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Biofouling: The Journal of Bioadhesion and Biofilm
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The specific degree-of-polymerization of A-type
proanthocyanidin oligomers impacts Streptococcus
mutans glucan-mediated adhesion and transcriptome
responses within biofilmsGuoping Feng
a, Marlise I. Klein
a, Stacy Gregoire
a, Ajay P. Singh
b, Nicholi Vorsa
bc&
Hyun Kooad
aCenter for Oral Biology, University of Rochester Medical Center , Rochester , NY , USA
b
Department of Plant Biology and Plant Pathology , Rutgers University , New Brunswick ,NJ , USAcPhilip E. Marucci Center for Blueberry and Cranberry Research and Extension, Rutgers
University , Chatsworth , NJ , USAdDepartment of Microbiology and Immunology , University of Rochester Medical Center ,
Rochester , NY , USA
Published online: 22 May 2013.
To cite this article:Guoping Feng , Marlise I. Klein , Stacy Gregoire , Ajay P. Singh , Nicholi Vorsa & Hyun Koo (2013) Thespecific degree-of-polymerization of A-type proanthocyanidin oligomers impacts Streptococcus mutans glucan-mediated
adhesion and transcriptome responses within biofilms, Biofouling: The Journal of Bioadhesion and Biofilm Research, 29:6,
629-640, DOI: 10.1080/08927014.2013.794456
To link to this article: http://dx.doi.org/10.1080/08927014.2013.794456
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The specic degree-of-polymerization of A-type proanthocyanidin oligomers impacts
Streptococcus mutans glucan-mediated adhesion and transcriptome responses within bio
lmsGuoping Fenga, Marlise I. Kleina, Stacy Gregoirea, Ajay P. Singhb, Nicholi Vorsab,c and Hyun Kooa,d*
aCenter for Oral Biology, University of Rochester Medical Center, Rochester, NY, USA; bDepartment of Plant Biology and PlantPathology, Rutgers University, New Brunswick, NJ, USA; cPhilip E. Marucci Center for Blueberry and Cranberry Research andExtension, Rutgers University, Chatsworth, NJ, USA;
dDepartment of Microbiology and Immunology, University of Rochester Medical
Center, Rochester, NY, USA
(Received 15 January 2013;nal version received 28 March 2013)
Cranberry A-type proanthocyanidins (PACs) have been recognized for their inhibitory activity against bacterial adhesionand biolm-derived infections. However, the precise identication of the specic classes of degree-of-polymerization(DP) conferring PACs bioactivity remains a major challenge owing to the complex chemistry of these avonoids. In thisstudy, chemically characterized cranberries were used in a multistep separation and structure-determination technique toisolate A-type PAC oligomers of dened DP. The inuences of PACs on the 3D architecture of biolms and
Streptococcus mutans-transcriptome responses within biolms were investigated. Treatment regimens that simulatedtopical exposures experienced clinically (twice-daily, 60 s each) were used over a saliva-coated hydroxyapatite biolmmodel. Biolm accumulation was impaired, while specic genes involved in the adhesion of bacteria, acid stress toler-ance, and glycolysis were affected by the topical treatments (vs the vehicle-control). Genes (rmpC, mepA, sdcBB, andgbpC) associated with sucrose-dependent binding of bacteria were repressed by PACs. PACs of DP 4 and particularlyDP 8 to 13 were the most effective in disrupting bacterial adhesion to glucan-coated apatitic surface (>85% inhibition vsvehicle control), and gene expression (eg rmpC). This study identied putative molecular targets of A-type cranberryPACs in S. mutans while demonstrating that PAC oligomers with a specic DP may be effective in disrupting theassembly of cariogenic biolms.
Keywords:Streptococcus mutans; biolm; cranberry; proanthocyanidin; adhesion
Introduction
Biolms are highly organized and structured microbial
communities enmeshed in an extracellular matrixcomprised mainly of polysaccharides and proteins
(Flemming & Wingender 2010). Biolms are the prevail-
ing microbial lifestyle in natural niches, causing many
diseases in humans including those occurring in the
mouth, such as dental caries (OToole et al. 2000;
Hall-Stoodley et al. 2004). Dental caries is one of the
most prevalent and costly biolm-dependent oral
diseases worldwide (Marsh 2003).
In the mouth, the assembly of cariogenic biolms is a
prime example of the consequences arising from interac-
tions between bacteria (and their products) and dietary
sugars on pellicle-coated tooth surfaces. Within the
complex oral microbiome, Streptococcus mutans (one of
the main species responsible for dental caries) is not
always the most abundant organism. However, it can rap-
idly orchestrate the formation of cariogenic biolms when
sucrose becomes available (Paes Leme et al. 2006; Klein
et al. 2012). S. mutans-derived glucosyltransferases (Gtfs)
are present in the tooth pellicle and on microbial surfaces,
producing exopolysaccharides (EPS) in situ (Schilling &
Bowen 1992; Vacca-Smith & Bowen 1998; Gregoire et al.2011). The EPS formed on surfaces provides binding sites
for bacteria (particularly for S. mutans) promoting local
accumulation of microbes while forming a diffusion-limit-
ing polymeric matrix that protects embedded bacteria
(Stewart & Franklin 2008; Xiao et al. 2012).
In parallel, sucrose (and other sugars) is also
fermented by S. mutans and other acidogenic bacteria
within the EPS-rich matrix, creating acidic microenviron-
ments across the biolm and at the attachment surface
(Xiao et al. 2012). The low pH environment facilitates
EPS production while acid-tolerant and acidogenic ora
prosper within biolms, ensuring continued accumulation
of EPS and acids that promote biolm accretion andlocalized dissolution of the adjacent tooth enamel (Paes
Leme et al. 2006). Once the biolm is established, the
resident bacteria become recalcitrant to antimicrobial
therapies, making them difcult to remove while
facilitating their virulence expression (Lewis 2001).
*Corresponding author. Email: Hyun_Koo@urmc.rochester.edu1
Present address: Department of Food Science, Cornell University, Ithaca, NY 14853, USA.
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Therefore, interventions that target EPS-mediated
bacterial binding and biolm assembly could disrupt the
pathogenesis of dental caries in a highly precise manner.
Cranberries are widely cultivated and consumed, par-
ticularly in the USA. Cranberry fruit is a rich source of
various classes of avonoids including avonols, antho-
cyanins, and proanthocyanidins (PACs; polymeric
avan-3-ols), and offers signicant therapeutic potential (Cote
et al. 2010). Cranberry juice and aqueous extracts have
been recognized for their anti-adhesion and anti-biolm
activity against several bacterial pathogens including the
uropathogenic Escherichia coli and Helicobacter pylori
(Howell et al. 1998; Burger et al. 2000; Liu et al. 2006;
Koo, Duarte, et al. 2010). The extracts also affected the
swarming activity of Pseudomonas aeruginosa (OMay
& Tufenkji 2011). PACs appear to be the main bioactive
avonoid. PACs in cranberry are predominantly found in
oligomeric forms (up to 13 monomeric units) with at
least one A-type double interavan linkage [epicatechin-
(4 8, 2O 7)-epicatechin] (Foo et al. 2000a).This double linkage affords conformational rigidity to
PACs and appears to play a role in their anti-adhesion
bioactivity (Foo et al. 2000b; Howell et al. 2005). A-type
PACs are found in high concentrations uniquely in cran-
berries. A-type PACs appear to be more bioactive than
B-type PACs, which lack the second interavan linkage,
and which are found in other tannin-rich foods (Yanagida
et al. 2000; Foo et al. 2000b; Howell et al. 2005;
Gregoire et al. 2007).
Previous studies have shown that PACs-containing
extract is highly effective in inhibiting the synthesis of
EPS by surface-adsorbed Gtfs, and impairing the accumu-
lation of biolms ofS. mutans on apatitic surfaces (Duarteet al. 2006). Recently, it was reported that topical applica-
tion of an extract containing cranberry PACs reduced the
incidence of dental caries in vivo (Koo, Duarte, et al.
2010). Interestingly, the PACs neither affected bacterial
growth in vitro nor the number of viable populations
in vivo (Duarte et al. 2006; Koo, Duarte, et al. 2010).
Although previous studies have shown the anti-adhesion/
anti-biolm properties of cranberry extracts, the identity of
the bioactive PACs and their molecular targets against S.
mutansremain to be elucidated. The isolation of individual
PAC oligomers from cranberries is challenging due to their
complex chemistry and multiple degrees-of-polymeriza-
tion (DP) with similar mass to charge ratios. Yet, it is a
critical step for further elucidation of the mechanisms of
action and standardization/characterization of this natural
product, both of which are key factors for the successful
development of useful therapies to treat human diseases
(Schmidt et al. 2007).
In this study, a chemically characterized cranberry cul-
tivar which is propagated clonally and consists of a single
genotype was used. A step-wise chromatographic isola-
tion method yielded a highly reproducible PACs-enriched
fraction and individual PACs. It was found that cranberry
PACs modulate the expression of several virulence factors
associated with sucrose-dependent adhesion. The bioac-
tivity of individual PACs on the S. mutans transcriptome
and bacterial adhesion varied depending on their DP. The
data provide insights into the potential therapeutic targets
and the identity of cranberry PACs with high potency,which could be used to design new therapies to be evalu-
ated in vivo.
Materials and methods
Cranberry source and extraction procedures
The fruit source for the PAC oligomers was from the
cranberry cultivar Stevens, which was grown and
harvested from the variety trial at the PE Marucci Center,
Rutgers University, Chatsworth, NJ. Stevens is well-
dened genetically (Fajardo et al. 2012), and is a widely
grown commercial cranberry cultivar with a high avo-
noid content (Vvedenskaya & Vorsa 2004). Cranberrieswere grown under standard (and monitored) nutritional
and cultivation conditions. Plants of the cultivar
Stevens in eld plots were ngerprinted using DNA
SCAR proling to conrm the genotype and to verify
that there was no contamination by genetic off-types
(Polashock & Vorsa 2002).
Isolation and purication of cranberry A-type PACs
oligomers
Fruit was harvested early in the season, prior to fruit
anthocyanin synthesis, and used for the isolation A-type
PACs. The cranberry PACs-containing fraction was
obtained as described previously (Koo, Duarte, et al.2010). The puried fraction contained oligomers (210),
and polymers 11, 12, and 13, and was devoid of any
otheravonoid contaminants (eg avonols and anthocya-
nins). Individual PACs were further isolated and puried
using a method published previously based on HPLC
and diol gravity column chromatography (Singh et al.
2009). The purity of all isolated PAC DP classes was
>95% (w/w) as determined using liquid chromatography-
mass spectrometry (LC-MS-MS) and matrix-assisted
laser desorption/ionization time-of-ight mass spectrome-
try (MALDI-TOF-MS). The treatment concentrations
used for the microarray analysis were 1.5 mg ml1 of the
puried PAC only-fraction, which was selected based on
a previous study (Koo, Duarte, et al. 2010). For compar-
ing the bioactivity of the various PAC molecules of dif-
ferent DP, the treatment concentration was 100M for
DP 2, DP 3, DP 4, DP 56 (combined 1:1 equimolar),
DP 89 (combined 1:1), DP 1011 (combined 1:1), and
DP 1213 (combined 1:1), which was the approximate
amount present in the PAC extract. All compounds were
dissolved in 15% ethanol in 2.5 mM potassium phos-
phate buffer, which was also used as a vehicle control;
630 G. Fenget al.
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treatments with 15% ethanol did not affect the viability
of cells of S. mutans in a biolm when compared to
untreated controls. The pH of the treatment solutions
was maintained at 6.5 0.2. Based on stability studies
(data not shown) the isolated PACs were found to be
stable at pH 6.5, with enhanced solubility.
Preparation and treatment of the biolm
Biolms of S. mutans were formed on saliva coated
hydroxyapatite (sHA) surfaces (12.7 mm in diameter,
1 mm in thickness, Clarkson Chromatography Products
Inc., South Williamsport, PA) as detailed elsewhere
(Koo, Xiao, et al. 2010). S. mutans UA159 (ATCC
700610), a cariogenic oral pathogen whose genome has
been sequenced (Ajdic et al. 2002) was used in this
study. The biolms were grown in ultra-ltered
(10 kDaMW cut-off membrane; Prep/Scale, Millipore,
MA) buffered tryptone-yeast extract broth (UFTYE;
2.5% tryptone and 1.5% yeast extract with the addition
of 4.35 g l1
of potassium phosphate and 1 g l1
ofMgSO47H2O, pH 7.0) with 1% sucrose at 37C and
5% CO2. Briey,S. mutans in exponential growth phase
was inoculated into 2.8 ml of UFTYE + 1% sucrose
containing sHA discs placed vertically in a custom-made
holder. Biolms were allowed to form on sHA discs
without interruption for the rst 20 h. Subsequently, the
biolms were treated twice with each of the test agents
or vehicle control, at 10 am (22 h-old) and 4 pm (28 h-
old), with an additional treatment the following morning
(10 am; 46h-old). The biolms were exposed to the
treatments for 60 s, dip-washed in sterile saline solution
(0.89% w/v NaCl) to remove excess agents, and then
transferred to fresh culture medium (Koo, Duarte, et al.2010). The biolm was analyzed after 44 h using confo-
cal microscopy (Olympus FV 1000, Olympus, Tokyo,
Japan) to examine the effects on the overall 3D architec-
ture after receiving the initial topical treatments. The
gene expression prole was determined 4 h after the last
topical treatment at 46 h (Figure 1), to evaluate the
impact of PACs on S. mutans within the accumulated
biolms post-treatment. The time-points represent the
most active period of the biolm development process in
this model, and were selected based on recent studies on
the dynamics of the S. mutans transcriptome during bio-
lm development and in response to topically applied
agents (Klein et al. 2010; Falsetta et al. 2012).
Confocal laser scanning microscopy of biolms
The overall effect of topical applications of the PACs
fraction on the 3D architecture, and the amount of EPS
and bacteria biomass within intact biolms was assessed
using confocal uorescence imaging (Xiao et al. 2012).
EPS was labeled via incorporation of the Alexa Fluor
647 dextran conjugate (MW 10 kDa; absorbance/uores-
cence emission maxima of 647/668nm), while cells of
S. mutans were stained with SYTO 9 (485/498 nm)
(Molecular Probes Inc., Eugene, OR) as detailed else-
where (Xiao et al. 2012). Imaging was performed using
an Olympus FV 1000 two-photon laser scanning micro-scope (Olympus, Tokyo, Japan) equipped with a 10
(0.45 numerical aperture) water immersion objective
lens. Each biolm was scanned at 5, randomly selected,
positions on the microscope stage and the confocal
image series were generated by optical sectioning at each
of these positions. Three independent experiments were
conducted. The step size of z-series scanning was 2 m.
The confocal images were analyzed using software for
simultaneous visualization and quantication of EPS and
bacterial cells within intact biolms (Koo, Xiao, et al.
2010; Xiao et al. 2012). Amira 5.4.1 software (Visage
Imaging, San Diego, CA) was used to create 3D render-
ings of each structural component (EPS and bacteria) tovisualize the architecture of the biolm. COMSTAT was
used to calculate the biomass and average thickness of
the treated biolms (Heydorn et al. 2000).
Isolation and purication of RNA from the biolm
RNA was extracted and puried using standard protocols
optimized for biolms (Cury & Koo 2007). The RNA
Figure 1. The experimental design for the treatment and analysis of biolms ofS. mutans.
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integrity numbers of puried samples used for microarray
and RT-qPCR were P9 as determined by microcapillary
electrophoresis and Agilent 2100 Bioanalyzer (Agilent
Technologies, Santa Clara, CA) (Schroeder et al. 2006).
Puried RNA samples were stored in RNase-free water at
80 C. The RNA used as a normalization reference for
microarray analysis was isolated from planktonicS. mutans UA159 with the same method.
Microarray experiments: cDNA synthesis, labeling, and
microarray hybridization
Whole genomic proling was conducted using S. mutans
UA159 microarrays (version 3) provided by the J. Craig
Venter Institute (JCVI). Details about the arrays are
available at http://pfgrc.jcvi.org/index.php/microarray/array_
description/Streptococcus_mutans/version3.html . The exper-
imental RNAs from biolms and the reference RNA were
used to generate cDNA according to the protocol provided
by JCVI athttp://pfgrc.jcvi.org/index.php/microarray/pro-
tocols.html. Puried experimental cDNAs were coupledwith indocarbocyanine (Cy3)-dUTP, while reference
cDNA was coupled with indodicarbocyanine (Cy5)-dUTP
(Amersham, GE Healthcare, Little Chalfont, UK). Hybrid-
izations were carried out at 42 C for 17 h with a Maui
hybridization system (Biomicro Systems, Salt Lake City,
UT). The slides were then washed according to JCVI
protocols and scanned using a GenePix 4000B scanner
(Molecular Devices, Sunnyvale, CA) at 532 nm (Cy3
channel) and 635 nm (Cy5 channel).
Microarray data analysis
After the slides were scanned, single-channel images
were loaded into JCVI Spotnder software (http://www.tm4.org/spotnder.html) and overlaid. A spot grid was
created according to JCVI specications and then manu-
ally adjusted to t all spots within the grid. The intensity
values of each spot were measured and saved into .mev
les. Data were normalized using LOWESS and standard
deviation regularization with default settings, followed by
in-slide replicate analysis using the JCVI microarray data
analysis software MIDAS (http://www.tm4.org/midas.
html). Spots that were agged as having either low inten-
sity values or low signal saturation were discarded auto-
matically. The statistical analysis was carried out using
BRB-ArrayTools (version 3.8.1; http://linus.nci.nih.gov/
BRB-ArrayTools.html) with a cutoffp value of 0.001 for
class prediction and of 0.001 and 0.01 for class compari-
son. A total of 4 microarray slides pairs were selected by
BRB for class comparison analysis. MDV (available from
LANL Oralgenhttp://www.oralgen.lanl.gov/) was used to
assign gene names and functional classes to identied
genes, as described previously (Klein et al. 2010). Genes
were then sorted in Microsoft Excel to identify those with
an absolute fold-change P1.6 (up-regulation), or 60.8
(down-regulation) compared to the vehicle control. The
transcriptome data were organized into the following
biological themes: EPS (genes involved in the production
of the biolm matrix), biolm/adhesion (genes involved
in the formation of the biolm that do not play a specic
role in matrix production), glycolytic pathways (genes
with known functions in sugar metabolism), stress (genes
with known functions in the stress response, includingoxidative stress and acid tolerance response), regulators
(genes with known regulatory functions), others (with
known biological functions but which have an unknown
role in the formation of a biolm/infection), and genes
encoding hypothetical proteins (genes lacking an identi-
ed function). Furthermore, the potency of the transcrip-
tional change was accounted for by sorting genes
according to the magnitude of their fold-change (treat-
ment/vehicle). cDNA microarray data have been depos-
ited in the NCBI Gene expression omnibus (GEO)
database (http://www.ncbi.nlm.nih.gov/geo) under GEO
accession number GSE40172.
Reverse transcription quantitative PCR (RT-qPCR)
RT-qPCR was performed to validate and verify further
the expression of specic genes selected from microarray
data analysis. cDNAs were synthesized from 1g of
puried RNA using a BioRad iScript cDNA synthesis
kit (Bio-Rad Laboratories, Inc., Hercules, CA). RNA
samples without reverse transcriptase were included as a
negative control. The resulting cDNA and negative con-
trols were amplied by a MyiQ qPCR detection system
with iQ SYBR Green supermix (Bio-Rad Laboratories,
Inc., CA, USA) and specic primers (Table 1). When
Taqman probes were available, cDNAs and controls were
amplied using a Bio-Rad CFX96 system (Bio-RadLaboratories). The 16S rRNA primers/TaqMan probes
were run separately, and primers/TaqMan probes for
other specic targets were combined and used in a multi-
plex setting. For reactions with only one TaqMan probe
(used for target 16S rRNA), the iQ Supermix (BioRad)
was used. For multiplex reactions (gtfB, gtfC, gtfD and
fruA and, dexA and ftf), the iQ Multiplex Powermix
(BioRad) was employed. Primer and TaqMan probes
sequences can be found in Table 1. Standard curves were
used to determine the relative number of cDNA mole-
cules, which were normalized to the relative number of
16S rRNA cDNA in each sample, as described previ-
ously (Yin et al. 2001). 16S rRNA served as a reference
gene (Klein et al. 2010). These values were used to
determine the fold-change between each treated sample
and the vehicle control. The MIQE guidelines (Bustin
et al. 2009) were followed for quality control of the data
generated and for data analysis.
Glucan-mediated bacterial adherence
The bacterial adherence assay was performed as described
previously (Koo et al. 2006). The HA surfaces were
632 G. Fenget al.
http://pfgrc.jcvi.org/index.php/microarray/array_description/Streptococcus_mutans/version3.htmlhttp://pfgrc.jcvi.org/index.php/microarray/array_description/Streptococcus_mutans/version3.htmlhttp://pfgrc.jcvi.org/index.php/microarray/protocols.htmlhttp://pfgrc.jcvi.org/index.php/microarray/protocols.htmlhttp://www.tm4.org/spotfinder.htmlhttp://www.tm4.org/spotfinder.htmlhttp://www.tm4.org/spotfinder.htmlhttp://www.tm4.org/spotfinder.htmlhttp://www.tm4.org/midas.htmlhttp://www.tm4.org/midas.htmlhttp://linus.nci.nih.gov/BRB-ArrayTools.htmlhttp://linus.nci.nih.gov/BRB-ArrayTools.htmlhttp://www.oralgen.lanl.gov/http://www.ncbi.nlm.nih.gov/geohttp://www.ncbi.nlm.nih.gov/geohttp://www.oralgen.lanl.gov/http://linus.nci.nih.gov/BRB-ArrayTools.htmlhttp://linus.nci.nih.gov/BRB-ArrayTools.htmlhttp://www.tm4.org/midas.htmlhttp://www.tm4.org/midas.htmlhttp://www.tm4.org/spotfinder.htmlhttp://www.tm4.org/spotfinder.htmlhttp://pfgrc.jcvi.org/index.php/microarray/protocols.htmlhttp://pfgrc.jcvi.org/index.php/microarray/protocols.htmlhttp://pfgrc.jcvi.org/index.php/microarray/array_description/Streptococcus_mutans/version3.htmlhttp://pfgrc.jcvi.org/index.php/microarray/array_description/Streptococcus_mutans/version3.html8/13/2019 Pac Cranberri
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coated with claried human saliva (sHA) and glucans
were formed in situ by surface-adsorbed GtfC in the pres-
ence of 100 mM sucrose (Koo et al. 2006) for 4 h. This
process ensures that sHA is completely coated with
glucans (glucan-coated sHA) as determined by scanning
electron and confocal uorescence microscopy. Cells of
S. mutans (1109 cells ml1) were then treated with the
aqueous extract of PAC or PAC molecules of DP 213,
washed and incubated with glucan-coated sHA (Koo et al.
2006). After incubation for 30 min, the beads were washed
and the number of adherent bacteria was determined with
scintillation counts (Schilling & Bowen 1992).
Statistical analyses
Exploratory analysis (EDA test) was performed to assess
the normality of the data sets. For microarray analysis,
pair-wise comparison was performed to test treatment vs
vehicle control. Type I error of p < 0.05 for one-way
analysis of variance was used to reject the null hypothe-
sis for biolm structural quantitation, RT-qPCR, and
adhesion assays. SAS version 9.2 (SAS Institute, Cary
NC) was used for all analyses.
Results and discussion
This study isolated A-type PACs of various DP classes
from a standardized single cranberry genotype, cultivar
Stevens, which has a high content of avonoids
(Vvedenskaya & Vorsa 2004). The Stevens variety has
also been characterized genetically (Polashock & Vorsa
2002; Fajardo et al. 2012) and the major avonoid
classes synthesized during fruit maturation determined
Table 1. Primers and TaqMan probes used for RT-qPCR.
GenBank ID Gene Primer sequence (forward and reverse)TaqMan probe sequence and dual-labeledprobes (reporters and quenchers) References
16S rRNA ACCAGAAAGGGACGGCTAAC CTAACGCAATAAGCACTCCGCCTGG Xiao et al. (2012)TAGCCTTTTACTCCAGACTTTCCTG 5 FAM/3 BHQ-1
SMU.1004 gtfB AAACAACCGAAGCTGATAC ATTGGCTGCATTGCTATCATCA
CAATTTCTTTTACATTGGGAAG 5 HEX/3 BHQ-1SMU.1005 gtfC CTCTGACTGCTACTGATACAAG AGCAACATCTCAACCAACCGCC
CCGAAGTTGTTGTTGGTTTAAC 5 Cal Fluor Red 610/3 BHQ-2SMU.910 gtfD AGCACAAACTTCTGAAGAGC CCTGTGCTTCTTCTGCTTGCTT
CAGCTTTTGCCTGTGTTAAAG 5 Quasar 670/3 BHQ-2SMU.2042c dexA TATTTTAGAGCAGGGCAATCG ACGCCAGTCATCCTCAACCGCA
AACCTCCAATAGCAGCATAAC 5 Quasar 705/3 BHQ-2SMU.78 fruA AACAACTGCTGCTGATACTG TCTGGCTGCTGTCTTCTGTTCT
CTGCGGTTTCTTGAGATGAC 5 FAM/3 BHQ-1SMU.2028c ftf CTGACATAACTACGCCAAAG CGCAATCTTACGAGCCTGTTCTGTT
TGCTTAAATTAATACCAGCTTC 5 HEX/3 BHQ-1
SMU.80 hrcA AATTAGGACTCTTAGAGAAGGC NA This studyAACATCCTGTTCGTCCAA
SMU.270 rmpC TTGGCTTCTTCTTCAATA NA
TAACCATCAAGGTAACTGSMU.148 adhE GCATCATTGGTGAAGATA NA
GTTAGTTGTTGGTACGATSMU.2109 mepA CTAGTGAGTGGACATTAG NA
CTAATAGGTGAGGCTTACSMU.671 citZ CAACGGTAAGTCTATTAGG NA
TAATGGTCGGTATCTTGGSMU.1396 gbpC CTAAGTATGGCGATCATG NA
ATATTGGTTGTCCTTAGCSMU.1933c sdcBB TCCTTGGATAGAATAGATAC NA
AAGGCTGATAAGTATACCSMU.629 sodA ATGACGCACTTGAACCATAT NA
CTGACGAATATCCGCTGG
SMU.562 clpE GCGACTATTCATCTATATGC NA Kajfasz et al. (2009)
GTCCATTTTCAGGATCTG
SMU.940 patB CTTGTCTTTCATCGGTGTTCATAC NA Falsetta et al. (2012)GGTCGCTCGTTTGATTGTTATTAC
Note: NA, not applicable. All oligonucleotides were designed using the Beacon designer program (Premier Biosoft, Palo Alto, CA), and were synthe-sized and supplied by Integrated DNA Technologies (Coralville, IA).
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(Vvedenskaya & Vorsa 2004); enabling the timing of the
fruit harvest to maximize the extraction and isolation of
PACs. Such approaches eliminate the confounding
genetic variation that result in the chemical and bioactiv-
ity variation commonly associated with natural products
research, while helping to identify precisely the bioactive
molecules and the putative targets.
Topical applications of PACs affect biolm
accumulation and 3D architecture
The impact of a chemically characterized and puried
PACs-containing fraction on EPS-mediated biolm
assembly was examined. The clinical conditions of typi-
cal exposure of exogenously introduced therapeutic
agents in the mouth were simulated by applying the
extract topically twice daily for brief exposures (60s)
after the initial formation of the biolm (22 h). The typi-
cal architecture of the biolm prior to treatment (22 h) is
shown in Figure S1. [Supplementary material is available
via a multimedia link on the online article webpage.]The biomass and thickness of both the EPS (red) and
adherent bacteria (green) in biolms treated with PACs
were signicantly less than in vehicle-treated biolms
(Table 2), which resulted in defective accumulation of
the biolm and altered the 3D architecture (Figure 2).
Confocal images revealed a marked (albeit not com-
plete) impairment of EPS-matrix development (Figure 2),
which contained approximately 3.5 times less EPS with
reduced thickness vs vehicle-treated biolms (Table 2).
The data agree well with effective inhibition of glucan
synthesis by surface-adsorbed GtfB and GtfC by cran-
berry PACs (Duarte et al. 2006). Such effects can inu-
ence the pattern of bacterial binding and accumulation toa great extent. Glucans produced on the pellicle by sur-
face-adsorbed GtfC promote the initial adhesion of bacte-
ria while the polymers formed by GtfB bound to the
bacterial surface helps further aggregation, forming cohe-
sive microcolonies (Xiao et al. 2012). This sucrose-
dependent mechanism is highly favorable for S. mutans
as this bacterium expresses multiple glucan-binding
proteins (Sato et al. 1997; Lynch et al. 2007).
The uorescence images show that the formation and
further accumulation of new microcolonies (green) were
disrupted following treatment with PACs (vs vehicle
control), probably as a result of a reduction in the
synthesis of EPS in situ. The deletion of both gtfB and
gtfC in S. mutans completely impaired the ability of the
mutant strain to assemble microcolonies even in the
presence of sucrose (Xiao et al. 2012). However, it is
possible that bacterial adhesion to EPS may be impaired
as large areas of EPS were devoid of any attached cellsor cell clusters (Figure 2).
The development of microcolonies enmeshed in the
EPS is critical for the expression of biolm virulence.
These structures act as diffusion-limiting barriers facili-
tating the creation of highly acidic microenvironments at
the attachment surface (Xiao et al. 2012), which may
promote demineralization of the adjacent tooth-enamel.
The few sparsely distributed microcolonies remaining in
the biolms treated with PACs appear to be larger (with
a higher EPS to bacteria ratio) than those in the
vehicle-treated biolms (and vs biolms pre-treated at
22 h; Figure S1), suggesting that microcolony develop-
ment was not completely inhibited. However, it is alsoapparent that there is much less EPS surrounding these
microcolonies, particularly in the outer layers (Figure 2).
Such biolm architecture, with large areas devoid of
microcolonies, would be less capable of maintaining an
acidic pH at the attachment surface (Xiao et al. 2012).
Whether the remaining microcolonies can cause enamel
demineralization awaits further evaluation.
Previous studies have shown that extracts containing
PACs have no effect on the growth and viability of
S. mutans (Koo, Duarte, et al. 2010) and Candida
albicans (Feldman et al. 2012). However, it is possible
that PACs could affect the expression of EPS synthesiz-
ing Gtfs or the EPS-mediated bacterial adhesion factors,which are critical for the assembly of microcolonies.
Gene expression byS. mutans within biolms post
treatment with PACs
Microarrays and multiplex RT-qPCR were used to charac-
terize how topical application of PACs inuences the S.
mutans transcriptome within the biolm. RT-qPCR was
necessary to assess the transcriptional proles of EPS-
associated genes (gtfBCD, ftf, dexA, and fruA), as micro-
arrays have low condence at detecting mRNA levels,
particularly if genes are repressed (Klein et al. 2010). One
Table 2. Biomass and average thickness values of bacterial cells and EPS in PACs-treated biolms.
Treatment
Biomass (m3 m2) Average thickness (m)
Bacteria EPS Bacteria EPS
Vehicle control 28.93 3.61a 68.18 7.92a 98.93 13.3a 101.2 10.53a
PACs 4.31 2.68b 19.88 7.23b 5.89 3.98b 20.82 8.51b
Note: Biolms were treated topically with vehicle control and PACs-containing fraction. Values labeled with a different letter in each column are statis-tically different (p < 0.0001,n = 3).
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Figure 2. Representative 3D rendered images of biolms of S. mutans following topical treatment with the vehicle control (A), andwith the cranberry PACs-containing fraction (B). The EPS channel is in red, bacterial cells are in green. Scale bars = 100m.
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hundred and nineteen genes whose expression was differ-
entially regulated in response to PACs were detected. To
evaluate the data generated from microarray and RT-qPCR
analyses, genes were organized into categories relevant to
the formation of biolms of S. mutans, tness and viru-
lence expression (see Materials and methods).
On balance there were far more repressed thaninduced genes (Figure 3), suggesting that upon exposure
to cranberry PACs, genes associated with many cellular
activities during biolm development were down-regu-
lated rather than up-regulated. The expression levels of
selected genes detected by microarray analysis were vali-
dated by RT-qPCR (Table 3). The microarray data
showed that a large number of genes affected by PACs
have an unknown function related to the formation of
biolm or expression of virulence. It is currently being
investigated whether these genes have any signicant
role in the development of biolms of S. mutans. For
example, patB (SMU.940) expression was highly affected
following treatment with PACs. Recently, a knockoutmutant of patB from S. mutans UA159 has been gener-
ated; initial analysis suggests that the gene deletion does
not cause a defect in growth, but that the formation of
biolm is reduced in the mutant strain with reduced
amounts of EPS (vs the parental strain) (Falsetta et al.
2012). Although further characterization is needed, it
appears that patB may be involved with EPS synthesis
during the formation of a biolm. It is possible thatpatB
may be induced to compensate for the lack of EPS in
the biolms treated with PACs.
PACs affects expression of sucrose-dependent adhesionfactors but not the expression of EPS-related enzymes
The EPS (glucans) produced from sucrose by Gtfs, pres-
ent on tooth pellicle and bacterial surfaces, act as bind-
ing sites (Schilling & Bowen 1992) promoting specic
and enhanced adhesion by S. mutans through membrane-
associated glucan-binding proteins and other co-factors
(Banas & Vickerman 2003). This study examined
whether PACs disrupt the transcription of relevant genes
associated with these sucrose-dependent processes.
The data show that a major group of sucrose-depen-
dent adhesion factors are repressed after treatment with
PACs (Table 3). These factors include gbpC, rmpC,mepA and sdcBB, which play important roles in bacterial
adherence and subsequent formation of biolms of
S. mutans in the presence of sucrose. The gene gbpC
encodes glucan-binding protein C which has been identi-
Table 3. Selected genes from PACs-treated biolms revealed as being differentially expressed (vs vehicle control) by microarrayanalysis.
GenBank ID Gene Functional category Microarray RT-qPCR
SMU.1396 gbpC Biolm/adhesion 0.81 0.83
SMU.270 rmpC Biolm/adhesion 0.68 0.64
SMU.2109 mepA Biolm/adhesion 0.72 0.70
SMU.1933c sdcBB Biolm/adhesion 0.74 0.69
SMU.148 adhE Glycolytic pathway 0.7 0.72
SMU.671 citZ Glycolytic pathway 0.67 0.75
SMU.80 hrcA Stress (acid) 0.76 0.89SMU.629 sodA Stress (oxidative) 1.6 1.56
SMU.562 clpE Stress (acid) 1.75 1.56
SMU.940 patB Other 1.7 2.78
Note: These genes were selected for RT-qPCR validation. Biolms were treated topically with vehicle control and PACs-containing fraction. Valuesmarked with an asterisk () are signicantly different from biolms treated with vehicle control (p < 0.05,n =3).
Figure 3. Functional categories affected by treatment with PACs. Number of repressed (left panel) and induced (right panel) genesofS. mutans in biolms exposed to PACs-containing fraction (n = 4). Genes are grouped into specic functional categories (biologicalthemes) as listed in the center of the graph. Genes whose fold of change in expression was 60.8 orP1.6 (p < 0.05) are included.
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ed as a critical contributor to the development of bio-
lms of S. mutans and the maintenance of 3D biolm
architecture (Sato et al. 1997; Lynch et al. 2007). MepA,
rmpC, and sdcBB were identied as membrane-associ-
ated sucrose-dependent adhesion factors in addition to
the Gbp family (Tao & Tanzer 2002). Insertional inacti-
vation of these genes resulted in a de
ciency in bacterialadhesion in vitro and colonization of tooth surface
in vivo in the presence of sucrose, despite normal Gtf
production and activity. This is the rst report showing
the molecular impact of cranberry PACs on the expres-
sion of biolm-related adhesion factors by S. mutans.
In contrast, this study did not nd signicant
differences in the expression of genes associated with
EPS synthesis (gtfBCD and ftf) and degradation (dexA
and fruA) between the vehicle control and PACs treat-
ments (Figure S2). Therefore, the overall reduction of
EPS synthesis may be due primarily to inhibitory effects
of the PACs directly on the enzymatic activity of GtfB
and GtfC as reported by previous studies (Duarte et al.2006; Gregoire et al. 2007; Koo, Duarte, et al. 2010)
and not the repression of transcription.
The extracellular effects on glucan synthesis com-
bined with transcriptional repression of sucrose-mediated
adhesion provide a fundamental interpretation of previ-
ous data showing that crude extracts of cranberry reduce
bacterial adherence to glucan-coated apatitic surfaces
and formation of biolms without antibacterial activity
(Duarte et al. 2006; Koo et al. 2006; Gregoire et al.
2007; Koo, Duarte, et al. 2010).
PACs affects expression of other relevant genes
associated with the virulence of biolmsAnother group of repressed genes were identied, that
are linked with glycolysis and the acid stress response in
S. mutans (Table 3). The expression of adhE and citZ
was down-regulated by the PACs fraction. The genes
ahdE and citZ encode alcohol-acetaldehyde dehydroge-
nase and citrate synthase, which are important metabo-
lism-related enzymes in the glycolytic pathways. AdhE
catalyzes the reaction of converting acetyl-CoA to etha-
nol and CoA (Kessler et al. 1992). The cit operon
encodes proteins for the citrate pathway and for the syn-
thesis of the glutamate in minimal medium where the
organic nitrogen source is limited (Cvitkovitch et al.
1997). The gene citZ was also found to be a stress-
responsive gene to osmolarity (Chia et al. 2001). The
gene hrcA also appears to be repressed after exposure to
the PACs fraction (albeit it was not conrmed via
RT-qPCR). HrcA is a transcriptional regulator of dnaK
and groE, which encode important chaperones that play
critical roles in helping S. mutans to cope with acidic
stress (Jayaraman et al. 1997; Lemos et al. 2001, 2007).
Conversely, clpEand sodAwere induced by treatment
with the PACs. ClpE belongs to the Clp system which
consists of proteases for degrading or chaperoning incor-
rectly folded proteins under acidic conditions (Kajfasz
et al. 2009). Gene sodA encodes superoxide dismutase
which provides protection against oxidative stress (Nakay-
ama 1992). The up-regulation of these genes may suggest
mobilization of some of the stress response factors in
order for the cells in the bio
lms treated with PACs, whichare less protected by EPS, to cope with environmental
stresses. Collectively, these transcriptional changes may
explain why treatment with PACs reduced (albeit moder-
ately) the acid production and acid tolerance of biolms of
S. mutans(Duarte et al. 2006; Gregoire et al. 2007).
Bioactivity of isolated cranberry PACs with varying DP
The major disruptive effects caused by topical applica-
tions of cranberry PACs fraction appear to be on the
assembly of the EPS-matrix and EPS-mediated processes
involved in the adhesion of S. mutans and the develop-
ment of a biolm. Several sucrose-dependent adhesion
factors were repressed following topical exposure toPACs. However, the cranberry PACs contained in the
fraction are a mixture of molecules with variable DP.
The PACs range in DP from 2 to 13 epicatechin units.
Therefore, the individual bioactivity and the role of the
DP on S. mutans binding to glucan-coated surfaces and
on gene expression in biolms were assessed.
Glucan-mediated bacterial adhesion
The inuence of compounds of varying DP on bacterial
binding to glucan-coated apatitic surfaces was investi-
gated. Specic PAC oligomers with DP >8 were potent
Figure 4. The adhesion of cells of S. mutans to a glucan-coated apatitic surface. Bacterial cells were treated withisolated PAC molecules of DP 213. The percentage binding ofbacteria was calculated relative to vehicle control treated cells(control= 100%). Values with 3 and 4 asterisks are signicantlydifferent from the control at a level ofp < 0.001 andp < 0.0001,respectively (n = 3). All the values among different DPs aresignicantly different from each other (p < 0.05), exceptbetween DP 89 and DP 1213 (p > 0.05).
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inhibitors of the adhesion of S. mutans (as high as 85%
inhibition, Figure 4). Generally, the bioactivity of PACs
increase with higher numbers of epicatechin units in the
molecule, but this relationship is not linear. There is an
increased effectiveness from DP 2 to 4 and DP 5 to DP
13. However, a clear distinction in bioactivity was
observed for molecules of higher DP (DP >8) whencompared to smaller oligomers (DP 2 to DP4) (Figure 4).
The inhibitory activity from highest to lowest effect (vs
vehicle control) was DP 1011 (85.9%, percentage of
inhibition), DP 89 (65.7%), DP 1213 (58.3%), DP 4
(28.7%), and DP 3 (15.7%). None of the isolated PACs
caused bacterial aggregation (as observed under phase-
contrast microscope) and precipitation.
The failure of DP 56 to inhibit adhesion is particu-
larly intriguing, especially considering the overall trend
of increased effectiveness with higher DP. Oligomers can
exist in various conformations as to whether units are
linked by C(4)C(8) vs C(4)C(6) bonds, and the loca-
tion of the double A-type linkage. Additional studiesshall identify the linkages of DP 56 oligomers relative
to those of DP 34 and DP >6 to determine potential
differences in the structure/conformation of these DP
classes, which could be linked with bioactivity.
Gene expression
The genes rmpC and hrcA were selected based on the
microarray analysis, and relevance to sucrose-dependent
biolm formation and aciduricity (Jayaraman et al. 1997;
Tao & Tanzer 2002). The results from RT-qPCR analysis
followed a similar trend of inhibition as observed with
bacterial adhesion assays. There is trend of increasedeffectiveness between DP 2 and 4, and from DP 5 to 13
(albeit mostly incremental) (Figure 5). For the PACs with
a DP ranging from 2 to 4 epicatechin units, the reduction
in the expression of rmpC and hrcA appeared to be
correlated with increasing DP, with DP 4 being the most
effective (Figure 5). The PACs with a DP 5 to 1011 dis-
played similar activities. There was a signicant increase
in repression following treatment with the DP 1213
class. The expression prole of these two genes indicated
that DP 4 and DP 1213 were the most effective cran-
berry PACs in modulating the expression of selected
genes ofS. mutans in biolms. It is also apparent that the
specic isolated PACs are more active than the PACsfraction in disrupting the gene expression of S. mutans
(particularly hrcA). The extract contains a mixture of
active and less-active PACs, which may affect the overall
bioactivity (vs individual compounds). In addition, the
isolated PACs were devoid of any signicant effects on
the viability of cells ofS. mutansin biolms (Figure S3).
Collectively, the data show some evidence of the
structure-activity relationship of cranberry PAC mole-
cules. There is a trend of increased bioactivity with
higher DP (number of epicatechin units), particularly in
glucan-mediated bacterial adhesion. But this relationship
appears to be present in two DP ranges: DP 2 to 4 and
DP 5 to 13. In each range, DP 4 and DP 1213 (or DP
1011 for adhesion assay) were the most effective mole-
cules. PACs are well recognized for their interactions
with proteins (Bennick 2002; Jobstl et al. 2004). Procy-
anidin dimers linked through a C(4)C(8) interavanoid
bond were reported to have greater tannin specic bind-
ing activity than dimers with C(4)C(6) linkage (de
Freitas & Mateus 2001). Besides having the A-type
interavanoid linkage, cranberry trimers were all identi-
ed to have the C(4)C(8) linkage (Foo et al. 2000b).
Larger and more complex polyphenols (DP >8) interact
more strongly and have greater protein-binding activity
than smaller oligomers (Baxter et al. 1997).It is plausible that the oligomers of differing DP in
this study exert their effect by more than one mecha-
nism. The effect on adhesion could be an outcome of the
effect on gene expression as well as the direct interaction
of PACs with glucan-producing enzymes (Gtfs) and with
glucan-binding proteins associated with the surface of
S. mutans (Gbps). The DP 34 classes may exert most
of the activity on gene expression, whereas DP >8
involves direct proteinPAC interactions. It is possible
Figure 5. The expression of S. mutans genes rmpC and hrcAin biolms treated topically with isolated PAC molecules of DP2 to 13. The values marked with 1, 2, 3, and 4 asterisks aresignicantly different from the control at a level of p < 0.05,p < 0.01, p < 0.001, and p < 0.0001, respectively. For rmpC, thevalue for DP 2 is signicantly different from all others at a
level of p < 0.05. The values for DP 3, DP 4, DP 5
6, DP 8
9and DP 1011 are signicantly different from DP 2 and DP1213 (p < 0.05) but not from each other (p > 0.05). The valuefor DP 1213 is signicantly different from all others at a levelof p < 0.05. For hrcA, the value for DP 2 is signicantlydifferent from DP 3, DP 4, and DP 1213 (p < 0.05). The valuefor DP 3 is signicantly different from DP2 to DP 1213(p < 0.05). The value for DP 4 is signicantly different fromDP 2, DP 56, DP 89, DP 1011 and DP 1213 (p < 0.05).The values for DP 56, DP 89, and DP 1011 aresignicantly different from DP 2, DP 4, and DP 1213(p < 0.05) but not from DP 3 or from each other (p > 0.05).
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that the reduced effect on adhesion with the DP 56
class is that it has lesser impact on both of these mecha-
nisms. The exact reason why these compounds have
such a structure-activity relationship is unclear. It could
be a result of the interplay between the DP with other
factors, including the molecular structure itself (eg the
position of A-type double linkage), the location of C(4)
C(6)/C(4)(8) linkages and the size of the molecules,
which may cause strong steric hindrance. How these fac-
tors interact with each other and inuence bioactivity
needs to be investigated in future studies.
Overall, the mechanisms of action by which topical
applications of fractions containing A-type PACs disrupt
the accumulation of biolms are intriguing, as the expres-
sion of several sucrose-dependent bacterial adhesion fac-
tors were repressed while those associated with EPS-
synthesis were unaffected. However, PACs can inhibit
the activity of EPS-producing enzymes extracellularly
(Duarte et al. 2006; Koo, Duarte, et al. 2010). Further-
more, the expression of specic genes associated withacid stress and glycolysis was also repressed. Further
analysis with puried compounds revealed that oligomers
with DP 4 and DP 8 to 13 were the most active in dis-
rupting EPS (glucan)-mediated bacterial adhesion and
gene expression, while dimers and those with DP 56
were generally inactive. The identication of the com-
pounds of highest bioactivity could facilitate the effective
design of anti-biolm therapies based on cranberry PACs,
while providing the basis for additional structure-activity
studies to further elucidate the mechanisms of action of
these promising non-bacteriocidal biolm inhibitors.
AcknowledgmentsThis work was supported by a NIH grant (5R01DE016139).The authors thank the Multiphoton Core Facility of theUniversity of Rochester Medical Center, for their technicalassistance. A.P.S. and N.V. are thankful to Sneha Shah andSusan Butterworth for their help in isolation of PACs.
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