Download Enzymic activity of salivary amylase when bound

FEMSMicrobiolo~Letters92 {1992)193-198
© 1992Federationof EuropeanMktobiologicalSocieties0378.1(D7/92/$05.00
Publishedby Elsevier
193
FEMSLE04848
Enzymic activity of salivary amylase when bound to the surface
of oral streptococci
Charles W.I. Douglas, Jason Heath and Justin P. Gwynn
Department of Oral Pathology. Universityof Sheffiehl, SheJ']ieht, UK
Received 16 December1991
Revisionreceived6 February 1992
Accepted7 FebruaryltJ92
Key words: Amylase; Streptococci; Saliva; Starch
1. SUMMARY
The enzymatic activity of salivary amylase
bound to the surface of several species of oral
streptococci was determined by the production of
acid from starch and by the degradation of maltotetraose to glucose in a coupled, spectrophotometric assay. Most strains able to bind amylase
exhibited functional enzyme on their surface and
produced acid from the products of amylolytic
degradation. These strains were unable to utilise
starch in the absence of salivary amylase. Two
strains failed to produce acid from starch, despite
the presence of functional salivary amylase, because they could not utilise maltose. Strains that
could not bind salivary amylase failed to produce
acid from starch. In no case was all the bound
salivary amylase active, and two strains of Streptococcus mitis which bound amylase did not exhibit any enzyme activity on their cell surface.
Correspondence to: C.W.I. Douglas, Department of Oral
Pathology,Schoolof ClinicalDentistry.ClaremontCrescent.
SheffieldSI0 2TA, UK.
The ability to bind amylase may confer a survival
advantage on oral bacteria which inhabit hosts
that consume diets containing starch.
2. INTRODUCTION
Certain species of oral streptococci are known
te bind salivary a-amylase to their cell surface
[ 1,2], This interaction appears to be largely species
specific [3,4], virtually irreversible under physiological conditions [1,2,5], and partly inhibited by
amylase substrates [2,5]. While the molecular
mechanisms of the interaction are not fully understood, the amylase receptor of one organism,
Streptococc'~s gordonii strain challis, has been
partially characterised [5]. However, very little is
known of the ecological significance of amylase
binding among oral bacteria.
The binding of salivary amylase to organisms
could be beneficial in several ways. It could, for
example, provide organisms with a mechanism for
attaching to the tooth surface. In vivo the tooth is
coated with a layer of adsorbed salivary proteins
(the acquired pellicle), including amylase, and so
194
binding to amylase in pellicle could provide one
mechanism for attaching to the tooth surface
[1,2,6]. However, such a mechanism seems unlikely given the high affinity salivary amylase in
solution has for its streptococcal receptor [1,2].
An alternative might be to assist cells in avoiding
the host defences. Coated cells may not appear
'foreign' to the host. Finally, amylase binding may
have a nutritive function, providing organisms
with the ability to utilise starch-related substrates
without requiring to synthesise their own amylase. Support for this hypothesis comes from observations that two strains of S. gordonii produce
acid from starch only when they have salivary
amylase bound to their cell surface [5,7]. Here we
extend these observations and show that not all
streptococci bind amylase in the same way.
3. MATERIALS AND METHODS
3. i. Bacteria and culture conditions
Eighteen strains e'. oral streptococci were used
in the study, representing the species S. gordonii
(NCTC 7868 (challis), Blackburn), S. sanguis
(NCTC 7863, SK96), 'S. crista' (CR3, CR311,
AKI, CC5a), 'S. pat:~sanguis" (MGH413, ATCC
15911), S. mitis (NCTC 12261, K208, OS51, OP51,
NCTC 10712) and S. oralis (CN3410, ATCC 981 i,
NCTC 7864). Organisms were maintained or,
blood agar at 4°C.
3.2. Salit'a
Whole saliva from three individuals was stimulated by chewing Parafilm (American Can) and
saliva was cleared by centrifugation at 27 000 × g
for 15 min at 8°C. Clarified saliva was used as the
source of amylase.
3.3. Coath)g with amylase
Bacteria were cultured in 20 ml of Brain Heart
Infusion broth (Oxoid) overnight at 37°C, washed
twice in 50 mM phosphate buffer, pl-I 6.5, and
adjusted to a cell density of approximately 5 × 10~
cells per ml. Ten ml of each cell suspension was
then centrifuged and the pellets were resuspended in 1(30/xl of clarified saliva. These mixtures were gently agitated at room temperature
for 30 min before washing the cells four times
with 135 mM KCI, pH 7.0, and resuspending in 1
ml of the same solution. Another 10 ml of each
cell suspension was similarly treated but with KCI
rather than saliva.
3. 4. Acid production
Acid production during incubation of the bacterial suspension at 37°C with various substrates
was monitored using a Corning 120 pH meter.
The pH of each suspension (1 mi) was followed
continuously for 5 min before addition of substrates and then for a further 10-rain period. The
final pH reached was subtracted from the mean
starting pH and the change in pH was converted
into ~tmol of H + by calculation. The main substrate used was potato starch (Sigma; boiled to
dissolve and adjusted to pH 7.0 with NaHCO 3,
final concentration 5 mg/ml), but in some experiments dextrin, maltoheptaose, maltotriose, maltose or glucose (Sigma; 2 mg/ml final concentration) was used. All substrates were dissolved in
KCI solution.
3.5. Amylase assay
Amylase activity was assayed at room temperature using a coupled assay kit (Sigma) with maitotetrao~e as amylase substrate, Amylase was
measured in 50-t~l aliquots of clarified saliva after
dilution (1:50 or 1 : 100) in phosphate buffer. For
amylase activity on bacterial surfaces, the cell
suspensions described in Section 3.3. were diluted
1:10 (final suspension approx. 2.5 × l0 s cells/ml)
for use and 50-p.I aliquots were added to 1 ml of
assay reagent. 'Uncoated' cells were used as controls. The enzyme reaction was followed continuously at 340 nm in a spectrophotometer and one
unit of amylase activity was defined as the amount
of enzyme which yielded one mmoi of NADH per
ml per rain under the test conditions.
3.6. Western blotting
in some experiments, cells were extracted with
500 #1 of 6 M urea after incubation with saliva
and washing. These extracts (25 tzl) were then
subjected to SDS polyacr.lamide gel electrophoresis and Western blotting [5] using antiamylase serum (Sigma; diluted 1:500) followed
195
by horseradish peroxidase-conjugated goat-antirabbit IgG (Sigma; diluted 1 : 1000).
4. RESULTS
4.1. Acid production
4.1.1. From starch. Previous work, using a simple screening procedure, had established that 12
of the strains tested were able to bind amylase
from saliva [3]. Of these 12 strains, 8 produced
significant amounts of acid from starch when they
had been coated with salivary amylase, whereas
no acid was produced when the organisms had
not been in contact with saliva (Table !). Four
strains, S. mitis NCTC 12261, OPS1 and K205
and 'S. crista' CC5a, failed to produce acid from
starch when coated with amylase. Of 5 strains
that were not able to bind amylase, none produced acid from starch.
Table I
Acid production from starch by cells coated with salivary
amylaseand by controlcells
pH Change"
With amyLse No amylase
Amylase binding strains
S, gordonii challis
S. gordonii Blackburn
39.5
45.4
02
0.1
"S. crista" CR3
"S. crista' CR311
43A
16.1)
0.3
11.2
'S. crista' CCSa
'S. ¢rista' AKI
"S. parasanguis" MGH413
0.2
21.8
46.6
0.1
0.6
0.4
S. mitis NCTC 10712
26,1
S, mitis NCTC 12261
0.2
S. mitis OP51
0.5
S. mitis OS51
18.8
S, mitis K208
0
Non-binding strains
S, sanguis NCTC 7863
0.4
S, sanguis SK96
0,3
'S. parasanguis' ATCC 1591 i 0.3
0,1
0
0
0.1
0
O.I
0. I
0.2
S, oralis NCTC7864
0,1
0.1
S, oralis ATCC 9811
S. oralis CN3410
0,4
0.4
0.1
0.1
pH change expressed as hydrogen ion conceutration (.amol
m l - I ) . Values are means of two measurements of pH
change during a 10-rain incubation with 0.5% starch at
37°C.
Table 2
Production of acid from malt(~e
Strain
pH change"
S. gordonii challis
S. sanguis NCTC 7863
"S. crista" CC5a
S. mitis NCTC 12261
S. mitis OP51
S. mills K208
39.7
21,5
3,1
16.4
0.6
I11.1
~' pH expressed as hydrogen ion concentration (~amol ml-t),
Values at,,: means of two measurements of pH change
during r; 10-rainincubationwith 2 mgml- i malto~ at 37~C.
4. L 2 From other substrates. S. gordonii challis
produced acid from dextrin and maltoheptaose,
but only when coated with amylase, whereas acid
was produced from maltose (Table 2) and from
mattotriose and glucose (data not shown) irrespc~.tive of the presence of amylase. Five additional strains were examined for their ability to
produce acid from maltose (Table 2). 'S. crista"
CCSa and S. mitis OP51 failed to produce acid
from maltose, explaining their lack of acid production from starch. $. mitis NCTC 12261 and S.
mitis K208 were able to utilise maltose readily
(Table 2), although they did not produce acid
from starch.
4.Z Amylase actit'io'
Clarified saliva from three donors contained a
mean of 68,86 + !.9 U/ml of amylase (mean of
three determinations each) and all but 0.02-0.08
U/ml of en~me was removed from the saliva by
incubation with amylase-binding streptococci
(data not shown). In no case was all of the amylase activity that had disappeared from the saliva
subsequently detected on the surface of the bacteria. The level of amylase activity present was
similar in the saliva from different donors, and
amylase was always in excess when coating cells.
Two S. mitis strains, NCTC 12261 and K208, did
not exhibit any cell-associated salivary amylase
activity (Table 3), while three other S. mitis strains
(NCTC 10712, O1151 and OS51) had substantial
levels of bound amylase activity. It was confirmed
that NCTC 12261 and K208 had bound salivary
amylase to their cell surface, by detection of the
196
Table 3
5. DISCUSSION
Amylase activity on cells after incubation in saliva
S. gordonii challis
S. gordonii challis
(heat killed)
"S. crista' CCSa
S. mitis NCTC 10712
S. mitis NCTC 12661
S. mitis K2.08
S. mitis OS51
S. mitis OP51
Amylase
(U ml- t) ,,
% Activity h
47.4
50.9
69.0
74.0
40,5
28.0
0
0
49.6
40.4
59.0
45.5
0
0
65.5
59.0
:' Units of amylase activity associated with cells after incubation in saliva.
i, Amylase activity associated with the bacteria, expressed as a
percentage of the amount of enzyme that had disappeared
from saliva during incubation with the bacteria.
enzyme immunologically in extracts (6 M urea) of
saliva-coated cells by Western blotting (Fig. 1).
However, the amount of amylase recovered from
cells by this method was low, particularly in the
case of NCTC 12261.
1
23
I
I
I
4
5
6 7
I
I
I
I
Fig. 1. Western blot showing amylase present in whole saliva
(lane 1), saliva after absorption with S. mitis strains K208
(lane 2) and NCTC 12261 (lane 5), an SDS extract of 'salivacoated' S. mitis strain K208 (lane 3J and NCTC 12661 (lane 6)
and a 6 M urea extract of similarly coated cells (K2.08. lane 4;
NCTC 1226i, lane 7). The blot was probed with anti-amylase
serum followed by horseradish peroxidase-conjugated goatanti-rabbit lgG.
The work described here shows that salivary
amylase is enzymatically active when bound to
the surface of oral streptococci and that the resultant amylolytic products of starch degradation
can be utilised by most organisms, with the subsequent production of acid. In contrast, strains of
oral streptococci that do not bind salivary amylase could not utilise starch appreciably, at least
within the time-scale of these experiments. This
confirms previous observations with two S. gordonii strains [5,7], and shows that several other
strains and species behave similarly.
The fact that certain streptococci can produce
acid from starch in the presence of salivary amylase in vitro might suggest that the phenomenon
could be a contributory factor to dental caries. It
is recognised that cooked starch and cooked flour
preparations can cause a pH drop in dental plaque
[9], perhaps partly by the mechanism described
here. However, despite much debate about the
cariogenicity of starch and starch-containing
foodstuffs, human studies have not implicated
starch as being significantly caries-promoting
[9,10].
Scannapieco et al. [7] reported that 90% of the
salivary amylase bound to S. gord~mii strain challis was cnzymatically active, while here we found
only 69%, using the same assay metho~t. This is a
higher level of activity than we have reported
previously (19%) but different assay methods were
used for this estimation. The enzyme assay used
here was not influenced by metabolic activity of
the cells, since heat-killed cells exhibited the same
amount of absorbed amylase activity as viable
cells (Table 3), but clearly the method employed
for assaying cell-bound amylase is an important
factor influencing the results obtained. Other
strains assayed for bound salivary amylase activity
showed varying levels of enzyme activity (45.565.6%) depending on the strain but none exhibited all of the enzyme which had been adsorbed
from saliva. Interestingly, 'S. crista" CC5a and S.
mitis OP51 showed a high level of cell-associated
salivary amylase activity although they did not
produce acid from starch, presumably because of
their relative inability to utilise maltose.
197
All of these data suggest that the bulk of the
salivary amylase molecules bind to the bacteria
via a portion of the enzyme other than its active
site. However, two strains of $. mitis (NCTC
12261 and K208) have been described here which
did not exhibit any cell associated amylase activity, despite having removed all of the enzyme
from a portion of saliva. It is possible that in
these cases the amylase is being degraded by the
bacteria, but this cannot be the full explanation
because some of the enzyme can be recovered
from the cells by extracting with 6 M urea. Alternatively, NCTC 12261 and K208 might bind amylase via its active site or the amylase molecule
might have undergone a conformational change
upcn binding to the bacteria, rendering it inactive. All of these possibilities require further investigation.
Since, for most strains, the binding of salivary
amylase to their cell surface confers on them the
ability to uti!ise starch, it seems reasonable to
speculate that these organisms have evolved a
mechanism for utilising a nutrient, without having
to synthesise their own hydrolytic enzyme, simply
taking advantage of the availability of a host's
secreted enzyme. However, it is puzzling why
some organisms (viz. S. mitis NCTC 12261 and
"11"~O~,....
o1..o uld bind the enzyme and then not take
K2,,u,
advantage of its activity. Of course, at present it
is not known whether organisms with the same
amylase.binding characteristics as NCTC 12261
and K208 exist naturally or whether they are
variants that have been accidentally selected by
laboratory culture. An alternative explanation for
the amylase-binding phenotype is that amylase
.
could have an antimicrobial function in the oral
cavity. It is known that certain pathogenic species
of bacteria are inhibited by a-amylase, or by its
action on starch [11,12], but there are no data
available as yet concerning similar effects on
membexs of the resident oral flora, Finally, it is
possible that bacteria coated with up to 30000
molecules of salivary amylase [2] may be relatively
'hidden' from the host's immune system, but
clearly further work will be required to establish
the true ecological significance of the amylasebinding phenotype.
REFERENCES
[l] Douglas, C.W.I. 0983) Arch. Oral Biol. 28, 567-573.
[2] Scannapieco, F.A., Bergey, E.J., Reddy, M.S. and Levine,
MJ. (1989) Infect. lmmun. 57, 2853-2863.
[31 Douglas, C.W.I., Pea~, A.A. and While)', R.A. (1990)
FEMS Microbiol. Lett. 66, 193-198.
14] Kilian, M. and Nyvad, B. 0990) J, Clin. Microbiol. 28,
2576-977.
[5] Douglas. C,W.I. (1990)J. Dent. Res. 69, 1746-t752.
[6] Orstavik, D. and Kraus, F.W. (1973) J. Oral Pathol. "~'~
68-76.
[7] Scannapicco, F,A., BhandD', K., Ramasubbu, N. and
Levine, M.J. (1990) Biochem, Biophys, Res. Comm, 173,
1109-1115.
[8] Mormann, J.E. and Muhlemann, H.R. (19811 Caries Res.
15, 166-175.
19] Gustafsson, B.E., Quesnel, C.E,, Lanke, S.L., Lundvist.
C.. Grahnen, H.. Bonow, B.E. and Krass¢. B. (1954)
Acta. Odont, Scand, I I, 232-364,
{10] Scheinin, A., Makkinen. K.K. and Ylitalo, K. (1975) Acta.
Odont. Stand. 33 (suppl. 70), 67-104.
~| I] Mellersh, A.. Clark. A, and Hafiz, S. (1979) Br. J. Vener.
Dis. 55, 21.)-23,
[121 Bortner, C.A., Miller, R.D. and Arnold, R.A. (1983)
Infect. Immun. 41, 44-49.