Download Some effects of Fluoride on the IgA Protease of the Oral Bacterium

3 12s Biochemical Society Transactions ( 1 996) 24
Some effects of Fluoride on the IgA Protease of the Oral
Bacterium Streptocaccns sanguis.
ROBERT DUGUID' and BERNARD SENIOR
Departments of Conservative Dentistry' and Medical
Microbiology, University of Dundee, DDl4HR. Scotland, U.K.
The importance of Immunoglobulin A (IgA) in conferring
specific immunity on mucosal surfaces lining the human
alimentary, respiratory and urogenital tracts is now widely
accepted 111. There is convincing evidence that several
pathogenic bacteria that colonise mucosal surfaces produce IgA
proteases and that IgA proteases contribute to the virulence of
these bacteria [2,3]. Recent evidence suggests that the majority
of pioneer oral streptococci that colonise the mouth before tooth
eruption produce IgA protease [4] and it has long been known
that Streptucuccus sunguis, a pioneer organism of the tooth
surface, is a producer of IgA protease [51.
IgA proteases vary in their catalytic mechanisms and include
serine-, metalb and cysteine-type proteases [6].IgA proteases
produced by oral streptococci are metallopmteinases [6] which
cleave specifically the achain within a sequence of 13 amino
acids present in the hinge region of human IgA molecules of the
IgAl subclass [7l.
Any agent which reduces the IgA protease activity of oral
bacteria is likely to reduce the virulence of these bacteria and to
affect their ability to colonise oral mucosae and tooth surfaces.
There is evidence that levels of specific IgA antibodies influence
the number of oral streptococci colonising the tooth surface [8].
In these experiments the effect of fluoride on the IgA protease
activity of Srreprucoccus sanguis has been studied.
Streptucuccus sanguis (NCTC 7863) was inoculated by a swab
onto the surface of sterile semipermeable membranes placed
aseptically on the surface of blood agar plates and incubated at
37°C for 48 h. This allows bacterial growth on the membrane
surface together with the retention of secreted enzyme. Two
principal types of experiment were performed. In order to
determine the effect of fluoride on IgA protease activity, the
membranes were removed and the bacterialenzyme mixture
harvested into a minimal volume of 50 mM Tris-azide buffer
(pH 8.0). The bacterial suspension was centrifuged and the
supernate, containing IgA protease collected for assay.
IgA protease activity was determined by incubating 25 pl
culture supemate containing enzyme, 15 pl IgA in Tris-azide
buffer and a range of fluoride concentrations ranging from 1 to
500 mM fluoride in Tris-azide buffer, for 72 h at 37°C.
Following SDS polyacrylamide gel electrophoresis and overnight
Western blotting on cellulose nitrate, the membrane was stained
with 0.2% Ponceau S to detect molecular weight markers and
then incubated with goat anti-human IgA (achain specific)
alkaline phosphatase conjugate for 2 h and was developed using
nitroblue tetrazolium and bromochloroindolyl phosphate. The
results showed no evidence of any inhibition of Streptucuccus
sanguis IgA protease activity at any concentration of fluoride
tested. Even at 500 mM fluoride no inhibitory effect of fluoride
on IgA protease activity was observed.
To determine whether fluoride had any effect on the
production of IgA protease by the bacteria as opposed to an
effect on the activity of enzyme already produced - Streptucuccus
sunguis was inoculated into Todd-Hewitt Broth and grown
overnight at 37°C. The bacterial culture was then centrifuged,
the cells then washed in saline to remove any IgA protease. and
equal amounts of a dense suspension of the washed cells
inoculated into Todd-Hewitt Broth containing a range of fluoride
Concentrations from 1 to 500 mM and incubated for 48 h at
-
37°C. After incubation the cultures were centrifuged and the
supernate incubated with IgA prior to SDS PAGE, Western
blotting and development as described above. IgA protease
activity was seen following incubation of the bacteria in 0, 1 , 5
and 10 mM fluoride but not when the bacteria were incubated in
50, 100 and 500 mM fluoride. These results suggest that IgA
protease production is inhibited at high concentrations of
fluoride.
There are three ways in which fluoride may affect the
production and activity of bacterial IgA protease. The first is by
a direct effect on enzyme activity. The results of these
experiments suggest that fluoride has no direct effect on
Streptucuccus sunguis IgA protease and that the enzyme has a
high degree of tolerance to fluoride. The second possible effect
of fluoride on IgA protease may be on the synthesis and/or
export of the enzyme from the bacteria. The results here suggest
that fluoride completely inhibits IgA protease synthesis and/or
export at concentrations above 10 mM. A third possible effect of
fluoride is a direct effect on bacterial growth. Any reduction in
bacterial numbers produced by fluoride will also reduce the
amount of IgA protease produced by bacteria at that site.
Streptucuccus sanguis is quantitatively one of the most
important bacteria of the tooth surface environment. The 1gA
protease activity of this organism is believed to reduce
susceptibility to the host mucosal defence mechanisms and may
also benefit other bacteria colonising the same local
microenvironment. This could include some of the non-IgA
protease producing bacteria such as the dental caries-producing
organisms Streptococcus mutam and Streptococcus sobrim.
Thus if IgA-protease activity or production at the tooth surface
was affected by fluoride then not only IgA protease-producing
bacteria but also a number of other bacteria colonising this site
may loose their protection against host mucosal defence
mechanisms. These results exclude a direct effect of fluoride on
IgA protease activity but do raise the possibility that, either by
an effect on synthesis or export of the enzyme, fluoride may
reduce the virulence and colonisation ability of all the flora at the
tooth surface.
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(1983) Ann. NY Acad. Sci. 409,612-624
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in Medical Microbiology 2, 200-207
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(1994) Infect. Immun. 62, 2165-2168
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J. Immunol. 113, 289-291
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8. Taubman, M.A. (1992) in Contemporary Oral
Microbiology d Immunology (Slots, J. & Taubman,
M.A., eds.) pp 535-541,Mosby Year Book, St Louis, 11.
USA