Infection and Immunity, August 2000, p. 4834-4837, Vol. 68, No. 8
0019-9567/00/$04.00+0
Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892
Received 23 March 2000/Returned for modification 27 April 2000/Accepted 26 May 2000
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ABSTRACT |
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Attachment of Streptococcus gordonii to the acquired pellicle of the tooth surface involves specific interactions between bacterial adhesins and adsorbed salivary components. To study saliva-regulated gene expression in S. gordonii, we used random arbitrarily primed PCR (RAP-PCR). Bacteria were incubated in either brain heart infusion medium or saliva. Total RNA from both conditions was purified and RAP fingerprinted and then PCR amplified with an arbitrary primer. The differentially displayed DNA fragments were cloned, sequenced, and analyzed using the BLAST search network service. Three DNA products were up-regulated. One was identified as that of the sspA and -B genes, which encode the salivary agglutinin glycoprotein-binding proteins SspA and SspB of S. gordonii; another had 79% identity with the Lactococcus lactis clpE gene, encoding a member of the Clp protease family; and the third product showed no significant homology to known genes. Five down-regulated genes were identified which encode proteins involved in bacterial metabolism. We have shown, for the first time, direct induction of sspA and -B in S. gordonii by human saliva.
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TEXT |
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Streptococcal adhesion to oral surfaces results partially from initial binding of cells to absorbed salivary components, which are tightly bound to the mineral matrix of human enamel and form the salivary pellicle. Bacterial binding to salivary proteins and glycoproteins is relevant to initial binding events for which genes have been identified by transposon mutagenesis (22), as well as to accumulation of bacteria in the complex biofilm of mature dental plaque. The streptococcal salivary adhesins that have been described in the most detail are the LraI family polypeptides and the antigen I/II family of polypeptides (15, 31). Antigen I/II polypeptides bind a mucin-like salivary component named salivary agglutinin glycoprotein (SAG) in a lectin-like interaction (4).
Although much progress has been made in defining the role of saliva in oral microbial ecology, little is known concerning the physiological response of bacteria following binding to a salivary component. The objective of this research was to identify the genes regulated by contact between oral bacteria and salivary molecules of the conditioning film or salivary pellicle on the enamel surface. We chose to study Streptococcus gordonii DL1 because it is genetically transformable, it binds salivary components, and it is an early colonizer of the clean enamel surface.
To study saliva-regulated gene expression in S. gordonii, we used random arbitrarily primed PCR (RAP-PCR), a method adapted from the differential-display (DD) PCR method (29). We identified eight saliva-regulated cDNA fragments, seven of which had sequence identity to known genes. Sequence analysis showed that five down-regulated products encode proteins involved in bacterial metabolism. Of the three up-regulated genes, one has been found to be 95% identical to the SAG-binding cell surface adhesin SspA and SspB (antigen I/II family)-encoding genes of S. gordonii.
S. gordonii DL1 (Challis) was cultured anaerobically (BBL
GasPak system; Becton Dickinson Microbiology System, Cockeysville, Md.)
at 37°C in brain heart infusion (BHI) medium (Difco Laboratories, Detroit, Mich.). Escherichia coli Epicurian Coli XL10-Gold
Ultracompetent Cells (Stratagene, La Jolla, Calif.) were used to clone
differentially expressed RAP-PCR products. Transformation and
identification of bacterial colonies that contain recombinant plasmids
were performed in accordance with standard protocols (27).
Fresh stimulated whole saliva samples were collected from six or more
healthy persons. The donors were not on medication or ill, nor had they
eaten or drunk in the 60 min prior to saliva collection. Saliva
secretion was stimulated by Parafilm chewing, and the saliva was
collected on ice. Dithiothreitol (2.5 mM final concentration) was added to the collected saliva, and the mixture was stirred for 20 min at
4°C. The saliva was centrifuged at 5,000 × g, and
the supernatant fluid was filtered through a 0.22-µm-pore-size
polyethersulfone filter. Resulting sterile saliva samples were kept
frozen at
20°C until used. An overnight culture of S. gordonii was diluted 1:11 in fresh BHI medium or 1:11 in dilute
saliva (whole saliva at 1:4 in sterile water). Bacteria were cultured
for 2 h anaerobically (GasPak) at 37°C. After 2 h in either
BHI medium or saliva, bacteria were harvested by centrifugation
(2,500 × g, 10 min, 4°C). The pellet was washed with
diethylpyrocarbonate-treated water, resuspended in Ultraspec RNA
reagent solution (Biotecx Laboratories Inc., Houston, Tex.), and
transferred to a Multimix Tube containing a commercially prepared
mixture of 0.1-, 1.4-, and 4-mm-diameter silica-ceramic beads (Bio 101, Inc., Vista, Calif.). The tube was shaken in a FastPrep FP120 bead
beater (Bio 101) at top speed for 45 s and placed on ice, and the
lysate was clarified by centrifugation (2,500 × g, 2 min, 4°C). RNA was isolated from the supernatant by the procedure of
Lunsford (24). Subsequently, the RNA samples were treated
with DNase I for 15 min at 37°C using RQ1 DNase (Promega, Madison,
Wis.). The integrity of the RNA was assessed by electrophoresis in
accordance with standard protocols (27). Each reverse
transcription reaction was performed as recommended by the manufacturer
(Stratagene) with 2 µg of S. gordonii DL1 total RNA and
the chosen arbitrary primer, A3
(5'-AATCTAGAGCTCTCCTGG-3'). Reactions for each condition were done in triplicate. As a negative control to identify products amplified in subsequent steps as a result of residual genomic DNA
contamination, an identical reaction mixture without Moloney murine
leukemia virus reverse transcriptase was done. The second DNA strand
was synthesized and PCR amplified in the presence of [
-33P]dATP (DuPont NEN, Boston, Mass.) in a
thermocycler (Perkin-Elmer Cetus, Norwalk, Conn.) as follows: 1 low-stringency cycle (94°C for 1 min, 36°C for 5 min, and 72°C
for 5 min), 40 high-stringency cycles (94°C for 1 min, 50°C for 2 min, and 72°C for 2 min), and an elongation step (72°C for 10 min).
Following PCR amplification, the reaction mixture was combined with
stop buffer containing formamide, xylene cyanol, and bromophenol blue
(USB Corp., Cleveland, Ohio) and heated at 80°C for 2 min. Each
reaction was electrophoresed in adjacent lanes of a CastAway precast
4.5% acrylamide-7 M urea sequencing gel (Stratagene). The gel was
dried and radioautographed using Kodak X-Omat AR film (Eastman Kodak
Co., Rochester, N.Y.). The autoradiogram was aligned with the gel;
bands of interest were cut from the gel and placed into a centrifuge
tube. The DNA was eluted in 1× TE buffer (Digene, Beltsville, Md.) for
1 h at 60°C and then incubated overnight at room temperature.
The sample was centrifuged, and 2 µl of the eluate was amplified
using primer A3 and the 40-cycle high-stringency RAP-PCR
program. No radioactive deoxynucleoside triphosphates were included.
The products were analyzed on a 2% (wt/vol) agarose gel. The RAP-PCR
products were extracted from the agarose gel with a QIAquick gel
extraction kit (Qiagen, Santa Clarita, Calif.) and purified using a Bio
101 Geneclean Spin kit. The RAP-PCR products were blunt ended by mixing with Pfu DNA polymerase and deoxynucleoside triphosphates
(Stratagene). The blunt-ended DNA products were cloned in cloning
vector pPCR-Script Amp SK(+) (Stratagene). Cloned DNAs were sequenced
using the Perkin-Elmer Applied Biosystems 377XL automated DNA
sequencer. Sequence analysis was performed by the BLAST search
algorithm (10). A Northern blot was prepared in accordance
with standard protocols (27) following electrophoretic
separation of 5 µg of total RNA isolated from each condition (BHI
medium or saliva). The blot was probed with the DNA fragment purified
from the sequencing gel, reamplified, and
-32P
radiolabeled by random primer extension (Lofstrand Labs Limited, Gaithersburg, Md.).
The RNA purification method used gave high-quality RNA with no
detectable degradation of rRNA by gel electrophoresis (data not shown).
A single primer, A3, was selected from a reverse
transcription-tested set of five commercial primers (Stratagene) with
RNA from S. gordonii DL1 under the BHI medium condition,
although all five primers yielded PCR products of useful size for
future differential display experiments (data not shown). The RAP-PCRs
were done in triplicate on the same sample. This experiment with primer
A3 was repeated twice with different RNA preparations, and
similar results were obtained. Total RNA (represented by cDNA) of
S. gordonii DL1 incubated with saliva was compared to the
BHI medium condition. Eight bands appeared to be differentially
expressed (Fig. 1). Five products were
down-regulated, and three were up-regulated. Cloned inserts of the
RAP-PCR products ranged in size from about 200 to 1,000 bp. The DNA
sequence of the eight RAP-PCR products derived from differentially
expressed genes was compared to that in the GenBank database by using
the BLAST search algorithm to identify similarities to known sequences
(Table 1). Sequence analysis showed that
the down-regulated products have sequence similarity to proteins likely to be involved in bacterial metabolism in the shift from BHI medium to
saliva. For example, dihydrodipicolinate synthase (bands 2 and 4) is
involved in the pathway for the biosynthesis of diaminopimelate and
lysine (2), glucose kinase (band 5) is involved in glucose metabolism (9), and the PhoH protein (band 6) is normally
induced by phosphate starvation (30). Bacteria in saliva are
in the presence of less freely metabolizable nutrient than when in BHI medium, which has much low-molecular-weight nutrient. For the up-regulated products, band 3 was particularly overexpressed and was
95% identical to a region common to both sspA and
sspB, which encode the SAG-binding cell surface adhesin
proteins SspA and SspB (antigen I/II family) of S. gordonii
(6). These adhesins recognize multiple ligands, and they
mediate a wide range of streptococcal adherence properties, including
binding to salivary agglutinin glycoproteins (11, 25, 26),
type 1 collagen (23, 28), and other microbial cells,
including Actinomyces naeslundii (6, 16),
Candida albicans (12), and Porphyromonas
gingivalis (1, 21). The band 7 product was 79%
identical to the Lactococcus lactis clpE gene that encodes a
member of the Clp protease family (13). Band 8 showed no
identity to any known gene.
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The up-regulated 438-bp RAP-PCR product corresponding to
sspA and -B (Fig. 1, band 3; Table 1) was
reamplified and used as a probe for the Northern blot (Fig.
2). The mRNA corresponding to
sspA and -B (approximately 4.5 kb) was present in
RNA purified from cells in contact with saliva (Fig. 2, lane 2) but was
not detectable in RNA purified from cells suspended in BHI medium (Fig.
2, lane 1). The 4.5-kb transcript has the predicted size corresponding
to the translation of the sspA and -B product of approximately 1,500 amino acid (aa) residues (6).
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S. gordonii is the only oral streptococcal species so far identified that expresses two antigen I/II proteins. Mature SspA (1,542 aa residues) and SspB (1,462 aa residues) are the products of tandemly arranged chromosomal genes that are independently transcribed (6). In previous studies, SspA and -B were detected in BHI medium-grown cells, suggesting that sspA and sspB are constitutively expressed (5, 8, 16). In our experiments with BHI medium, the RAP-PCR product corresponding to sspA and -B was weak even after 40 cycles of PCR amplification (Fig. 1, band 3). In addition, the Northern blot did not show any detectable level of sspA or -B mRNA from BHI medium-grown cells, suggesting that the level of expression is low under these conditions.
The present study is the first to show up-regulation of sspA and -B transcription in the presence of saliva. The 438-bp RAP-PCR up-regulated product detected in DL1 corresponds to a region with a sequence present in both sspA and sspB in S. gordonii M5. Structural and transcriptional start site differences between the promoters of sspA and sspB in S. gordonii M5 indicate that sspA is regulated differently from sspB in S. gordonii M5 (7). It would be interesting to study the differential expression of sspA and sspB in S. gordonii DL1, but only a 2,347-bp region containing the 3' end of sspA, the intergenic region, and the 5' end of sspB has been sequenced in this strain (accession no. U40027) (6). The 438-bp RAP-PCR product is not homologous to this region. Sequencing of these two genes from S. gordonii DL1 is necessary to further analyze potential differential regulation of sspA and sspB in this streptococcus.
Survival of streptococci in the oral cavity may be dependent on their ability to adhere tightly to host tissue surfaces and to evade the host defenses in this open-flow system. We show that the transcription of the SAG-binding adhesin sspA and -B genes is directly induced when S. gordonii DL1 is placed in contact with saliva; this behavior may influence binding and colonization of the tooth surface. Early colonization can occur not only by direct binding of S. gordonii to saliva-coated surfaces (3, 19) but also by coadhesion of S. gordonii and other saliva-coated cells (3, 17), as well as by coaggregation of S. gordonii and other saliva-coated partners, including streptococci (18) and actinomyces (20). Our findings support the consideration of S. gordonii as a primary colonizer and as an anchor during biofilm development of dental plaque.
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ACKNOWLEDGMENTS |
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This research was supported by the Conseil Regional de Bretagne (France).
We thank M. Gilmore for extensive advice on DD. Thanks also to D. Demuth, H. Jenkinson, and D. Wall for helpful suggestions and R. Andersen for technical assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: National Institutes of Health/NIDCR, Bldg. 30, Room 310, 30 Convent Dr., MSC 4350, Bethesda, MD 20892-4350. Phone: (301) 496-1497. Fax: (301) 402-0396. E-mail: pkolenbrander{at}dir.nidcr.nih.gov.
Editor: E. I. Tuomanen
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REFERENCES |
|---|
|
|
|---|
| 1. | Brooks, W., D. R. Demuth, S. Gil, and R. J. Lamont. 1997. Identification of a Streptococcus gordonii SspB domain that mediates adhesion to Porphyromonas gingivalis. Infect. Immun. 65:3753-3758[Abstract]. |
| 2. |
Chen, N. Y.,
S. Q. Jiang,
D. A. Klein, and H. Paulus.
1993.
Organization and nucleotide sequence of the Bacillus subtilis diaminopimelate operon, a cluster of genes encoding the first three enzymes of diaminopimelate synthesis and dipicolinate synthase.
J. Biol. Chem.
268:9448-9465 |
| 3. |
Ciardi, J. E.,
G. F. A. McCray,
P. E. Kolenbrander, and A. Lau.
1987.
Cell-to-cell interaction of Streptococcus sanguis and Propionibacterium acnes on saliva-coated hydroxyapatite.
Infect. Immun.
55:1441-1446 |
| 4. |
Demuth, D. R.,
P. Berthold,
P. S. Leboy,
E. E. Golub,
C. A. Davis, and D. Malamud.
1989.
Saliva-mediated aggregation of Enterococcus faecalis transformed with a Streptococcus sanguis gene encoding the SSP-5 surface antigen.
Infect. Immun.
57:1470-1475 |
| 5. |
Demuth, D. R.,
C. A. Davis,
A. M. Corner,
R. J. Lamont,
P. S. Leboy, and D. Malamud.
1988.
Cloning and expression of a Streptococcus sanguis surface antigen that interacts with a human salivary agglutinin.
Infect. Immun.
56:2484-2490 |
| 6. | Demuth, D. R., Y. Duan, W. Brooks, A. R. Holmes, R. McNab, and H. F. Jenkinson. 1996. Tandem genes encode cell-surface polypeptides SspA and SspB which mediate adhesion of the oral bacterium Streptococcus gordonii to human and bacterial receptors. Mol. Microbiol. 20:403-413[Medline]. |
| 7. | Demuth, D. R., Y. Duan, H. F. Jenkinson, R. McNab, S. Gil, and R. J. Lamont. 1997. Interruption of the Streptococcus gordonii M5 sspA/sspB intergenic region by an insertion sequence related to IS1167 of Streptococcus pneumoniae. Microbiology 143:2047-2055[Abstract]. |
| 8. | Demuth, D. R., M. S. Lammey, M. Huck, E. T. Lally, and D. Malamud. 1990. Comparison of Streptococcus mutans and Streptococcus sanguis receptors for human salivary agglutinin. Microb. Pathog. 9:199-211[CrossRef][Medline]. |
| 9. | Enright, M. C., and B. G. Spratt. 1998. A multilocus sequence typing scheme for Streptococcus pneumoniae: identification of clones associated with serious invasive disease. Microbiology 144:3049-3060[Abstract]. |
| 10. | Gish, W., and D. J. States. 1993. Identification of protein coding regions by database similarity search. Nat. Genet. 3:266-272[CrossRef][Medline]. |
| 11. |
Hajishengallis, G.,
T. Koga, and M. W. Russell.
1994.
Affinity and specificity of the interactions between Streptococcus mutans antigen I/II and salivary components.
J. Dent. Res.
73:1493-1502 |
| 12. | Holmes, A. R., R. McNab, and H. F. Jenkinson. 1996. Candida albicans binding to the oral bacterium Streptococcus gordonii involves multiple adhesin-receptor interactions. Infect. Immun. 64:4680-4685[Abstract]. |
| 13. |
Ingmer, H.,
F. K. Vogensen,
K. Hammer, and M. Kilstrup.
1999.
Disruption and analysis of the clpB, clpC, and clpE genes in Lactococcus lactis: ClpE, a new Clp family in gram-positive bacteria.
J. Bacteriol.
181:2075-2083 |
| 14. |
Jenkinson, H. F.,
R. A. Baker, and G. W. Tannock.
1996.
A binding-lipoprotein-dependent oligopeptide transport system in Streptococcus gordonii essential for uptake of hexa- and heptapeptides.
J. Bacteriol.
178:68-77 |
| 15. | Jenkinson, H. F., and D. R. Demuth. 1997. Structure, function and immunogenicity of streptococcal antigen I/II polypeptides. Mol. Microbiol. 23:183-190[CrossRef][Medline]. |
| 16. |
Jenkinson, H. F.,
S. D. Terry,
R. McNab, and G. W. Tannock.
1993.
Inactivation of the gene encoding surface protein SspA in Streptococcus gordonii DL1 affects cell interactions with human salivary agglutinin and oral actinomyces.
Infect. Immun.
61:3199-3208 |
| 17. | Kolenbrander, P. E., R. N. Andersen, K. M. Kazmerzak, and R. J. Palmer, Jr. Coaggregation and coadhesion in oral biofilms. In D. Allison, P. Gilbert, H. Lappin-Scott, and M. Wilson (ed.), Community structure and co-operation in biofilms, in press. Cambridge University Press, Cambridge, United Kingdom. |
| 18. |
Kolenbrander, P. E.,
R. N. Andersen, and L. V. H. Moore.
1990.
Intrageneric coaggregation among strains of human oral bacteria: potential role in primary colonization of the tooth surface.
Appl. Environ. Microbiol.
56:3890-3894 |
| 19. | Kolenbrander, P. E., L. Dû, M. Aspiras, S. Li, K. Kazmerzak, R. Wu, and R. Andersen. Spatial organization and
contact-induced gene expression in biofilms of Streptococcus
gordonii Challis. In D. Martin and J. Tagg (ed.),
Streptococci and streptococcal diseases entering the new millennium,
in press.
|
| 20. |
Kolenbrander, P. E., and C. S. Phucas.
1984.
Effect of saliva on coaggregation of oral Actinomyces and Streptococcus species.
Infect. Immun.
44:228-233 |
| 21. | Lamont, R. J., S. Gil, D. R. Demuth, D. Malamud, and B. Rosan. 1994. Molecules of Streptococcus gordonii that bind to Porphyromonas gingivalis. Microbiology 140:867-872[Abstract]. |
| 22. |
Loo, C. Y.,
D. A. Corliss, and N. Ganeshkumar.
2000.
Streptococcus gordonii biofilm formation: identification of genes that code for biofilm phenotypes.
J. Bacteriol.
182:1374-1382 |
| 23. | Love, R. M., M. D. McMillan, and H. F. Jenkinson. 1997. Invasion of dentinal tubules by oral streptococci is associated with collagen recognition mediated by the antigen I/II family of polypeptides. Infect. Immun. 65:5157-5164[Abstract]. |
| 24. | Lunsford, R. D. 1995. Recovery of RNA from oral streptococci. BioTechniques 18:412-413[Medline]. |
| 25. |
Moisset, A.,
N. Schatz,
Y. Lepoivre,
S. Amadio,
D. Wachsmann,
M. Schöller, and J.-P. Klein.
1994.
Conservation of salivary glycoprotein-interacting and human immunoglobulin G-cross-reactive domains of antigen I/II in oral streptococci.
Infect. Immun.
62:184-193 |
| 26. |
Nakai, M.,
N. Okahashi,
H. Ohta, and T. Koga.
1993.
Saliva-binding region of Streptococcus mutans surface protein antigen.
Infect. Immun.
61:4344-4349 |
| 27. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 28. | Sciotti, M. A., I. Yamodo, J. P. Klein, and J. A. Ogier. 1997. The N-terminal half part of the oral streptococcal antigen I/IIf contains two distinct binding domains. FEMS Microbiol. Lett. 153:439-445[CrossRef][Medline]. |
| 29. |
Shepard, B. D., and M. S. Gilmore.
1999.
Identification of aerobically and anaerobically induced genes in Enterococcus faecalis by random arbitrarily primed PCR.
Appl. Environ. Microbiol.
65:1470-1476 |
| 30. | Song, B. H., and J. Neuhard. 1989. Chromosomal location, cloning and nucleotide sequence of the Bacillus subtilis cdd gene encoding cytidine/deoxycytidine deaminase. Mol. Gen. Genet. 216:462-468[CrossRef][Medline]. |
| 31. | Whittaker, C. J., C. M. Klier, and P. E. Kolenbrander. 1996. Mechanisms of adhesion by oral bacteria. Annu. Rev. Microbiol. 50:513-552[CrossRef][Medline]. |
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