AEM Accepted Manuscript Posted Online 5 June 2015 Appl. Environ. Microbiol. doi:10.1128/AEM.01180-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1 2
Extracellular Glycoside Hydrolase Activities in the Human Oral Cavity
3
Taichi Inui,a# Lauren C. Walker,b§ Michael W.J. Dodds,a A. Bryan Hanleyc
4 5 6
Wm. Wrigley Jr. Company, Chicago, Illinois 60642, USAa; School of Chemistry, University of Edinburgh, Edinburgh, Scotland, UKb, The Knowledge Transfer Network, The Roslin Institute,
7 8 9 10 11 12 13 14 15 16 17 18
Midlothian, Scotland, UKc
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Keywords: Oral Ecology, glycosidase, extracellular enzyme, ectoenzyme
Running Head: Glycoside Hydrolases in Oral Cavity #Address correspondence to Taichi Inui, [email protected] §Current address Anheuser-Busch InBev, Edinburgh, Scotland, UK Supplementary data: Following supplementary data may be found in the online version of this article: Table S1. Known glycoside hydrolase families among the tested oral micro-organisms in this study Table S2. Primers used for qPCR experiment
1
23
Abstract
24
Carbohydrate availability shifts when bacteria attach to a surface and form biofilm. When
25
salivary planktonic bacteria form an oral biofilm, a variety of polysaccharides and glycoproteins
26
would be the primary carbon sources, however simple sugar availabilities would be limited due
27
to low diffusion from saliva to biofilm. We hypothesized bacterial glycoside hydrolase (GH)
28
activities would be higher in a biofilm compared to saliva in order to maintain metabolism in a
29
low sugar–high glycoprotein environment. Salivary bacteria from 13 healthy individuals were
30
used to grow in vitro biofilm using two separate media, one with sucrose and the other limiting
31
carbon sources to a complex carbohydrate. All six GHs measured showed higher in vitro
32
activities when grown in the media with complex carbohydrate being the sole carbon source. We
33
then collected saliva and overnight dental plaque samples from the same individuals and
34
measured ex vivo activities for the same six enzymes to determine how oral microbial utilization
35
of glycoconjugates shifts between the planktonic phase in saliva and the biofilm phase in
36
overnight dental plaque. Overall higher GH activities were observed in plaque samples, in
37
agreement with in vitro observation. A similar pattern was observed in GH activity profiles
38
between in vitro and ex vivo data. 16S DNA analysis showed that plaque sample had a higher
39
abundance of microorganisms with larger number of GH gene sequences. These results suggest
40
differences in sugar catabolism between the oral bacteria located in the biofilm compared to
41
those in saliva.
42
2
43
INTRODUCTION
44
The oral cavity is a dynamic environment. For instance both salivary flow and the level of
45
exogenous dietary carbon sources increase during ingestion of food whereas both are at a
46
minimum during sleep. Therefore the oral microbiota may be influenced by alterations in the
47
host environment, especially by changes in nutrient supply, local pH, and redox potential (1).
48
One notable example is that the microbial community becomes more acidogenic in the presence
49
of sugar (2). Endogenous salivary glycoconjugates become the most significant carbon source
50
for oral microorganisms when an exogenous dietary carbon source is scarce. Under those
51
circumstances Carbohydrate-Active enZymes (CAZymes) play an important role in the
52
adaptation of microorganisms (3-6). These CAZymes include glycosyltransferases and glycoside
53
hydrolases (GHs). The type and availability of carbon sources also influence the development of
54
biofilm at stages throughout the biofilm formation as microorganisms utilize the available carbon
55
sources as nutrients, as building blocks for extracellular polysaccharide production, and as the
56
point of attachment (7).
57
carbohydrates by GH enzymes is one mechanism whereby oral microbes adapt to different
58
carbon sources between the salivary planktonic phase, where exogenous simple sugars are
59
generally more available, and the oral biofilm phase where, in the absence of exogenous simple
60
sugars, several alternative, more complex endogenous salivary glycoconjugates are the principal
61
carbon source (8-11).
62 63 64
We speculated that the breakdown of complex, endogenous
Our hypotheses for the present study were that 1. GH activities are higher under conditions where the primary carbon sources are limited to complex glycan conjugates. 3
65
2. Levels of GH enzymes would be higher in dental plaque than in saliva due to the shift in
66
microbial population between saliva and plaque.
67
Here we report the GH levels between conditions where carbon source availabilities
68
include simple sugars (saliva) and limited to complex glycoconjugates (dental plaque). Our first
69
experiment was to test if carbohydrate availability will influence GH activities in in vitro oral
70
biofilm, followed by ex vivo experiment in which GH activities were measured in saliva and
71
overnight dental plaque. In both in vitro and ex vivo experiments, the samples were separated
72
into cell-free supernatant and cellular pellets by centrifuge in order to monitor the enzyme
73
activities that are secreted and those on cell-surfaces.
74 75
MATERIALS AND METHODS
76
In vitro Biofilm Growth
77
Gum base stimulated saliva samples were collected from 13 healthy volunteers (7 females and 6
78
males, age 24 – 45) at 9 am. Subjects were in good general health based on medical history and
79
not being treated for medical or dental condition or taking antibiotics or other medications,
80
which might influence salivary secretion. Subjects refrained from eating, drinking, and oral
81
hygiene for at least 2 hours prior to the collection.
82
immediately treated with phenylmethanesulfonylfluoride (PMSF, final concentration 1 mM,
83
Sigma-Aldrich, St. Louis, MO). Samples were then centrifuged at 12000 xg for 30 min at 4 °C.
84
The pellet was suspended in phosphate buffered saline (pH 7.4) to a final OD600 of 0.600. The
85
pellet suspension was divided and inoculated for biofilm growth in two different media in
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parallel. Hydroxyapatite disks (Clarkson Chromatography Products, South Williamsport, PA)
Samples were collected on ice and
4
87
were coated for 1 hour at room temperature in 24 well plates (BD Bioscience, Franklin Lakes,
88
NJ) with pooled supernatant to form salivary pellicle. Pellet suspensions from each individual
89
were then inoculated separately onto disks and grown for 72 hours at 37 °C in either peptone-
90
yeast base media containing 5% sucrose (sucrose media) or 1.25 % hog gastric mucin in
91
phosphate buffered saline (mucin media) (12). The sucrose media has been shown to be capable
92
of growing a more diversified community with a microbial profile closer to that of the salivary
93
microbiota when inoculated with pooled human saliva. On the other hand, mucin media would
94
represent the environment where the carbon source is limited to complex glycoconjugates. The
95
media were refreshed every 24 hours. The spent media was centrifuged at 12000 xg for 30 min at
96
4 °C after which the obtained supernatant was filter sterilized for assay of GH activity. After 72
97
hour growth the biofilm was collected and weighed. It was then centrifuged to remove residual
98
media and resuspended in 50mM N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid
99
(TES) buffer, pH 7.4 to a final concentration of 5.0 mg pellet mL-1.
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Ex vivo Sample Collection
101
Saliva collection and processing were done the same way to the point of centrifugation from the
102
same volunteers as the above. The supernatant and the pellet were weighed, followed by having
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the pellet resuspended in 50 mM TES buffer to a final concentration of 5.0 mg pellet mL-1. Both
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supernatant of pellet suspension were subjected for further analyses.
105
Overnight grown plaque samples were collected from the same 13 volunteers on a
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separate day after abstaining from normal oral hygiene and consumption of food or drinks for the
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previous 12 hours. A Hayman style micro spatula (Thermo Fisher Scientific Inc., Waltham, 5
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MA) was used to scrape the tooth surface to collect supragingival and marginal gingival plaque
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samples from buccal surfaces of the maxillary and mandibular anterior ten teeth, i.e. second
110
premolar to second premolar. The collected plaque was weighed immediately and centrifuged at
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12000 xg for 30 min at 4 °C. The pellet and plaque fluid were then separated, weighed, and
112
treated as previously described for saliva samples.
113
Total Protein Analysis/Turbidity of Plaque suspension
114
Total protein was measured using Bradford reagent (Bio-Rad, Hercules, CA). Turbidity was
115
measured at 600 nm using the same instrument.
116
GH Activities
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GH activities were measured separately for each individual using black 96-well microtiter plates
118
(BD Bioscience). Following 1 hour incubation at 37 °C, sample reactants (pellets and
119
supernatants from in vitro biofilm, saliva, and plaque in TES buffer) were sonicated and
120
vortexed, and diluted 10 fold with TES buffer. This dilution factor was determined based on
121
preliminary experiment to determine zero-order region. 50 μL aliquots of diluted reactants were
122
added to 20 μL of 4-methylumbelliferone (4MU)-substrate solution in 100 mM citric acid-
123
Na2HPO4 buffer (pH 5.4) to a final concentration of 4 μM. Each well contained one of the
124
following 4MU linked glycosides as substrates; 4MU-α-D-NAc-neuraminic acid, 4MU-α-L-
125
fucoside, 4MU- β-D-glucoside, 4MU-β-D-galacatoside, 4MU-NAc-β-D-glucosaminide, 4MU-
126
NAc-β-D-galactosaminide (Sigma-Aldrich).
127
previously detected in oral specimens (13). Plates were then incubated for 1hour at 37 °C before
These substrates were selected based on GH
6
128
the reaction was quenched with 130 μL of 500 mM Na2CO3-NaHCO3 buffer (pH 10.3).
129
Fluorescence was measured using Victor X3 multilabel reader (ex/em = 355/460 nm)
130
(PerkinElmer, Waltham, MA). Two negative controls were used, namely 20 μL citric acid
131
Na2HPO4 buffer (4MU substrate blank) and 50 μL TES buffer (reactant blank). The positive
132
control contained 20 μL of authentic β-glucosidase solution from almonds (Sigma-Aldrich).
133
Known concentrations of 4MU solutions were used to plot a standard curve. The detection limit
134
was 10 nM (signal-to-noise ratio >3) and the range of linearity was 62.5 nM – 4 μM. All assays
135
were run in triplicate and data were expressed in terms of unit activity per mg of protein after
136
adjusting the dilution factors (unit defined as release of 1 μmol 4MU per minute).
137
quantitative Polymerase Chain Reaction (qPCR) Analysis
138
The
139
Fusobacterium nucleatum,
140
Streptococcus gordonii,
141
Treponema denticola.
142
(QIAGEN Inc., Valencia CA), and analyzed by qPCR analyses using CFX-96 Real-Time System
143
coupled with C1000 Thermal cycler (BioRad) in accordance to the manufacturers’ instruction.
144
See Table S2 for the primer sequences.
145
Statistical Analysis
146
All statistical analyses were performed using Microsoft Excel. The statistical significance for
147
GH activities was estimated for each enzyme at a 95% confidence interval using Welch’s t-test
following
species
were
tested:
Aggregatibacter actinomycetemcomitans,
Porphyromonas gingivalis, Streptococcus mutans,
Prevotella intermedia, and
Streptococcus salivarius,
16S DNA was extracted from the cellular pellets using QIAcube
7
148
for in vitro results and 2-way ANOVA for ex vivo results. The statistical significance for qPCR
149
results was estimated by paired t-test at a 95% confidence interval.
150 151
RESULTS
152
In vitro biofilm GH activities
153
Overall, higher GH activities were found in spent mucin media supernatant except for
154
glucosidase at 24 and 48 hour time points, as well as fucosidase at 24 hour time point (Fig. 1 and
155
Table 1). These two enzymes showed continuous increase in their activities for 72 hours
156
whereas the other four GH activities peaked at 48 hour. At 72 hour, fucosidase, sialidase, and
157
glucosaminidase were found at higher activity in supernatant than in cell bound form (Table 1).
158
Glucosaminidase activity was found to be the highest for both secreted and cell bound form
159
among the 6 measured GH, followed by galactosaminidase activity.
160
Salivary GH activities
161
Saliva samples, 4.0 mL from each donor, were centrifuged to give cellular and cell-free
162
supernatant. The average total amounts of cellular pellet and supernatant were 141.3 mg and
163
3815.1 mg, respectively. The average total protein concentrations were 713.58 μg/mL for the
164
supernatant and 215.40 μg/mL for cellular pellet resuspension, respectively.
165
turbidities (OD600) were 0.012 for supernatant and 1.027 for cellular pellet. The removal of
166
cellular material from supernatant was ensured by filter sterilization. Both cellular suspension
167
and cell-free supernatant were assayed for GH activities (Fig. 2).
The average
8
168
The activity of β-glucosaminidase was found to be the highest of all in both pellet (cell-
169
bound) and supernatant (secreted) in the saliva sample. Secreted fucosidase, hexosaminidase,
170
and sialidase were found present in supernatant while no activity was detected for galactosidase
171
or glucosidase in supernatant. Secreted fucosidase activity level was equivalent with that of cell-
172
bound form.
173
Overnight Plaque GH activities
174
Overnight grown biofilm samples were collected from the same donors and separated into
175
cellular pellet and cell-free supernatant by centrifugation immediately after collection. The total
176
amount of cellular pellet and supernatant were 70.66 mg and 30.99 mg respectively. The total
177
protein concentrations were 365.40 μg/mL for the supernatant and 278.13 μg/mL for cellular
178
pellet resuspension. The turbidity values (OD600) were 0.018 for supernatant and 1.078 for
179
cellular pellet.
180
sterilization. Both cellular suspension and cell-free supernatant were assayed for glycosidase
181
activities (Fig. 3).
The removal of cellular material from supernatant was ensured by filter
182
The overall enzymatic activities for all six enzymes were higher in overnight plaque by
183
10-50 fold compared to those in saliva. All six enzymes were present in both cell-bound and
184
secreted forms, including hexosidases the secreted form of which was absent in saliva. However
185
the activity ratio of secreted form to cell bound form were lower in fucosidase, compared to that
186
in saliva. In contrast to saliva sample, the secreted forms were observed for galactosidase and
187
glucosidase in overnight plaque. Indeed, the highest activity observed in the secreted form was
188
that of glucosidase which was more than a third of the activity of its cell bound counterpart. 9
189
Two way ANOVA showed the mean activities were statistically different at α=0.05
190
across four ex vivo sample groups for all enzymes.
191
qPCR Analysis
192
All species showed 1-4 log increase in overnight plaque compared to saliva, except for
193
S. salivarius which showed slight decrease (Fig. 4). The total numbers of 16S gene copy were
194
1.90 x 106 for saliva and 8.41 x 107 for plaque, respectively. In saliva sample the two most
195
abundant species detected, S. salivarius and F. nucleatum accounted for 98.3 and 1.1% of the
196
total of 8 measured species respectively. In plaque sample the three most abundant species,
197
namely F. nucleatum, S. gordonii, and S. mutans accounted for 50.3, 24.8, and 18.2%,
198
respectively. S. gordonii showed the highest increase in numbers to 105 copies in biofilm.
199
Paired t-test showed the number of DNA copies for all species are significantly different between
200
saliva and biofilm except for S. salivarius.
201 202
DISCUSSION
203
In vitro biofilm GH activity levels
204
Our results supported the first hypothesis that higher activities of GH were observed in biofilm
205
grown in mucin media where the only carbon source available was complex carbohydrate. This
206
is in agreement with previous reports in which single species Streptococci showed upregulation
207
of the GH when fermented in absence of simple carbon sources (4, 5) The activities of four out
208
of six enzymes peaked at 48 hours in agreement with a previous study where GH of S. gordonii
209
culture were down regulated during exponential growth phase compared to the stationary phase 10
210
(4). This would suggest that at least some species of oral bacteria are able to shift from a
211
preferred metabolite (glucose) to a more complex carbohydrate (mucin) when conditions require.
212
Ex vivo GH activity levels in overnight plaque and saliva
213
The results agreed with the second hypothesis in which overall higher activities of GH occurred
214
in biofilm samples in comparison to saliva samples. While the sample size is small in this study,
215
these results are in line with what have been reported previously on microbiota in saliva and
216
dental plaque: the dental plaque environment favours organisms that are able to metabolise
217
complex organic molecules (14, 15).
218
Bacteria with the largest numbers of GH genes are found to be most prevalent in dental
219
plaque and this appears to correlate with higher GH activity. Among the measured
220
microorganisms, S. gordonii and P. gingivalis harbour the largest number of GH coding
221
sequences as well as the most types of GH enzymes.
222
Pr. intermedia and Tr. denticola have genes for β-N-acetyl-hexosamindiase (GH20) and α-
223
fucosidase (GH29). On the other hand, the most abundant GHs in S. salivarius, which accounted
224
for the majority of salivary species, are α-amylase, lysozyme, and sucrase (Table S1). Our
225
results cannot determine what percentage of the cell-bound GH activities in saliva was derived
226
from bacteria; however the enzymes in host cells are expressed at low levels and are associated
227
primarily with intra-cellular metabolism, and hence are not excreted to a significant extent.
228
GH activity profiles between secreted and cell bound forms
These organisms, along with
11
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Beside overall enzyme activity levels, the results also showed that secretion patterns of GHs
230
differ in the conditions where carbon source is limited to complex carbohydrates. Our results are
231
in line with previous reports in that 1. all six GHs were present in saliva of healthy adults (16,
232
17); and 2. β-D-galactosidase activity was absent in salivary supernatant while those of β-D-
233
hexosaminidase and sialidase were present (18).
234
activities were consistently the highest among the measured GH in our samples, both in vitro and
235
ex vivo. β-N-Ac-glucosaminidase and glucosidase have been reported as the predominant GHs
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in healthy human dental plaque (13). The presence of both cell-bound and secreted forms of
237
sialidase is also in agreement with previous reports (19, 20).
The cell bound form of glucosaminidase
238
Both fucosidase and sialidase were present in secreted form in saliva. Although secreted
239
form of sialidase has been reported, this is the first report on secreted fucosidase to the authors’
240
best knowledge. The presence of secreted forms of GH, especially fucosidase and sialidase, can
241
be explained by the modification of terminal glycoside moiety to provide a better scaffold for
242
bacterial adhesion (21, 22).
243
activities in saliva suggests that alternative hexose sugar production in response to external
244
glycosides is not significantly induced. Our in vitro experiment showed the onset of glucosidase
245
activity in mucin media occurred at a later time point compared to other 5 GH activities. The
246
presence of secreted hexosidases in biofilm is initially counterintuitive, because the sugars
247
produced extracellularly are considered as the preferred carbon source for microorganisms and
248
will be available for other species in the biofilm. However since the trigger for production of
249
GH will be the energy requirements of the bacteria producing the enzyme, a more global benefit
250
to the biofilm may be an example of kin discrimination (23). Because there is limited diffusion
The complete lack of secreted glucosidase and galactosidase
12
251
in biofilm, the beneficiaries of such a secreted enzyme could be limited to those in close
252
proximity to the producer, which are more likely to be genetically related to each other (24). It is
253
noteworthy that secreted form of glucosidase was detected in biofilm grown in nutrient rich
254
sucrose media at an earlier time point than that grown in mucin media in our in vitro experiment.
255
Future study focusing on how secreted GHs are induced and distributed in biofilm may provide a
256
clue in how oral ecology maintains homeostasis despite constant perturbations in healthy
257
individuals.
258
Our results showed a reactive shift of glycoside hydrolase activities in microbial
259
community between planktonic and sessile phases in healthy adults. While further studies are
260
necessary to confirm our finding with a larger number of subjects, the marked increase in activity
261
of several GHs in plaque compared to saliva may serve as potential biomarkers for phenotypic
262
changes associated with dental plaque formation.
263 264
ACKNOWLEDGEMENTS
265
Authors thank Dr. Xiaoqun Mo, Dr. Panagiota Tsatsos, and Ms. Lisa Tran at Wm. Wrigley Jr.
266
Co. for technical support.
267 268
REFERENCES
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1. 2. 3.
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Harty DWS, Mayo JA, Cook SL, Jacques NA. 2000. Environmental regulation of glycosidase and peptidase production by Streptococcus gordonii FSS2. Microbiology 146:1923-1931. Homer KA, Whiley RA, Beighton D. 1994. Production of specific glycosidase activities by Streptococcus intermedius strain UNS35 grown in the presence of mucin. J Med Microbiol 41:184-190. Bradshaw DJ, Homer KA, Marsh PD, Beighton D. 1994. Metabolic cooperation in oral microbial communities during growth on mucin. Microbiology 140 (Pt 12):34073412. Bowden GH, Li YH. 1997. Nutritional influences on biofilm development. Adv Dent Res 11:81-99. Renzi F, Manfredi P, Dol M, Fu J, Vincent S, Cornelis GR. 2015. Glycan-Foraging Systems Reveal the Adaptation of Capnocytophaga canimorsus to the Dog Mouth. mBio 6:e02507-02514. Stoodley P, Wefel J, Gieseke A, Debeer D, von Ohle C. 2008. Biofilm plaque and hydrodynamic effects on mass transfer, fluoride delivery and caries. J Am Dent Assoc 139:1182-1190. McLean JS, Ona ON, Majors PD. 2008. Correlated biofilm imaging, transport and metabolism measurements via combined nuclear magnetic resonance and confocal microscopy. ISME J 2:121-131. Beyenal H, Lewandowski Z. 2002. Internal and external mass transfer in biofilms grown at various flow velocities. Biotechnol Prog 18:55-61. Tian Y, He X, Torralba M, Yooseph S, Nelson KE, Lux R, McLean JS, Yu G, Shi W. 2010. Using DGGE profiling to develop a novel culture medium suitable for oral microbial communities. Mol Oral Microbiol 25:357-367. Wickström C, Svensäter G. 2008. Salivary gel-forming mucin MUC5B--a nutrient for dental plaque bacteria. Oral Microbiol Immunol 23:177-182. Keijser BJF, Zaura E, Huse SM, van der Vossen JMBM, Schuren FHJ, Montijn RC, ten Cate JM, Crielaard W. 2008. Pyrosequencing analysis of the Oral Microflora of healthy adults. J Dent Res 87:1016-1020. Cantarel BL, Lombard V, Henrissat B. 2012. Complex Carbohydrate Utilization by the Healthy Human Microbiome. PLoS One 7:e28742. Knas M, Choromanska M, Karaszewska K, Dudzik D, Waszkiel D, BorzymKluczyk M, Zaniewska A, Zwierz K. 2007. Activity of lysosomal exoglycosidases in saliva of patients with HIV infection. Adv Med Sci 52:186-190. Waszkiewicz N, Szajda SD, Jankowska A, Waszkiewicz M, Kepka A, Konarzewska B, Szulc A, Snarska J, Zwierz K. 2009. Catabolism of salivary glycoconjugates in acute ethanol intoxication. Med Sci Monit 15:CR413-417. Quinn MO, Miller VE, Dal Nogare AR. 1994. Increased salivary exoglycosidase activity during critical illness. Am J Respir Crit Care Med 150:179-183. Corfield T. 1992. Bacterial sialidases--roles in pathogenicity and nutrition. Glycobiology 2:509-521. 14
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328
15
329
Fig. 1: Relative in vitro biofilm GH activities. Overall activities were higher in mucin media (solid
330
lines) than in sucrose media (dotted lines) with exceptions in F: α-L-fucosidase at 24 hour supernatant and G: β-D-
331 332
glucosidase at 24 and 48 hours supernatant samples. The other enzymes are coded as, S: β-D-NAc-sialidase, Ga: β-
333
*: p < 0.05 and **: p < 0.01. The scales are not equal for all enzymes. See Table 1 for the values of enzymatic
334
activities.
D-galactosidase, GN: β-D-NAc-glucosaminidase, and GaN: β-D-NAc-galactosaminidase. Asterisk marks denote as
16
335
Fig. 2: Salivary GH activities. Open bars represent the activities in cell-bound enzymes whereas the filled
336
bars represent those in cell-free supernatant. The highest activity was found for cell-bound glucosaminidase.
337 338 339
Fucosidase was found to have the overall lowest activity. No secreted glucosidase or galactosidase activities were detected in saliva supernatant. The values are the average of triplicated measures from 13 individuals.
17
340 341
Fig. 3: Biofilm GH activities. Open bars represent the activities in cell-bound enzymes whereas the filled
342
supernatant. Glucosidase showed the highest activity in supernatant. The ratios of activities between secreted and
343
cell-bound forms were higher than saliva sample except for fucosidase. The values are the average of triplicated
344 345
measures from 13 individuals.
bars represent those in supernatant. Unlike saliva sample, glucosidase and galactosidase activities were found in
18
346 347
Fig. 4: qPCR analysis. The solid bars show the number of DNA copies found in the saliva sample whereas the
348
A. actinomycetemcomitans and S. gordonii in saliva sample. All measured species showed statistical difference
349
between samples except for S. salivarius
open bars show those in the biofilm sample. There were no measureable copies of DNA detected for
19
Table 1: in vitro GH activities A: 24 h supernatant β-D-N-Ac-sialidase α-L-Fucosidase β-D-Glucosidase β-D-Galactosidase β-D-N-Ac-Glucosaminidase β-D-N-Ac-Galactosaminidase
Mucin media
α-L-Fucosidase β-D-Glucosidase β-D-Galactosidase β-D-N-Ac-Glucosaminidase β-D-N-Ac-Galactosaminidase
Min
Max
Avg
SD
Min
258.69
58.4
192.24
365.49
101.61
19.1
66.65
123.23
1 2
Extracellular Glycoside Hydrolase Activities in the Human Oral Cavity
3
Taichi Inui,a# Lauren C. Walker,b§ Michael W.J. Dodds,a A. Bryan Hanleyc
4 5 6
Wm. Wrigley Jr. Company, Chicago, Illinois 60642, USAa; School of Chemistry, University of Edinburgh, Edinburgh, Scotland, UKb, The Knowledge Transfer Network, The Roslin Institute,
7 8 9 10 11 12 13 14 15 16 17 18
Midlothian, Scotland, UKc
19 20 21 22
Keywords: Oral Ecology, glycosidase, extracellular enzyme, ectoenzyme
Running Head: Glycoside Hydrolases in Oral Cavity #Address correspondence to Taichi Inui, [email protected] §Current address Anheuser-Busch InBev, Edinburgh, Scotland, UK Supplementary data: Following supplementary data may be found in the online version of this article: Table S1. Known glycoside hydrolase families among the tested oral micro-organisms in this study Table S2. Primers used for qPCR experiment
1
23
Abstract
24
Carbohydrate availability shifts when bacteria attach to a surface and form biofilm. When
25
salivary planktonic bacteria form an oral biofilm, a variety of polysaccharides and glycoproteins
26
would be the primary carbon sources, however simple sugar availabilities would be limited due
27
to low diffusion from saliva to biofilm. We hypothesized bacterial glycoside hydrolase (GH)
28
activities would be higher in a biofilm compared to saliva in order to maintain metabolism in a
29
low sugar–high glycoprotein environment. Salivary bacteria from 13 healthy individuals were
30
used to grow in vitro biofilm using two separate media, one with sucrose and the other limiting
31
carbon sources to a complex carbohydrate. All six GHs measured showed higher in vitro
32
activities when grown in the media with complex carbohydrate being the sole carbon source. We
33
then collected saliva and overnight dental plaque samples from the same individuals and
34
measured ex vivo activities for the same six enzymes to determine how oral microbial utilization
35
of glycoconjugates shifts between the planktonic phase in saliva and the biofilm phase in
36
overnight dental plaque. Overall higher GH activities were observed in plaque samples, in
37
agreement with in vitro observation. A similar pattern was observed in GH activity profiles
38
between in vitro and ex vivo data. 16S DNA analysis showed that plaque sample had a higher
39
abundance of microorganisms with larger number of GH gene sequences. These results suggest
40
differences in sugar catabolism between the oral bacteria located in the biofilm compared to
41
those in saliva.
42
2
43
INTRODUCTION
44
The oral cavity is a dynamic environment. For instance both salivary flow and the level of
45
exogenous dietary carbon sources increase during ingestion of food whereas both are at a
46
minimum during sleep. Therefore the oral microbiota may be influenced by alterations in the
47
host environment, especially by changes in nutrient supply, local pH, and redox potential (1).
48
One notable example is that the microbial community becomes more acidogenic in the presence
49
of sugar (2). Endogenous salivary glycoconjugates become the most significant carbon source
50
for oral microorganisms when an exogenous dietary carbon source is scarce. Under those
51
circumstances Carbohydrate-Active enZymes (CAZymes) play an important role in the
52
adaptation of microorganisms (3-6). These CAZymes include glycosyltransferases and glycoside
53
hydrolases (GHs). The type and availability of carbon sources also influence the development of
54
biofilm at stages throughout the biofilm formation as microorganisms utilize the available carbon
55
sources as nutrients, as building blocks for extracellular polysaccharide production, and as the
56
point of attachment (7).
57
carbohydrates by GH enzymes is one mechanism whereby oral microbes adapt to different
58
carbon sources between the salivary planktonic phase, where exogenous simple sugars are
59
generally more available, and the oral biofilm phase where, in the absence of exogenous simple
60
sugars, several alternative, more complex endogenous salivary glycoconjugates are the principal
61
carbon source (8-11).
62 63 64
We speculated that the breakdown of complex, endogenous
Our hypotheses for the present study were that 1. GH activities are higher under conditions where the primary carbon sources are limited to complex glycan conjugates. 3
65
2. Levels of GH enzymes would be higher in dental plaque than in saliva due to the shift in
66
microbial population between saliva and plaque.
67
Here we report the GH levels between conditions where carbon source availabilities
68
include simple sugars (saliva) and limited to complex glycoconjugates (dental plaque). Our first
69
experiment was to test if carbohydrate availability will influence GH activities in in vitro oral
70
biofilm, followed by ex vivo experiment in which GH activities were measured in saliva and
71
overnight dental plaque. In both in vitro and ex vivo experiments, the samples were separated
72
into cell-free supernatant and cellular pellets by centrifuge in order to monitor the enzyme
73
activities that are secreted and those on cell-surfaces.
74 75
MATERIALS AND METHODS
76
In vitro Biofilm Growth
77
Gum base stimulated saliva samples were collected from 13 healthy volunteers (7 females and 6
78
males, age 24 – 45) at 9 am. Subjects were in good general health based on medical history and
79
not being treated for medical or dental condition or taking antibiotics or other medications,
80
which might influence salivary secretion. Subjects refrained from eating, drinking, and oral
81
hygiene for at least 2 hours prior to the collection.
82
immediately treated with phenylmethanesulfonylfluoride (PMSF, final concentration 1 mM,
83
Sigma-Aldrich, St. Louis, MO). Samples were then centrifuged at 12000 xg for 30 min at 4 °C.
84
The pellet was suspended in phosphate buffered saline (pH 7.4) to a final OD600 of 0.600. The
85
pellet suspension was divided and inoculated for biofilm growth in two different media in
86
parallel. Hydroxyapatite disks (Clarkson Chromatography Products, South Williamsport, PA)
Samples were collected on ice and
4
87
were coated for 1 hour at room temperature in 24 well plates (BD Bioscience, Franklin Lakes,
88
NJ) with pooled supernatant to form salivary pellicle. Pellet suspensions from each individual
89
were then inoculated separately onto disks and grown for 72 hours at 37 °C in either peptone-
90
yeast base media containing 5% sucrose (sucrose media) or 1.25 % hog gastric mucin in
91
phosphate buffered saline (mucin media) (12). The sucrose media has been shown to be capable
92
of growing a more diversified community with a microbial profile closer to that of the salivary
93
microbiota when inoculated with pooled human saliva. On the other hand, mucin media would
94
represent the environment where the carbon source is limited to complex glycoconjugates. The
95
media were refreshed every 24 hours. The spent media was centrifuged at 12000 xg for 30 min at
96
4 °C after which the obtained supernatant was filter sterilized for assay of GH activity. After 72
97
hour growth the biofilm was collected and weighed. It was then centrifuged to remove residual
98
media and resuspended in 50mM N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid
99
(TES) buffer, pH 7.4 to a final concentration of 5.0 mg pellet mL-1.
100
Ex vivo Sample Collection
101
Saliva collection and processing were done the same way to the point of centrifugation from the
102
same volunteers as the above. The supernatant and the pellet were weighed, followed by having
103
the pellet resuspended in 50 mM TES buffer to a final concentration of 5.0 mg pellet mL-1. Both
104
supernatant of pellet suspension were subjected for further analyses.
105
Overnight grown plaque samples were collected from the same 13 volunteers on a
106
separate day after abstaining from normal oral hygiene and consumption of food or drinks for the
107
previous 12 hours. A Hayman style micro spatula (Thermo Fisher Scientific Inc., Waltham, 5
108
MA) was used to scrape the tooth surface to collect supragingival and marginal gingival plaque
109
samples from buccal surfaces of the maxillary and mandibular anterior ten teeth, i.e. second
110
premolar to second premolar. The collected plaque was weighed immediately and centrifuged at
111
12000 xg for 30 min at 4 °C. The pellet and plaque fluid were then separated, weighed, and
112
treated as previously described for saliva samples.
113
Total Protein Analysis/Turbidity of Plaque suspension
114
Total protein was measured using Bradford reagent (Bio-Rad, Hercules, CA). Turbidity was
115
measured at 600 nm using the same instrument.
116
GH Activities
117
GH activities were measured separately for each individual using black 96-well microtiter plates
118
(BD Bioscience). Following 1 hour incubation at 37 °C, sample reactants (pellets and
119
supernatants from in vitro biofilm, saliva, and plaque in TES buffer) were sonicated and
120
vortexed, and diluted 10 fold with TES buffer. This dilution factor was determined based on
121
preliminary experiment to determine zero-order region. 50 μL aliquots of diluted reactants were
122
added to 20 μL of 4-methylumbelliferone (4MU)-substrate solution in 100 mM citric acid-
123
Na2HPO4 buffer (pH 5.4) to a final concentration of 4 μM. Each well contained one of the
124
following 4MU linked glycosides as substrates; 4MU-α-D-NAc-neuraminic acid, 4MU-α-L-
125
fucoside, 4MU- β-D-glucoside, 4MU-β-D-galacatoside, 4MU-NAc-β-D-glucosaminide, 4MU-
126
NAc-β-D-galactosaminide (Sigma-Aldrich).
127
previously detected in oral specimens (13). Plates were then incubated for 1hour at 37 °C before
These substrates were selected based on GH
6
128
the reaction was quenched with 130 μL of 500 mM Na2CO3-NaHCO3 buffer (pH 10.3).
129
Fluorescence was measured using Victor X3 multilabel reader (ex/em = 355/460 nm)
130
(PerkinElmer, Waltham, MA). Two negative controls were used, namely 20 μL citric acid
131
Na2HPO4 buffer (4MU substrate blank) and 50 μL TES buffer (reactant blank). The positive
132
control contained 20 μL of authentic β-glucosidase solution from almonds (Sigma-Aldrich).
133
Known concentrations of 4MU solutions were used to plot a standard curve. The detection limit
134
was 10 nM (signal-to-noise ratio >3) and the range of linearity was 62.5 nM – 4 μM. All assays
135
were run in triplicate and data were expressed in terms of unit activity per mg of protein after
136
adjusting the dilution factors (unit defined as release of 1 μmol 4MU per minute).
137
quantitative Polymerase Chain Reaction (qPCR) Analysis
138
The
139
Fusobacterium nucleatum,
140
Streptococcus gordonii,
141
Treponema denticola.
142
(QIAGEN Inc., Valencia CA), and analyzed by qPCR analyses using CFX-96 Real-Time System
143
coupled with C1000 Thermal cycler (BioRad) in accordance to the manufacturers’ instruction.
144
See Table S2 for the primer sequences.
145
Statistical Analysis
146
All statistical analyses were performed using Microsoft Excel. The statistical significance for
147
GH activities was estimated for each enzyme at a 95% confidence interval using Welch’s t-test
following
species
were
tested:
Aggregatibacter actinomycetemcomitans,
Porphyromonas gingivalis, Streptococcus mutans,
Prevotella intermedia, and
Streptococcus salivarius,
16S DNA was extracted from the cellular pellets using QIAcube
7
148
for in vitro results and 2-way ANOVA for ex vivo results. The statistical significance for qPCR
149
results was estimated by paired t-test at a 95% confidence interval.
150 151
RESULTS
152
In vitro biofilm GH activities
153
Overall, higher GH activities were found in spent mucin media supernatant except for
154
glucosidase at 24 and 48 hour time points, as well as fucosidase at 24 hour time point (Fig. 1 and
155
Table 1). These two enzymes showed continuous increase in their activities for 72 hours
156
whereas the other four GH activities peaked at 48 hour. At 72 hour, fucosidase, sialidase, and
157
glucosaminidase were found at higher activity in supernatant than in cell bound form (Table 1).
158
Glucosaminidase activity was found to be the highest for both secreted and cell bound form
159
among the 6 measured GH, followed by galactosaminidase activity.
160
Salivary GH activities
161
Saliva samples, 4.0 mL from each donor, were centrifuged to give cellular and cell-free
162
supernatant. The average total amounts of cellular pellet and supernatant were 141.3 mg and
163
3815.1 mg, respectively. The average total protein concentrations were 713.58 μg/mL for the
164
supernatant and 215.40 μg/mL for cellular pellet resuspension, respectively.
165
turbidities (OD600) were 0.012 for supernatant and 1.027 for cellular pellet. The removal of
166
cellular material from supernatant was ensured by filter sterilization. Both cellular suspension
167
and cell-free supernatant were assayed for GH activities (Fig. 2).
The average
8
168
The activity of β-glucosaminidase was found to be the highest of all in both pellet (cell-
169
bound) and supernatant (secreted) in the saliva sample. Secreted fucosidase, hexosaminidase,
170
and sialidase were found present in supernatant while no activity was detected for galactosidase
171
or glucosidase in supernatant. Secreted fucosidase activity level was equivalent with that of cell-
172
bound form.
173
Overnight Plaque GH activities
174
Overnight grown biofilm samples were collected from the same donors and separated into
175
cellular pellet and cell-free supernatant by centrifugation immediately after collection. The total
176
amount of cellular pellet and supernatant were 70.66 mg and 30.99 mg respectively. The total
177
protein concentrations were 365.40 μg/mL for the supernatant and 278.13 μg/mL for cellular
178
pellet resuspension. The turbidity values (OD600) were 0.018 for supernatant and 1.078 for
179
cellular pellet.
180
sterilization. Both cellular suspension and cell-free supernatant were assayed for glycosidase
181
activities (Fig. 3).
The removal of cellular material from supernatant was ensured by filter
182
The overall enzymatic activities for all six enzymes were higher in overnight plaque by
183
10-50 fold compared to those in saliva. All six enzymes were present in both cell-bound and
184
secreted forms, including hexosidases the secreted form of which was absent in saliva. However
185
the activity ratio of secreted form to cell bound form were lower in fucosidase, compared to that
186
in saliva. In contrast to saliva sample, the secreted forms were observed for galactosidase and
187
glucosidase in overnight plaque. Indeed, the highest activity observed in the secreted form was
188
that of glucosidase which was more than a third of the activity of its cell bound counterpart. 9
189
Two way ANOVA showed the mean activities were statistically different at α=0.05
190
across four ex vivo sample groups for all enzymes.
191
qPCR Analysis
192
All species showed 1-4 log increase in overnight plaque compared to saliva, except for
193
S. salivarius which showed slight decrease (Fig. 4). The total numbers of 16S gene copy were
194
1.90 x 106 for saliva and 8.41 x 107 for plaque, respectively. In saliva sample the two most
195
abundant species detected, S. salivarius and F. nucleatum accounted for 98.3 and 1.1% of the
196
total of 8 measured species respectively. In plaque sample the three most abundant species,
197
namely F. nucleatum, S. gordonii, and S. mutans accounted for 50.3, 24.8, and 18.2%,
198
respectively. S. gordonii showed the highest increase in numbers to 105 copies in biofilm.
199
Paired t-test showed the number of DNA copies for all species are significantly different between
200
saliva and biofilm except for S. salivarius.
201 202
DISCUSSION
203
In vitro biofilm GH activity levels
204
Our results supported the first hypothesis that higher activities of GH were observed in biofilm
205
grown in mucin media where the only carbon source available was complex carbohydrate. This
206
is in agreement with previous reports in which single species Streptococci showed upregulation
207
of the GH when fermented in absence of simple carbon sources (4, 5) The activities of four out
208
of six enzymes peaked at 48 hours in agreement with a previous study where GH of S. gordonii
209
culture were down regulated during exponential growth phase compared to the stationary phase 10
210
(4). This would suggest that at least some species of oral bacteria are able to shift from a
211
preferred metabolite (glucose) to a more complex carbohydrate (mucin) when conditions require.
212
Ex vivo GH activity levels in overnight plaque and saliva
213
The results agreed with the second hypothesis in which overall higher activities of GH occurred
214
in biofilm samples in comparison to saliva samples. While the sample size is small in this study,
215
these results are in line with what have been reported previously on microbiota in saliva and
216
dental plaque: the dental plaque environment favours organisms that are able to metabolise
217
complex organic molecules (14, 15).
218
Bacteria with the largest numbers of GH genes are found to be most prevalent in dental
219
plaque and this appears to correlate with higher GH activity. Among the measured
220
microorganisms, S. gordonii and P. gingivalis harbour the largest number of GH coding
221
sequences as well as the most types of GH enzymes.
222
Pr. intermedia and Tr. denticola have genes for β-N-acetyl-hexosamindiase (GH20) and α-
223
fucosidase (GH29). On the other hand, the most abundant GHs in S. salivarius, which accounted
224
for the majority of salivary species, are α-amylase, lysozyme, and sucrase (Table S1). Our
225
results cannot determine what percentage of the cell-bound GH activities in saliva was derived
226
from bacteria; however the enzymes in host cells are expressed at low levels and are associated
227
primarily with intra-cellular metabolism, and hence are not excreted to a significant extent.
228
GH activity profiles between secreted and cell bound forms
These organisms, along with
11
229
Beside overall enzyme activity levels, the results also showed that secretion patterns of GHs
230
differ in the conditions where carbon source is limited to complex carbohydrates. Our results are
231
in line with previous reports in that 1. all six GHs were present in saliva of healthy adults (16,
232
17); and 2. β-D-galactosidase activity was absent in salivary supernatant while those of β-D-
233
hexosaminidase and sialidase were present (18).
234
activities were consistently the highest among the measured GH in our samples, both in vitro and
235
ex vivo. β-N-Ac-glucosaminidase and glucosidase have been reported as the predominant GHs
236
in healthy human dental plaque (13). The presence of both cell-bound and secreted forms of
237
sialidase is also in agreement with previous reports (19, 20).
The cell bound form of glucosaminidase
238
Both fucosidase and sialidase were present in secreted form in saliva. Although secreted
239
form of sialidase has been reported, this is the first report on secreted fucosidase to the authors’
240
best knowledge. The presence of secreted forms of GH, especially fucosidase and sialidase, can
241
be explained by the modification of terminal glycoside moiety to provide a better scaffold for
242
bacterial adhesion (21, 22).
243
activities in saliva suggests that alternative hexose sugar production in response to external
244
glycosides is not significantly induced. Our in vitro experiment showed the onset of glucosidase
245
activity in mucin media occurred at a later time point compared to other 5 GH activities. The
246
presence of secreted hexosidases in biofilm is initially counterintuitive, because the sugars
247
produced extracellularly are considered as the preferred carbon source for microorganisms and
248
will be available for other species in the biofilm. However since the trigger for production of
249
GH will be the energy requirements of the bacteria producing the enzyme, a more global benefit
250
to the biofilm may be an example of kin discrimination (23). Because there is limited diffusion
The complete lack of secreted glucosidase and galactosidase
12
251
in biofilm, the beneficiaries of such a secreted enzyme could be limited to those in close
252
proximity to the producer, which are more likely to be genetically related to each other (24). It is
253
noteworthy that secreted form of glucosidase was detected in biofilm grown in nutrient rich
254
sucrose media at an earlier time point than that grown in mucin media in our in vitro experiment.
255
Future study focusing on how secreted GHs are induced and distributed in biofilm may provide a
256
clue in how oral ecology maintains homeostasis despite constant perturbations in healthy
257
individuals.
258
Our results showed a reactive shift of glycoside hydrolase activities in microbial
259
community between planktonic and sessile phases in healthy adults. While further studies are
260
necessary to confirm our finding with a larger number of subjects, the marked increase in activity
261
of several GHs in plaque compared to saliva may serve as potential biomarkers for phenotypic
262
changes associated with dental plaque formation.
263 264
ACKNOWLEDGEMENTS
265
Authors thank Dr. Xiaoqun Mo, Dr. Panagiota Tsatsos, and Ms. Lisa Tran at Wm. Wrigley Jr.
266
Co. for technical support.
267 268
REFERENCES
269 270 271 272 273 274
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328
15
329
Fig. 1: Relative in vitro biofilm GH activities. Overall activities were higher in mucin media (solid
330
lines) than in sucrose media (dotted lines) with exceptions in F: α-L-fucosidase at 24 hour supernatant and G: β-D-
331 332
glucosidase at 24 and 48 hours supernatant samples. The other enzymes are coded as, S: β-D-NAc-sialidase, Ga: β-
333
*: p < 0.05 and **: p < 0.01. The scales are not equal for all enzymes. See Table 1 for the values of enzymatic
334
activities.
D-galactosidase, GN: β-D-NAc-glucosaminidase, and GaN: β-D-NAc-galactosaminidase. Asterisk marks denote as
16
335
Fig. 2: Salivary GH activities. Open bars represent the activities in cell-bound enzymes whereas the filled
336
bars represent those in cell-free supernatant. The highest activity was found for cell-bound glucosaminidase.
337 338 339
Fucosidase was found to have the overall lowest activity. No secreted glucosidase or galactosidase activities were detected in saliva supernatant. The values are the average of triplicated measures from 13 individuals.
17
340 341
Fig. 3: Biofilm GH activities. Open bars represent the activities in cell-bound enzymes whereas the filled
342
supernatant. Glucosidase showed the highest activity in supernatant. The ratios of activities between secreted and
343
cell-bound forms were higher than saliva sample except for fucosidase. The values are the average of triplicated
344 345
measures from 13 individuals.
bars represent those in supernatant. Unlike saliva sample, glucosidase and galactosidase activities were found in
18
346 347
Fig. 4: qPCR analysis. The solid bars show the number of DNA copies found in the saliva sample whereas the
348
A. actinomycetemcomitans and S. gordonii in saliva sample. All measured species showed statistical difference
349
between samples except for S. salivarius
open bars show those in the biofilm sample. There were no measureable copies of DNA detected for
19
Table 1: in vitro GH activities A: 24 h supernatant β-D-N-Ac-sialidase α-L-Fucosidase β-D-Glucosidase β-D-Galactosidase β-D-N-Ac-Glucosaminidase β-D-N-Ac-Galactosaminidase
Mucin media
α-L-Fucosidase β-D-Glucosidase β-D-Galactosidase β-D-N-Ac-Glucosaminidase β-D-N-Ac-Galactosaminidase
Min
Max
Avg
SD
Min
258.69
58.4
192.24
365.49
101.61
19.1
66.65
123.23
