polyphenol-rich extract from propolis reduces the expression and

Research ArticlePolyphenol-Rich Extract from Propolis Reduces the Expressionand Activity of Streptococcus mutansGlucosyltransferases at Subinhibitory Concentrations

Jorge Jesuacutes Veloz12 Nicolaacutes Saavedra1 Marysol Alvear3

Tomaacutes Zambrano1 Leticia Barrientos1 and Luis A Salazar1

1Center of Molecular Biology and Pharmacogenetics Scientific and Technological Bioresource Nucleus (BIOREN)Universidad de La Frontera Avenida Francisco Salazar 01145 4811230 Temuco Chile2Departamento de Ciencias Biologicas y Quımicas Facultad de Ciencias Universidad San Sebastian Campus Los LeonesLota 2465 7510157 Providencia Santiago Chile3Departamento de Ciencias Quımicas y Recursos Naturales Facultad de Ingenierıa y Ciencias Universidad de La FronteraAvenida Francisco Salazar 01145 Temuco Chile

Correspondence should be addressed to Luis A Salazar luissalazarufronteracl

Received 10 December 2015 Accepted 8 March 2016

Academic Editor Kimon A Karatzas

Copyright copy 2016 Jorge Jesus Veloz et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Tooth decay is an infectious disease whose main causative agent identified is Streptococcus mutans (S mutans) Diverse treatmentshave been used to eradicate this microorganism including propolis To date it has been shown that polyphenols from Chileanpropolis inhibit S mutans growth and biofilm formation However the molecular mechanisms underlying this process are unclearIn the present study we assessed the effect of Chilean propolis on the expression and activity of the glycosyltransferases enzymesand their related genes Polyphenol-rich extract from propolis inhibited gene expression of glycosyltransferases (GtfB GtfC andGtfD) and their related regulatory genes for exampleVicKVicR andCcpA Moreover the treatment inhibited glucosyltransferasesactivity measured by the formation of sucrose-derived glucans Additionally an inhibitory effect was observed in the expressionof SpaP involved in sucrose-independent virulence of S mutans In summary our results suggest that Chilean propolis hasa dose-dependent effect on the inhibition of genes involved in S mutans virulence and adherence through the inhibition ofglucosyltransferases showing an anticariogenic potential of polyphenols from propolis beyond S mutans growth inhibition

1 Introduction

Streptococcus mutans (S mutans) is considered to be theprincipal causative agent of human dental caries among awide variety of microorganisms detected in the oral cavity [12] Although the etiology and factors related to dental cariesdevelopment are well known this pathology remains a publichealth issue due to its increased prevalence in recent reports[3] Therefore the search for new therapeutic strategiesholds great interest Polyphenols from different botanicalsources have emerged as promissory therapeutic agents dueto their wide range of biological activities [4] Propolis

is a resinous substance collected by bees (Apis mellifera)from different vegetable species [5] Several pharmacologicalproperties have been described for the extracts of propo-lis such as antidiabetogenic antiatherogenic antimicrobialand antifungal properties which are related mainly to thepolyphenols content of propolis samples [6ndash9] Since chem-ical studies have determined a relationship between propoliscomposition and the region from where propolis is collectedtogether with the plant source of each area [10 11] the bio-logical activities of propolis can vary depending on the sameconditions aforementioned We have previously reportedantimicrobial activity of Chilean propolis for S mutans [7]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 4302706 7 pageshttpdxdoiorg10115520164302706

2 BioMed Research International

and the inhibition of biofilm formation by these microor-ganisms [12] However the underlyingmolecularmechanisminvolved in this inhibitory effect by Chilean propolis remainsunclear

The capacity of S mutans to form a biofilm on the teethsurface is considered to be the most important virulencemechanism related to its cariogenicity which involvessucrose-dependent and sucrose-independent processesGlucosyltransferases (Gtfs) are involved in the sucrose-dependent mechanism allowing S mutans the synthesis ofboth water-soluble and water-insoluble glucans from sucrose[13 14] These sucrose-derived glucans favor bacterial adhe-sion to the tooth enamel by binding to hydroxyapatite miner-als (HA) and enabling the interaction between microorgan-isms Gtfs from S mutans differ on the types of glucans theycan synthetize and the roles performed in biofilm formationGtfB synthetizes insoluble glucans with 120572-13-linkages thatfacilitates cell aggregation in stable biofilms by mediatingthe interaction between S mutans bacteria In contrast GtfDformsmainly soluble glucans with 120572-16-linkages that containan hydrophobic domain allowing the interaction withsalivary proteins in the pellicle GtfC produces both solubleand insoluble glucans showing the highest affinity for HAamong other Gtfs [15] The expression of Gtfs is influencedby environmental factors of the oral cavity for example pHand carbohydrate availability but it is also affected by theexpression of other regulatory genes such as vicRvicK whichbelongs to the vicRKX operon in the S mutans chromosome[16] and the catabolite control proteinA (CcpA) [17] Regard-ing the sucrose-independent mechanism it is less importantthan sucrose-dependent mechanisms but it also participatesin the primary adherence process in biofilm developmentinvolving the interaction between salivary agglutinins and thesurface protein adhesin SpaP (III antigen) on the S mutansbacterial wall which is encoded by the SpaP gene [18 19]

The present study aimed to evaluate the effect of sub-bactericide concentrations of polyphenol-rich extract fromChilean propolis on the expression of glucosyltransferasesand regulatory genes as a possible mechanism underlying thepropolis inhibitory effect on S mutans

2 Materials and Methods

21 Polyphenol-Rich Extract from Propolis Propolis was col-lected in spring of 2008 from La Araucanıa Chile Thesample was crushed in cold 30 grams was dissolved in100mL of ethanol (70) and mixed in constant agitationfor 7 days at room temperature Subsequently polyphenol-rich extract of propolis (PEP) was filtered with Whatman20 and centrifuged at 327 g for 20 minutes at 4∘C Laterthe solvent evaporated in 60∘C for 2 hours in a Rotavaporevaporator (Buchi R-210 Germany) and dissolved for 24 hwith sterile DMSO to obtain 50 ww extract concentratepropolis extract (EP)

22 Determination of Total Phenolic Content To quantifythe total phenolic content of polyphenol-rich extract frompropolis we used the colorimetric Folin-Ciocalteu assay

Briefly 100 120583L of sample (PEP or calibrator solution) wasmixed with 100 120583L of distilled water and 2mL of Folin-Ciocalteu reagent (Merck Germany) The resultant solutionwas gently mixed and incubated during 8 minutes Finally3mL of sodium carbonate 20 (wv) was added in a 25mLflask and incubated for 2 hours The absorbance of thesolution was at measured 760 nm in a microplate readerFor the calibration curve we used standard solutions ofpinocembrin-galangin in a proportion of 2 1 [11] Thusthe results of quantification are expressed in equivalents ofpinocembrin-galangin mixture (120583gmLminus1)

23 Streptococcus mutans Isolation and Culture ConditionsStreptococcus mutans strains were obtained from salivarysamples of children with active tooth decayThe samples werecultured in petri plates with Columbia agar enriched withsucrose (1) in an anaerobic container (Becton DickinsonandCompany NYUSA)Then culture plates were incubatedat 37∘C and 5 of CO

2for 24 hours Finally S mutans

was identified using polymerase chain reaction (PCR) aspreviously described [20]

24 Quantification of Relative Gene Expression in S mutansCultures under Polyphenol-Rich Extract Treatment To eval-uate the activity of the PEP on glucosyltransferases andother virulence-related genes S mutans was cultured intrypticase soy broth and treatments were applied using amacrodilution sensitivity test scheme with PEP concentra-tions ranging between 01 and 16120583gmLminus1Moreover culturesexposed to DMSO and chlorhexidine were used as a vehiclecontrol and positive control respectively S mutans cultureswere incubated during 24 hours in the conditions abovedescribed [21] Total RNA was isolated from cultures usingTRIzol reagent (Ambion USA) according to manufacturerrsquosinstructions Then cDNA was synthesized using a reversetranscriptase reaction starting from 1 120583g of total RNA andusing 200 ng of random primers and 200U of RevertAid M-MuLV Reverse Transcriptase (Promega Madison WI USA)in a final volume of 20120583L Glucosyltransferases B C and Dand VicK VicR SpaP and CcpA genes were amplified usingreal time PCR in a StepOne Real Time PCR System (LifeTechnologies USA)

For real time PCR amplification we used 05120583L of eachforward and reverse primers (200 nM) 125 120583L of Fast SYBRGreen Master Mix (Life Technologies USA) 1 120583L of cDNAsample and RNAse-free water up to complete 25 120583L of finalvolume The amplification program was performed in thefollowing conditions initial denaturation at 95∘C for 10minutes and 40 cycles of 15 seconds at 95∘Cwith an extensionstage at 60∘C for 1 minute Results were analyzed by thecomparative cq method (2minusΔΔCq) [22] using the 16S rRNAof S mutans as reference gene All these experiments werecompleted by triplicate and results were expressed as relativeexpression in arbitrary units The sequence of primers usedfor this analysis was GtfB 51015840-AGC AAT GCA GCC AATCTA CAA AT-31015840 and 51015840-ACG AAC TTT GCC GTT ATTGTC A-31015840 GtfC 51015840-CTC AAC CAA CCG CCA CTG TT-31015840and 51015840-GGT TTA ACG TCA AAA TTA GCT GTA TTA-31015840

BioMed Research International 3

GtfD 51015840-CTT TGGTTCAGACGGTGTTG-31015840 and 51015840-CTGCTT TTGACT TGT TTT CCG-31015840 SpaP 51015840-CAG TACCTGACTTGATAATAACACC-31015840 and 51015840-TCCCTGCAAGAATCA CTC AGA A-31015840 VicR 51015840-CGC AGT GGC TGA GGAAAA TG-31015840 and 51015840-ACC TGT GTG TGT CGC TAA GTGATG-31015840 VicK 51015840-CAC TTT ACG CAT TCG TTT TGC C-31015840and 51015840-CGT TCT TCT TTT TCC TGT TCG GTC-31015840 CcpA51015840-CCG TGAAGCGGGAGTGTCCA-31015840 and 51015840-TGCCAAACC ACG CGC CAC AG-31015840 16S rRNA 51015840-TGG AAA CGATAG CTA ATA CCG CAT A-31015840 and 51015840-TAA TAC AAC GCAGGT CCA TCT ACT A-31015840

25 Estimation of Glucosyltransferases Enzymatic Activity Toobtain a crude extract of glucosyltransferases from culturesthe cells were suspended in a potassium phosphate buffer(20mM) at pH 68 and then centrifuged at 10 000 g at4∘C The enzymes were precipitated with 80 sulfate ofammonium solution The pellet was resuspended in 500 120583Lof buffer potassium phosphate for later ultrafiltration in a10KDa and 3000 MWCO Vivaspin500 (GE Healthcare LifeScience USA) Finally a phenylmethanesulfonyl fluoridesolution PMSF (1mM) and sodium azide at concentration of002 were added [23] For the enzymatic reaction 50 120583L ofcrude glucosyltransferases extract and 50 120583L of sterile sucrose(01M) in potassium phosphate buffer containing PEP inconcentrations ranging from 01 to 16120583gmLminus1 were addedThese solutions were incubated at 37∘C for 6 hours in 96-well microplates After incubation the content of each wellwas transferred to a microtube and centrifuged at 10 000 gduring 10 minutes For estimation of water-soluble glucansthe centrifugated liquid was transferred into a microplateand mixed with 10 120583L of concentrate sulfuric acid and 10 120583Lof phenol Finally the microplates were incubated at 95∘Cand the absorbance was quantified at 490 nm in a microplatereader For water-insoluble glucans assessment the pelletobtained after centrifugation was dissolved in 300 120583L ofsodium hydroxide (1M) and the absorbance was quantifiedin the same conditions [24] These assays were performedby triplicate and results were expressed as means plusmn standarddeviation

26 Statistical Analysis Statistical analyses were performedusing computational software package Prism 5 (Graph PadSoftware Inc San Diego USA) The values for comparisonof multiple means of individual experiments were estimatedby statistical analysis of variance (ANOVA) Significant dif-ferences were considered at 119901 lt 005

3 Results

31 Total Polyphenols Content of Polyphenol-Rich Extract fromPropolis The total phenolics content in PEP was quantifiedin equivalence of pinocembrin-galangin mixture by theFolin-Ciocalteu reaction to obtain the starting concentrationand perform appropriate treatment dilutions for gene expres-sion and glucosyltransferases assays Thus PEP solution wasdetermined to contain a concentration of 137753120583gmLminus1 oftotal polyphenols

32 Effect of Polyphenol-Rich Extract from Propolis on RelativeGene Expression in S mutans Cultures To evaluate the effectof treatment with polyphenols on gene expression of gluco-syltransferases and regulatory genes cultured S mutans wastreated using four noninhibitory concentrations of PEP (0102 04 and 08 120583gmLminus1) and one bactericide concentration(16 120583gmLminus1) as death control Values were selected consider-ing the minimum inhibitory and bactericide concentrationsobtained for the same extract [12] Moreover chlorhexidinewas used as positive control due to its well-known bactericideeffect on S mutans strains As expected chlorhexidine andbactericide concentration of PEP showed a significant reduc-tion on glucosyltransferases or regulatory genes expressionand no effect of vehicle was observed on the expression ofall evaluated genes Moreover all remaining concentrationsof PEP used as treatments inhibited the expression of GtfBGtfC GtfD and SpaP (Figure 1) Similarly the expression ofregulatory genes VicK and CcpA was reduced in S mutansunder PEP treatment at all tested concentrations Howeverthat inhibitory effect was significant only from 02120583gmLminus1for VicR gene (Figure 2)

33 Effect of Polyphenol-Rich Extract from Propolis on For-mation of Water-Soluble and Water-Insoluble Glucans by Smutans Cultures The formation of water-soluble and water-insoluble glucans by S mutans under PEP treatment wasassessed in supernatant and pellet of treated cultures In bothcases glucans formationwas significantly reduced at all testedconcentrations compared with vehicle-treated cells used ascontrol (Figure 3)

4 Discussion

Different studies have been conducted evaluating the effectof polyphenolic compounds with the ability to inhibit Strep-tococcus mutans growth to describe novel therapeutic alter-natives for dental caries treatment These polyphenols havebeen obtained from several botanical resources includingplants [25] fruits [26] green tea [27] and propolis [28 29]The antibacterial effect of Chilean propolis has been previ-ously determined showing minimal inhibitory concentra-tions (MIC) and minimal bactericide concentrations (MBC)of about 09 and 13 120583gmLminus1 respectively Moreover it wasreported that polyphenols from propolis inhibited biofilmformation by S mutanswhen using subinhibitory concentra-tions which probably involves the regulation of physiologicalmechanisms related to Smutans virulence [12] In the presentstudy we evaluated the effect of subinhibitory concentrationsof a polyphenol-rich extract from propolis previously usedto inhibit biofilm growth and formation by S mutans on theexpression of virulence genes and glucan formation by Gtfsenzymes The most important virulence factor of S mutansis its ability to accumulate on dental plaque by formationof biofilm which is mediated by three distinct Gtfs (GtfBGtfC and GtfD) acting by extracellular water-soluble andwater-insoluble glucans synthesis from diet sucrose Teethcolonization occurs by using different pathways in which thebacteria can generate an attachment on the tooth surface by


lowast lowast

lowast lowast lowastlowastRe

lativ

e exp

ress

ion

(AU

)

00

05

10

DM

SO

Con

trol

Chlo

rhex

idin

e 02 04 08 1601

Polyphenols (120583g mLminus1)

(a)

lowast

lowast

lowast

lowast lowast lowast

Relat

ive e

xpre

ssio

n (A

U)

00

05

10

DM

SO

Con

trol

Chlo

rhex

idin

e 02 04 08 1601


(b)

lowast

lowastlowast

lowast

lowast

lowast

Relat

ive e

xpre

ssio

n (A

U)

00

05

10

DM

SO

Con

trol

Chlo

rhex

idin

e 02 04 08 1601


(c)

lowastlowastlowastlowast

lowast

lowast

Relat

ive e

xpre

ssio

n (A

U)

00

05

10

DM

SO

Con

trol

Chlo

rhex

idin

e 02 04 08 1601


(d)

Figure 1 Effect of polyphenol-rich extract from propolis on the expression of genes related with S mutans virulence (a) GtfB (b) GtfC (c)GtfD (d) SpaP Statistical significance was determined by ANOVA and Dunnettrsquos multiple comparison test using nontreated cells as controllowast

119901 lt 005

the antigenic protein SpaP (cPAc or antigen III) togetherwith the development of acidic conditions in the oral cavitythrough Gtfs activities by glucan synthesis allowing mineraltooth dissolution and cellular aggregation in biofilm structure[30 31] After exposition to polyphenols gene expressionof glucosyltransferases showed different levels of repressiondepending on the concentration of polyphenols tested Theexpression of genes encoding GtfB and GtfC showed astronger inhibition in our model being repressed more than40 and 60 respectively at concentrations between 01 andup to 08 120583gmLminus1 with a dose-dependent effect Similarly Smutans cultures exposed to polyphenols showed that GtfDwas inhibited in approximately 40 or more at subinhibitoryconcentrations starting from 02120583gmLminus1 Since these Gtfsare decisive in biofilm formation through the synthesis ofinsoluble glucans in virulent phenotypes its inhibition canresult in reduced biofilm formation which is consistent with

what is previously published for Chilean propolis treatment[12 32 33] Gtfs are regulated at the transcriptional levelin response to environmental conditions such as pH car-bohydrate availability and cell density Moreover S mutansdisplays open reading frames (Orf) for Gtfs regulation theseregulatory factors are associated with two-component regu-latory systems (TCSs) as VicKVicR [34 35] ConcordantlyPEP treatment results in the inhibition of VicK and VicRexpression however the effect was not dose-dependentsuggesting thatGtfs inhibitionmight involve other regulatorymechanisms VicK that corresponds to a membrane-boundsensor histidine kinase (HK) showed a highly decreasedexpression in approximately more than 50 compared tocontrol in gene expression quantification assays HKs com-ponents are bifunctional proteins having both kinase andphosphatase activities VicR belongs to the OmpR family ofRRs it is conformed by a helix-turn-helixDNAbindingmotif


lowast lowast lowast lowastlowast lowastRe

lativ

e exp

ress

ion

(AU

)

00

05

10

DM

SO

Con

trol

Chlo

rhex

idin

e 02 04 08 1601


(a)

lowast

lowast

lowastlowast

lowast

Relat

ive e

xpre

ssio

n (A

U)

00

05

10

DM

SO

Con

trol

Chlo

rhex

idin

e 02 04 08 1601


(b)

lowastlowastlowast

lowast

lowast

lowast

Relat

ive e

xpre

ssio

n (A

U)

00

05

10

DM

SO

Con

trol

Chlo

rhex

idin

e 02 04 08 1601


(c)

Figure 2 Effect of polyphenol-rich extract from propolis on the expression of regulatory genes involved in S mutans virulence mechanisms(a) VicK (b) VicR (c) CcpA Statistical significance was determined by ANOVA and Dunnettrsquos multiple comparison test using nontreatedcells as control lowast119901 lt 005

and it is involved upstream in virulence genes includingGtfB GtfC and GtfD The biochemistry of the VicRVicKTCS is important to understand antimicrobial resistancemechanisms and for the development of new potentialtherapies [36] Carbon Catabolite Repression (CCR) genessuch as CcpA can also regulate S mutans metabolism andadherence on tooth colonization and biofilm formation inte-grating complex regulatory networks that activate or silencesome genes in response to carbon source and availability[37] Gene expression of S mutans showed important CcpAinhibition following PEP treatment reaching under 30 ofexpression in relation to control cells at low concentrationsof PEP in a dose-dependent manner CcpA is critical forgtfB and gtfC expression because CcpA inactivation resultsin a marked decrease in the levels of Gtfs promoter activity[38] suggesting that inhibitory dose-dependent effect of PEPon Gtfs expression might be triggered by its repression

Moreover enzymatic activity of Gtfs estimated by insolubleand soluble glucans formation was also affected by PEP atsubinhibitory concentrations confirming a functional effectof Gtfs inhibition SpaP expression was also reduced by PEPtreatment contributing to biofilm inhibition by a sucrose-independent mechanism and may lead to the anticariogenicaction of polyphenols [18 19]

The polyphenolic compounds contained in Chileanpropolis are diverse and include a variety of flavonoids andphenolic acids [12] Among the principal components foundwithin Chilean propolis pinocembrin stands out for its highcontent [7] This compound has been identified as a promis-ing pharmacological candidate mainly for being describedwith numerous biological activities including antimicrobialanti-inflammatory antioxidant and anticancer effects [39]Since the present study did not include the evaluation ofindividual polyphenols additional studies are required to


Water-soluble glucansWater-insoluble glucans

Glucan synthesis

lowast

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lowastlowast

0

1

2

3

OD490

nm

01 02 04 08 16ControlPolyphenols (120583g mLminus1)

Figure 3 Effect of polyphenol-rich extract from propolis on themetabolic activity of glucosyltransferases Statistical significancewas determined by ANOVA andDunnettrsquos multiple comparison testusing nontreated cells as control lowast119901 lt 005

identify the compound responsible of the modulatory effectsshown by PEP treatment

5 Conclusion

In summary our results suggest that Chilean propolis hasa dose-dependent effect on the inhibition of genes involvedin S mutans virulence and adherence through the inhibitionof glucosyltransferases showing an anticariogenic potentialof polyphenols from propolis beyond S mutans growthinhibition

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

The authors would like to express their gratitude to CON-ICYT and the financial support of DIUFRO Project DI10-0031

References

[1] M Matsumoto-Nakano K Fujita and T Ooshima ldquoCompari-son of glucan-binding proteins in cariogenicity of Streptococcusmutansrdquo Oral Microbiology and Immunology vol 22 no 1 pp30ndash35 2007

[2] J A Aas B J Paster L N Stokes I Olsen and F E DewhirstldquoDefining the normal bacterial flora of the oral cavityrdquo Journalof Clinical Microbiology vol 43 no 11 pp 5721ndash5732 2005

[3] R A Bagramian F Garcia-Godoy and A R Volpe ldquoTheglobal increase in dental caries A pending public health crisisrdquoAmerican Journal of Dentistry vol 22 no 1 pp 3ndash8 2009

[4] D Vauzour A Rodriguez-Mateos G Corona M J Oruna-Concha and J P E Spencer ldquoPolyphenols and human health

prevention of disease andmechanisms of actionrdquoNutrients vol2 no 11 pp 1106ndash1131 2010

[5] S Castaldo and F Capasso ldquoPropolis an old remedy used inmodern medicinerdquo Fitoterapia vol 73 supplement 1 pp S1ndashS62002

[6] J B Daleprane V da Silva Freitas A Pacheco et al ldquoAnti-atherogenic and anti-angiogenic activities of polyphenols frompropolisrdquo Journal of Nutritional Biochemistry vol 23 no 6 pp557ndash566 2012

[7] L Barrientos C L Herrera G Montenegro et al ldquoChemicaland botanical characterization of chilean propolis and biolog-ical activity on cariogenic bacteria Streptococcus mutans andStreptococcus sobrinusrdquo Brazilian Journal of Microbiology vol44 no 2 pp 577ndash585 2013

[8] N Saavedra L Barrientos C L Herrera M Alvear GMontenegro and L A Salazar ldquoEffect of Chilean propolison cariogenic bacteria Lactobacillus fermentumrdquo Ciencia eInvestigacion Agraria vol 38 no 1 pp 117ndash125 2011

[9] H Koo P L Rosalen J A Cury Y K Park M Ikegakiand A Sattler ldquoEffect of Apis mellifera propolis from twoBrazilian regions on caries development in desalivated ratsrdquoCaries Research vol 33 no 5 pp 393ndash400 1999

[10] V Bankova ldquoChemical diversity of propolis and the problem ofstandardizationrdquo Journal of Ethnopharmacology vol 100 no 1-2 pp 114ndash117 2005

[11] M Popova V Bankova D Butovska et al ldquoValidated methodsfor the quantification of biologically active constituents ofpoplar-type propolisrdquo Phytochemical Analysis vol 15 no 4 pp235ndash340 2004

[12] J J Veloz N Saavedra A Lillo M Alvear L Barrientosand L A Salazar ldquoAntibiofilm activity of chilean propolis onStreptococcus mutans is influenced by the year of collectionrdquoBioMed Research International vol 2015 Article ID 291351 6pages 2015

[13] T Ooshima MMatsumura T Hoshino S Kawabata S Sobueand T Fujiwara ldquoContributions of three glucosyltransferasesto sucrose-dependent adherence of Streptococcus mutansrdquoJournal of Dental Research vol 80 no 7 pp 1672ndash1677 2001

[14] Z T Wen and R A Burne ldquoFunctional genomics approach toidentifying genes required for biofilm development by Strepto-coccus mutansrdquo Applied and Environmental Microbiology vol68 no 3 pp 1196ndash1203 2002

[15] W Krzysciak A Jurczak D Koscielniak B Bystrowska and ASkalniak ldquoThe virulence of Streptococcus mutans and the abil-ity to form biofilmsrdquo European Journal of Clinical Microbiologyand Infectious Diseases vol 33 no 4 pp 499ndash515 2014

[16] M D Senadheera B Guggenheim G A Spatafora et al ldquoAVicRK signal transduction system in Streptococcus mutansaffects gtfBCD gbpB and ftf expression biofilm formation andgenetic competence developmentrdquo Journal of Bacteriology vol187 no 12 pp 4064ndash4076 2005

[17] L Zeng S C Choi C G Danko A Siepel M J Stanhopeand R A Burne ldquoGene regulation by CcpA and cataboliterepression explored by RNA-Seq in Streptococcusmutansrdquo PLoSONE vol 8 no 3 Article ID e60465 2013

[18] S-J Ahn S-J Ahn Z T Wen L J Brady and R A BurneldquoCharacteristics of biofilm formation by Streptococcus mutansin the presence of salivardquo Infection and Immunity vol 76 no 9pp 4259ndash4268 2008

[19] L J Brady S E Maddocks M R Larson et al ldquoThe changingfaces of Streptococcus antigen III polypeptide family adhesinsrdquoMolecular Microbiology vol 77 no 2 pp 276ndash286 2010


[20] LA Salazar CVasquez AAlmuna et al ldquoMolecular detectionof cariogenic streptococci in salivardquo International Journal ofMorphology vol 26 no 4 pp 951ndash958 2008

[21] CLSI ldquoMethods for dilution antimicrobial susceptibility testfor bacteria that grow aerobically approved standardmdasheighteditionrdquo CLSI Document M07-A8 Clinical and LaboratoryStandards Institute (CLSI) Wayne Pa USA 2009

[22] T D Schmittgen and K J Livak ldquoAnalyzing real-time PCR databy the comparative CT methodrdquo Nature Protocols vol 3 no 6pp 1101ndash1108 2008

[23] H Koo P L Rosalen J A Cury Y K Park and W HBowen ldquoEffects of compounds found in propolis on Strep-tococcus mutans growth and on glucosyltransferase activityrdquoAntimicrobial Agents andChemotherapy vol 46 no 5 pp 1302ndash1309 2002

[24] B Islam S N Khan I Haque M Alam M Mushfiq and A UKhan ldquoNovel anti-adherence activity ofmulberry leaves inhibi-tion of Streptococcus mutans biofilm by 1-deoxynojirimycin iso-lated fromMorus albardquo Journal of Antimicrobial Chemotherapyvol 62 no 4 pp 751ndash757 2008

[25] J Smullen G A Koutsou H A Foster A Zumbe and D MStorey ldquoThe antibacterial activity of plant extracts containingpolyphenols against Streptococcus mutansrdquoCaries Research vol41 no 5 pp 342ndash349 2007

[26] S Duarte S Gregoire A P Singh et al ldquoInhibitory effectsof cranberry polyphenols on formation and acidogenicity ofStreptococcus mutans biofilmsrdquo FEMSMicrobiology Letters vol257 no 1 pp 50ndash56 2006

[27] W Shumi M A Hossain D-J Park and S Park ldquoInhibitoryeffects of green tea polyphenol epigallocatechin gallate (EGCG)on exopolysaccharide production by Streptococcus mutansundermicrofluidic conditionsrdquo Biochip Journal vol 8 no 3 pp179ndash186 2014

[28] S A Liberio A L A Pereira M J A M Araujo et al ldquoThepotential use of propolis as a cariostatic agent and its actions onmutans group streptococcirdquo Journal of Ethnopharmacology vol125 no 1 pp 1ndash9 2009

[29] S Duarte P L Rosalen M F Hayacibara et al ldquoThe influenceof a novel propolis on mutans streptococci biofilms and cariesdevelopment in ratsrdquo Archives of Oral Biology vol 51 no 1 pp15ndash22 2006

[30] R O Mattos-Graner M H Napimoga K Fukushima M JDuncan and D J Smith ldquoComparative analysis of Gtf isozymeproduction and diversity in isolates of Streptococcus mutanswith different biofilm growth phenotypesrdquo Journal of ClinicalMicrobiology vol 42 no 10 pp 4586ndash4592 2004

[31] Y Terao R Isoda J Murakami S Hamada and S KawabataldquoMolecular and biological characterization of gtf regulation-associated genes in Streptococcus mutansrdquo Oral Microbiologyand Immunology vol 24 no 3 pp 211ndash217 2009

[32] S Biswas and I Biswas ldquoRegulation of the glucosyltransferase(gtfBC) operon by CovR in Streptococcus mutansrdquo Journal ofBacteriology vol 188 no 3 pp 988ndash998 2006

[33] P-MChen J-Y Chen and J-S Chia ldquoDifferential regulation ofStreptococcus mutans gtfBCD genes in response to copper ionsrdquoArchives of Microbiology vol 185 no 2 pp 127ndash135 2006

[34] A Yoshida and H K Kuramitsu ldquoMultiple Streptococcusmutans genes are involved in biofilm formationrdquo Applied andEnvironmental Microbiology vol 68 no 12 pp 6283ndash62912002

[35] R N Stipp R B Goncalves J F Hofling D J Smith and RO Mattos-Graner ldquoTranscriptional analysis of gtfB gtfC and

gbpB and their putative response regulators in several isolatesof Streptococcus mutansrdquo Oral Microbiology and Immunologyvol 23 no 6 pp 466ndash473 2008

[36] E Ayala J S Downey L Mashburn-Warren D B SenadheeraD G Cvitkovitch and S D Goodman ldquoA biochemical charac-terization of the DNA binding activity of the response regulatorVicR from streptococcus mutansrdquo PLoS ONE vol 9 no 9Article ID e108027 2014

[37] J Abranches MM Nascimento L Zeng et al ldquoCcpA regulatescentralmetabolism and virulence gene expression in Streptococ-cus mutansrdquo Journal of Bacteriology vol 190 no 7 pp 2340ndash2349 2008

[38] C M Browngardt Z T Wen and R A Burne ldquoRegM isrequired for optimal fructosyltransferase and glucosyltrans-ferase gene expression in Streptococcus mutansrdquo FEMS Micro-biology Letters vol 240 no 1 pp 75ndash79 2004

[39] A Rasul F M Millimouno W Ali Eltayb M Ali J Li andX Li ldquoPinocembrin a novel natural compound with versatilepharmacological and biological activitiesrdquo BioMed ResearchInternational vol 2013 Article ID 379850 9 pages 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

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and the inhibition of biofilm formation by these microor-ganisms [12] However the underlyingmolecularmechanisminvolved in this inhibitory effect by Chilean propolis remainsunclear

The capacity of S mutans to form a biofilm on the teethsurface is considered to be the most important virulencemechanism related to its cariogenicity which involvessucrose-dependent and sucrose-independent processesGlucosyltransferases (Gtfs) are involved in the sucrose-dependent mechanism allowing S mutans the synthesis ofboth water-soluble and water-insoluble glucans from sucrose[13 14] These sucrose-derived glucans favor bacterial adhe-sion to the tooth enamel by binding to hydroxyapatite miner-als (HA) and enabling the interaction between microorgan-isms Gtfs from S mutans differ on the types of glucans theycan synthetize and the roles performed in biofilm formationGtfB synthetizes insoluble glucans with 120572-13-linkages thatfacilitates cell aggregation in stable biofilms by mediatingthe interaction between S mutans bacteria In contrast GtfDformsmainly soluble glucans with 120572-16-linkages that containan hydrophobic domain allowing the interaction withsalivary proteins in the pellicle GtfC produces both solubleand insoluble glucans showing the highest affinity for HAamong other Gtfs [15] The expression of Gtfs is influencedby environmental factors of the oral cavity for example pHand carbohydrate availability but it is also affected by theexpression of other regulatory genes such as vicRvicK whichbelongs to the vicRKX operon in the S mutans chromosome[16] and the catabolite control proteinA (CcpA) [17] Regard-ing the sucrose-independent mechanism it is less importantthan sucrose-dependent mechanisms but it also participatesin the primary adherence process in biofilm developmentinvolving the interaction between salivary agglutinins and thesurface protein adhesin SpaP (III antigen) on the S mutansbacterial wall which is encoded by the SpaP gene [18 19]

The present study aimed to evaluate the effect of sub-bactericide concentrations of polyphenol-rich extract fromChilean propolis on the expression of glucosyltransferasesand regulatory genes as a possible mechanism underlying thepropolis inhibitory effect on S mutans

2 Materials and Methods

21 Polyphenol-Rich Extract from Propolis Propolis was col-lected in spring of 2008 from La Araucanıa Chile Thesample was crushed in cold 30 grams was dissolved in100mL of ethanol (70) and mixed in constant agitationfor 7 days at room temperature Subsequently polyphenol-rich extract of propolis (PEP) was filtered with Whatman20 and centrifuged at 327 g for 20 minutes at 4∘C Laterthe solvent evaporated in 60∘C for 2 hours in a Rotavaporevaporator (Buchi R-210 Germany) and dissolved for 24 hwith sterile DMSO to obtain 50 ww extract concentratepropolis extract (EP)

22 Determination of Total Phenolic Content To quantifythe total phenolic content of polyphenol-rich extract frompropolis we used the colorimetric Folin-Ciocalteu assay

Briefly 100 120583L of sample (PEP or calibrator solution) wasmixed with 100 120583L of distilled water and 2mL of Folin-Ciocalteu reagent (Merck Germany) The resultant solutionwas gently mixed and incubated during 8 minutes Finally3mL of sodium carbonate 20 (wv) was added in a 25mLflask and incubated for 2 hours The absorbance of thesolution was at measured 760 nm in a microplate readerFor the calibration curve we used standard solutions ofpinocembrin-galangin in a proportion of 2 1 [11] Thusthe results of quantification are expressed in equivalents ofpinocembrin-galangin mixture (120583gmLminus1)

23 Streptococcus mutans Isolation and Culture ConditionsStreptococcus mutans strains were obtained from salivarysamples of children with active tooth decayThe samples werecultured in petri plates with Columbia agar enriched withsucrose (1) in an anaerobic container (Becton DickinsonandCompany NYUSA)Then culture plates were incubatedat 37∘C and 5 of CO

2for 24 hours Finally S mutans

was identified using polymerase chain reaction (PCR) aspreviously described [20]

24 Quantification of Relative Gene Expression in S mutansCultures under Polyphenol-Rich Extract Treatment To eval-uate the activity of the PEP on glucosyltransferases andother virulence-related genes S mutans was cultured intrypticase soy broth and treatments were applied using amacrodilution sensitivity test scheme with PEP concentra-tions ranging between 01 and 16120583gmLminus1Moreover culturesexposed to DMSO and chlorhexidine were used as a vehiclecontrol and positive control respectively S mutans cultureswere incubated during 24 hours in the conditions abovedescribed [21] Total RNA was isolated from cultures usingTRIzol reagent (Ambion USA) according to manufacturerrsquosinstructions Then cDNA was synthesized using a reversetranscriptase reaction starting from 1 120583g of total RNA andusing 200 ng of random primers and 200U of RevertAid M-MuLV Reverse Transcriptase (Promega Madison WI USA)in a final volume of 20120583L Glucosyltransferases B C and Dand VicK VicR SpaP and CcpA genes were amplified usingreal time PCR in a StepOne Real Time PCR System (LifeTechnologies USA)

For real time PCR amplification we used 05120583L of eachforward and reverse primers (200 nM) 125 120583L of Fast SYBRGreen Master Mix (Life Technologies USA) 1 120583L of cDNAsample and RNAse-free water up to complete 25 120583L of finalvolume The amplification program was performed in thefollowing conditions initial denaturation at 95∘C for 10minutes and 40 cycles of 15 seconds at 95∘Cwith an extensionstage at 60∘C for 1 minute Results were analyzed by thecomparative cq method (2minusΔΔCq) [22] using the 16S rRNAof S mutans as reference gene All these experiments werecompleted by triplicate and results were expressed as relativeexpression in arbitrary units The sequence of primers usedfor this analysis was GtfB 51015840-AGC AAT GCA GCC AATCTA CAA AT-31015840 and 51015840-ACG AAC TTT GCC GTT ATTGTC A-31015840 GtfC 51015840-CTC AAC CAA CCG CCA CTG TT-31015840and 51015840-GGT TTA ACG TCA AAA TTA GCT GTA TTA-31015840





3 Results




4 Discussion



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5 Conclusion


Competing Interests


Acknowledgments


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Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





3 Results




4 Discussion



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5 Conclusion


Competing Interests


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Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


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5 Conclusion


Competing Interests


Acknowledgments


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Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



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5 Conclusion


Competing Interests


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Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Glucan synthesis

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5 Conclusion


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Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

polyphenol-rich extract from propolis reduces the expression and

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