Apr 14, 1988 - John Wright PSG, Inc., Littleton, Mass. 6. Bowen, W. H. 1968. The trace element requirements of cario- genic and non-cariogenic streptococci.
Vol. 56, No. 8
INFECTION AND IMMUNITY, Aug. 1988, p. 2205-2207 0019-9567/88/082205-03$02.00/0 Copyright © 1988, American Society for Microbiology
NOTE
Expression of the Glucan-Binding Lectin of Streptococcus cricetus Requires Manganous Ion D. DRAKE,' K. G. TAYLOR,2 AND R. J. DOYLE'* Departments of Microbiology and Immunology' and Chemistry,2 Health Science Center, University of Louisville, Louisville, Kentucky 40292 Received 9 February 1988/Accepted 14 April 1988
Streptococcus cricetus AHT exhibited a requirement for manganese for growth and expression of the glucan-binding lectin. While low concentrations of manganese (0.1 to 10 ,uM) were able to support growth, higher concentrations (>100 ,uM) were required for full expression of the glucan-binding lectin. The manganous-aquo ion may be important in cellular adhesion and accumulation processes in dental plaque. ature for 90 min with sodium Chelex 100 (Bio-Rad Laboratories, Richmond, Calif.) at a concentration of 1 g of resin per 3 g of dry medium. Subsequently, the media were filtered twice and stirred with mixed-bed resin AG501-X8 (Bio-Rad) under identical conditions. Media prepared under these conditions were termed treated media. Buffers and trace metal stocks were prepared with Chelex-extracted distilled water. All glassware was acid cleaned (1.0 N HCI) prior to use. GBL activity was determined in a quantitative aggregation-rate assay. Briefly, high-molecular-weight glucan T2000 (Sigma) was added to washed cell suspensions at a final concentration of 10 pLg/ml. The decrease in turbidity was
Evidence from epidemiological studies implies a role for certain trace metals in cariogenesis (1, 7, 8, 10, 11). Elements such as aluminum, selenium, and strontium have been associated with a low incidence of dental caries (7, 11), whereas an increased prevalence of caries appears to be correlated with high concentrations of manganese, copper, and cadmium (1, 4, 5, 7, 8, 10). Studies of trace element content in water supplies have suggested a strong cariogenic potential for manganese (1, 5, 10). Additionally, examination of the composition of whole human enamel in the western United States revealed that, regardless of origin, increased concentrations of manganese were always associated with a higher incidence of caries (8). It is known that most microorganisms require manganese as a trace element (13, 14). Manganese is required for sporulation in bacilli (14), serves as a cofactor for various enzymes such as superoxide dismutase (3, 12), and functions as an O3 scavenger in Lactobacillus plantarum (3). Manganese is also an important growth factor for the oral streptococci and lactobacilli. Stamer et al. (15) demonstrated that more than 88% of lactic acid bacteria tested exhibited a requirement for manganese. Bowen (6) showed that manganese was the only transition metal required for growth of cariogenic and noncariogenic streptococci. A more recent study examining the effects of various trace metals on growth of Streptococcus mutans in a chemostat has confirmed the absolute requirement for manganese (2). Streptococcus cricetus and other oral streptococci produce a cell surface glucan-binding lectin (GBL) which has been implicated as a possible virulence component in cellular adhesion and accumulation processes in dental plaque (9). We now show that expression of the GBL is directly dependent on the manganese content of the growth medium. S. cricetus AHT (serotype a) was used throughtout this study. Bacteria were incubated statically at 37°C in 5% CO2 for all experiments. Trypticase soy broth (TSB; BBL Microbiology Systems, Cockeysville, Md.) was initially treated with yeast invertase (Sigma Chemical Co., St. Louis, Mo.) to eliminate possible sucrose contamination. To reduce trace metal content, growth media were treated according to the following protocol. Media were first mixed at room temper*
monitored spectrophotometrically (Spectronic 20; Bausch & Lomb, Inc., Rochester, N.Y.). Data were subjected to pseudo-first-order rate kinetics analysis with rate constants obtained from slopes of best-fitting lines (8a). Results of the first series of experiments are shown in Table 1. In this study, media were supplemented with EDTA (Sigma) to eliminate trace metals. Growth of S. cricetus and expression of GBL occurred only in media supplemented with MnSO4. Addition of other trace metals, singularly or in combination with EDTA, would not support growth. These data are therefore in agreement with previous observations (6). Cultivation of S. cricetus in treated media yielded similar results. Bacteria were able to grow in such media, but determination of GBL activity revealed a significant decrease in lectin expression (Fig. 1). Bacteria grown in media deficient in manganese but supplemented with iron (1.0 mM) or magnesium (1.0 mM) elicited low levels of GBL activity. In contrast, treated medium supplemented with manganese (1.0 mM) exhibited GBL activity comparable to that of nontreated controls. The capacity for treated media to support growth of S. cricetus in the absence of an exogenous source of manganese, which EDTA-supplemented media could not do, appeared to be due to carry-over of manganese with the inoculum. Support for this premise was obtained when cells cultured under manganese-deficient conditions as described above failed to grow when transferred to freshly treated medium (data not shown). Similar results were obtained in a study by Martin et al. (12). To study the relationship between manganese concentration, growth, and expression of GBL, S. cricetus AHT cells were cultured in treated media without a manganese supple-
Corresponding author. 2205
2206
NOTE
INFECT. IMMUN.
100
1.0
GBL activityc (10-'/min)
90
0.9
80
0.8
70 I
0.7
60 -
0.6 Lw
501-
0.5 4
0 _o
401-
0.4 °
0 -J co CD
30 F
0.3
20
0.2
TABLE 1. Effects of various trace metals on growth of S. cricetus AHT in EDTA-supplemented mediaa Trace metal
A
addedb
None (control)d MgSO4 FeSO4 ZnSO4 Co(C2H302)2 Ni(NO3)2
0.75
2.7
0.0
0.0
0.0 0.0 0.0
I
0.0 0.0
0.0 0.0
c
CuS04
0.0
0.0
CaC12 Combinatione MnSO4
0.0 0.0 0.72
0.0 0.0
0.0
3.2
a EDTA was added to TSB at a final concentration of 1.0 mM. Bacteria were incubated statically in 5% C02 at 37°C for 16 to 18 h. b Each trace metal was added at a final concentration of 1.0 mM. c Rate of glucan T2000-induced aggregation. First-order rate constants were obtained from slopes of best-fitting lines. d Bacteria were cultured in TSB without EDTA. Supplementation with all the trace metals except MnSO4 in TSB-EDTA at a final concentration each of 0.1 mM. All of these assays were conducted using nearly identical cell densities (1.2 to 1.4 A520, 1.25-cm path length).
ment to render them manganese starved. Cells from this culture were used as inocula for treated media supplemented with various concentrations of manganese. Culture turbidity and relative GBL activity were determined following overnight incubation. The results are shown in Fig. 2. The addition of 100 p,M Mn2+ supported cell growth and GBL activity essentially equivalent to that of nontreated controls. Supplementation of treated media with 10 ,uM manganese supported similar cell growth, but a significant decrease in GBL expression was observed when compared with that of controls. Moreover, the addition of manganese at 1.0 and 0.1 p.M caused only slightly reduced growth yields but 65 and 90% decreases, respectively, in GBL activity. Therefore, it
-0.4
C _lr-
1
2
3
4
-Mn
5
TIME (minutes) FIG. 1. Rate of glucan T2000-induced aggregation of S. cricetus AHT. Cells were cultured in treated TSB supplemented with MnSO4, FeSO4, or MgSO4 at a final concentration of 1.0 mM. Bacteria were harvested by centrifugation and suspended in phosphate-buffered saline (20 mM potassium phosphate, 0.15 M NaCl [pH 7.2]). A, Absorbance at times shown; AO, initial absorbance
reading.
ol o.-
10
z
Cf-
K
0.1 0.1
1.0
10.0
100.0
[MnSO4 ]pM FIG. 2. Determination of the growth of S. cricetus AHT and expression of the GBL in treated TSB supplemented with manganese.
appears that manganese not only serves as a growth factor for S. cricetus but also has a role in stimulating the expression of the GBL. Other studies have suggested possible mechanisms for the cariogenicity of manganese. Martin et al. (12) presented evidence that S. mutans may employ manganese as a cofactor for superoxide dismutase. Beighton (4) described studies in which manganese stimulated the carbohydrate metabolism of S. mutans in vitro. Additionally, it was shown that significant increases in caries levels of rats monoinfected with S. mutans Ingbritt occurred when manganese was added to their drinking water. The results from the studies reported herein suggest an additional explanation for the cariogenic potential of manganese, i.e., the stimulation of GBL. How manganese stimulates GBL activity is at present not well understood, but research not reported here has indicated that manganese may serve as a cofactor, suggesting that the GBL is a manganoprotein (manuscripts in
preparation). The results indicate that expression of GBL activity is significantly enhanced by the transition metal manganese. This may be of considerable importance in the pathogenesis of dental caries and underscores the need for development of therapeutic agents to limit the uptake of manganese by the oral streptococci. LITERATURE CITED 1. Adkins, B. L., and F. L. Losee. 1970. A study of the covariation of dental caries prevalence and multiple trace element content of water supplies. N.Y. State Dent. J. 36:618-622. 2. Aranha, H., R. C. Strachan, J. E. L. Arceneaux, and B. R. Byers. 1982. Effect of trace metals on growth of Streptococcus mutans in a Teflon chemostat. Infect. Immun. 35:456-460. 3. Archibald, F. 1986. Manganese: its acquisition by and function in the lactic acid bacteria. Crit. Rev. Microbiol. 13:63-109. 4. Beighton, D. 1982. The influence of manganese on carbohydrate
NOTE
VOL. 56, 1988
metabolism and caries induction by Streptococcus mutans strain Ingbritt. Caries Res. 16:189-192. 5. Beighton, D. 1983. Manganese, p. 237-244. In M. E. J. Curzon, T. W. Curtress, and A. F. Gardner (ed.), Trace elements and dental disease. Postgraduate dental handbook series, vol. 9. John Wright PSG, Inc., Littleton, Mass. 6. Bowen, W. H. 1968. The trace element requirements of cariogenic and non-cariogenic streptococci. Arch. Oral Biol. 13:713714. 7. Curzon, M. E. J. 1983. Epidemiology of trace elements and dental caries, p. 11-30. In M. E. J. Curzon, T. W. Curtress, and A. F. Gardner (ed.), Trace elements and dental disease. Postgraduate dental handbook series, vol. 9. John Wright PSG, Inc., Littleton, Mass. 8. Curzon, M. E. J., and F. L. Losee. 1978. Dental caries and trace element composition of whole human enamel: western United States. J. Am. Dent. Assoc. 96:819-822. 8a.Drake, D., K. G. Taylor, A. S. Bleiweis, and R. J. Doyle. 1988. Specificity of the glucan-binding lectin of Streptococcus cricetus. Infect. Immun. 56:1864-1872. 9. Gibbons, R. J., and R. J. Fitzgerald. 1969. Dextran-induced agglutination of Streptococcus mutans, and its potential role in
10. 11.
12.
13. 14. 15.
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the formation of microbial dental plaques. J. Bacteriol. 98:341346. Glass, R. L., K. J. Rothman, F. Espinol, H. Velex, and N. J. Smith. 1973. The prevalence of human dental caries and waterborne trace metals. Arch. Oral Biol. 18:1099-1104. Little, M. F., and K. Barrett. 1976. Strontium and fluoride content of surface and inner enamel versus caries prevalence in the Atlantic coast of the United States of America. Caries Res. 10:297-307. Martin, M. E., R. C. Strachan, H. Aranha, S. L. Evans, M. L. Salin, B. Welch, J. E. L. Arceneaux, and B. R. Byers. 1984. Oxygen toxicity in Streptococcus mutans: manganese, iron, and superoxide dismutase. J. Bacteriol. 159:745-749. Schmid, J., and G. Auling. 1987. Manganese transport in Brevibacterium ammoniagenes ATCC 6872. J. Bacteriol. 169:33853387. Silver, S., and P. Jasper. 1977. Manganese transport in microorganisms, p. 105-149. In E. D. Weinberg (ed.), Microorganisms and minerals. Marcel Dekker, Inc., New York. Stamer, J. R., M. N. Albury, and C. S. Pederson. 1964. Substitution of manganese for tomato juice in the cultivation of lactic acid bacteria. Appl. Microbiol. 12:165-168.
