Department of Oral Biology, State University of New York at Buffalo, 3435 Main Street, Buffalo, NY 14214-3092, USA
Correspondence
Peter M. Vesey
p.m.vesey{at}ncl.ac.uk
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ABSTRACT |
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Present address: Oral Biology, School of Dental Sciences, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4BW, UK.
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INTRODUCTION |
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It has been shown by scanning electron microscopic investigations that oral treponemes and rod-like bacteria are the main morphological agents at the advancing front of subgingival plaque in sites of periodontitis (Saglie et al., 1982). Oral spirochaetes have been implicated in the aetiology of periodontal diseases (Loesche, 1988
, 1993
), and their numbers strongly correlate with the severity of periodontal inflammation. Treponema denticola is a small helically shaped obligate anaerobic spirochaete frequently isolated from the periodontal pocket and correlated with the severity of periodontal disease (Simonson et al., 1988
; Haffajee & Socransky, 1994
; Asai et al., 2002
). Another major Gram-negative rod bacterium which has been associated with periodontitis is Porphyromonas gingivalis (Slots & Genco, 1984
). It has been shown previously in vitro that there is a symbiotic relationship between these two periodontopathogens for nutrients (Grenier, 1992
). The interaction between T. denticola and P. gingivalis in biofilm formation could play an important role in the initial stages of the onset of periodontal disease. Furthermore, no information is currently available regarding biofilm formation by any spirochaete.
In T. denticola the cytoplasmic filaments, a ribbon-like structure, span the cytoplasm at all stages of the cell division process. It has been suggested that the cytoplasmic filament protein, CfpA, may be involved in chromosome structure, segregation, or the cell division process in T. denticola 33520 (Izard et al., 2001). Here, we report the construction of a T. denticola 35405 cfpA mutant and investigate its possible role in biofilm formation.
The structure and motility of treponemes such as T. denticola is an unusual feature which enables motility in a highly viscous environment (Kimsey & Spielman, 1990; Klitorinos et al., 1993
). Like other spirochaetes, the T. denticola flagellar filaments are located within the periplasmic space. A non-motile mutant, lacking flagellar hooks and flagella, was previously constructed by insertionally inactivating the flgE gene by allelic exchange (Li et al., 1996
). It was therefore of interest to utilize this mutant to assess the role of motility in biofilm formation by T. denticola. We also speculated that chemotaxis may be an important factor during the initial development of biofilm formation. A T. denticola mutant defective in the T. denticola methyl-accepting chemotaxis A (dmcA) gene has been shown to be defective in chemotaxis toward nutrients (Kataoka et al., 1997
). Therefore, this mutant was also examined to investigate the role of chemotaxis in mixed biofilm formation with P. gingivalis.
As earlier studies have found that the cell surface Msp may serve as a T. denticola adhesin (Fenno et al., 1996), it was also of interest to examine the role of this putative outer sheath protein in mixed biofilm formation. Likewise, the prolyl-phenylalanine-specific serine protease (dentilisin; also called chymotrypsin-like protease) is a major extracellular protease produced by T. denticola (Fenno et al., 1998
). The gene encoding this protease, prtP, has been isolated and inactivated (Ishihara et al., 1998
) and the attenuated protease-deficient mutant was compared to the T. denticola type strain in its ability to form a mixed biofilm. It has already been suggested that dentilisin participates in adhesion to epithelial cells (Leung et al., 1996
) and also affects the organization of the Msp protein (Ishihara et al., 1998
).
The present study investigated the interaction between the two periodontopathogens P. gingivalis and T. denticola in an in vitro biofilm model. The aim of this study was first to devise a procedure to accurately quantitate T. denticola in a biofilm and second to identify the genes involved in mixed biofilms of T. denticola and P. gingivalis using a panel of T. denticola mutants. These results suggest that T. denticola requires several factors to form mixed biofilms with P. gingivalis.
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METHODS |
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Construction of the cfpA mutant in T. denticola 35405.
A T. denticola 35405 cfpA mutant was constructed by electroporation using the DraDentErm 8 plasmid (unnamed in Izard et al., 2001; supplied by J. Izard, New York State Department of Health, Albany, NY, USA) which contained a cloned cfpA-20H fragment from T. denticola 33520 disrupted at the DraI site by intergration of the ErmF/AM cassette (Izard et al., 2001
). Studies in our laboratory have shown that the Erm cassette does not produce polar mutations in T. denticola when cloned in the same direction as the inactivated gene. Briefly, 12 µg non-methylated plasmid DNA was used to electroporate T. denticola ATCC 35405 competent cells produced by growing 100 ml T. denticola to an OD600 of 0·3. The cells were washed three times with ice-cold 10 % glycerol and resuspended in a final volume of 2 ml electroporation buffer. Electroporation was performed using 100 µl cells and 1 µl plasmid DNA in cuvettes with a 1 mm gap using a Bio-Rad Gene Pulser at 1·8 kV, 200
and 25 µF (Bio-Rad). The time constant was 4·3 ms. Immediately following electroporation 2 ml TYGVS was added followed by overnight anaerobic incubation at 37 °C. Cells were then grown on TYGVS plates with SeaPlaque, 0·7 % (w/v) (low-melting-point) agarose (Bio Whittaker) and 40 µg erythromycin ml1. After 7 days, colonies were visible and isolated. Disruption of the cfpA gene was confirmed using PCR with cfpA probes. Confirmation of mutant construction was carried out using PCR amplification outside the region of gene interruption using primers DENTCF4 (5'-AAATCGCTACCCTTCTTGATG-3') located in the cfpA sequence and DENTCF5 (5'-GCAGCCAAATCGTTAAAG-3') located outside the cloned sequence in the 3' end of the cfpA sequence. For PCR to confirm the relative position of the antibiotic resistance cassette we used DENTCF4 and ERMBGLF (Limberger et al., 1999
). An additional PCR to confirm the position of the antibiotic resistance cassette used ERMBGLR (Limberger et al., 1999
) and DENTCF5.
Quantification of T. denticola using SAAPNA assays.
To accurately quantify the number of T. denticola attached to a surface or incorporated into a biofilm we needed to first establish a convenient and accurate procedure for quantification. In this regard the dentilisin activity of the oral spirochaete was utilized. The fluorogenic chymotrypsin substrate II, Suc-Ala-Ala-Pro-Phe-AMC (SAAPNA) (Calbiochem), was dissolved in 50 mM Tris/HCl, pH 8, to a concentration of 12·59 mM. For analysis, 12 µl SAAPNA substrate was added to 138 µl cell suspension in a 96-well microtitre plate and read in a Bio-Tek FLx 800 microplate fluorescence reader using excitation at 360 nm and emission at 460 nm. In each assay the SAAPNA activity of an aliquot of cells at an OD660 of 0·2 in PBS (10 mM potassium phosphate, 0·9 % NaCl, pH 7·4) was measured to normalize the chymotrypsin activity of the mutants to T. denticola 35405. The results are presented as relative fluorescence units (r.f.u.).
Attachment of T. denticola to fibronectin.
T. denticola was collected from a 5-day-old liquid culture by centrifugation (10 000 g), washed once in PBS and adjusted to an OD660 of 0·2 (Haapasalo spectrophotomoter) in PBS, corresponding to 5x108 cells ml1 as determined with a PetroffHausser bacteria counter (Hausser & Son). Fibronectin (Sigma; 100 µl) was used to coat polystyrene 96-well plates at a concentration of 1 mg ml1 for 12 h at 5 °C. After washing three times with 200 µl PBS, 100 µl T. denticola ATCC 35405 (OD660 of 0·2 in PBS) was added, incubated and washed again. The number of attached cells was then determined using the SAAPNA assay outlined above.
Biofilm formation.
An in vitro static biofilm model was used to quantify biofilm formation by T. denticola and P. gingivalis. A P. gingivalis biofilm was initially formed in 96-well polyvinylchloride (PVC) plates by inoculating 10 ml BHI+haemin with a 100 µl P. gingivalis 381 overnight starter culture and then aliquoting the culture at 100 µl per well. The plate was incubated at 37 °C anaerobically overnight. The following day planktonic P. gingivalis cells were removed by washing three times with 200 µl PBS. The P. gingivalis biofilm was then incubated with 100 µl T. denticola cells from an overnight culture resuspended in fresh TYGVS (OD660 of 0·2).
Quantification of T. denticola using viability counts.
The procedure outlined above for the production of an in vitro biofilm was followed with some modifications. Prior to the final washing step, a 25 µl aliquot of planktonic cells was removed from the wells of the microtitre plate. Dilutions of the planktonic cells were made in PBS and spread onto TYGVS+low-melting-point SeaPlaque agarose (0·8 % w/v) containing rifampicin (1 µg ml1) selective medium. The wells were then washed three times with sterile PBS and resuspended with 100 µl PBS. The biofilm was removed by mechanical disruption with a pipette tip and a homogeneous suspension was achieved by repeatedly pipetting PBS into the well containing the biofilm. Removal of the biofilm was evaluated by staining any residual biofilm with crystal violet dye. Dilutions of the biofilm were made in PBS and spread onto TYGVS+low-melting-point SeaPlaque agarose (0·8 % w/v) containing rifampicin (1 µg ml1) selective medium. The results were obtained in duplicate and the mean number of c.f.u. determined.
Scanning electron microscopy (SEM).
Microscope glass coverslips were incubated in the wells of 6-well polystyrene microtitre plates with a 1 : 10 dilution of a P. gingivalis 381 overnight culture in 2 ml TSB medium and incubated anaerobically at 37 °C for 8 h. The cover slips were then washed with 3 vols PBS and the preformed P. gingivalis biofilm was incubated with T. denticola 35045 resuspended in PBS to an OD660 of 0·2 and incubated anaerobically at 37 °C for 1 h.
The mixed biofilm was prepared for SEM using the following protocol. The biofilm was washed three times with PBS and fixed using 2·5 % glutaraldehyde in 0·1 M sodium cacodylate buffer, pH 7·4, for 30 min at room temperature. The biofilm was then washed three times for a total of 10 min with sodium cacodylate buffer, 0·2 M pH 7·4, followed by sequential dehydration in increasing concentrations of ethanol, 5 min in each (25, 50, 70, 90 and 100 %). A final dehydration step in hexamethyl disilazane (HMDS) (Sigma) for 30 min was included, the biofilm was then coated with carbon and viewed using SEM (Hitachi S-4000 FESEM) operating at 15 kV.
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RESULTS |
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Attachment of T. denticola to fibronectin was determined at three time points, 1, 2 and 3 h. When quantified using the SAAPNA substrate the biofilms gave r.f.u. values of 13 225±2212, 10 611±645 and 4288±329, respectively. Analysis of the kinetics of biofilm formation indicated that T. denticola 35405 colonization of fibronectin-coated plates decreased over a 3 h period. This was undoubtedly the result of the degradation of fibronectin by dentilisin of strain 35405 (Ishihara et al., 1996) as the opposite observation was made with the dentilisin mutant. In contrast, when T. denticola was incubated with a preformed P. gingivalis biofilm the numbers of T. denticola recovered from the mixed biofilm after 5, 11·5 and 27 h gradually increased. When quantified using SAAPNA substrate, r.f.u. values of 1193±188, 4349±435 and 8160±633, respectively, were obtained. Therefore, T. denticola biofilm formation on preformed P. gingivalis biofilms was examined in greater detail.
The number of T. denticola used in the biofilm studies was standardized to an OD660 of 0·2. However, due to the highly sensitive SAAPNA substrate and detection system used there was a large variance in r.f.u. values on different days, largely due to variation in the fluorescence reader sensitivity setting. In contrast, quantification of T. denticola in biofilms run in triplicate and then quantified simultaneously in 96-well microtitre plates gave consistent results.
Biofilm formation by T. denticola mutants
To examine the genetic basis for initial T. denticola biofilm formation, the role of several potential colonization-dependent genes of the oral spirochaete in mixed biofilm formation was examined. We constructed a T. denticola 35405 cfpA mutant, PVUB7, which was confirmed by PCR to have a disrupted cfpA gene, resulting in reduced migration in TYGVS plates when compared to T. denticola 35405 (data not shown). These findings were consistent with the findings of Izard et al. (2001) and the corresponding T. denticola 33520 cfpA mutant. When the cfpA mutant was compared with parental strain 35405, it was observed that the mutant was attenuated in forming biofilms on fibronectin (70·1 % reduction) as well as P. gingivalis-coated surfaces (89·2 % reduction) when compared to ATCC 35405. Therefore, though localized cytoplasmically (Izard et al., 2001
), this filamentous protein affects biofilm formation. The cfpA mutant, and other T. denticola mutants tested, all displayed similar planktonic growth rates.
Chemotaxis, specifically involving the methyl-accepting chemotaxis protein DmcA, did not appear to play a significant role in the initial attachment of T. denticola into a mixed biofilm with P. gingivalis (Fig. 2). Recently, a novel leucine-rich repeat protein gene, lrrA, has been identified in our laboratory (A. Ikegami & H. K. Kuramitsu, unpublished data). This putative lipoprotein has been demonstrated to mediate interactions between strain 35405 and other oral bacteria, including P. gingivalis. A T. denticola lrrA mutant was constructed by insertional inactivation using a suicide plasmid with the cassette in the same orientation as the lrrA gene. However, only a slight reduction in the ability of the lrrA mutant to form a mixed biofilm was noted (Fig. 2
). By contrast, the major surface protein of T. denticola, Msp, appears to play a major role in biofilm formation with P. gingivalis since the msp mutant was markedly attenuated in mixed biofilm formation (Fig. 2
). Furthermore, the flgE mutant was also unable to initiate a biofilm (Fig. 2
) with P. gingivalis and displayed reduced attachment to fibronectin (data not shown). These results suggest that in the in vitro mixed biofilm model the motility of T. denticola and its major outer sheath protein are both important factors in the formation of a mixed biofilm with P. gingivalis. Longer term biofilms with T. denticola mutants that are impaired in the initial stages of biofilm formation did not demonstrate an increased incorporation of T. denticola into the mature biofilm (data not shown).
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DISCUSSION |
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The present results have also suggested the important role of several T. denticola genes in the formation of mixed biofilms with P. gingivalis. The cytoplasmic filament protein, CfpA, appears to play a key role in this process. Strain 35405 mutants which are defective in the cfpA gene are attenuated in colonizing preformed biofilms of P. gingivalis 381. The mutant appears to be also defective in spreading on agarose plates as observed for a similar mutant constructed in strain 33520 (lzard et al., 2001). However, the motility of the cfpA mutant compared to parental strain 35405 did not appear to be altered when viewed under dark field microscopy. Whether or not the reduction in biofilm formation is related to the altered spreading property of the mutant in relatively viscous media still remains to be determined. Since spreading is considered to be an important factor in the development of biofilms (O'Toole et al., 2000
), this may be a reasonable explanation for this property of the mutant. Moreover, it is interesting that a cytoplasmic protein can affect the spreading properties of the organism in relatively viscous media. Whether or not this is a direct or indirect effect on this property is still uncertain.
The motility of T. denticola appears to play a role in mixed biofilm formation since the flgE mutant, which is non-motile (Li et al., 1996), is markedly attenuated in colonizing preformed P. gingivalis biofilms. It may be that motility is required to overcome the repulsive forces present on the surface of P. gingivalis biofilms. However, we cannot rule out the possibility that some other defect in the flgE mutant is responsible for such alterations.
Although the methyl-accepting chemotaxis protein DmcA does not appear to play a role in forming mixed biofilms with P. gingivalis, the importance of chemotaxis cannot be ruled out as DmcA is not the only methyl-accepting chemotaxis protein which exists in T. denticola (Kataoka et al., 1997).
The Msp protein of strain 35405 also appears to play a major role in forming mixed biofilms with P. gingivalis. The msp mutant was markedly deficient in forming mixed biofilms in the static in vitro system. This is not surprising since this surface protein has been proposed to serve as a major adhesin of these organisms (Fenno et al., 1996). Interestingly, since dentilisin was demonstrated to be involved in the organization of Msp into the outer sheath of T. denticola (Ishihara et al., 1998
), the prtP mutant was not attenuated in mixed biofilm formation as was the msp mutant. This suggests that the PrtP-mediated alteration of Msp maturation is not required for interaction with P. gingivalis in forming mixed biofilms.
Recently, a mutant defective in the LrrA leucine-rich repeat protein of T. denticola has been constructed and demonstrated to show increased co-aggregation with P. gingivalis 381 (A. Ikegami & H. K. Kuramitsu, unpublished data). Therefore, the present observation that the lrrA mutant was only moderately altered in forming mixed biofilms with P. gingivalis is compatible with this property.
The present results demonstrating that T. denticola can form mixed biofilms with preformed biofilms of P. gingivalis in vitro may be relevant to subgingival plaque formation by these periodontopathic organisms. An earlier in situ localization study of both organisms in subgingival plaque using monospecific antibodies has demonstrated that T. denticola is present in layers which appear to be exterior to P. gingivalis (Kigure et al., 1995). This may suggest a sequential colonization of the subgingival region: attachment by P. gingivalis to other early colonizers followed by subsequent colonization by the oral spirochaete. However, since other organisms known to interact with both organisms are also present in subgingival plaque (i.e. Fusobacterium nucleatum) other interactions (Kolenbrander, 1988
) may also play important roles in the incorporation of both organisms into subgingival plaque. Clearly, since dental plaque is composed of a variety of organisms, a multiplicity of interactions are likely to be involved in oral biofilm formation. Nevertheless, the present investigation suggests one of the interactions which could play an important role in the initial colonization of the gingival margin by T. denticola.
Future work in our laboratory is under way to develop an alternative method for studying the relationship between P. gingivalis and T. denticola using confocal scanning laser microscopy. Mixed biofilm formation in a continuous flow cell biofilm model will also be evaluated.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Asai, Y., Jinno, T., Igarashi, H., Ohyama, Y. & Ogawa, T. (2002). Detection and quantification of oral treponemes in subgingival plaque by real time PCR. J Clin Microbiol 40, 33343340.
Dawson, J. R. & Ellen, R. P. (1994). Clustering of fibronectin adhesins towards Treponema denticola tips upon contact with immobilized fibronectin. Infect Immun 62, 22142221.[Abstract]
Fenno, J. C., Muller, K. H. & McBride, B. C. (1996). Sequence analysis, expression, and binding activity of recombinant major outer sheath protein (Msp) of Treponema denticola. J Bacteriol 178, 24892497.[Abstract]
Fenno, J. C., Wong, G. L., Hannam, P. M. & McBride, B. C. (1998). Mutagenesis of outer membrane virulence determinants of the oral spirochete Treponema denticola. FEMS Microbiol Lett 163, 209215.[CrossRef][Medline]
Grenier, D. (1992). Nutritional interactions between two suspected periodontopathogens, Treponema denticola and Porphyromonas gingivalis. Infect Immun 60, 52985301.[Abstract]
Haffajee, A. D. & Socransky, S. S. (1994). Microbial etiological agents of destructive periodontal diseases. Periodontol 2000 5, 78111.
Ishihara, K., Miura, T., Kuramitsu, H. K. & Okuda, K. (1996). Characterization of the Treponema denticola prtP gene encoding a prolyl-phenyalanine-specific protease (dentilisin). Infect Immun 64, 51785186.[Abstract]
Ishihara, K., Kuramitsu, H. K., Miura, T. & Okuda, K. (1998). Dentilisin activity affects the organization of the outer sheath of Treponema denticola. J Bacteriol 180, 38373844.
Izard, J., Samsonoff, W. A. & Limberger, R. J. (2001). Cytoplasmic filament-deficient mutant of Treponema denticola has pleiotropic defects. J Bacteriol 183, 10781084.
Kataoka, M., Li, H., Arakawa, S. & Kuramitsu, H. (1997). Characterization of a methyl-accepting chemotaxis protein gene, dmcA, from the oral spirochete Treponema denticola. Infect Immun 65, 40114016.[Abstract]
Kigure, T., Saito, A., Seida, K., Yamada, S., Ishihara, K. & Okuda, K. (1995). Distribution of Porphyromonas gingivalis and Treponema denticola in human subgingival plaque at different periodontal pocket depths examined by immunochemical methods. J Periodontol Res 30, 332341.[Medline]
Kimsey, R. B. & Spielman, A. (1990). Motility of Lyme disease spirochetes in fluids as viscous as the extracellular matrix. J Infect Dis 162, 12051208.[Medline]
Klitorinos, A., Noble, P., Siboo, R. & Chan, E. C. (1993). Viscosity-dependent locomotion of oral spirochetes. Oral Microbiol Immunol 8, 242244.[Medline]
Kolenbrander, P. E. (1998). Intergeneric coaggregation among human oral bacteria and ecology of dental plaque. Annu Rev Microbiol 42, 627656.
Kolenbrander, P. E., Roxannan, A., Blehert, D. S., Egland, P. G., Foster, J. S. & Palmer, R. J., Jr (2002). Communication among oral bacteria. Microbiol Mol Biol Rev 66, 486505.
Leung, W. K., Haapasalo, M., Uitto, V.-J., Hannam, P. M. & McBride, B. C. (1996). The surface proteinase of Treponema denticola may mediate attachment of the bacteria to epithelial cells. Anaerobe 2, 3946.[CrossRef]
Li, H., Ruby, J., Charon, N. & Kuramitsu, H. (1996). Gene inactivation in the oral spirochete Treponema denticola: construction of an flgE mutant. J Bacteriol 178, 36643667.[Abstract]
Limberger, R. J., Slivienski, L. L., Izard, J. & Samsonoff, W. A. (1999). Insertional inactivation of Treponema denticola tap1 results in a nonmotile mutant with elongated flagellar hooks. J Bacteriol 181, 37433750.
Loesche, W. J. (1988). The role of spirochetes in periodontal disease. Adv Dent Res 275283.
Loesche, W. J. (1993). Bacterial mediators in periodontal disease. Clin Infect Dis 16 (suppl. 44), S203S210.
O'Toole, G. A., Kaplan, H. B. & Kolter, R. (2000). Biofilm formation as microbial development. Annu Rev Microbiol 54, 4979.[CrossRef][Medline]
Pratt, L. A. & Kolter, P. (1998). Genetic analysis of Escherichia coli biofilm formation: roles of flagella, motility, chemotaxis and type I pili. Mol Microbiol 30, 285293.[CrossRef][Medline]
Saglie, R., Newman, M. G., Carranza, F. A. & Pattison, G. L. (1982). Bacterial invasion of gingiva in advanced periodontitis in humans. J Periodontol 53, 217222.[Medline]
Simonson, L. G., Goodman, C. H., Bial, J. J. & Morton, H. E. (1988). Quantitative relationship of Treponema denticola to severity of periodontal disease. Infect Immun 56, 726728.[Medline]
Slots, J. & Genco, R. J. (1984). Black-pigmented Bacteroides species, Capnocytophaga species, and Actinobacillus actinomycetemcomitans in human periodontal disease: virulence factors in colonization, survival, and tissue destruction. J Dent Res 63, 412421.[Medline]
Received 3 October 2003;
revised 4 February 2004;
accepted 8 March 2004.
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