archives of oral biology 53 (2008) 1003–1010

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journal homepage: www.intl.elsevierhealth.com/journals/arob

Electron microscopic detection and activity of

glucosyltransferase B, C, and D in the in situ formed pellicle

C. Hannig a,*, A. Ruggeri b, B. Al-Khayer c, P. Schmitz a, B. Spitzmüller a,

D. Deimling a, K. Huber c, W. Hoth-Hannig c, W.H. Bowen d, M. Hannig c
a
Department of Operative Dentistry and Periodontology, University of Freiburg, Hugstetter Str. 55, D-79106 Freiburg, Germany
b
Department of SAU & FAL, University of Bologna, Bologna, Italy
c
Clinic of Operative Dentistry, Periodontology and Preventive Dentistry, University Hospital, Saarland University,
Building 73, D-66421 Homburg/Saar, Germany
d
Center for Oral Biology and Eastman Department of Dentistry, 601 Elmwood Avenue, Box 611,
University of Rochester Medical Center, Rochester, NY 14642, USA

article info abstract

Article history: Objective: Glucosyltransferases (GTFs) represent a virulence factor of mutans streptococci. The
Received 9 January 2008 aim of the present in situ study was to investigate the distribution of different GTF-isoforms
Received in revised form in the pellicle.
18 March 2008 Design: Bovine enamel slabs were fixed on buccal and palatal sites of individual splints worn
Accepted 17 April 2008 by five subjects for 30 and 120 min to allow pellicle formation. Pellicle specimens were
processed for transmission electron microscopy (TEM) and field emission in-lens scanning
electron microscopy (FEI-SEM). Gold-immunolabelling was used for detection of GTF-iso-
Keywords: forms B, C and D. Furthermore, glucosyltransferase activity of 3-, 30- and 120-min pellicles
Glucosyltranferases was tested via determination of fructose release.
Enzymes Results: All isoforms of the enzyme were found to be randomly distributed within all layers
Pellicle of the pellicle. In cross-sections (TEM), GTF D was the most abundant isoform. More labelled
Immunolabelling molecules were detected on buccal sites compared with palatal surfaces, the number of
Biofilm molecules detected increased with time. The amount of GTF B, C and D found on the pellicle
Isoform surface by FEI-SEM showed no correlation with pellicle formation time or localisation in the
Activity oral cavity. Overall, GTF D was detected more frequently on the surface than GTF B and C. All
Fructose pellicles tested showed GTF-activity.
Conclusion: The study shows for the first time the presence of the GTF-isoforms B, C and D
within all layers of the in situ formed pellicle. This emphasises the impact of streptococcal
products on the composition of the pellicle and illustrates a mechanism used by bacteria to
colonize dental surfaces.
# 2008 Elsevier Ltd. All rights reserved.

1. Introduction acquired pellicle in the literature.1,2 On the one hand,

pellicles possess protective properties, serve as an anti-
The initial oral biofilm that is formed within seconds on all erosive barrier, and harbour antibacterial properties.1–3 On
solid substrata exposed to the oral fluids is referred to as the other hand, they contain specific receptors for bacterial

* Corresponding author. Tel.: +49 761 270 4888; fax: +49 761 270 4762.
E-mail address: christian.hannig@uniklinik-freiburg.de (C. Hannig).
0003–9969/$ – see front matter # 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.archoralbio.2008.04.005
1004 archives of oral biology 53 (2008) 1003–1010
adherence and serve as the starting point of plaque
formation.4 Pellicles are composed of proteins, glycoproteins
and mucins.1,2 A considerable amount of in situ pellicle
proteins consist of salivary enzymes such as lysozyme, a-
amylase and carbonic anhydrases.3 These enzymes are
adsorbed onto dental surfaces in an active conformation.3,5
Surprisingly, beside these mammalian enzymes, bacterial
glucosyltransferases (GTFs) are present in the in situ formed
pellicle.5,6 Glucosyltransferases (EC 2.4.1.5) are extracellular
enzymes synthesizing glucans from disaccharides, mainly
from saccharose, utilising the a-glucosidic bond between
fructose and glucose.7–9 They are released not only by S.
mutans species 9,10 but also by S. sanguis.11 There is clear
evidence for three isoenzymes of GTF within the pellicle: GTF
B, C and D.7,12 However, these three isoforms were only
investigated in experimental pellicles gained in vitro from
salivary samples until now.7,12
GTF-activity increases continuously during initial pellicle
formation.5 Interestingly, GTF-activities are enhanced in the
immobilised state as shown for GTF C and D, respectively in
contrast to other pellicle bound enzymes such as amylase or
lysozyme.3,7
GTF B catalyses the synthesis of an insoluble glucan
from sucrose, composed mainly of a1,3-glucose moieties.
GTF D synthesizes a water-soluble glucan of predominatly
a1,6-linked glucosyl units. Glucan polymers yielded by GTF
C contain both a1,3- and a1,6-linked glucans.7–9 GTF B, C,
and D have the same basic structure.13 They are composed
of a signal peptide of approximately 38 amino acids at the
amino terminus followed by a variable domain of approxi-
mately 200 amino acids characteristic for each isoform. GTF
B contains 1475 amino acids and is highly hydrophilic.14
Also GTF D with its 1475 amino acids is of high hydro-
philicity.15 GTF C which is composed of 1375 amino
acids has a more amphiphilic character due to small
hydrophobic domains.7,16 This conformational difference
of GTF C may confer to the high affinity of GTF C to
hydroxyapatite.7 Due to the kinetic and other character-
istics, being quite similar to those of GTF in the in situ
pellicle, GTF C was assumed to be the predominant isoform
in the acquired pellicle.7,12
However, despite these studies on glucosyltransferase-
isoforms and their activity, there is no information on
their distribution and localisation in the in situ formed
pellicle.
Previous studies indicated clearly that the immunolabel-
ling technique offers the opportunity to visualize the
distribution and localisation of certain proteins within the
pellicle in cross-sectional views as well as on the pellicle’s
surface.17,18 Therefore, the aim of the present in situ study was
to evaluate the previously identified pellicle-bound GTF-
isoforms B, C, and D with this well-established method to
get further insights into the ultrastructure of the pellicle with
special focus on this bacterial virulence factor. It was therefore
determined which isoenzymes of GTF are present within the
different layers of the in situ pellicle and on the pellicle’s
surface. Thereby, it was hypothesized that GTF C is the
predominant GTF-isoform within the pellicle. Furthermore,
the activity of glycosyltransferase immobilised at the pellicle
surface was measured.
2. Materials and methods
2.1. Subjects and specimens
Five healthy volunteers, members of the laboratory staff,
participated in the study. Visual oral examination was carried
out by an experienced dentist. The subjects showed no signs of
gingivitis or caries. Informed written consent had been given
by the subjects about participation in the study. The study
design was reviewed and approved by the Ethics Committees
of the universities of Göttingen (proposal 16/6/05) and
Homburg (proposal 52/05) in Germany.
Cylindrical enamel slabs (diameter 5 mm, 19.63 mm2 sur-
face area, height 1.5 mm) were prepared from labial surfaces
of bovine incisors of 2-year-old cattle. The surfaces were
polished by wet grinding with abrasive paper (400–4000 grit).
The smear layer on the slabs was removed by ultrasonication
with NaOCl for 3 min.18–20 Afterwards, the samples were
disinfected in ethanol (70%) for another 3 min, washed in
distilled water and stored in aqua dest. for 24 h before
exposure in the oral cavity.18
2.2. Pellicle formation
For in situ pellicle formation, individual upper jaw splints were
vacuum-formed from 1.5-mm-thick methacrylate foils. Cav-
ities were prepared in the buccal and palatal aspects of the
splints at the sites of the premolars and the 1st molar. The
slabs were fixed on the splints using polyvinyl siloxane
impression material (President light body, Coltene, Switzer-
land), exposing only the surfaces of the enamel slabs to the
oral fluids. The splints were carried intraorally for 30 and
120 min, to allow pellicle formation on the specimens’
surfaces. Each subject carried two samples per site (buccal/
palatal) and intraoral exposure time (30/120 min) for the FEI-
SEM as well as for TEM experiments. Volunteers refrained
from eating and drinking 2 h before insertion of the splints and
during intraoral exposure.
After intraoral exposure, the slabs were immediately
removed from the splints and thoroughly rinsed with running
tap water for 5 s in order to remove non-adsorbed salivary
remnants. Separate samples were prepared for the three
isoenzymes GTF B, C and D.
2.3. Immunolabelling technique
2.3.1. TEM (transmission electron microscopy)
Detection of the different GTF-isoforms in cross-sectional
views was achieved with TEM. After intra-oral exposure, the
pellicle-coated enamel slabs were fixed in 4% paraformal-
dehyde/0.1% glutaraldehyde for 2 h at 4 8C. Before embed-
ding in LR-White resin (London Resin company, Theale,
Berks, UK) dehydration took place in an ascending
series of ethanol. The enamel part of the embedded
specimens was dissolved by decalcification using 1 M HCl,
and re-embedding took place with Araldite CY 212 (Serva,
Heidelberg, Germany). A Mikrostar 458 diamond knife
(Mikrotechnik, Bensheim, Germany) fixed in an Ultracut E
microtome (Reichert, Heidelberg, Germany) was used to cut
ultrathin sections in series. The ultrathin sections were
 archives of oral biology 53 (2008) 1003–1010
mounted on 300-mesh nickel grids (Plano, Wetzlar, Ger-
many).
Detection of glucosyltansferases was conducted by two-
step labelling. The primary antibodies were rabbit polyclonal
immunoglobulins against GTF B, C, and D, respectively. These
polyclonal antibodies were made monospecific to GTF B, C or
D, respectively, as described previously.21 Since the primary
antibodies were not conjugated with a marker, 10-nm gold-
labelled anti-rabbit-immunoglobulin G (Biotrend, Cologne,
Germany) was used as secondary antibody for visualization by
transmission electron microscopy. Primary and secondary
antibodies were diluted 1:500 for the labelling experiments. At
least five ultrathin sections of each specimen were gold-
immunolabelled as described in detail previously.17,18 Briefly,
the ultrathin sections were pretreated with NH4Cl, incubated
with the primary antibodies against GTF B, C, D, respectively,
washed in phosphate buffered saline (PBS) and treated with
bovine serum albumin (BSA). Subsequently, the sections were
incubated with the 10 nm-gold-labelled secondary antibodies.
This procedure was followed by PBS rinsing and contrasting
with uranylacetate. All steps of the protocol were performed at
room temperature. The TEM analysis was performed in an EM
902 microscope (Zeiss, Oberkochen, Germany) at an 80,000-
fold magnification. The 10-nm gold particles were counted
within at least two randomly chosen ultrathin sections from
each individual specimen. The number of gold globules was
determined on a length of 100 mm. This was performed in
consecutive steps of 1 mm, according to the width of the TEM
screen. From the data of all five subjects, the mean values and
standard deviations of gold particle numbers were calculated
for the 30-min and 120-min pellicles formed buccally and
palatally, respectively. Characteristic images of the pellicle
layer were photo-documented using a magnification of 68,000-
fold.
2.3.2. FEI-SEM (field emission in lens scanning electron
microscopy)
The detection of GTF B, C and D on the pellicle’s surface was
performed by FEI-SEM analysis.17,18 In contrast to the TEM
analysis, the two-step gold-immunolabelling took place
immediately after intraoral exposure without fixation of the
pellicle according to the protocol described above. Both
antibodies were diluted 1:50 for the labelling experiments.
In order to stabilize the pellicle and the binding of the
antibodies, the sections were incubated for 1 min in 1%
glutaraldehyde. After gold-immunolabelling the specimens
were dehydrated in an ascending series of ethanol and critical-
point dried.
Specimens were fixed on stubs afterwards and coated with
a 5-nm-thick layer of evaporated carbon using a Balzer
Medical 010 Multicoating System (BAL-TEC). The microscopic
analysis took place in a FEI-SEM JEOL JSM-890 (JEOL, Tokyo,
Japan) as well as in a FEI Phillips XL 30 ESEM/FEG (FEI,
Eindhoven, The Netherlands) using SEI and BEI detectors
separately or in mixed mode.18 Characteristic and represen-
tative images of the pellicle layer were recorded at a
magnification of 40,000-fold. In each specimen, the gold
particles were counted as number per mm2 on 10 randomly
distributed images of different parts of the specimen’s surface.
From the data of all five subjects, the mean values and
1005
standard deviations of gold particle numbers per mm2 were
calculated for the 30-min and 120-min pellicles formed
buccally and palatally, respectively.
2.3.3. Controls
Control specimens were processed as follows for both TEM
and FEI-SEM:
1. Specimens not exposed to the oral cavity were labelled
using the above-mentioned gold-immunolabelling proce-
dure.
2. Specimens exposed to the oral cavity were incubated
without the primary antibody in phosphate buffer and
then with the secondary antibody.
Control specimens were free of gold globules.
2.4. Determination of the enzyme activity
Glucosyltransferase transfers the a-glucosidic residue from
sucrose to form glucans. Thereby, fructose is released as
byproduct that can be measured in an enzymatic assay.
Enamel slabs were exposed to the oral fluids of five subjects
for 3, 30 or 120 min at buccal or oral positions, respectively.
Each subject carried six specimens per subgroup. These six
specimens were pooled and incubated in 1 ml reaction buffer
for 4 h at 37 8C; the composition of the reaction buffer was
500 mmol/l sucrose, 40 mmol/l dextran 9000, 50 mmol/l KCl,
1 mmol/l CaCl2, 0.1 mmol/l MgCl2, 0.35 mmol/l K2HPO4, and
0.65 mmol/l KH2PO4.5
For determination of fructose release, a volume of 110 ml
was removed from the reaction tubes, the enzymes were
inactivated at 80 8C for 15 min and centrifuged for 5 min. A
volume of 100 ml was transferred to the fructose-assay.22
The fructose concentration was determined photometri-
cally using a test kit (# 10139106035, Boehringer, Mannheim,
Germany).
D-Glucose and D-fructose are phosphorylated by hexoki-
nase yielding glucose-6-phospate or fructose-6-phosphate,
respectively. Thereby, ATP is converted to ADP. In the
presence of glucose-6-phosphate dehydrogenase, glucose-6-
phosphate is oxidized by NADP to gluconate-6-phosphate with
the formation of NADPH. When this substrate is converted
completely and the reaction finished, phospho-glucose
isomerase is added to change fructose-6-phosphate to
glucose-6-phosphate. Glucose-6-phosphate reacts with NADP
forming D-gluconate-6-phosphate and NADPH. The amount of
NADPH released in this reaction is stoichiometric to the
amount of D-fructose and can be monitored at a wavelength of
340 nm.
The assay was carried out in a Tecan Infinite M 200
spectrometer (Tecan, Munich, Germany).
The activity was calculated as amount of fructose release
per cm2 pellicle-coated surface and min[mmol fructose/
min  cm2 or mU/cm2].
2.5. Statistics
The influence of pellicle formation time, isoform and
localisation on the amount of gold particles detected or
1006 archives of oral biology 53 (2008) 1003–1010

Fig. 1 – Results of transmission electron microscopy (TEM): detection of gold-labelled GTF-molecules (B, C and D) in cross-
sections of the pellicle. Pellicle formation took place on buccal and palatal sites of the upper premolars and 1st molar, n = 5
subjects. MV W S.D., counted gold globules/100-mm pellicle length.

enzyme activity, respectively, was evaluated with ANOVA
( p  0.05). In addition, the Scheffé-procedure was carried out
(SPSS 15.0).
3. Results
All in situ pellicles demonstrated gold-labelled molecules of
the three GTF-isoforms B, C and D. However, TEM as well as
FEI-SEM analyses revealed high intraindividual and inter-
individual variability concerning the number of gold-labelled
molecules detected (Figs. 1 and 2).
3.1. TEM
The TEM experiments offer a cross-sectional view on the
pellicle’s ultrastructure and distribution of the gold-labelled
molecules. Typical TEM pictures are given in Fig. 3. All pellicles
were of a fine granular structure and showed an electron-
dense basal layer. In addition, buccal pellicles showed
agglomerates of proteins with diameters ranging from 100
to 250 nm. The thickness of the pellicle layer increased with
the formation time and differed considerably between buccal
and palatal specimens. Buccally formed pellicles ranged
between 20 and 120 nm in thickness after 30 min of formation
time, and between 40 and 300 nm after 120 min, respectively.
The thickness of palatal pellicles ranged between 20 and
60 nm after 30 min, and between 20 and 80 nm after 2 h of
intraoral exposure.
All GTF-isoforms were present in every layer of the pellicle
including the electron-dense basal layer. The detected GTF-
molecules of all three isoforms were found to be randomly
distributed in all layers of the pellicle. However, GTF D was
shown to be the most abundant GTF-isoform in the pellicle as

Fig. 2 – Results of FEI-SEM: detection of gold-labelled GTF-molecules (B, C and D) on the surface of the pellicle. Pellicle
formation took place on buccal and palatal sites of the upper premolars and 1st molar, n = 5 subjects. MV W S.D., counted
gold globules/mmS2.
 archives of oral biology 53 (2008) 1003–1010 1007

Fig. 3 – TEM-pictures of 30-min in situ pellicles. Labelled GTF-molecules (arrows) can be distinguished well. The salivary
pellicle is characterized by a fine granular structure. A 10-nm electron-dense basal layer is detectable in the micrographs.
Micelle-like agglomerates of adsorbed proteins are found in buccally formed pellicles. (a) GTF B, 120 min buccal; (b) GTF D,
120 min buccal; (c) GTF B, 30 min palatal; (d) GTF D, 120 min palatal. Black scale bar: 100 nm, original magnification: 68,000-
fold.

Fig. 4 – FEI-SEM pictures of 30-min in situ pellicles. GTF-molecules (arrows) can be distinguished well. Note the globular
structure of the pellicle surface. Magnification: 40,000-fold, (a) GTF B, 30 min buccal; (b) GTF D, 30 min palatal, length of the
scale bar: 200 nm.

compared with GTF B and C (Fig. 1). Over all samples, For all isoforms, a clear impact of pellicle formation time
significantly more GTF D molecules were detected as and localisation in the oral cavity on the amount of labelled
compared with GTF B and C (ANOVA, Scheffé-procedure, GTF-molecules was observed (Fig. 1). More labelled molecules
p < 0.001). were detected on buccal sites than on palatal surfaces and the

Fig. 5 – Activity of glucosyltransferase immobilised in the in situ pellicle layer as measured via release of fructose, n = 30
enamel slabs per subgroup, five subjects.
1008 archives of oral biology 53 (2008) 1003–1010
mean number of labelled GTF-molecules increased with
pellicle formation time. This influence was significant for
GTF B and D (ANOVA: GTF B p = 0.001, GTF C p = 0.87, GTF D
p = 0.029).
The amount of the different GTF-isoforms detected within
buccal 30-min pellicles as well as in palatal and buccal 120-
min pellicles differed significantly ( p < 0.05). For palatal
30-min specimens, there was no significant impact of the
GTF-isoform on the amount of detectable GTF-molecules.
3.2. FEI-SEM
The FEI-SEM data give information on the superficially
adsorbed GTF-molecules (Figs. 2 and 4). The GTF-molecules
of the three isoforms were distributed randomly, but the high
standard deviation has to be considered. Over all formation
times and localisations, GTF D was detected more frequently
on the surface than GTF B and C (ANOVA: p < 0.001, Scheffé-
procedure: GTF B vs. GTF D: p < 0.001, GTF C vs. GTF D:
p = 0.029). For all GTF-isoforms, there was a significant impact
of localisation and formation time on the amount of labelled
GTF-molecules (ANOVA: p < 0.001). However, the findings
were inconsistent.
3.3. GTF-activity
GTF-activity was determined via release of fructose. All
pellicle samples tested exposed GTF-activity at the surface.
There was no significant impact of pellicle formation time or
localisation in the oral cavity on the GTF-activity (ANOVA,
n.s.). However, after 120 min in tendency higher activities
were observed (Fig. 5).
4. Discussion
In the present study, GTF-molecules of different isoforms were
visualized in the in situ pellicle. The adopted method based on
gold-immunolabelling has proven success in previous inves-
tigations on detection of distinct proteins in the pellicle layer
such as lysozyme, amylase or carbonic anhydrases.17,18
Bovine enamel slabs were used for in situ pellicle formation
in accordance with former studies on enzymes in the acquired
pellicle.18,23,24 It is easier to harvest enamel slabs of homo-
genous quality from bovine than from human teeth.
In addition to the electron microscopic techniques, the
immobilised glucosyltransferase activity was measured.
Typically, GTF immobilised to enamel or hydroxyapatite is
monitored by direct scintillation counting using [14C]-gluco-
sylsucrose as a substrate.5,7 However, another option is to
determine the amount of fructose released due to conversion
of sucrose with highly specific fructose assays.22,25 A well
established and precise fructose assay was used for this
purpose.22
To the best knowledge of the authors, the present study
shows for the first time the presence of the GTF-isoforms B, C
and D within all layers of the in situ formed pellicle as well as
on its outer surface. GTF-molecules of all isoforms were even
detected in the electron-dense basal layer. Previous studies on
the GTF-isoforms and activity were usually based on experi-
mental pellicles, only, formed on hydroxyapatite in vitro from
human saliva.7,12
Furthermore, pellicle bound GTF-activity was demon-
strated for five individuals. Already after 3 min of pellicle
formation, considerable amounts of activity were present at
the surface. In a previous in situ study using hydroxyapatite
discs, GTF-activity at the pellicle’s surface increased in a time-
dependent manner over a 20-min period.5 However, in the
present study, there was no clear influence of pellicle
formation time on the enzymatic activity. The high intraindi-
vidual and interindividual variation observed when evaluating
GTF with enzymatic and gold-immunolabelling techniques is
in good accordance with other in situ or in vivo investigations
on proteins and enzymes in the acquired pellicle.23,26,27
In contrast to the hypothesis of the present study, GTF D
seems to be the most abundant isoform in the pellicle. In
previous studies, salivary GTF C was shown to have functional
properties more similar to those of pellicles’ GTF if compared
with GTF B and D, and it was assumed to be the predominant
isoform of GTF in the in situ pellicle, whereas GTF D was
expected to be of lower relevance.7,12 However, both GTF C and
D have enhanced activities when immobilised in experimental
pellicles7,12 corresponding to the present FEI-SEM findings
indicating a slight dominance of GTF D. The binding properties
of the different isotypes to saliva-coated hydroxyapatite and
to bacteria have been investigated previously.28 Thereby GTF B
was shown to have a high affinity to bacterial surfaces
whereas GTF C and D did not bind to bacterial surfaces. On the
other hand, on saliva-coated hydroxyapatite, GTF C yielded
the highest affinity and GTF D exposed the most binding sites
despite a low affinity to the surface. Interestingly, the binding
of GTF B to hydroxyapatite is enhanced by GTF C meaning a
positive cooperativity.28 These in vitro findings offer an
explanation for the detection of all GTF-isoforms in the
present in situ study.
The high number of GTF D molecules detected in the
present study despite the low affinity to saliva-coated
hydroxyapatite may indicate a specific transient role of this
isoform in plaque formation by providing a primer for other
isoforms.28 GTF D synthesizes a water-soluble glucan of
predominatly a1,6-linked glucosyl units enhancing bacterial
activity.8,9
Generally, pellicle thickness increases with time.18,29
Furthermore, buccal pellicles are thicker than palatal ones.2,29
As expected, the amount of labelled GTF-molecules detected
in cross-sectional views by TEM increased in a time-
dependent manner, and less labelled molecules were detected
on palatally formed pellicles, respectively. However, after
120 min only about 40% more labelled GTF-molecules were
detected in cross-sections compared to the 30-min pellicles.
This indicates that protein adsorption during pellicle forma-
tion is not a linear process. It is rather a dynamic equilibrium
of adsorption and desorption than a constant immobilisation
of biomolecules.2,3 On the other hand, the amount of labelled
GTF-molecules and the GTF-activity detected on the pellicle’s
surface were not influenced clearly by localisation or pellicle
formation time as observed already with other pellicle
proteins.17,18
Along with amylase,23 the superficially exposed glucosyl-
tranferases also yield significant enzymatic activities.5,6 The
 archives of oral biology 53 (2008) 1003–1010
simultaneous presence of GTFs and amylase in an active
conformation even in the very early stages of pellicle
formation suggests interaction of these two enzymes in the
immobilised state.3,12 Glucans synthesized by GTFs promote
adhesion of S. mutans.30,31 The glucan synthesis is increased if
starch hydrolysates are supplied by amylase.12 However,
surprisingly GTF reduces amylase activity and GTF and
amylase seem to compete for binding sites in the pellicle,12
whereas GTFs themselves expose a higher enzymatic activity
in the immobilised state.5–7 Due to the manifold interactions
of amylase and GTF in bacterial metabolism and microbial
adhesion, it may be postulated that both enzymes have
identical epitopes.2,12
Host salivary defence mechanisms such as IgA, lysozyme
or agglutinin aggravate bacterial adherence to solid sub-
strata, particularly during the initial phase of oral biofilm
formation.1,2 The host proteins seem to win the race for the
surface and only very few bacteria are found to be adhered to
the teeth during the first 120 min of biofilm formation.20,32
Despite these host defence mechanisms focussing mainly on
the bacterial cells, the micro-organisms manage to immo-
bilise virulent GTF enzymes along with host proteins in an
active conformation into the initial pellicle, promoting
bacterial adherence to glucans formed on the tooth surface.5
After some hours, the amount of bacteria in the biofilm
increases.33 Accordingly, GTFs can be regarded as key factors
for bacterial colonization and supragingival plaque forma-
tion.5,12,21
GTFs may be seen as an example of very effective bacterial
adaptation to physiological biofilms of hosts. This applies for
the GTF-isoforms B, C and D, respectively, as shown in the
present data. Future research on bacterial biofilm formation
must consider all GTF-isoforms due to their key role even in
early bacterial adherence.
5. Conclusions
The study shows for the first time the presence of the GTF-
isoforms B, C and D within in all layers of the in situ formed
pellicle and on the pellicle’s surface. Already after 3 min,
considerable GTF-activity is present in the pellicle layer. This
emphasises the high impact of cariogenic streptococci on the
composition of the pellicle and means optimal adaptation to
the host defence mechanisms.
Acknowledgement
This study was supported in part by the German Research
Foundation (DFG proposals HA 2718/3-3, HA 5192/1-2).
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