Department of Oral Biology, Box 357132, University of Washington, Seattle, WA 98195, USA1
Bacterin Inc., Bozeman, MT 59717, USA2
Center for Biofilm Engineering, Montana State University, Bozeman, MT 59717, USA3
Department of Biochemistry, University of Pennsylvania School of Dental Medicine, Philadelphia, PA 19104, USA4
Author for correspondence: Richard J. Lamont. Tel: +1 206 543 5477. Fax: +1 206 685 3162. e-mail: lamon{at}u.washington.edu
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ABSTRACT |
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Keywords: minor fimbriae, antigen I/II, coadhesion
Abbreviations: BAR, SspB adherence region; CSLM, confocal scanning laser microscopy
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INTRODUCTION |
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Colonization of the plaque biofilm by P. gingivalis is contingent upon a variety of factors, including reduced oxygen tension, sufficient nutritional resources and an appropriate substrate for adhesion (Bradshaw et al., 1998 ; Lamont & Jenkinson, 2000
; Marsh, 1989
, 1994
). Furthermore, as a secondary colonizer of plaque, P. gingivalis is likely to encounter tissue surfaces that are already colonized with antecedent bacteria and their products. Consistent with these constraints, P. gingivalis has been shown to adhere to primary colonizing organisms such as S. gordonii and related streptococcal species, Actinomyces naeslundii, and to other Gram-negative secondary colonizing organisms such as Fusobacterium nucleatum, Treponema denticola and Bacteroides forsythus (Kinder & Holt, 1989
; Kolenbrander & Andersen, 1989
; Lamont et al., 1992
; Schwarz et al., 1987
; Yao et al., 1996
). Coadhesion of P. gingivalis with these organisms involves specific adhesinreceptor interactions. For example, a galactose-specific lectin mediates adherence of F. nucleatum to P. gingivalis (Shaniztki et al., 1997
). The coadhesion of P. gingivalis and S. gordonii is multimodal and involves at least two distinct adhesinreceptor pairs. The major and minor fimbriae of P. gingivalis both have been shown to bind to S. gordonii cells (Chung et al., 2000
; Lamont et al., 1993
). The major fimbriae, which may extend up to 3 µm from the cell surface, are composed of the FimA protein (Yoshimura et al., 1984
) and interact with a component of S. gordonii that has not yet been identified. In contrast, the minor fimbriae extend only 0·10·5 µm from the cell surface (Hamada et al., 1996
) and contribute to coadhesion by binding to the Ssp polypeptides (Brooks et al., 1997
; Demuth et al., 2001
). FimA and the minor fimbrial protein (designated Mfa1) are genetically and antigenically distinct (Arai et al., 2000
), suggesting that these two adhesins differ both structurally and functionally.
The Ssp proteins are members of the antigen I/II family of streptococcal surface proteins that are highly conserved in overall structure and primary sequence across all the human oral streptococcal species (Jenkinson & Demuth, 1997 ). However, despite the high degree of structural similarity, P. gingivalis adheres to streptococci and interacts with antigen I/II proteins in a species-specific manner. Our previous studies have shown that P. gingivalis adheres avidly to the SspA and SspB polypeptides of S. gordonii, but does not interact with the SpaP protein, the homologue of SspA/B expressed by Streptococcus mutans (Brooks et al., 1997
). Adherence is mediated by a discrete domain, designated BAR (SspB adherence region), comprising amino acid residues 11671250 of SspB, which is fully conserved between SspB and SspA (Brooks et al., 1997
; Demuth et al., 2001
). Within BAR, Asn1182 and Val1185 have been suggested to confer a unique secondary structural motif that is recognized and bound by the P. gingivalis minor fimbrial protein (Demuth et al., 2001
). This structural motif (and Asn1182 and Val1185) is essential for adherence and is not conserved in SpaP polypeptide.
Using an open flow chamber under conditions of low shear force, Cook et al. (1998) showed that subsequent to its adherence to S. gordonii, P. gingivalis rapidly accretes to form a biofilm consisting of structures resembling towering microcolonies separated by fluid-filled channels. Although adherence is clearly a first step that is necessary for accretion to occur, it is not known how the two independent fimbrial-mediated adherence mechanisms of P. gingivalis contribute to biofilm formation. In this study, we show that the formation of P. gingivalis biofilms exhibits the same species specificity observed in minor-fimbriae-mediated adherence, suggesting that the Mfa1-mediated interaction of P. gingivalis with the streptococcal Ssp polypeptides may drive biofilm development. Moreover, P. gingivalis biofilms do not form on an Ssp null mutant of S. gordonii which retains the ability to interact with FimA, or when the mfa1 gene of P. gingivalis has been disrupted. In addition, recombinant Enterococcus faecalis strains expressing chimeric SspB/SpaP proteins containing BAR support biofilm growth, whereas hybrid proteins without BAR do not. SspB proteins containing site-specific mutations of essential functional amino acid residues of BAR do not permit P. gingivalis biofilm development. These results suggest that the interaction of the minor fimbriae with Ssp is sufficient to allow P. gingivalis to form biofilms on a streptococcal substrate.
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METHODS |
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Construction of P. gingivalis minor-fimbriae-deficient mutant.
P. gingivalis SMF1 was created by homologous recombination between P. gingivalis 33277 chromosomal DNA and a suicide plasmid carrying an internal fragment of the mfa1 gene as follows. A 0·85 kb internal fragment of mfa1 was amplified by PCR with the primers 5'-TATCCGAGGCCAATGCTATC-3' and 5'-GCATCAAGAAGTTGGGCTTC-3'. Amplification conditions were denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and elongation at 72 °C for 3 min, for 30 cycles. The product was cloned into the PCR cloning vector pCR11 (Invitrogen). A BamHIXbaI fragment was excised and ligated into the shuttle vector pVA3000 that contains the antibiotic resistance cassette ermAM-ermF (Lee et al., 1996 ). The resulting plasmid was conjugated into P. gingivalis 33277 as described previously (Park & Lamont, 1998
). Transconjugants with erythromycin resistance were selected. Confirmation that the plasmid had integrated into the mfa1 gene was achieved by Southern blotting and loss of mRNA expression was established by RT-PCR using the primers defined above.
Interbacterial binding assay.
Coadhesion between P. gingivalis and S. gordonii was measured with the nitrocellulose blot assay described previously (Lamont et al., 1992 ). Briefly, S. gordonii cells were suspended in buffered KCl (5 mM KCl, 2 mM K2PO4, 1 mM CaCl2, pH 6·0) and 108 bacteria were deposited on nitrocellulose paper in a dot-blot apparatus. The blot was washed three times in KCl containing 0·1% Tween 20 (KCl-Tween). The adsorbed bacteria were subsequently incubated for 2 h at room temperature with [3H]thymidine-labelled P. gingivalis strains 33277 or SMF1 (mean c.p.m. per cell, 5x10-4) suspended in KCl-Tween. After washing to remove unbound organisms, the experimental areas of the nitrocellulose were excised and adherence was quantified by scintillation spectroscopy.
Biofilm formation.
Biofilm studies were carried out essentially as described by Cook et al. (1998) . Briefly, standard glass coverslips were coated with centrifugation-clarified pooled human saliva for 30 min at 37 °C and washed three times with sterile PBS. Saliva-coated glass coverslips were mounted under aseptic conditions in a polycarbonate flow cell (0·6x1·0 cm) with an attached peristaltic pump. Flow cells were inoculated initially with the streptococcal or enterococcal strains (107 cells ml-1) at a flow rate of 4·1 ml h-1 for 2 h (S. gordonii) or 4 h (S. mutans and E. faecalis). S. mutans and E. faecalis adhere less efficiently to saliva surfaces in comparison to S. gordonii, hence the longer incubation period. The formation of a 7080% confluence bacterial monolayer was confirmed by phase-contrast microscopy. The flow cells were then subjected to a secondary inoculation of P. gingivalis (107 cells ml-1) under similar flow conditions. The total length of exposure of streptococci or enterococci to P. gingivalis was 4 h, which is less than the doubling time for P. gingivalis cells when grown in rich broth. Thus, under these conditions the accumulation of P. gingivalis biofilms is a result of accretion of planktonic cells rather than growth of bound cells. Biofilms were visualized with a Bio-Rad MRC600 confocal scanning laser microscope with an Olympus IMT-2 inverted light microscope and a MS plan 60x 1.4 NA objective. For confocal scanning laser microscopy (CSLM), streptococci and enterococci were stained with 10 mg hexidium iodide ml-1 and P. gingivalis was stained with 10 mg fluorescein ml-1. CSLM was first used in the reflected white light mode to directly observe biofilm formation over the course of the experiments. A representative area of the coverslip was then selected and observed under reflected laser light of 488, 546 and 647 nm. A series of fluorescent optical sections were collected to determine the depth of the bacterial layers and/or microcolonies and to assemble a three-dimensional view of the biofilms using the Slicer (Fortner Research) imaging program. Under these conditions of biofilm formation, P. gingivalis cells were observed to gradually and continuously accumulate on the substratum over the 4 h inoculation period. Sloughing and reattachment of biofilm microcolonies was not observed and no significant auto-aggregation of planktonic P. gingivalis cells occurred.
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RESULTS |
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DISCUSSION |
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Our studies have focused on the adherence of P. gingivalis to streptococci and the subsequent formation of biofilms on the streptococcal substrate. Adherence of P. gingivalis is mediated by at least two distinct adhesinreceptor pairs (Brooks et al., 1997 ; Chung et al., 2000
; Lamont et al., 1993
) involving both the FimA and Mfa1 proteins, the predominant polypeptides composing the major and minor fimbriae of P. gingivalis, respectively. Both adhesins have been shown to mediate adherence to S. gordonii under static conditions in vitro. Several lines of evidence from the present investigation suggest that the development of P. gingivalis biofilms on a S. gordonii substratum is dependent primarily on the interaction of minor fimbriae with streptococcal antigen I/II. First, the development of P. gingivalis biofilms exhibited the same species specificity as was previously demonstrated for minor-fimbriae-mediated adherence to streptococci in vitro. Second, biofilms did not develop on S. gordonii OB219, a strain that does not express the antigen I/II receptor for the minor fimbrial adhesin but which retains the ability to interact with the major fimbrial adhesin. Thus, FimA-mediated adhesion alone is insufficient to induce the formation of biofilms. Third, a minor-fimbriae-deficient mutant of P. gingivalis was impaired in its ability to bind to S. gordonii and was unable to form biofilms. The structural motif (BAR) of antigen I/II that is recognized by Mfa1 also was shown to be essential for the development of P. gingivalis biofilms. Recombinant E. faecalis strains expressing chimeric SspB/SpaP proteins lacking BAR or expressing SspB site-specific mutants lacking essential residues of BAR did not support biofilm development. These results demonstrate that wild-type P. gingivalis (FimA+, Mfa1+) are unable to form biofilms on streptococci or enterococcal constructs that do not possess the appropriate antigen I/II determinants, even if the cells are capable of interacting with FimA. Thus, individual components of the multimodal binding interaction appear to have discrete and independent roles. Consistent with this concept, Love et al. (2000)
found FimA binding to S. gordonii to be independent of SspA or SspB and that a FimA mutant was able to co-invade dentinal tubules with S. gordonii.
While our results indicate that the Mfa1SspB-mediated interaction is necessary for biofilm development, they do not completely exclude the possibility of a role for the other adhesinreceptor pairs that participate in the multimodal P. gingivalisS. gordonii coadhesion event. These molecules could serve either to allow initial low-affinity contact between the organisms or to help stabilize the interaction between adhered organisms. The long major fimbriae that can extend up to 3 µm from the cell surface would be suitable candidates to effect relatively long-range, low-affinity interactions with the streptococci. The proximal association between the two organisms would then allow the close range, higher affinity Mfa1SspB interactions that induce biofilm formation. The role of the major fimbriae will be more apparent when the identification and distribution of their cognate receptor is accomplished. This model of P. gingivalisS. gordonii coadhesion and biofilm development is represented in Fig. 7. Longer distance interactions, primarily mediated by the major fimbriae, facilitate localization of cells of P. gingivalis and S. gordonii. Multimodal coadhesion then occurs involving both Mfa1SspB and FimAreceptor interactions. The engagement of Mfa1 with SspB initiates a signal transduction event within P. gingivalis that results in cells that are primed for biofilm formation. Theoretically, the transformation to a biofilm ready state could entail expression of a surface receptor that permits autoaggregation of P. gingivalis and stimulates other P. gingivalis cells to produce the same receptor. Alternatively, or additionally, a soluble signalling molecule may be released by P. gingivalis that stimulates cells to accumulate on the solid phase.
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The maintenance of the complex oral biofilm community is thought to depend on a series of cohesive interactions that stabilize plaque structure and facilitate nutritional interrelationships (Kolenbrander & London, 1993 ; Rosan & Lamont, 2000
). Moreover, the adherence event itself can transduce information into the bacterial cell regarding the substrate and environment in which the organism has localized (Cleary & Retnoningrum, 1994
; Cornelis & Van Gijsegem, 2000
; McNab & Jenkinson, 1998
). In the case of BAR-mediated adhesion, P. gingivalis responds by accretion into microcolonies. This process can be postulated to involve intracellular signalling pathways within P. gingivalis, an example of which has recently been described (Hayashi et al., 2000
), and modulation of gene expression. The complete range of phenotypic changes that accompany biofilm formation remains to be determined.
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ACKNOWLEDGEMENTS |
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Received 23 January 2002;
revised 12 February 2002;
accepted 18 February 2002.