Effects of Antibodies on Adherence and Cell-Associated Glucan Production by Streptococcus mutans Cells R. T. EVANS, R. J. GENCO, and F. G. EMMINGS* Department of Oral Biology, State University of New York at Buffalo, School of Dentistry, Buffalo, New York 14226, USA
Previous investigations have shown that antibodies offer protection against the infection of teeth by Streptococcus mutanst and subsequent caries.2,3 Investigations in our laboratories have shown that monkeys immunized against S mutans secrete antibodies to these organisms in their parotid fluid as well as serum antibodies.' When challenged with the immunizing strain, these monkeys show a noticeably reduced level of oral infection with the immunizing strain, but not when challenged with a non-cross-reacting strain of S mutans, which demonstrates that specific antibodies play a role in preventing dental infection by S mutans. Investigations by Taubman and Smith3 in rats immunized with S mutans showed increased levels of salivary antibody and a reduced level of infection by S mutans as well as a reduction in dental caries in the immunized rats. Antibodies may have a variety of effects on S mutans including bacteriocidal effects and opsonization as well as a reduction of adherence and an altering of bacterial metabolism. Antibody-mediated reduction in the adherence of S mutans to the tooth surface may result in the inhibition of infection and subsequent protection against S mutans-induced caries. Adherence inhibition brought about by antibodies to S mutans could occur as a result of one of the following reasons: the antibody could prevent synthesis of dextran by glucosyltransferase; the antibody could inhibit the binding of glucosyltransferase or the glucosyltransferase-dextran complexes to receptors on the S mutans cell surface; or the antibody could bind to other bacterial This investigation was supported, in part, by USPHS Research Grants DE-04061, DE-0167, and DE-02814. 0 Present address: Department of Clinical Dentistry, Strong Memorial Hospital, Rochester, NY 14642.
surface antigens and thereby change its surface properties, thus interfering with adherence of the organism to the tooth. Antibodies may effect organisms in other ways that could result in reduced cariogenicity, for example, antibodies may block the production of acids or other metabolic products necessary for virulence, or antibodies could lead to the removal of the organisms by specific bacteriocidal or opsonizing effects. The present investigations examine the effects of serum antibodies on adherence and on the synthesis of cell-associated glucans (CAG), both of which are important for the expression of cariogenicity by S mutans.4,5
Materials and Methods S mutans strains E-49, FA-1, GS-5, 6715, and LM-7, representing Bratthall serotypes a, b, c., d, and e, respectively, were used.a The organisms were maintained in frozen deep cultures and transferred in Trypticase soy broth (TSB) before use. Cultures used for testing in the adherence assay were grown overnight in TSB, and the cell density adjusted to an optical density (OD) of 0.10 at 540 nm. Antiserums to these organisms were prepared in New Zealand white rabbits by intravenous administration of washed, viable cells that were adjusted to an OD of about 108 cells/ml based on McFarland's standards. Injections were administered in graduated doses of 0.25, 0.5, 0.75, and 1.0 ml (1.0 ml given three times) for a total of six injections during a two-week period. The rabbits were bled one week after the final injection. Booster immunizations, when required, were given as a single 1-ml, intravenous injection of 108 cells/ml, and the a Obtained from Dr. Robert Fitzgerald, Veterans Hospital, Miami, Fla.
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EVANS, GENCO, AND EMMINGS
rabbits were bled one week later. Serum was stored in aliquots at -20 C. Antiserums to rabbit immunoglobulins were prepared by immunizing goats with purified rabbit IgG.6 Inhibition of S mutans adherence by rabbit antiserums was measured according to modification of the procedure of Olson, Bleiweiss, and Small.7 Briefly, this method estimates the adherence of cells to glass surfaces by measuring the OD of adherent and nonadherent cells suspended in sodium hydroxide. By combining the OD for the adherent and nonadherent cells, total growth can be estimated. Inhibition of adherence was calculated by comparing the OD measured for the glass-adherent cells in the presence of the immune serum with those of the glass-adherent cells measured in the presence of the preimmune serum. The effect of antiserum on total growth was determined by comparing the total OD of the cells growing in the presence and the absence of the immune serum. In order to measure the effects of antiserum to S mutans on the production of CAG, sucrose that was labeled with 14C in the glucose moiety only was added to the culture medium. After 18 hours of incubation, the nonadherent cells were decanted and the adherent cells were washed two times with 0.15 3l sodium chloride, 0.02 M sodium phosphate, buffered at a pH of 7.4 (PBS), and the washings were combined with the decanted cells. The adherent cells were removed from the glass surface and the
cell-associated radioactivity was solubilized by incubating the cells in 0.5 N sodium hydroxide for three to five minutes. The OD540tim of the sodium hydroxide suspension was measured as an indication of the number of adherent cells. After extraction, the cells were removed by centrifugation and the supernate radioactivity measured. This radioactivity routinely represented 80 to 90% of the 14C label associated with the cells. Radioactivity was measured by liquid scintillation counting using scintillation fluid containing 4% CAB-O-Sil.8 There was little or no quenching obtained under these conditions of counting. The radioactive counts were corrected for OD (cell density) by dividing the counts per minute by the OD of the cells. Antibody-mediated inhibition of 14C glucose incorporation into CAG was calculated by comparing the radioactivity of cells grown in the presence of the immune serum as compared to that for cells grown in the presence of either the preimmune serum from the same rabbit or the nonimmune serum. Figure 1 shows the protocol used to determine both adherence of S mutans cells to glass surfaces as well as cell-associated, radioactive glucan production. Results The effect of antiserums on adherence and incorporation of radioactivity into CAG of S mutans strain 6715 is given in Table 1. There is noticeable inhibition of adherence at antiserum dilutions from 1:10 to 1:160
S MUTANS GROWN IN
MEDIUM WITH ANTISERUM AND
I
14C
SUCROSE
DECANT
ADHERENT CELLS 0.5N NoOH-QD ADHERENT CELLS
NON-ADHERENT CELLS CENTRIFUGE SUPERNATE
CENTRIFUGE CELLS SUPERNATE O.D. NON-ADHERENT ICAG OF ADHERENTI
CELLS
0.5N NaOH
|
|CELLS
CELLSI
CENTRIFUGE CELLS
C
SUPERNATE CAG OF NON-ADHERENT CELLS FIG 1
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ANTIBODY EFFECTS ON S MUTANS
Yol 55 1976 TABLE 1 EFFECrS OF RABBIT ANTISERUM TO S MUTANS
STRAIN 6715 ON ADHERENCE AND CAG BY S MUTANS STRAIN 6715 CELLS CAG Antiserum Dilution
Adherence
Adherent Cells
Nonadherent Cells
(%)
(%)
(%)
1:10
-80*
1:20 1:40 1:80 1:160 1:320 1:640 1:1,280
-84 -90 -90 -96
-82 -66 -30 - 8 +60 +34 +22 +32
-90 -88 -80
-22 -10 0
-80
-76 -86
-72 3
-
* Percent decrease (-) or increase (+) compared to normal rabbit serum controls.
and significant but reduced levels of inhibition of adherence at the 1:320 and 1:640 dilutions. The effect of antiserums on the production of CAG was different when adherent to nonadherent cells were compared. Adherent cells seemed to be much less sensitive to the effect of serums, since inhibition of CAG was noted only up to a dilution of 1:80, although the inhibition of CAG production of nonadherent cells was detected with antiserum dilutions up to 1:640. Another interesting feature that was noted consistently was the increase in CAG production at the higher dilutions of antiserums. It seems that some factor in the serum is increasing glucan formation on the adherent cells whereas at the same dilution these serums effected a noticeable reduction in glucan synthesis by the nonadherent cells. To determine if the effect of antiserums on the
reduction of CAG by adherent cells was mediated by immunoglobulins (presumably antibodies), the effects of rabbit serum were tested before and after absorption with goat arntiserum to rabbit immunoglobulin. The goat antiserum was used at concentrations previously found to precipitate most of the rabbit immunoglobulins. Rabbit antiserum to strain 6715 when incubated with cells after absorption with saline or normal goat serum resulted in a 60 to 68% reduction of CAG production by adherent cells. In contrast, if the same antiserum was pretreated with goat antirabbit immunoglobulin antiserum, the reduction of CAG production by adherent cells was not significantly reduced (Table 2). These results demonstrate that the inhibition of CAG by antiserums to whole cells of S mutans strain 6715 is mediated by an immunoglobulin. The low level of inhibition of CAG observed when the goat antirabbit immunoglobulin serum was used suggests that this serum has naturally occurring antibodies to S mutans. Since inhibition of adherence and CAG synthesis by antiserum to S mutans appears to be antibody mediated, it should show a specificity characteristic of immunologic reactions. Specificity was tested in a series of experiments; the results are given in Table 3. Antiserums to strains of the d serotype (S mutans strains 6715, KIR, OMZ176, and SL-1) were tested for their ability to inhibit adherence and CAG production by S mu tans strain 6715. As can be seen from this table, noticeable inhibition of adherence and CAG production was brought about by the antiserum to S mutans strain 6715 against S mutans strain 6715 cells as
TABLE 2 ABSORsrION OF INHIBITORY AcrIvrry OF ANTISERUMS OF S MUTANS BY ANTI-IMMUNOGLOBULIN ANTIBODIES Rabbit Serum
Anti-6715t Anti-6715 Anti-6715 PRSt PRS PRS
%o Reduction 14C-
Serum Dilution
1:34 1:34 1:34 1:34 1:34 1:34
C129
Absorbent
Saline Normal goat Goat antirabbit Ig Goat antirabbit Ig Normal goat Saline
Incorporation by Adherent Cells
68 60 12 13 0 0
* Corrected for cell numbers. t Rabbit antiserum to whole cells of S mutans strain 6715. $ PRS, preimmune rabbit serum from the same rabbit in which the anti-S mutans strain 6715 was prepared. Downloaded from jdr.sagepub.com at UNIV OF MICHIGAN on July 17, 2015 For personal use only. No other uses without permission.
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EVANS, GENCO, AND EMMINGS
TABLE 3 INHIBITION OF S MUTANS STRAIN 6715 ADHERENCE AND CAG BY ANTISERUMS TO GROUP D STRAINS
Antiserum
Anti-6715 (d)
*
S mutans
Adherence Inhibition
CAG Inhibition
Test Strain
(%)
(%)
87 98
95
92 70
98 82
96
97
25 15
Previous investigations have shown that antibodies offer protection against the infection of teeth by Streptococcus mutanst and subsequent caries.2,3 Investigations in our laboratories have shown that monkeys immunized against S mutans secrete antibodies to these organisms in their parotid fluid as well as serum antibodies.' When challenged with the immunizing strain, these monkeys show a noticeably reduced level of oral infection with the immunizing strain, but not when challenged with a non-cross-reacting strain of S mutans, which demonstrates that specific antibodies play a role in preventing dental infection by S mutans. Investigations by Taubman and Smith3 in rats immunized with S mutans showed increased levels of salivary antibody and a reduced level of infection by S mutans as well as a reduction in dental caries in the immunized rats. Antibodies may have a variety of effects on S mutans including bacteriocidal effects and opsonization as well as a reduction of adherence and an altering of bacterial metabolism. Antibody-mediated reduction in the adherence of S mutans to the tooth surface may result in the inhibition of infection and subsequent protection against S mutans-induced caries. Adherence inhibition brought about by antibodies to S mutans could occur as a result of one of the following reasons: the antibody could prevent synthesis of dextran by glucosyltransferase; the antibody could inhibit the binding of glucosyltransferase or the glucosyltransferase-dextran complexes to receptors on the S mutans cell surface; or the antibody could bind to other bacterial This investigation was supported, in part, by USPHS Research Grants DE-04061, DE-0167, and DE-02814. 0 Present address: Department of Clinical Dentistry, Strong Memorial Hospital, Rochester, NY 14642.
surface antigens and thereby change its surface properties, thus interfering with adherence of the organism to the tooth. Antibodies may effect organisms in other ways that could result in reduced cariogenicity, for example, antibodies may block the production of acids or other metabolic products necessary for virulence, or antibodies could lead to the removal of the organisms by specific bacteriocidal or opsonizing effects. The present investigations examine the effects of serum antibodies on adherence and on the synthesis of cell-associated glucans (CAG), both of which are important for the expression of cariogenicity by S mutans.4,5
Materials and Methods S mutans strains E-49, FA-1, GS-5, 6715, and LM-7, representing Bratthall serotypes a, b, c., d, and e, respectively, were used.a The organisms were maintained in frozen deep cultures and transferred in Trypticase soy broth (TSB) before use. Cultures used for testing in the adherence assay were grown overnight in TSB, and the cell density adjusted to an optical density (OD) of 0.10 at 540 nm. Antiserums to these organisms were prepared in New Zealand white rabbits by intravenous administration of washed, viable cells that were adjusted to an OD of about 108 cells/ml based on McFarland's standards. Injections were administered in graduated doses of 0.25, 0.5, 0.75, and 1.0 ml (1.0 ml given three times) for a total of six injections during a two-week period. The rabbits were bled one week after the final injection. Booster immunizations, when required, were given as a single 1-ml, intravenous injection of 108 cells/ml, and the a Obtained from Dr. Robert Fitzgerald, Veterans Hospital, Miami, Fla.
C127 Downloaded from jdr.sagepub.com at UNIV OF MICHIGAN on July 17, 2015 For personal use only. No other uses without permission.
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EVANS, GENCO, AND EMMINGS
rabbits were bled one week later. Serum was stored in aliquots at -20 C. Antiserums to rabbit immunoglobulins were prepared by immunizing goats with purified rabbit IgG.6 Inhibition of S mutans adherence by rabbit antiserums was measured according to modification of the procedure of Olson, Bleiweiss, and Small.7 Briefly, this method estimates the adherence of cells to glass surfaces by measuring the OD of adherent and nonadherent cells suspended in sodium hydroxide. By combining the OD for the adherent and nonadherent cells, total growth can be estimated. Inhibition of adherence was calculated by comparing the OD measured for the glass-adherent cells in the presence of the immune serum with those of the glass-adherent cells measured in the presence of the preimmune serum. The effect of antiserum on total growth was determined by comparing the total OD of the cells growing in the presence and the absence of the immune serum. In order to measure the effects of antiserum to S mutans on the production of CAG, sucrose that was labeled with 14C in the glucose moiety only was added to the culture medium. After 18 hours of incubation, the nonadherent cells were decanted and the adherent cells were washed two times with 0.15 3l sodium chloride, 0.02 M sodium phosphate, buffered at a pH of 7.4 (PBS), and the washings were combined with the decanted cells. The adherent cells were removed from the glass surface and the
cell-associated radioactivity was solubilized by incubating the cells in 0.5 N sodium hydroxide for three to five minutes. The OD540tim of the sodium hydroxide suspension was measured as an indication of the number of adherent cells. After extraction, the cells were removed by centrifugation and the supernate radioactivity measured. This radioactivity routinely represented 80 to 90% of the 14C label associated with the cells. Radioactivity was measured by liquid scintillation counting using scintillation fluid containing 4% CAB-O-Sil.8 There was little or no quenching obtained under these conditions of counting. The radioactive counts were corrected for OD (cell density) by dividing the counts per minute by the OD of the cells. Antibody-mediated inhibition of 14C glucose incorporation into CAG was calculated by comparing the radioactivity of cells grown in the presence of the immune serum as compared to that for cells grown in the presence of either the preimmune serum from the same rabbit or the nonimmune serum. Figure 1 shows the protocol used to determine both adherence of S mutans cells to glass surfaces as well as cell-associated, radioactive glucan production. Results The effect of antiserums on adherence and incorporation of radioactivity into CAG of S mutans strain 6715 is given in Table 1. There is noticeable inhibition of adherence at antiserum dilutions from 1:10 to 1:160
S MUTANS GROWN IN
MEDIUM WITH ANTISERUM AND
I
14C
SUCROSE
DECANT
ADHERENT CELLS 0.5N NoOH-QD ADHERENT CELLS
NON-ADHERENT CELLS CENTRIFUGE SUPERNATE
CENTRIFUGE CELLS SUPERNATE O.D. NON-ADHERENT ICAG OF ADHERENTI
CELLS
0.5N NaOH
|
|CELLS
CELLSI
CENTRIFUGE CELLS
C
SUPERNATE CAG OF NON-ADHERENT CELLS FIG 1
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ANTIBODY EFFECTS ON S MUTANS
Yol 55 1976 TABLE 1 EFFECrS OF RABBIT ANTISERUM TO S MUTANS
STRAIN 6715 ON ADHERENCE AND CAG BY S MUTANS STRAIN 6715 CELLS CAG Antiserum Dilution
Adherence
Adherent Cells
Nonadherent Cells
(%)
(%)
(%)
1:10
-80*
1:20 1:40 1:80 1:160 1:320 1:640 1:1,280
-84 -90 -90 -96
-82 -66 -30 - 8 +60 +34 +22 +32
-90 -88 -80
-22 -10 0
-80
-76 -86
-72 3
-
* Percent decrease (-) or increase (+) compared to normal rabbit serum controls.
and significant but reduced levels of inhibition of adherence at the 1:320 and 1:640 dilutions. The effect of antiserums on the production of CAG was different when adherent to nonadherent cells were compared. Adherent cells seemed to be much less sensitive to the effect of serums, since inhibition of CAG was noted only up to a dilution of 1:80, although the inhibition of CAG production of nonadherent cells was detected with antiserum dilutions up to 1:640. Another interesting feature that was noted consistently was the increase in CAG production at the higher dilutions of antiserums. It seems that some factor in the serum is increasing glucan formation on the adherent cells whereas at the same dilution these serums effected a noticeable reduction in glucan synthesis by the nonadherent cells. To determine if the effect of antiserums on the
reduction of CAG by adherent cells was mediated by immunoglobulins (presumably antibodies), the effects of rabbit serum were tested before and after absorption with goat arntiserum to rabbit immunoglobulin. The goat antiserum was used at concentrations previously found to precipitate most of the rabbit immunoglobulins. Rabbit antiserum to strain 6715 when incubated with cells after absorption with saline or normal goat serum resulted in a 60 to 68% reduction of CAG production by adherent cells. In contrast, if the same antiserum was pretreated with goat antirabbit immunoglobulin antiserum, the reduction of CAG production by adherent cells was not significantly reduced (Table 2). These results demonstrate that the inhibition of CAG by antiserums to whole cells of S mutans strain 6715 is mediated by an immunoglobulin. The low level of inhibition of CAG observed when the goat antirabbit immunoglobulin serum was used suggests that this serum has naturally occurring antibodies to S mutans. Since inhibition of adherence and CAG synthesis by antiserum to S mutans appears to be antibody mediated, it should show a specificity characteristic of immunologic reactions. Specificity was tested in a series of experiments; the results are given in Table 3. Antiserums to strains of the d serotype (S mutans strains 6715, KIR, OMZ176, and SL-1) were tested for their ability to inhibit adherence and CAG production by S mu tans strain 6715. As can be seen from this table, noticeable inhibition of adherence and CAG production was brought about by the antiserum to S mutans strain 6715 against S mutans strain 6715 cells as
TABLE 2 ABSORsrION OF INHIBITORY AcrIvrry OF ANTISERUMS OF S MUTANS BY ANTI-IMMUNOGLOBULIN ANTIBODIES Rabbit Serum
Anti-6715t Anti-6715 Anti-6715 PRSt PRS PRS
%o Reduction 14C-
Serum Dilution
1:34 1:34 1:34 1:34 1:34 1:34
C129
Absorbent
Saline Normal goat Goat antirabbit Ig Goat antirabbit Ig Normal goat Saline
Incorporation by Adherent Cells
68 60 12 13 0 0
* Corrected for cell numbers. t Rabbit antiserum to whole cells of S mutans strain 6715. $ PRS, preimmune rabbit serum from the same rabbit in which the anti-S mutans strain 6715 was prepared. Downloaded from jdr.sagepub.com at UNIV OF MICHIGAN on July 17, 2015 For personal use only. No other uses without permission.
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TABLE 3 INHIBITION OF S MUTANS STRAIN 6715 ADHERENCE AND CAG BY ANTISERUMS TO GROUP D STRAINS
Antiserum
Anti-6715 (d)
*
S mutans
Adherence Inhibition
CAG Inhibition
Test Strain
(%)
(%)
87 98
95
92 70
98 82
96
97
25 15
