Article

Effects of Sucrose on the Extracellular Matrix of Plaque-Like Biofilm Formed in vivo, Studied by Proteomic Analysis

Piracicaba Dental School, UNICAMP, Piracicaba, Brazil.
Caries Research (Impact Factor: 2.28). 11/2008; 42(6):435-43. DOI: 10.1159/000159607
Source: PubMed
ABSTRACT
Previous studies have shown that sucrose promotes changes in the composition of the extracellular matrix (ECM) of plaque-like biofilm (PLB), but its effect on protein expression has not been studied in vivo. Therefore, the protein compositions of ECM of PLB formed with and without sucrose exposure were analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). For this purpose, a crossover study was conducted during two phases of 14 days each, during which a volunteer wore a palatal appliance containing eight enamel blocks for PLB accumulation. In each phase, a 20% sucrose solution or distilled and deionized water (control) were extraorally dripped onto the blocks 8x/day. On the 14th day, the PLB were collected, the ECM proteins were extracted, separated by two-dimensional gel electrophoresis, digested by in-gel trypsin and MALDI-TOF MS analyzed. In the ECM of PLB formed under sucrose exposure, the following changes compared with the control PLB were observed: (1) the presence of upregulated proteins that may be involved in bacterial response to environmental changes induced by sucrose and (2) the absence of calcium-binding proteins that may partly explain the low inorganic concentration found in ECM of PLB formed under sucrose exposure. The findings showing that sucrose affected the ECM protein composition of PLB in vivo provide further insight into the unique cariogenic properties of this dietary carbohydrate.

Full-text

Available from: Adriana Franco Paes Leme
Fax +41 61 306 12 34
E-Mail karger@karger.ch
www.karger.com
Original Paper
Caries Res 2008;42:435443
DOI: 10.1159/000159607
Effects of Sucrose on the Extracellular
Matrix of Plaque-Like Biofilm Formed
in vivo, Studied by Proteomic Analysis
A.F. Paes Leme
a
C.M. Bellato
b
G. Bedi
c
A.A. Del Bel Cury
a
H. Koo
c
J.A. Cury
a
a
Piracicaba Dental School, UNICAMP, and
b
CENA/University of São Paulo, Piracicaba , Brazil;
c
University of Rochester Medical Center, Rochester, N.Y. , USA
binding proteins that may partly explain the low inorganic
concentration found in ECM of PLB formed under sucrose
exposure. The findings showing that sucrose affected the
ECM protein composition of PLB in vivo provide further in-
sight into the unique cariogenic properties of this dietary
carbohydrate. Copyright © 2008 S. Karger AG, Basel
Sucrose is the most cariogenic carbohydrate because it
is acidogenic and serves as substrate for extracellular
polysaccharide (EPS) synthesis by cariogenic bacteria.
Previous studies have shown that this carbohydrate pro-
motes biochemical (proteins, polysaccharides, ions)
changes in the extracellular matrix (ECM) composition
of plaque-like biofilm (PLB) [Cury et al., 2000]. Also,
these effects are dependent on sucrose concentration
[ Aires et al., 2006], frequency of exposure [Ccahuana-
Vásquez et al., 2007] and time of PLB formation [Vale et
al., 2007]. The changes in the ECM composition of PLB
are strongly associated with the transition from health to
disease, and the carbohydrates are key environmental
factors influencing the ECM composition [Cury et al.,
2000; Ribeiro et al., 2005]. However, the composition of
ECM proteins has not been explored, even though it
could potentially play significant roles in the pathogenic-
ity of PLB [as reviewed by Paes Leme et al., 2006].
Key Words
Dental plaque Gel electrophoresis, two-dimensional
Mass spectrometry Sucrose
Abstract
Previous studies have shown that sucrose promotes changes
in the composition of the extracellular matrix (ECM) of
plaque-like biofilm (PLB), but its effect on protein expres-
sion has not been studied in vivo. Therefore, the protein com-
positions of ECM of PLB formed with and without sucrose
exposure were analyzed by two-dimensional gel electro-
phoresis and matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry (MALDI-TOF MS). For this
purpose, a crossover study was conducted during two phas-
es of 14 days each, during which a volunteer wore a palatal
appliance containing eight enamel blocks for PLB accumula-
tion. In each phase, a 20% sucrose solution or distilled and
deionized water (control) were extraorally dripped onto the
blocks 8 ! /day. On the 14th day, the PLB were collected, the
ECM proteins were extracted, separated by two-dimension-
al gel electrophoresis, digested by in-gel trypsin and MALDI-
TOF MS analyzed. In the ECM of PLB formed under sucrose
exposure, the following changes compared with the control
PLB were observed: (1) the presence of upregulated proteins
that may be involved in bacterial response to environmental
changes induced by sucrose and (2) the absence of calcium-
Received: February 6, 2008
Accepted after revision: August 6, 2008
Published online: October 3, 2008
Prof. Jaime A. Cury
Piracicaba Dental School, PO Box 52
13414-903 Piracicaba, SP (Brazil)
Tel./Fax +55 19 2106 5302
E-Mail jcury@fop.unicamp.br
© 2008 S. Karger AG, Basel
Accessible online at:
www.karger.com/cre
Page 1
Paes Leme /Bellato /Bedi /Del Bel Cury /
Koo
/Cury
Caries Res 2008;42:435–443
436
Indeed, preliminary data have suggested that sucrose
and its glucose and fructose moieties induce different
changes in the ECM protein composition of PLB formed
in situ [Cury et al., 2000], but the proteins were not iden-
tified. Other studies have explored in vitro the cellular
protein composition of Streptococcus mutans to under-
stand the physiological responses of this cariogenic bac-
teria under a variety of environmental conditions [Qui-
vey et al., 2000; Svensäter et al., 2000; Wilkins et al., 2002;
Welin et al., 2003; Len et al., 2004a, b]. However, little is
known about the ECM protein composition of PLBs
formed in experimental conditions that mimic saliva
properties (salivary flow, buffer capacity, clearance, min-
erals and protein content), availability of nutrients as well
as diversity, selection and competition of microbial spe-
cies found in the oral cavity, which are relevant to explain
the biological properties of PLBs formed in vivo.
Therefore, using proteomic approaches, we evaluated
the protein composition of ECM of PLB formed with and
without sucrose exposure, to increase our understanding
of the cariogenic properties of this most consumed sugar.
A preliminary report of major differences in the levels of
calcium binding proteins has previously been reported
[Paes Leme et al., 2006].
Materials and Methods
Experimental Design
This study was approved by the Research and Ethics Commit-
tee of the Piracicaba Dental School (UNICAMP, Piracicaba, SP,
Brazil), and involved a crossover in situ design [Cury et al., 1997]
conducted in two phases of 14 days each. A healthy 26-year-old
volunteer wore an acrylic palatal appliance containing eight hu-
man dental enamel blocks [Hara et al., 2003, for details] and 20%
sucrose solution or distilled and deionized water (control) was
extraorally dripped onto the blocks 8 times a day during each
phase of 14 days ( fig. 1 a, b). The dental PLB formed on the enam-
el blocks were collected ( fig. 1 c) 10 h after the last exposure to
treatment and subjected to protein extraction and analysis. At
least two distinct experiments were performed for each treatment
with samples obtained from the same volunteer.
Extraction of ECM Protein of PLB
Extracellular proteins were extracted according to Cury et al.
[2000]. The PLBs were treated with 50 l of 0.1
N NaOH [Fox and
Dawes, 1970] containing 1 m
M EDTA [Iacono et al., 1982] for each
milligram of PLB wet weight for 1 h at 0
° C. The extracts were
centrifuged (3,000 g ) for 30 min at 4
° C and 3 vol of ice-cold ace-
tone were added to the supernatant to precipitate and concentrate
the proteins extracted. After standing overnight at –20
° C, the
pellets were collected by centrifugation (3,000 g ) at 4
° C for 30 min
and after evaporation of residual acetone the pellets were resus-
pended in 0.125
M Tris-HCl, pH 6.8 plus 0.25 ml of protease in-
hibitor cocktail (Calbiochem) per gram PLB and stored at –20
° C.
Contaminants present in the extract, mainly polysaccharide,
were eliminated using the 2D clean up kit (GE Healthcare) and
protein concentration was determined [Bradford, 1976].
2-Dimensional Electrophoresis and Image Analysis
The first-dimension (isoelectric focusing, IEF) and second-
dimension electrophoresis were performed according to Bellato
et al. [2004] using 20 g of proteins for both experimental condi-
tions. IEF was conducted with Immobiline Dry Strip pH 4–7 (18
cm). Strips were rehydrated for 8 h at 20
° C with 400 l IEF solu-
tion (8
M urea, 4% CHAPS, 70 m M DTT, 0.8% ampholytes and
0.006% bromophenol blue) in an IPGphor system (GE Health-
20% sucrose
or
D.D. water
5 min
8×/day
(14 days)
a
b
c
(15th day)
Day 0 Day 14
1 mm Dental biofilm
Mesh
1 mm
Slab
Wax
Device
Dental biofilm
collection
Extracellular protein
extraction and
proteomic analyses
Fig. 1. Schematic illustration of the experimental design. D.D. = Distilled and deionized.
Color version available online
Page 2
Proteomic Analysis of Dental
Plaque-Like Biofilm
Caries Res 2008;42:435–443
437
care) with current limit 50 A/strip until focusing reached 70
kVh. After focusing, the proteins were reduced and alkylated by
sequential incubation in the following solutions: (1) 20 mg/ml
DTT in 0.05
M Tris-HCl, pH 8.4, 2% SDS, 30% glycerol, 6 M urea,
0.006% bromophenol blue and (2) 30 mg/ml iodoacetamide in the
same buffer. Then, the strips were directly applied to 818% gra-
dient polyacrylamide gels. Molecular-weight markers (Invitro-
gen) covering the 220- to 10-kDa range were applied at the basic
end of the IPG strips. Electrophoresis was carried out for 12 h at
10
° C. Following separation in the second dimension, the proteins
were visualized with silver staining [Blum et al., 1987]. The ex-
periment was repeated twice for each treatment to check repro-
ducibility and all gels were run in duplicate.
Eight gel (two separate experiments with controls and sucrose
treatment in duplicate) images were analyzed (ImageMaster 2D
Platinum software, version 5.0; GE Healthcare) as follows. Ob-
served masses for resolved proteins were calculated by comparing
their mobility with that of the molecular weight markers, and pI
values were calculated according to linearity of the IPG strips us-
ing the software. ImageMaster 2D Platinum detection parame-
ters, such as number of smooth, saliency, and minimum area,
were adjusted for every selected region of each gel to detect pro-
tein spots automatically. Subsequently, each protein spot received
an identification number, which was confirmed visually. Spots
found along the edges of the gels and streaked spots were not con-
sidered for further analysis. For each protein spot, the spot vol-
ume was calculated, according to the software manual, as above
spot border situated at 75% of the spot height (measured from the
peak of the spot), which permitted the automatic subtraction of
background. The percentage volume of each spot was determined
in relation to the total volume of all the spots in a gel. In order to
evaluate the reproducibility between the duplicates and between
independent experiments, the correlation coefficient was calcu-
lated according to the percent volume of paired spots [Huang,
2004]. Subsequently, a master gel image (average gel) was gener-
ated from matched gel sets for each condition by averaging the
volume of the spots. Individual spot volumes were expressed as
normalized volumes relative to the total detected spot volume.
Spots of each master gel of PLB formed in the presence and ab-
sence of sucrose were matched and the percent volume of spots
was compared. The protein spot levels were considered to have a
greater or smaller volume when there was at least a 1.5-fold dif-
ference [Wilkins et al., 2002, 2003].
Protein Digestion and Mass Spectrometry Identification
For identification, protein spots were excised, destained
[Gharahdaghi et al., 1999] and then digested with trypsin [Wil-
kins et al., 2001]. Tryptic peptides were analyzed using matrix-
assisted laser desorption/ionization time-of-flight mass spec-
trometry (MALDI-TOF MS; PerSeptive Biosystem Voyager DE-
STR) operated in the reflection-delayed extraction mode. Mass
lists were used to screen against database including Mascot
(www.matrixscience.com) and Protein Prospector (University
of California; www.prospector.ucsf.edu/ucsfhtml4.0/msfit.htm)
programs using the National Center for Biotechnology Informa-
tion nonredundant database (NCBI nr 2005.01.06). The parame-
ters for Prospector and Mascot searching were: (1) maximum
mass tolerance of 115 ppm, (2) one single miss cleavage, (3) car-
bamidomethylation of cysteines and (4) at least four peptides re-
quired to match. The candidate protein always possessed the
highest MOWSE (molecular weight search) score [Pappin et al.,
1993]. Since this study evaluated proteins in the PLB originating
from human saliva and oral microorganisms, and since the ge-
nome of some microorganisms is not currently undergoing se-
quencing, it was necessary to select some phylogenetically similar
species to perform this analysis [Wilkins et al., 2002; Len et al.,
2003].
R e s u l t s
Preliminary analyses using IPG strips between the pH
ranges 310 and 4.5–5.5 revealed that most of the protein
spots were concentrated within the pH range 47 (data
not shown). Thus, subsequent analyses for the extracel-
lular proteins extracted from the PLBs were evaluated
within the pH range 4–7.
Examination of the original two-dimensional gel elec-
trophoresis protein profiles showed 521.5 8 7.3 and 512.0
8 37.4 (mean 8 SD; n = 2) automatically detected spots
for PLBs formed in the absence (control) and presence of
sucrose, respectively. The image master gels created by
the software showed 445 spots detected in the control and
327 spots in sucrose-treated samples. The reproducibility
of image gels was evaluated by comparing the percent
volume of spots between gel duplicates and between in-
dependent experiments. The correlation coefficient be-
tween duplicates in the absence of sucrose was 0.904–
0.957 and in the presence of sucrose it was 0.8010.855.
For independent experiments, the correlation coefficient
was 0.7010.964 in the absence of sucrose and 0.810
0.940 in the presence of sucrose. Since the main source of
error associated with this form of quantification is bio-
logical variations, the variability in the number and per-
cent volume of spots in this study was minimized by re-
peating the experiment with the same volunteer, and by
running duplicate gels for the control and sucrose treat-
ments.
The comparative analysis based on the percent volume
of spots of the PLB formed in the absence or presence of
sucrose revealed 143 paired spots, of which 78 protein
spots with larger volumes and 19 spots with smaller vol-
umes in PLB formed in the presence of sucrose, while 46
protein spots showed a similar abundance in both condi-
tions ( fig. 2 a, b; tables 1 , 2 ). This comparative analysis
also showed 302 and 184 nonpaired spots in the absence
and presence of sucrose, respectively. The profiles of sal-
ivary and bacterial proteins in the matrix of the PLBs
were qualitatively and quantitatively different depending
on whether or not sucrose was applied during PLB forma-
tion ( tables 1 , 2 ).
Page 3
Paes Leme /Bellato /Bedi /Del Bel Cury /
Koo
/Cury
Caries Res 2008;42:435–443
438
2
7
8
9
14
15
16
11
12
19
24
18
22
20
38
39
45
48
31
29
33
32
46
4
7
kDa
220
90
50
40
30
25
20
15
10
pH
b
pH
kDa
220
90
50
40
30
25
20
15
10
1
3
4
5
6
10
13
25
23
27
26
28
30
35
36
34
37
48
49
40
42
43
50
51
44
54
21
19
53
52
47
41
4
7
a
2
7
8
9
11
16
15
17
20
22
29
32
38
39
4
5
6
10
13
17
28
21
50
35
54
14
Fig. 2. Two-dimensional electrophoresis
pattern of ECM proteins of PLB (20 g)
formed in the absence (
a ) or presence ( b )
of sucrose. ECM proteins were applied to
47 IPG strips followed by electrophoresis
on 8–18% polyacrylamide gel. The gels
were silver-stained. Spots indicated with
numbers were identified by in-gel trypsin
digestion and MALDI-TOF MS [reprint-
ed with permission of Paes Leme et al.,
2006].
Page 4
Proteomic Analysis of Dental
Plaque-Like Biofilm
Caries Res 2008;42:435–443
439
Most of the spots were excised from the gels and sub-
mitted to in-gel trypsin digestion for identification by
mass spectrometry. A total of 54 proteins were identified
from the PLB ( tables 1 , 2 ). Gel image analysis showed that,
in the PLB formed in the presence of sucrose, 17 proteins
were more abundant, 6 were less abundant and 4 proteins
showed similar abundance to the control group ( table 1 ).
Of the proteins identified, 20 were exclusively found in
ECM from the control group and 7 when the PLB was
formed in the presence of sucrose ( table 2 ).
Database search revealed that the proteins which were
identified in the PLB formed in the presence and absence
of sucrose were mainly calcium-binding proteins (spots
34, 36, 37, 42, 43, 49, 51, 52 and 53), proteins related to
binding properties (spots 23, 38, 39, 45, 46, 48, 50 and 54),
proteins associated with stress conditions (spots 2, 4, 5
and 6), proteins related to protein biosynthesis (spots 9,
14, 17 and 28), energy metabolism (spots 7, 8, 10, 11, 12,
20, 29 and 33), amino acid biosynthesis (spots 15 and 16)
and other proteins ( fig. 2 ; tables 1 , 2 ).
A number of isoforms were identified in both condi-
tions and these data indicated that the presence of iso-
forms, in terms of both the number and their abundance,
was altered under the two different conditions.
Table 1. Proteins assigned and their relative abundances in ECM of PLB formed in the presence of sucrose/control (fold change)
Spot
No.
a
Protein assigned Protein information
resource database code
b
Sequence
coverage
c
, %
Fold
change
d
16 Putative NADP-specific glutamate dehydrogenase 24379360
e
/24377287
f
(B) 38 36.33
38 Prolactin-induced protein
e
51094526 (M) 21 6.94
2 DnaK protein
e
15900431 (B) 15 6.67
19 Putative transposase
e
19746041 (B) 17 6.25
9 Translation elongation factor TU
e
15903386 (B) 31 5.40
15 Putative NADP-specific glutamate dehydrogenase 24379360
e
/24377287
f
(B) 29 3.94
20 Phosphotransferase system, mannose-specific EIIAB
e
15902305 (B) 14 3.75
7 Pyruvate kinase
e
42519006 (B) 27 3.63
29 Amino acid ABC transporter, ATP-binding protein
e
15900712 (B) 9 3.50
48 Prolactin-induced protein
e
51094526 (M) 45 3.26
39 Prolactin-induced protein
e
51094526 (M) 32 3.03
8 Pyruvate kinase
e
42519006 (B) 20 2.67
17 Elongation factor Tu 15599473
e
/9950496
f
(B) 20 2.61
22 Methionine synthase II (cobalamin-independent)
e
23002637 (B) 15 2.43
32 Hypothetical protein SMU.373
e
24378870 (B) 23 1.65
10 ATP synthase beta 15600747
e
/9951894
f
(B) 51 1.56
14 Tuf 38606905
e
/38606877
f
(B) 32 1.56
5 GroEL 15599581
e
/576779
f
(B) 25 1.26
28 Translation elongation factor Ts 46164365
e
/9949816
f
(B) 51 1.19
50 Prolactin-induced protein
e
51094526 (M) 36 1.19
4 GroEL 15599581
e
/576779
f
(B) 19 1.18
11 Enolase (isoform 1) 15900994
e
/15900994
f
(B) 21 0.61
21 Transporter, putative
e
34398072 (B) 9 0.58
13 Putative transposase
e
56707134 (B) 15 0.54
35 Putative ribonucleotide reductase (NrdI protein)
e
28896618 (B) 25 0.51
54 Prolactin-induced protein
e
51094526 (M) 45 0.38
6 GroEL 15599581
e
/576779
f
(B) 14 0.16
a
Refers to the proteins indicated in figure 2.
b
Proteins of bacterial (B) or mammalian (M) origin.
c
The percentage of amino acid coverage (peptides observed/theoretical number from sequence data given in database).
d
Fold change means the ratio of % volume of protein spot of PLB formed in the presence of sucrose/control; the expression is con-
sidered enhanced or diminished when the ratio is greater than 1.50- or lower than 0.66-fold, respectively [Wilkins et al., 2002,
2003].
e
Based on Protein Prospector program, the first candidate using a maximum of 115 ppm of mass tolerance.
f
Based on Mascot programs, the first candidate using a maximum of 115 ppm of mass tolerance.
Page 5
Paes Leme /Bellato /Bedi /Del Bel Cury /
Koo
/Cury
Caries Res 2008;42:435–443
440
Discussion
The formation of acid end-products during fermenta-
tion of dietary sugars by acidogenic bacteria is considered
the driving force to generate a cariogenic dental biofilm.
However, the effect of this stressful acidic environmental
condition on protein expression has been limited to in
vitro studies using S. mutans and the analyses have also
focused on cellular protein change. In the present study,
we investigated in vivo the protein change in the ECM of
PLB since sucrose, the most cariogenic dietary carbohy-
drate, promotes decreases in pH and is also a substrate for
EPS synthesis. The ECM has an important role in biofilm
structure and function [Sutherland, 2001].
To evaluate the protein composition of the ECM we
used short alkali extraction at low temperature to avoid
cellular membrane lysis [Fox and Dawes, 1970]. Alkali
extraction of ECM components from PLB formed in the
presence of sucrose is required to dissolve the high con-
centration of insoluble EPS usually found in this biofilm
[Cury et al., 2000], allowing the extraction of substances
that could be trapped in the matrix.
Therefore, the absence of calcium-binding proteins in
the ECM of PLB formed under exposure to sucrose ( ta-
Table 2. Proteins exclusively identified in ECM of PLB formed with and without sucrose exposure
Spot
No.
a
Conditions of PLB formation Protein information
resource database code
b
Sequence
coverage
c
, %
with sucrose without sucrose
12 Enolase (isoform 2)
d
15900994 (B) 14
18 Hypothetical protein Ip_0493
d
28377385 (B) 11
24 Enterotoxin
d
49484068 (B) 22
31 Hypothetical cytosolic protein
d
19704518 (B) 21
33 Phosphoglycerate mutase I
d
23003585 (B) 21
45 Prolactin-induced protein
d
51094526 (M) 22
46 Prolactin-induced protein
d
51094526 (M) 39
1 Lipase precursor
d
49484866 (B) 6
3 Putative type I restriction-modification
system, specificity determinant; restriction
endonuclease
d
24379345 (B) 7
23 ATP-binding protein, putative
d
33390966 (B) 12
25 Hypothetical protein
d
34762127 (B) 17
26 Thioesterase domain containing 1
d
8922871 (M) 19
27 GTPase
d
28378511 (B) 18
30 Hypothetical cytosolic protein
d
50914941 (B) 27
34 Calcium-binding protein 1 isoform 2
d
13929434 (M) 18
36 Calcium-binding protein 1 isoform 2
d
13929434 (M) 18
37 Calcium-binding protein 1 isoform 2
d
13929434 (M) 14
40 Hypothetical protein XP_498283
d
51467140 (M) 38
41 Putative chorismate mutase
d
24379707 (B) 32
42 S100 calcium-binding protein A9
d
4506773 (M) 39
43 S100 calcium-binding protein A9
d
4506773 (M) 48
44 Nucleoside diphosphate kinase 15599002
d
/9949980
e
(B) 56
47 Hypothetical protein SA V0447
d
15923437 (B) 30
49 S100 calcium-binding protein A9
d
4506773 (M) 47
51 S100 calcium-binding protein A9
d
4506773 (M) 42
53 S100 calcium-binding protein A9
d
4506773 (M) 31
52 S100 calcium-binding protein A9
d
4506773 (M) 31
a
Refers to the proteins indicated in figure 2.
b
Proteins of bacterial (B) or mammalian (M) origin.
c
The percentage of amino acid coverage (peptides observed/theoretical number from sequence data given in database).
d
Based on Protein Prospector program, the first candidate using a maximum of 115 ppm of mass tolerance.
e
Based on Mascot program, the first candidate using a maximum of 115 ppm of mass tolerance.
Page 6
Proteomic Analysis of Dental
Plaque-Like Biofilm
Caries Res 2008;42:435–443
441
ble 2 ) should not be due to their being trapped in the in-
soluble EPS. Thus, the presence of calcium-binding pro-
teins, exclusively in ECM of the PLB formed in the ab-
sence of sucrose, supports the view that these proteins, in
addition to plaque Ca binding [Rose et al., 1993], may act
as organic mineral reservoirs in PLB [Paes Leme et al.,
2006, for a review]. However, calcium-binding proteins
may have other functions which have not yet been re-
ported. On the other hand, prolactin-induced protein
was identified in both conditions; however, 2 isoforms
were uniquely found and 3 of them were found in greater
abundance in the matrix of PLB formed with sucrose ( ta-
bles 1 , 2 ).
It should be emphasized that several proteins found in
the ECM of PLB formed in vivo, such as enolase, transla-
tion elongation factors, pyruvate kinase, GroEL, DnaK,
and other proteins previously thought to be confined to
the cytosol are associated with the cell surface or secreted
into the external milieu [Joe et al., 1994; Pancholi and
Fischetti, 1998; Len et al., 2003; Wilkins et al., 2003; Len
et al., 2004a; Black et al., 2004; Nandakumar et al., 2005;
Paddick et al., 2006]. Therefore, the presence of these pro-
teins in the extracellular milieu may not have resulted
from cellular lysis during extraction of the extracellular
proteins.
Overall, our results showed that specific bacterial
stress response proteins are differentially expressed in
ECM of PLBs depending on the availability of sucrose
( tables 1 , 2; fig. 2 a, b). These proteins have been found in-
tracellularly in in vitro studies of S. mutans in response
to environmental changes [Svensäter et al., 2000; Quivey
et al., 2000; Wilkins et al., 2002; Welin et al., 2003; Len et
al., 2004a, b], and in the present study they were found in
the ECM. The extracellular presence of these proteins
may be a consequence of cell death, considering that the
PBL collected was 14 days old.
Proteins linked to carbohydrate metabolism, such as
pyruvate kinase and mannose-specific EIIAB, were up-
regulated in PBL formed under sucrose exposure ( table 1 )
in agreement with an in vitro report [Abranches et al.,
2006]. Enolase was also identified, one of the isoforms
being identified only in the presence of sucrose and the
other being downregulated in the presence of sucrose, but
the biological functions of the differential expression of
the isoforms remain to be clarified. The presence of these
proteins might be an indicator of enhanced carbohydrate
metabolism in the PLB formed in the presence of su-
crose.
The upregulation of ATP synthase beta chain in PLB
formed under sucrose exposure ( table 1 ) could reflect
bacterial adaptation to acidic stress conditions imposed
by eight decreases in pH per day. Even though this en-
zyme was found in ECM, the result agrees with the pre-
dominance of aciduric microorganisms in biofilms
formed in situ [Pecharki et al., 2005; Ribeiro et al., 2005;
Tenuta et al., 2006]. Also, the stress conditions promoted
by low pH induced upregulation of DnaK in the PLB
formed under sucrose exposure ( table 1 ) in agreement
with Jayaraman et al. [1997], who showed increased levels
of dnaK mRNA and intracellular DnaK in S. mutans in a
continuous chemostat culture in response to acid shock.
Furthermore, most proteins involved in the translation
function were also upregulated, such as the translation
elongation factors (EF-Tu). Thus, in addition to their reg-
ular function in translation elongation, these proteins
could behave like chaperones toward protein folding and
protection from stress as previously reported in Esche-
richia coli [Caldas et al., 1998], which could explain their
higher levels under the low pH conditions maybe caused
by sucrose fermentation ( table 1 ).
However, GroEL, another chaperone, behaved differ-
ently from DnaK, since it was downregulated in PLB
formed in the presence of sucrose ( table 1 ). This protein
is elevated in the intracellular compartment of S. mutans
subjected to an acidic environment [Wilkins et al., 2002;
Len et al., 2004a]. Apparently, the different isoforms of
GroEL found may be regulated by different pathways un-
der different stress conditions as in our study; the control
PLB was formed under nutrient-limited conditions, con-
firming a previous report [Len et al., 2003].
Interestingly, NADP-specific glutamate dehydroge-
nase was the protein most highly expressed under sucrose
exposure ( table 1 ). This enzyme is involved in the intra-
cellular metabolism of amino acids [Wilkins et al., 2001,
2002]. The ammonia produced could be an alternative
mechanism used by aciduric bacteria to increase the pH
of acidified cytoplasm. The reason for the extracellular
occurrence of this protein and its role in this lo cation re-
main to be investigated, but the fact that it was 30-fold
more highly expressed in PLB under sucrose exposure
suggests that its presence in the ECM is not an artifact of
cell lysis because the control PLB was extracted under the
same conditions. It can be speculated whether this pro-
tein could act as an adhesin by binding to immobilized
host and bacterial proteins through the glutamate-bind-
ing domain [Joe et al., 1994].
Many bacterial or salivary protein isoforms were iden-
tified in this study. However, neither the role nor the na-
ture of different isoforms, which possibly resulted from
processing by bacterial proteases or posttranslational
Page 7
Paes Leme /Bellato /Bedi /Del Bel Cury /
Koo
/Cury
Caries Res 2008;42:435–443
442
modification, is clear yet. For instance, protein phospha-
tases, which are important in the phosphorylation pro-
cess and signal transduction in other organisms [Mukho-
padyay et al., 1999; Vijay et al., 2000], were previously
found to be more abundant at low pH [Wilkins et al.,
2003] and they could have an effect on the PLB formed
under sucrose exposure.
It should be emphasized that the proteins found in the
PLB could originate from the host and different bacteria;
therefore, it was necessary to include several species such
as Homo sapiens, Actinomyces , Fusobacterium nuclea-
tum , Lactobacillus , Porphyromonas gingivalis , Prevotella
intermedia , Streptococcus anginosus , Streptococcus equi,
Streptococcus gordonii , Streptococcus mitis, S. mutans ,
Streptococcus oralis , Streptococcus pneumoniae , Strepto-
coccus pyogenes , Streptococcus salivarius , Streptococcus
sanguinis , Streptococcus sobrinus , Neisseria subflava ,
Veillonella parvula , and other species, such as Bacillus
subtilis , Staphylococcus aureus , and also Pseudomonas
aeruginosa to improve the protein identification. How-
ever, the fact that the majority of plaque bacteria are not
represented in the sequence databases could explain the
lack of the identification of many spots.
In summary, these results showed that sucrose in-
duced marked changes in ECM protein composition of
PLB formed in vivo. The data provide further insights
into how the biochemical and microbiological changes
induced by sucrose affect the ECM composition at a pro-
teomic level. The composition of bacteria- and host-de-
rived proteins in the ECM may be modulated by the avail-
ability of sucrose, but additional studies should be done
using other dietary carbohydrates to check whether this
effect is unique to this most cariogenic dietary carbohy-
drate. Also, in addition to the ECM composition, the in-
tracellular or total protein composition of PLB formed in
vivo should be evaluated as a control.
A c k n o w l e d g m e n t s
This study was supported by FAPESP (99/07185-7; 02/00293-3;
03/01536-0), CNPq (472392/03-4) and Protein Core Facility grant
NIH RR14682. We thank Dr. Peter Baker of Protein Prospector
and Dr. David Beighton of King’s College London for their assis-
tances with the Prospector program to help identify the proteins,
Dr. Anderson T. Hara from the Indiana University for designing
figure 1 , and Dr. Solange M.T. Serrano for critically reviewing the
manuscript. The manuscript was based on the thesis submit-
ted by the first author to the Faculty of Dentistry of Piracicaba,
UNICAMP, SP, Brazil, as a partial fulfillment of the requirements
of the Doctorate Program in Dentistry, concentration in cariol-
ogy.
References
Abranches J, Candella MM, Wen ZT, Baker HV,
Burne RA: Different roles of EIIABMan and
EIIGlc in regulation of energy metabolism,
biofilm development, and competence in
Streptococcus mutans . J Bacteriol 2006;
188:
3748–3756.
Aires CP, Tabchoury CP, Del Bel Cury AA, Koo
H, Cury JA: Effect of sucrose concentration
on dental biofilm formed in situ and on
enamel demineralization. Caries Res 2006;
40: 28–32.
Bellato CM, Garcia AKM, Mestrinelli F, Tsai
SM, Machado MA, Meinhardt LW: The in-
duction of differentially expressed proteins
of Xylella fastidiosa with citrus extract. Braz
J Microbiol 2004;
35: 235–242.
Black C, Allan I, Ford SK, Wilson M, McNab R:
Biofilm-specific surface properties and pro-
tein expression in oral Streptococcus sanguis .
Arch Oral Biol 2004;
49: 295–304.
Blum H, Beier H, Gross HJ: Improved silver
staining of plant proteins, RNA and DNA in
polyacrylamide gels. Electrophoresis 1987;
8:
93–99.
Bradford MM: A rapid and sensitive method for
the quantitation of microgram quantities of
protein utilizing the principle of protein-dye
binding. Anal Biochem 1976;
72: 248–254.
Caldas TD, Yaagoubi AEI, Richarme G: Chaper-
one properties of bacterial elongation factor
EF-Tu. J Biol Chem 1998;
273: 11478–11482.
Ccahuana-Vásquez RA, Tabchoury CP, Tenuta
LM, Del Bel Cury AA, Vale GC, Cury JA: Ef-
fect of frequency of sucrose exposure on den-
tal biofilm composition and enamel demin-
eralization in the presence of fluoride. Caries
Res 2007;
41: 9–15.
Cury JA, Rebello MAB, Del Bel Cury AA: In situ
relationship between sucrose exposure and
the composition of dental plaque. Caries Res
1997;
31: 356–360.
Cury JA, Rebelo MAB, Del Bel Cury AA, Der-
byshire MTVC, Tabchoury CPM: Biochemi-
cal composition and cariogenicity of dental
plaque formed in the presence of sucrose or
glucose and fructose. Caries Res 2000;
34:
491–497.
Fox DJ, Dawes C: The extraction of protein ma-
trix from human dental plaque. Arch Oral
Biol 1970;
15: 1069–1077.
Gharahdaghi F, Weinberg CR, Meagher DA,
Imai BS, Mische SM: Mass spectrometric
identification of proteins from silver-stained
polyacrylamide gel: a method for the remov-
al of silver ions to enhance sensitivity. Elec-
trophoresis 1999;
20: 601–605.
Hara AT, Queiroz CS, Paes Leme AF, Serra MC,
Cury JA: Caries progression and inhibition
in human and bovine root dentine in situ.
Caries Res 2003;
37: 339–344.
Huang CM: Comparative proteomic analysis of
human whole saliva. Arch Oral Biol 2004;
49:
951–962.
Iacono VJ, Mackay BJ, Pollock JJ, Bolot PR,
Laqqenhein S, Grossbard BL, Rochon ML:
Roles of lysozyme in the host response to
periodontopathic organisms; in Genco RJ,
Mergenhagen SE (eds): Host-Bacterial Inter-
actions in Periodontal Diseases. Washing-
ton, American Society for Microbiology,
1982, pp 318–342.
Jayaraman GC, Penders JE, Burne RA: Tran-
scriptional analysis of the Streptococcus mu-
tans hrc A, grp E and dna K genes and regula-
tion of expression in response to heat shock
and environmental acidification. Mol Mi-
crobiol 1997;
25: 329–341.
Joe A, Murray CS, McBride BC: Nucleotide se-
quence of a Porphyromonas gingivalis gene
encoding a surface-associated glutamate de-
hydrogenase and construction of a gluta-
mate dehydrogenase-deficient isogenic mu-
tant. Infect Immun 1994;
62: 1358–1368.
Page 8
Proteomic Analysis of Dental
Plaque-Like Biofilm
Caries Res 2008;42:435–443
443
Len ACL, Cordwell SJ, Harty DWS, Jacques NA:
Cellular and extracellular proteome analysis
of Streptococcus mutans grown in a chemo-
stat. Proteomics 2003;
3: 627–646.
Len ACL, Harty DWS, Jacques NA: Stress-re-
sponsive proteins are upregulated in Strepto-
coccus mutans during acid tolerance. Micro-
biology 2004a;150:
1339–1351.
Len ACL, Harty DWS, Jacques NA: Proteome
analysis of Streptococcus mutans metabolic
phenotype during acid tolerance. Microbiol-
ogy 2004b;150:
1353–1366.
Mukhopadyay SV, Kapatral V, Xu W, Chakra-
barty AM: Characterization of a Hank’s type
serine/threonine kinases and serine/threo-
nine phosphoprotein phosphatase in Pseu-
domonas aeruginosa . J Bacteriol 1999;
181:
6615–6622.
Nandakumar R, Nandakumar MP, Marten MR,
Ross JM: Proteome analysis of membrane
and cell wall associated proteins from Staph-
ylococcus aureus . J Proteom Res 2005;
4: 250–
257.
Paddick JS, Brailsford SR, Rao S, Soares RF, Kidd
EA, Beighton D, Homer KA: Effect of biofilm
growth on expression of surface proteins of
Actinomyces naeslundii genospecies 2. Appl
Environ Microbiol 2006;
72: 3774–3779.
Paes Leme AF, Koo H, Bellato CM, Bedi G, Cury
JA: The role of sucrose in cariogenic dental
biofilm formation – new insight. J Dent Res
2006;
85: 878–887.
Pancholi V, Fischetti VA: -Enolase, a novel
strong plasmin(ogen) binding protein on the
surface of pathogenic streptococci. J Biol
Chem 1998;
273: 14503–14515.
Pappin DJC, Hojrup P, Bleasby AJ: Rapid identi-
fication of proteins by peptide-mass finger-
printing. Curr Biol 1993;
3: 327–332.
Pecharki GD, Cury JA, Paes Leme AF, Tabchoury
CPM, Del Bel Cury AA, Rosalen PL, Bowen
WH: Effect of sucrose containing iron (II) on
dental biofilm and enamel demineralization
in situ. Caries Res 2005;
39: 123–129.
Quivey RG Jr, Kuhnert WL, Hahn K: Adaptation
of oral streptococci to low pH. Adv Microb
Physiol 2000;
42: 239–274.
Ribeiro CCC, Tabchoury CPM, Del Bel Cury
AA, Tenuta LMA, Rosalen PL, Cury JA: Ef-
fect of starch on the cariogenic potential of
sucrose. Br J Nutr 2005;
94: 44–50.
Rose RK, Dibdin GH, Shellis RP: A quantitative
study of calcium binding and aggregation in
selected oral bacteria. J Dent Res 1993;
72:
78–84.
Sutherland IW: The biofilm matrix – an immo-
bilized but dynamic microbial environment.
Trends Microbiol 2001;
9: 222–227.
Svensäter G, Sjögreen B, Hamilton IR: Multiple
stress responses in Streptococcus mutans and
the induction of general and stress-specific
proteins. Microbiology 2000;
146: 107–117.
Tenuta LM, Ricomini Filho AP, Del Bel Cury
AA, Cury JA: Effect of sucrose on the selec-
tion of mutans streptococci and lactobacilli
in dental biofilm formed in situ. Caries Res
2006;
40: 546–549.
Vale GC, Tabchoury CP, Arthur RA, Del Bel
Cury AA, Paes Leme AF, Cury JA: Temporal
relationship between sucrose-associated
changes in dental biofilm composition and
enamel demineralization. Caries Res 2007;
41: 406–412.
Vijay K, Brody MS, Freudlund E, Price CW: A
PP2C phosphatase containing a PAS domain
is required to convey signals of energy stress
to the sigmaB transcription factor of Bacillus
subtilis . Mol Microbiol 2000;
35: 180–188.
Welin J, Wilkins JC, Beighton D, Wrzesinski K,
Fey SJ, Mose-Larsen P, Hamilton IR, Sven-
säter G: Effect of acid shock on protein ex-
pression by biofilm cells of Streptococcus
mutans . FEMS Microbiol Lett 2003;
227: 287
293.
Wilkins JC, Beighton D, Homer KA: Effect of
acidic pH on expression of surface-associat-
ed proteins of Streptococcus oralis . Appl En-
viron Microbiol 2003;
69: 52905296.
Wilkins JC, Homer KA, Beighton D: Altered
protein expression of Streptococcus oralis
cultured at low pH revealed by two-dimen-
sional gel electrophoresis. Appl Environ Mi-
crobiol 2001;
67: 3396–3405.
Wilkins JC, Homer KA, Beighton D: Analysis of
Streptococcus mutans proteins modulated by
culture under acidic conditions. Appl Envi-
ron Microbiol 2002;
68: 2382–2390.
Page 9
  • Source
    • "1 Department of Epidemiology and Public Health, University College London, London, UK 2 London School of Hygiene and Tropical Medicine, London, UK, and World Obesity, London, UK mainly generated by a selective group of bacteria (Marsh 1999Marsh , 2003 Paes Leme et al. 2006; Takahashi and Nyvad 2011). Sucrose or its individual monosaccharide constituents together selectively promote mutans streptococci growth and other acidogenic and acid-tolerating species (Marsh 1999Marsh , 2003 Paes Leme et al. 2006; Vale et al. 2007; Paes Leme et al. 2008). Paes Leme et al. (2006) concluded that " sucrose causes major biochemical and physiological changes during the process of biofilm formation, which, in turn, enhance its caries-inducing properties. "
    [Show abstract] [Hide abstract] ABSTRACT: The importance of sugars as a cause of caries is underemphasized and not prominent in preventive strategies. This is despite overwhelming evidence of its unique role in causing a worldwide caries epidemic. Why this neglect? One reason is that researchers mistakenly consider caries to be a multifactorial disease; they also concentrate mainly on mitigating factors, particularly fluoride. However, this is to misunderstand that the only cause of caries is dietary sugars. These provide a substrate for cariogenic oral bacteria to flourish and to generate enamel-demineralizing acids. Modifying factors such as fluoride and dental hygiene would not be needed if we tackled the single cause-sugars. In this article, we demonstrate the sensitivity of cariogenesis to even very low sugars intakes. Quantitative analyses show a log-linear dose-response relationship between the sucrose or its monosaccharide intakes and the progressive lifelong development of caries. This results in a substantial dental health burden throughout life. Processed starches have cariogenic potential when accompanying sucrose, but human studies do not provide unequivocal data of their cariogenicity. The long-standing failure to identify the need for drastic national reductions in sugars intakes reflects scientific confusion partly induced by pressure from major industrial sugar interests. © International & American Associations for Dental Research 2015.
    Full-text · Article · Aug 2015 · Journal of dental research
  • Source
    • "Proteins are also considered an important component of the extracellular dental plaque matrix. There is evidence that the protein content in plaque fluid increases by >50% following exposure to sucrose [13], and changes in the proteome composition have been observed in response to sucrose [14]. However, it is not yet clear what effects these changes in protein content of the plaque matrix have on the structure and function of dental plaque. "
    [Show abstract] [Hide abstract] ABSTRACT: The extracellular matrix of microbial biofilms is critical for surface adhesion and nutrient homeostasis. Evidence is accumulating that extracellular DNA plays a number of important roles in biofilm integrity and formation on hard and soft tissues in the oral cavity. Here, we summarise recent developments in the field and consider the potential of targeting DNA for oral biofilm control. Copyright © 2015. Published by Elsevier Masson SAS.
    Full-text · Article · Apr 2015 · Microbes and Infection
  • Source
    • "In comparison, seven proteins were exclusively found in the plaque-like group with sucrose presence (isoform 2 of enolase, hypothetical protein ip_0493, enterotoxin, hypothetical cytosolic protein, phosphoglycerate mutase I, prolactininduced protein, and prolactin-induced protein) and 20 in the extracellular matrix from the control group. Briefly, insights regarding biochemical and microbiological changes by sucrose provide understanding of cariogenic mechanisms and extracellular matrix composition of plaque-like biofilm (Paes Leme et al., 2008). Proteomic tools were also used to analyze preventive aspects in dentistry, such as fluoride use. "
    [Show abstract] [Hide abstract] ABSTRACT: Despite all the dental information acquired over centuries and the importance of proteome research, the cross-link between these two areas only emerged around mid-nineties. Proteomic tools can help dentistry in the identification of risk factors, early diagnosis, prevention, and systematic control that will promote the evolution of treatment in all dentistry specialties. This review mainly focuses on the evolution of dentistry in different specialties based on proteomic research and how these tools can improve knowledge in dentistry. The subjects covered are an overview of proteomics in dentistry, specific information on different fields in dentistry (dental structure, restorative dentistry, endodontics, periodontics, oral pathology, oral surgery, and orthodontics) and future directions. There are many new proteomic technologies that have never been used in dentistry studies and some dentistry areas that have never been explored by proteomic tools. It is expected that a greater integration of these areas will help to understand what is still unknown in oral health and disease. J. Cell. Physiol. 228: 2271-2284, 2013. © 2013 Wiley Periodicals, Inc.
    Full-text · Article · Dec 2013 · Journal of Cellular Physiology
Show more