to a drop of pH
in the environment, which subsequently inhibits the growth of other microbial
competitors, making S. mutans a predominant species in the oral cavity.
Unlike other pathogens that live in the blood, cells or tissues that are of difficult
access, S. mutans lives in the tooth surface, which is easily and mechanically
accessible, thus, mechanical approaches for removing S. mutans can be
considered. However, S. mutans can tightly adhere to the tooth surface and its
efficient removal is difficult by mechanical method due to the formation of a

biofilm. Dental plaque biofilm is made by a community of oral microorganisms,
which adhere to the tooth surface, and S. mutans is the most predominant
species (Ajdić et al., 2002; Mitchell, 2003; Nobbs et al., 2009; Flemming et al.,
2010; Kolenbrander et al., 2010; Avilés-Reyes et al., 2017).
Figure 1. Dental caries and S. mutans. Dental caries is a result of tooth demineralization by acids
produced by microorganisms in the dental plaque. Dental plaque is a multi-species and
cross-kingdom biofilm in which S. mutants is the predominant species. Ca10(PO4)6(OH)2 is the
formula of hydroxyapatite, which is the primary mineral of tooth enamel.
Taken together, at least three indispensable factors play essential roles in the
cariogenicity of S. mutans. The first one is the acid production. Lactic acid
produced by S. mutans metabolic activity breakdowns the tooth enamel and
causes a pH decline of the surrounding environment, inhibiting the growth of
other species and making S. mutans the dominant species in the oral cavity. The
second one is the acid tolerance (Matsui et al., 2010; Smith et al., 2012;
Baker et al., 2017). Acid production results in a drop of pH in the local
environment. Therefore, S. mutans should be (and it actually is) able to respond
and adapt to this acid environment to survive. The third one is the biofilm
formation. The formation of a biofilm results in multiple benefits for S. mutans.

Indeed, biofilm formation allows S. mutans to tightly adhere to the tooth surface,
thus avoiding being removed. Moreover, its formation prevents the dilution
or/and neutralization of acids produced in the biofilm by water or saliva.
Furthermore, biofilm is helpful for adsorbing nutrition. Finally, biofilm formation
reduces the access of antibiotics to S. mutans, thus increasing antibiotic
resistance.
In this review, we summarized the available evidences regarding the inhibition of
S. mutans growth. We focus on the sources, structures and potential
mechanisms of action of these inhibitors. Beside small molecular compounds,
we also considered antibacterial peptides and protein inhibitors developed in this
field.
1. Small molecules currently used in dental cavity treatment
Currently, widely used anti-caries compounds include fluoride, chlorhexidine, and
xylitol (Figure 2). Most toothpastes contain between 0.22% and 0.312% fluoride,
usually in the form of sodium fluoride or sodium monofluorophosphate (Buzalaf
et al., 2011; Pandit et al., 2011; Takahashi et al., 2011; Liu et al., 2012; Nóbrega
et al., 2016; Demonte et al., 2017; Liao et al., 2017). Sodium fluoride came into
use to prevent tooth decay in the 1940s. The effects of fluoride on dental caries
can be divided into the following two: 1. Fluoride reduces tooth enamel decay by
the formation of fluorapatite and its incorporation into it; 2. Fluoride exhibits an
inhibitory action on S. mutans growth. The glycolytic pathway contributes to the
acid production by S. mutans. Fluoride inhibits the glycolytic pathway by directly
targeting the enolase enzyme (Takahashi et al., 2011). The accumulation of

internal polysaccharide when sugars are in excess helps S. mutans to respond
to starvation stress, thus contributing to cariogenicity by S. mutans. A recent
study showed that sodium monofluorophosphate inhibits internal polysaccharide
biosynthesis in S. mutans by targeting the S. mutans ADP-glucose
pyrophosphorylase (Demonte et al., 2017). Chlorhexidine is a broad-spectrum
antibiotic, came into medical use in the 1950s and it is the most widely used
antibacterial agent against S. mutans. Chlorhexidine is positively charged in
solution and can bind to the bacterial cell wall, damaging its cell membrane (Liu
et al., 2012; Coelho et al., 2017). Xylitol is a sugar alcohol used as a sweetener
and it attracted popular attention because evidence suggests that it reduces the
incidence of cavities. Several potential mechanisms of action are proposed for its
anti-caries activity. Xylitol is non-fermentable and cannot be metabolized into
lactic acid by S. mutans, as well as it cannot be used as an energy source
although being taken up into the cell to interfere with bacterial growth and
reproduction (Takahashi et al., 2011). Xylitol can promote the remineralization
of the tooth enamel by coordinating with and transporting polyvalent cations,
such as Ca2+.
Current anti-caries compounds mainly contribute to the prevention and reduction
of the incidence of dental caries. However, they have very limited success in the
treatment of already formed dental caries. In addition, they also face the
challenges of side-effects and drug resistance. These compounds possess low
selectivity, which may destroy the homeostasis of oral microbiome. Due to the
extensive use of fluoride, many microorganisms developed a resistance against
its effects. For example, fluoride antiporter protein mediates microbial fluoride

resistance by exporting fluoride ions (F-) to maintain a low F- level in the cell (Liao
et al., 2017). Therefore, the development of alternative inhibitors against S.
mutans growth is expected. In this review, we summarized current efforts on the
development of inhibitors against S. mutans growth.
Figure 2. Timeline of our understanding on the pathology of dental caries and development of
anti-caries agents. Current treatments for dental caries have limited success and face the
problems of having side-effects and developing drug resistance. Alternative antimicrobials against
S. mutans are required.
2. Natural products from plants
Natural products are rich sources of compounds with potential powers against
several diseases, including caries. Plants are the important source of natural
products (Koehn et al., 2005; Harvey et al., 2015). Numerous plant natural
products exhibit inhibitory activities against S. mutans growth. According to their
chemical structures, these compounds mainly include polyphenolic compounds,
especially flavonoids (Figure 3). However, their mechanism of action against S.
mutans survival is not very clear. The potential mechanisms include a specific
action on cell membrane and a modification of its permeability, leading to cell
death. At the transcriptional level, some compounds can induce the change in

the expression of genes involved in biofilm formation. Some compounds can
inhibit enzyme activity involved in acid production pathways. Interestingly, many
of these compounds can be found in tea, thus suggesting its potential anti-caries
effect (Jeon et al., 2011; Cheng et al., 2012; Abachi et al., 2016).
Earlier studies suggested that flavonoids exhibit inhibitory activities against S.
mutans growth. Flavonoids have the general structure of a 15-carbon skeleton,
which consists of two phenyl rings and heterocyclic ring. This carbon structure
can be abbreviated C6-C3-C6. Compounds in this category include apigenin
(Koo et al., 2003; Koo et al., 2005; Jeon et al., 2009), myricetin (Falsetta et al.,
2012), quercetin, kaempferol (Guan et al., 2012) and epigallocatechin gallate
(EGCG) (Xu et al., 2011; Bai et al., 2016). Apigenin affects the accumulation and
polysaccharide content of S. mutans biofilms without major impact on bacterial
viability (Koo et al., 2003; Koo et al., 2005; Jeon et al., 2009). Myricetin inhibits
the ability of S. mutans to assemble biofilms without altering cell viability. It
inhibits glucosyltransferase (Gtf) activity and reduces the expression of the gtfBC
gene cluster, which are involved in the synthesis of glucans (Falsetta et al., 2012).
Quercetin and kaempferol inhibit the growth of S. mutans and several other oral
bacteria. In addition, They also significantly inhibit acidogenicity, acidurity and
F-ATPase activity of S. mutans (Guan et al., 2012). EGCG inhibits various
cariogenic virulent factors of S. mutans at the transcriptional and enzymatic
levels, leading to reduced acidogenicity and compromised stress tolerance
(especially acid tolerance). Real-time PCR results demonstrated that EGCG
significantly suppresses the ldh, eno, atpD, and aguD genes, which encode
virulence factors that are involved in acid production and stress response of S.
mutans. Inhibition of the enzymatic activity of F1Fo-ATPase and lactate

dehydrogenase is also observed (Xu et al., 2011).

Figure 3. S. mutans growth inhibitors from plant natural products, marine natural products and
microbial natural products. Natural products from plants mainly include polyphenolic compounds,
especially flavonoids (EGCG, myricetin, apigenin, kaempferol, morin, and formononetin). Marine
natural products mainly include heterocyclic compounds (imidazole ring in bromoageliferin and 3F1;
oxazole ring in 5H6; furanone ring in furanones C-30) and fatty acids (DHA and EPA).

Several flavonoids, such as morin (Huang et al., 2014) and formononetin (Park
et al., 2017) inhibit the activity of Sortase A (SrtA). SrtA is a
membrane-associated transpeptidase responsible for the anchoring of
surface-exposed proteins to the cell wall envelope of gram-positive bacteria
(Hendrickx et al., 2011), including S. mutans. A mutant S. mutans strain lacking
srtA exhibits a decreased ability to colonize oral mucosa and teeth, consequently
reducing caries formation. Therefore, this enzyme represents a potential target in
the treatment and/or prevention of dental caries (Park et al., 2017). Morin is
another natural plant extract that inhibits S. aureus and S. mutans SrtA activity,
the latter inhibited by an IC50 of 27.2 ± 2.6 μM. S. mutans adheres to the tooth
surface through the sortase A (SrtA)-mediated cell wall anchored protein Pac.
Western blotting demonstrated that 30 μM morin induces Pac protein partial
release into the supernatant (Huang et al., 2014). trans-chalcone is the precursor
molecule of many flavonoids, and it inhibits S. mutans SrtA in vitro and biofilm
formation. Mass spectrometry revealed that trans-chalcone forms a Michael
addition adduct with the active site on SrtA cysteine (Wallock-Richards et al.,
2015). Recently, two new flavonoids, 7-hydroxy-6-methoxyflavanone and
formononetin, were found as strong inhibitors against S. mutans SrtA activity,
with IC50 values of 46.1 and 41.8 µM, respectively (Park et al., 2017).
Other polyphenols with inhibitory activity against S. mutans survival include
tannic acid (Sendamangalam et al., 2011), gallic acid (GA) (Kang et al., 2008,
Sendamangalam et al., 2011; Shao et al., 2015) and methyl gallate (MG) (Kang
et al., 2008; Kacergius et al., 2017). Tannic acid inhibits S. mutans biofilm

formation. Enzyme activity assay indicated that tannic acid exhibits an inhibitory
effect on two enzymes, glucosyltransferase and fructosyltransferase. These two
enzymes are involved in the synthesis of glucans and fructans, the important
components of extracellular polymers (Sendamangalam et al., 2011). Tannic acid
can be hydrolyzed into GA. GA can be found in many plants including tea leaves.
It has inhibitory effects on the growth of cariogenic and periodontopathic bacteria.
Moreover, GA inhibits the in vitro formation of S. mutans biofilms (Kang et al.,
2008; Sendamangalam et al., 2011; Shao et al., 2015). Methyl gallate is the
methyl ester of gallic acid. It has antibacterial and antibiofilm effects on S.
mutans. (Kang et al., 2008; Kacergius et al., 2017). In addition, MG inhibits the
acidogenicity of S. mutans (Kacergius et al., 2017).
Carvacrol and thymol are natural monoterpene phenols and can be found in the
plant Origanum vulgare L. Carvacrol and thymol IC50 values against S. mutans
were 65 and 54 µg/ml, respectively. The mechanism of action may be their
involvement in the permeabilization and depolarization of the cytoplasmic
membrane. Deformed and lysed cells as observed under scanning electron
microscope further confirm that thymol and carvacrol treatment results in the
lysis of the cells (Khan et al., 2017).
Other natural products from plants include curcumin (Hu et al., 2013a; Hu et al.,
2013b), α-mangostin (Nguyen et al., 2011; Nguyen et al., 2014) and trans-trans
farnesol (Koo et al., 2003; Jeon et al., 2011). Curcumin is an inhibitor of S.
aureus and S. mutans SrtA activity (Hu et al., 2013a) and it inhibits S. mutans
biofilm formation (Hu et al., 2013b). Curcumin exerts a strong inhibitory activity

against SrtA with a half maximal inhibitory concentration (IC50) of 10.2±0.7 μM.
α-mangostin is a xanthone purified from ethanolic extracts of the tropical plant
Garcinia mangostana L. α-Mangostin is a potent inhibitor of acid production by S.
mutans UA159. Studies on the mechanism of action suggest that α-mangostin is
a multitarget inhibitor. α-Mangostin is active against membrane enzymes,
including the F(H+)-ATPase and the phosphoenolpyruvate - sugar
phosphotransferase system. It also inhibits the glycolytic enzymes aldolase,
glyceraldehyde-3-phosphate dehydrogenase, and lactic dehydrogenase.
Glycolysis in intact cells in suspensions or biofilm formation is inhibited by
α-mangostin at concentrations of 12 and 120 µM, respectively. Other
α-mangostin inhibitory effects include (i) malolactic fermentation, involved in
alkali production from malate, and (ii) NADH oxidase, the major respiratory
enzyme for S. mutans (Nguyen et al., 2011; Nguyen et al., 2014). tt-farnesol is a
bioactive sesquiterpene alcohol commonly found in propolis (a beehive product)
and citrus fruits. It disrupts the ability of S. mutans to form biofilms. In general,
tt-farnesol significantly increases the membrane proton permeability and reduces
the glycolytic activity of S. mutans in the planktonic state and in biofilms. It may
also affect the competitiveness of S. mutans in a mixed-species environment by
primarily disrupting the membrane function and physiology of this bacterium
(Koo et al., 2003; Jeon et al., 2011). In addition, tt-farnesol inhibits acid
production, acid tolerance, and polysaccharide synthesis in S. mutans (Falsetta
et al., 2012).
Crude extracts and essential oils
Plants from traditional medicine, folk medicine, and herbal medicine represent

valuable resources for identifying bioactive compounds. The discovery of
artemisinin as anti-malaria drug from traditional Chinese medicine is one of the
most successful examples (Tu, 2011). Beside pure compounds, crude extracts
and the essential oils from plants are also investigated to identify alternative
antimicrobial agents against S. mutans (Morimoto-Yamashita et al., 2011;
Swadas et al., 2016; Gulube et al., 2016; Yadav et al., 2016; Tofiño-Rivera et al.,
2016; Lall et al., 2017; Janardhanan et al., 2017; Liu et al., 2017; Tardugno et al.,
2018). The constituents are unknown for most extracts and require further
activity-based fractionation to identify the active constituents. It is worthy of
attention that extracts from fruit, seed, fruit peel, plant leaves for tea beverages
and plants used as condiments in cooking exhibit an inhibitory action against S.
mutans growth.
3. Natural products from marine organisms
Marine organisms are another important source of natural products (Molinski et
al., 2009). Many marine natural products possess antibacterial and antibiofilm
activity on a wide range of bacteria. For example, marine sponge-derived natural
product bromoageliferin is a potent and versatile antibiofilm agent. Therefore, it
attracts the interest of researchers in their aim of discovering potential inhibitors
against S. mutants growth from marine natural products (Figure 3). Current
reported marine natural products with antibacterial activity against S. mutans
include oxazole derivatives (Chen et al., 2016), 2-Aminoimidazole (2-AI)
derivatives (Rogers et al., 2008; Liu et al., 2011; Pan et al., 2015; Garcia et al.,
2017), free fatty acids (Huang et al., 2010; Sun et al., 2017) and furanones (He et
al., 2012).

Oxazole derivatives
Oxazole and its natural product-related derivatives, especially those from marine
environment, possess significant biological activities, including anti-tumor,
antibacterial, anti-viral, antimalaria, and anthelmintic activities. Recently, an
oxazole derivative, named 5H6, was identified as a new inhibitor against S.
mutans survival. 5H6 inhibits S. mutans biofilm formation and adhesion without
affecting bacterial growth. In vivo study using a rat caries model showed that 5H6
disrupts the development of smooth-surface caries and sulcal-surface carious
lesions to the same extent as treatment with fluoride at 250 μg/mL. A study on
the mechanism of action suggested that 5H6 prevents the synthesis of
extracellular polysaccharides by antagonizing Gtfs (Chen et al., 2016).
2-AI derivatives
As mentioned above, marine sponge–derived bromoageliferin is a broad
spectrum antibiofilm agent. The 2-AI functional group plays key roles in its
antibiofilm activity. Therefore, many 2-AI derivatives were synthesized to identify
more effective and specific biofilm inhibitors. Encouragingly, numerous 2-AI
derivatives exhibit inhibitory activity against S. mutans biofilm formation (Liu et al.,
2011; Pan et al., 2015; Garcia et al., 2017). For example, 2-AI derivative 3F1
disperses mature S. mutans biofilm. However, 3F1 do not disperse biofilms
formed by the commensal species Streptococcus sanguinis or Streptococcus
gordonii. In addition, 3F1 treatment effectively prevents dental caries by
controlling S. mutans in a rat caries model without perturbing the oral microbiota.
These results suggest the specificity of 3F1 against S. mutans survival. The

mechanism of 3F1 action is not dependent on well-characterized biofilm factors,
including AgI/II, Gtfs, glucans, and eDNA, since they are often associated with
the initial development of S. mutans biofilm, while proteins and factors involved
in biofilm maintenance and maturation are largely unknown. Thus, 3F1 may
represent a target as a novel biofilm-related protein potentially inhibiting a crucial
interaction between S. mutans and the biofilm matrix in vitro (Garcia et al., 2017).
Free fatty acids
Unsaturated fatty acids such as docosahexaenoic acid (DHA) and
eicosapentaenoic acid (EPA) display antibacterial effects on mature S. mutans
biofilm (Huang et al., 2010; Sun et al., 2017). Free fatty acids are found naturally
at high levels in many marine organisms. DHA and EPA possess highly inhibitory
effects on various Gram-positive and Gram-negative bacteria survival. DHA and
EPA mainly affect the integrity of the bacterial plasma membrane, thereby
leading to bacteria damage and death. In addition, DHA or EPA may target
extracellular polysaccharides (EPS) synthesis, since an expression profile
showed the downregulation of gtfB and ftf gene, which encode enzymes that
catalyze the cleavage of sucrose to synthesize extracellular glucan and fructan
polysaccharides, facilitating bacteria adherence and aggregation.
Furanones
Furanones are natural inhibitors of quorum sensing (QS) signaling system, which
regulate biofilm formation in many species. The red marine alga D. pulchra
developed a defense mechanism to protect itself from extensive bacterial
colonization by producing brominated furanones as inhibitors. Synthetic

furanone C-30 inhibits S. mutans biofilm formation. The mechanism of action of
furanones is that these molecules, whose structures are similar to autoinducers
of QS signaling system, can bind to QS response regulators but fail to activate
them, thus inhibiting the QS system (He et al., 2012).
4. Microbial natural products
Microorganisms usually secrete molecules to antagonize competitors. Therefore,
many antibiotics were and are currently isolated from microbial natural products.
Several microbial natural products are inhibitors of S. mutans survival (Figure 3).
These compounds include walkmycin C (Eguchi et al., 2011), signermycin B
(Watanabe et al., 2012), carolacton (Kunze et al., 2010; Reck et al., 2011; Li et
al., 2013; Sudhakar et al., 2014) and vizantin (Takenaka et al., 2016).
The distinguishing feature of these compounds is that they target signaling
system of S. mutans. Walkmycin C inhibited the in vitro autophosphorylation
activity of three purified S. mutans histidine kinases, including VicK, CiaH, and
LiaS. Walkmycin C treatment represses biofilm formation, acid tolerance, and
competence of S. mutans (Eguchi et al., 2011). Signermycin B is identified as an
inhibitor of histidine kinase WalK. It targets the conserved dimerization domain of
WalK to inhibit autophosphorylation (Watanabe et al., 2012).
Carolacton is a secondary metabolite isolated from Sorangium cellulosum. It has
high activity against S. mutans biofilm formation. At a concentration of 10 nM
carolacton, biofilm damage is already at 35% under anaerobic conditions.
Planktonic growth of bacteria is only slightly impaired and no acute cytotoxicity

against mouse fibroblasts is observed (Kunze et al., 2010). A knock-out mutant
for comD, encoding a histidine kinase specific for the competence stimulating
peptide (CSP) is slightly less sensitive to carolacton than the wildtype (Kunze et
al., 2010). Competence related alternate sigma factor ComX expression is
strongly reduced by carolacton. A comparative time series microarray analysis
identified VicKRX and ComDE as two-component signal transduction system
and genes involved in cell wall metabolism, playing essential roles in the
response to carolacton treatment. A sensitivity testing of mutants with deletion of
all the 13 viable histidine kinases and the serine/threonine protein kinase PknB
of S. mutans identified only the ΔpknB deletion mutant as being insensitive to
carolacton treatment. A strong overlap between the regulon of PknB in S. mutans
and the genes affected by carolacton treatment was found. The results
suggested that carolacton acts by interfering with PknB-mediated signaling in
growing cells. The resulting altered cell wall morphology causes membrane
damage and cell death at low pH (Reck et al., 2011). Comparative proteome
analysis identified 192 proteins whose expression is affected by carolacton.
Function analysis of these proteins indicated that carolacton exerts its inhibitory
effects by disturbing peptidoglycan biosynthesis and degradation, thereby
causing damages to the integrity of the cell envelope, leading ultimately to cell
death (Li et al., 2013). A regulatory network analysis identified several regulators
involved in the response of S. mutans to carolacton (Sudhakar et al., 2014). It
was recently found that carolacton targets FolD, a key enzyme from the
folate-dependent C1 metabolism in S. pneumonia (Fu et al., 2017).
Vizantin is a molecule derived from trehalose-6,6′-dimycolate, a major

constituent of the outer surface membrane of the human pathogen
Mycobacterium tuberculosis. Vizantin was originally developed as a safe
immune-stimulating compound (Oda et al., 2014). Recently, vizantin has also
been found to have antibiofilm activity against S. mutans. Notably,
supplementation with sulfated vizantin up to 50 μM do not affect either bacterial
growth or biofilm formation, whereas 50 μM sulfated vizantin causes the
detachment of the biofilm from the surface. Sulfated vizantin at the concentration
of 50 μM upregulates gtfB and gtfC gene expression, but downregulates gtfD
gene expression, suggesting an altered architecture in the biofilm (Takenaka et
al., 2016).
5. Drug repositioning
Drug repositioning is a drug development strategy that tries to find new
applications of existing drugs (Ashburn and Thor, 2004). For example, a recent
study found the anti-cancer activity of the alcohol-abuse drug disulfiram (Skrott et
al., 2017). Drug repositioning strategy has been used to investigate inhibitors
against S. mutans growth in recent years (Figure 4). Several small-scale
drug-repositioning studies identified trimetrexate analogues (Zhang et al., 2015),
saxagliptin (De et al., 2016), zafirlukast (Gerits et al., 2017), clotrimazole,
econazole (Qiu et al., 2017) and reserpine (Zeng et al., 2017) as inhibitors
against S. mutans growth. Particularly, a latest high-throughput
drug-repositioning screening of 853 FDA-approved drugs identified 126 drugs
with inhibitory activity against planktonic S. mutans growth. These drugs were
further tested for activity against S. mutans biofilm and the results showed that
24 drugs inhibit biofilm formation, 6 drugs kill preexisting biofilms, 84 exhibit both

biofilm inhibition and killing activity, and 12 has no activity against biofilms
(Saputo et al., 2017a).
Figure 4. Examples of S. mutans growth inhibitors from drug-repositioning studies.
Trimetrexate analogs
Trimetrexate is a quinazoline derivative and targets human dihydrofolate
reductase (DHFR). A library of analogs based on trimetrexate was screened and
three new analogs that selectively inhibits S. mutans survival were identified. The
most potent inhibitor has an IC50 of 454.0±10.2 nM related to the biofilm
formation and 8.7±1.9 nM associated to DHFR activity in S. mutans. In contrast,
the IC50 of this compound for human DHFR is approximately 1000 nM,
a >100-fold decreased potency, demonstrating the high selectivity of the analog.
An analog that exhibited the least potency against S. mutans biofilm formation
has also the lowest activity towards S. mutans DHFR inhibition, further indicating
that inhibition of biofilms is related to a reduced DHFR activity (Zhang et al.,

2005).

Saxagliptin
Saxagliptin is used in the treatment of type 2 diabetes. It targets human
dipeptidyl peptidase-4 (DPP-4). S. mutans encodes an intracellular Xaa-Pro
dipeptidyl aminopeptidase (XPDAP) which is similar to the human DPP-4
enzyme. XPDAP is a narrow-range serine protease, which cleaves oligopeptides
with a proline as the penultimate residue from the N-terminus. Several studies
have suggested importance of XPDAP in utilization of proline-rich salivary
peptides as well as virulence in S. mutans (De et al., 2016). The inhibition
constants (Ki) of anti-human DPP-4 drugs sitagliptin, vildagliptin, and saxagliptin
against S. mutans XPDAP activity were determined, showing that saxagliptin
inhibits the activity of S. mutans XPDAP (Ki = 129 ± 16 μM) (De et al., 2016).
Zafirlukast
Zafirlukast is used for the chronic treatment of asthma. It targets human
leukotriene receptor antagonist (LTRA). Zafirlukast inhibits S. mutans growth
(MIC: 25 μM), biofilm formation (MBIC: 50 μM) and reduces preformed biofilm
(MBRC: 12.5 μM). In addition, zafirlukast exerts no toxicity on human osteoblasts
(Gerits et al., 2017).
Clotrimazole and Econazole
Dental plaque is not only a multi-species biofilm but also a cross-kingdom biofilm.
For example, C. albicans, a fungal microorganism, is frequently detected with
heavy infection by S. mutans in plaque-biofilms from children with

early-childhood caries. Furthermore, several recent studies suggested the
synergistic effects between S. mutans and C. albicans on biofilm formation
(Hwang et al., 2017; Ellepola et al., 2017). Therefore, compounds with inhibitory
activity against both S. mutans and C. albicans survival may have more effective
anti-caries activity. Based on this hypothesis, inhibitory effects of eight antifungal
drugs on S. mutans survival were determined in a recent study. Two antifungal
drugs, clotrimazole and econazole show an antibacterial activity on S. mutans
UA159 and S. mutans clinical isolates at 12.5 and 25 mg/L, respectively.
Clotrimazole and econazole can also inhibit S. mutans biofilm formation and
reduce the viability of preformed biofilm. In addition, they inhibit pH drop, lactate
acid production and acid tolerance in S. mutans. The antifungal effects of
clotrimazole and econazole are similar. They kill individual fungal cells by altering
the permeability of their cell wall. They bind to phospholipids in the cell
membrane and inhibit the biosynthesis of ergosterol and other sterols required
for cell membrane formation, leading to cell death via loss of intracellular
elements. As regard S. mutans, clotrimazole and econazole inhibit the
expression of S. mutans ldh gene, which is involved in the acid production.
However, their specific targets in S. mutans and their mechanism of action
underlining multiple inhibitory activities on S. mutans are not very clear and
require further analysis. Anyhow, this study provided a starting point in the
development of inhibitors that target two relevant species that contribute in the
formation of dental plaque biofilm (Qiu et al., 2017).
Reserpine
Bacterial efflux pumps are membrane proteins that can extrude substrates out of

the cells, including drugs, antibiotics, chemicals and signal molecules. Efflux
pumps play important roles in bacterial pathogenicity, antibiotic resistance and
biofilm formation. Two predicted ABC efflux pumps encoded by rcrP and rcrQ are
associated with cellular physiology, genetic competence, and acid and oxidative
stress tolerance of S. mutans. Therefore, inhibiting the efflux pump with its
inhibitors may suppress the virulence properties of S. mutans. Inhibitory activities
of several efflux pump inhibitors, including reserpine, verapamil hydrochloride,
thioridazine hydrochloride and sodium orthovanadate against S. mutans growth
were tested. Results indicated that reserpine suppresses acid tolerance, mutacin
production and transformation efficiency in S. mutans, and modifies biofilm
architecture and extracellular polysaccharide distribution. Studies on the
mechanism of action suggested that reserpine inhibits glycosyltransferase
activity in S. mutans. Results from quantitative real-time PCR demonstrated that
reserpine significantly alters QS expression profile and virulence-associated
genes (Zeng et al., 2017)
Doxercalciferol
Bacitracin is a polypeptide-type drug that interferes with cell wall synthesis.
Innate resistance to bacitracin is attributed to efflux by a conserved ABC-type
transporter, which is encoded by the mbrABCD operon in S. mutans.
Doxercalciferol is a vitamin D drug derivative that exhibits direct bactericidal
activity against the cariogenic S. mutans. In addition, through the use of a
combinatorial approach, doxercalciferol was shown to act synergistically with
bacitracin, suggesting that doxercalciferol may act in a bacitracin-related
pathway (Saputo et al., 2017b).

6. Target-based screening
Target-based screening strategy firstly identifies essential proteins involved
in the pathogenicity of bacteria. Secondly, the inhibitory activity of compounds
against target proteins was tested by an enzyme activity experiment or another
protein function experiment. If the three-dimensional structure of the target
protein is available, a structure-based screening is usually performed before the
experimental test. Afterwards, compounds with activity on target proteins are
evaluated by phenotype assays (Terstappen et al., 2007; Swinney and Anthony,
2011; Eder et al., 2014). One advantage of target-based screening is that the
screening can be easily performed in a high-throughput manner. In addition,
target proteins selection provides the opportunity for selecting unique proteins of
pathogens as candidate targets for screening, which is important for the
discovery of inhibitors with high specificity. This is particularly important for
inhibitory screening against S. mutans. The expected ideal inhibitor needs to be
antibacterial to S. mutans but without destroying of oral microbiome. Two
proteins, GtfC (Ren et al., 2015; Ren et al., 2016; Zhang et al., 2017) and QS
signaling protein ComA (Ishii et al., 2017) have been used in target-based
inhibitor screening against S. mutans.
Glucosyltransferase
Biofilm formation in S. mutans is promoted by major virulence factors known as
Gtfs, which synthesize adhesive EPS. A structure-based virtual screening of
approximately 150,000 commercially available compounds was performed
against the crystal structure of the glucosyltransferase domain of the GtfC

protein from S. mutans. Then, 51 chemically diverse lead compounds were
further screened for their inhibitory activities on EPS synthesis in vitro, resulting
in the identification of a quinoxaline derivative as a potential Gtf inhibitor. In vitro
assays showed that this compound is capable of inhibiting EPS synthesis and
biofilm formation in S. mutans by selectively antagonizing Gtfs instead of killing
the bacteria directly. Moreover, the in vivo anti-caries efficacy of the compound
was evaluated in a rat model. The results showed that the compound
significantly reduces the incidence and severity of smooth and sulcal surface
caries in vivo with a concomitant reduction in the percentage of S. mutans in the
animals’ dental plaque (Ren et al., 2016). In another study, a small molecule
library of 500,000 small molecule compounds was screened in silico against the
GtfC catalytic domain. A total of 90 compounds with diverse scaffolds which vary
in their functional groups, hydrophobicity, and H-bond accepting/donating
capacity were then purchased and subjected to in vitro biofilm assays using S.
mutans. Seven potent low micromolar inhibitors were identified. Two of these
compounds were the most potent, as they inhibit more than 85% of S. mutans
biofilms at 12.5 μM. Compounds #G16 and #G43 share several functional
groups including a nitro group, heterocyclic rings, and a polar carbonyl functional
property (Zhang et al., 2017).
S. mutans expresses 3 genetically distinct Gtfs, including GtfB, GtfC, and GtfD.
Recently, it was found that GtfB plays key roles in cross-kingdom biofilm
formation of S. mutans and C. albicans (Hwang et al., 2017; Ellepola et al., 2017).
This property makes GtfB a potential candidate target for a future inhibitor
screening.

QS signaling system protein ComA
The QS system is a bacterial cell-cell signal communication system mediated by
an inherent signal molecule called autoinducer. The ComABCDE pathway is a
QS system in S. mutans. Autoinducer peptides are processed from the precursor
ComC and concomitantly exported to the extracellular space by ComA and
ComB. The accumulated autoinducers bind to the membrane-bound receptor
kinase ComD, which subsequently phosphorylates the response regulator ComE
to activate the transcription of a specific set of genes, such as those essential for
the competence development and biofilm formation in S. mutans. ComA is a
bi-functional ATP-binding cassette (ABC) transporter that comprises three
domains: a N-terminal peptidase domain (PEP), a transmembrane domain, and
a C-terminal nucleotide-binding domain. A high-throughput screening system
using a fluorescence-labeled substrate was performed to identify inhibitors of
PEP in S. mutans. The first screening of 164,514 compounds yielded 951 hits
(0.58%) that inhibits PEP in S. mutans activity by >50% at a compound
concentration of 20 μM. These compounds were further subjected to screening
that examined inhibition against S. mutans biofilm formation. Finally, six
compounds were found to inhibit biofilm formation without inhibiting cell growth.
Two of the six compounds are quinuclidine derivatives, and the other four
compounds have no primary chemical structure in common (Ishii et al., 2017).
7. Antibacterial peptides and proteins
Besides small molecule inhibitors, antibacterial peptides were also investigated
for its activity against S. mutans survival. Compared to small molecule antibiotics,

the advantages of antibacterial peptides include broad-spectrum antimicrobial
activity, low toxicity to mammalian cell and less selection stress. Less selection
stress decreases the risk of antibiotic-resistant bacteria development, which is
one of the biggest challenges in the treatment of bacterial infection. In addition,
antibacterial peptides are encoded by genes and thus they can be easily
modified with genetic engineering techniques to obtain more derivatives
compared to small molecule derivatives obtained by chemical synthesis. Several
antibacterial peptides, such as nisin (Tong et al, 2010; Yamakami et al., 2013),
IMB-2 (Mai et al., 2011), decapeptide KSL (Liu et al., 2011), cyclic lipopeptide 4
(CLP-4) (Min et al., 2017) and lipopeptide C14KKc12K (Meir et al., 2017),
possess antimicrobial activity against S. mutans.
As we mentioned above, antimicrobial peptides are usually broad-spectrum. The
use of antimicrobial peptides in the oral cavity may disturb the oral microbiome
and cause unexpected problems. Therefore, it is better to improve the specificity
of antimicrobial peptides against S. mutans. The development of the
antimicrobial peptide IMB-2 is an enlightening work for this purpose. IMB-2 is
modified from a marine-derived, broad-spectrum antimicrobial peptide
pleurocidin. ComC signaling peptide pheromone (CSP) can bind to S. mutans
ComC. The foundation for creating IMB-2 is based on the addition of a targeting
domain of S. mutans ComC CSP to a killing domain of pleurocidin to generate a
target-specific antimicrobial peptide. The results showed that nearly 95% of S.
mutans bacteria lost their viability following exposure to fusion peptide IMB-2
(5.65 μM) for 15 min. In contrast, only 20% of S. sanguinis or S. gordonii bacteria
were killed following the same exposure. A S. mutans mutant defective in the

CSP receptor resulted in 60% survival following exposure to IMB-2, suggesting
that the targeted peptide predominantly bound to the CSP receptor to mediate
the killing of the wild-type strain (Mai et al., 2011).
The stability of the antimicrobial peptide in oral environment is another potential
problem for antimicrobial peptide inhibitors against S. mutans. Nisin, produced
by Lactococcus lactis, is an antibiotic peptide to effectively antagonize a broad
spectrum of Gram-positive bacteria. Nisin targets the membrane-bound
peptidoglycan precursor lipid II to inhibit peptidoglycan biosynthesis, leading to
membrane pore formation. Nisin displays an antimicrobial activity against
multiple oral microorganisms including S. mutans (Tong et al. 2010). The naked
form of nisin is sensitive to proteolytic degradation and oxidation. To improve its
stability, an encapsulated form of nisin within liposomes has been developed.
The concentration of nisin-liposome required for an efficacious inhibition of
glucan-biofilm synthesis is four times lower than that of naked nisin (Yamakami
et al., 2013).
Recently, two studies also tried to develop protein inhibitors against S. mutans
(Yang et al., 2016; Ito et al., 2017). Bacteriophage-encoded lysins are
peptidoglycan hydrolases that digest the bacterial cell wall, leading to the rapid
lysis and death of bacteria. ClyR, an engineered lysin, is active against all clinical
S. mutans isolates tested and S. mutans biofilms (Yang et al., 2016). Another
protein inhibitor against S. mutans is agaricus bisporus agglutinin (ABA), which
belongs to lectin. The mechanism of action of ABA is largely different from the
one of ClyR. Oral bacteria initiate biofilm formation by adhering to the tooth

surface through an interaction of a lectin-like bacterial protein with carbohydrate
chains on the pellicle. ABA binds to the carbohydrate chains and thus
competitively inhibits the attachment of S. mutans to carbohydrate chains on the
pellicle (Ito et al., 2017).
Conclusion and future perspectives
Dental caries remains a worldwide public health problem. S. mutans is the
primary pathogen in dental caries. Current anti-caries agents face the challenge
of side-effects and drug resistance. Therefore, alternative therapies are expected.
Recently years, many inhibitors against S. mutans were identified by phenotypic
or target-based screening. Most of these inhibitors come from natural products
and their derivatives. These compounds provide an important basis for the
development of new anti-caries therapies in the future. However, several works
also need to be addressed in the future. First, the inhibitory effects of many
compounds were only tested by in vitro assay. Their anti-caries ability in animal
model, influence on the oral microbiome and cytotoxicity need to be further
evaluated. Second, inhibitory mechanisms at the molecular level are largely
unknown for many of these compounds. Target identification of these inhibitors is
required for further understanding their mechanism of action. Last, many of the
currently proposed inhibitors are broad spectrum antimicrobials. Identifying
essential and unique proteins in S. mutans is required for uncovering more
candidate targets for a future target-base screening of inhibitors with high
specificity.

Acknowledgements
This work was supported by the Science, Technology and Innovation
Commission of Shenzhen Municipality (JCYJ20180306171604974), the Natural
Science Basic Research Plan in Shanxi Province of China (2018JQ3025), the
Fundamental Research Funds for the Central Universities (3102016QD022), the
National Natural Science Foundation of China (31300079) and the National Key
Research and Development Program of China (2018YFF01012104)
Disclosure statement
The authors do not have financial or commercial competing interests.
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Curr. Issues Mol. Biol. (2019) 32: 601-644. DOI: https://dx.doi.org/10.21775/cimb.032.

Progress of Antimicrobial Discovery Against

Shenzhen, Shenzhen 518057, Guangdong, P. R. China.

*Corresponding authors: [email protected], [email protected]

health issue. Streptococcus mutans is considered the most significant contributor

to dental caries. Its cariogenicity typically depends on its unique metabolic

formation. Currently used anti-caries therapies, such as fluoride and

chlorhexidine, are characterized by side-effects and drug resistance. Therefore,

the development of alternative inhibitors against S. mutans growth is urgently

derivatives from plants, marine organisms and microorganisms were studied to

evaluate their antibacterial activity against S. mutans. In addition,

caister.com/cimb 601 Curr. Issues Mol. Biol. (2019) Vol. 32

drug-repositioning base screening and target based high-throughput screening

were employed, resulting in inspiring progresses in recent years. In this review,

we summarized the available evidences regarding the inhibition of S. mutans

growth. We focus on the sources, structures and potential mechanism of action

of these inhibitors. Beside small molecular compounds, we also considered

antibacterial peptides and protein inhibitors developed in this field.

the population) have dental caries. Tooth decay is a result of demineralization of

sugars on the surface of the tooth are metabolized by microorganisms, which

carbohydrate metabolism, S. mutans produce lactic acid, leading to a drop of pH

in the environment, which subsequently inhibits the growth of other microbial

competitors, making S. mutans a predominant species in the oral cavity.

accessible, thus, mechanical approaches for removing S. mutans can be

efficient removal is difficult by mechanical method due to the formation of a

caister.com/cimb 602 Curr. Issues Mol. Biol. (2019) Vol. 32

biofilm. Dental plaque biofilm is made by a community of oral microorganisms,

2010; Kolenbrander et al., 2010; Avilés-Reyes et al., 2017).

produced by S. mutans metabolic activity breakdowns the tooth enamel and

causes a pH decline of the surrounding environment, inhibiting the growth of

Baker et al., 2017). Acid production results in a drop of pH in the local

environment. Therefore, S. mutans should be (and it actually is) able to respond

formation. The formation of a biofilm results in multiple benefits for S. mutans.

caister.com/cimb 603 Curr. Issues Mol. Biol. (2019) Vol. 32

or/and neutralization of acids produced in the biofilm by water or saliva.

Furthermore, biofilm is helpful for adsorbing nutrition. Finally, biofilm formation

reduces the access of antibiotics to S. mutans, thus increasing antibiotic

In this review, we summarized the available evidences regarding the inhibition of

S. mutans growth. We focus on the sources, structures and potential

mechanisms of action of these inhibitors. Beside small molecular compounds,

we also considered antibacterial peptides and protein inhibitors developed in this

1. Small molecules currently used in dental cavity treatment

Currently, widely used anti-caries compounds include fluoride, chlorhexidine, and

usually in the form of sodium fluoride or sodium monofluorophosphate (Buzalaf

inhibitory action on S. mutans growth. The glycolytic pathway contributes to the

acid production by S. mutans. Fluoride inhibits the glycolytic pathway by directly

targeting the enolase enzyme (Takahashi et al., 2011). The accumulation of

caister.com/cimb 604 Curr. Issues Mol. Biol. (2019) Vol. 32

internal polysaccharide when sugars are in excess helps S. mutans to respond

to starvation stress, thus contributing to cariogenicity by S. mutans. A recent

study showed that sodium monofluorophosphate inhibits internal polysaccharide

biosynthesis in S. mutans by targeting the S. mutans ADP-glucose

pyrophosphorylase (Demonte et al., 2017). Chlorhexidine is a broad-spectrum

antibacterial agent against S. mutans. Chlorhexidine is positively charged in

anti-caries activity. Xylitol is non-fermentable and cannot be metabolized into

lactic acid by S. mutans, as well as it cannot be used as an energy source

reproduction (Takahashi et al., 2011). Xylitol can promote the remineralization

of the tooth enamel by coordinating with and transporting polyvalent cations,

Current anti-caries compounds mainly contribute to the prevention and reduction

challenges of side-effects and drug resistance. These compounds possess low