The oral pathogen Porphyromonas gingivalis expresses a homolog of the internalin family protein InlJ. Inactivation of inlJ reduced monospecies biofilm formation by P. gingivalis. In contrast, heterotypic P. gingivalis-Streptococcus gordonii biofilm formation was enhanced in the InlJ-deficient mutant. The results indicate a nuanced role for InlJ in regulating biofilm accumulations of P. gingivalis

Capestany, Cindy A.

Jung, Il-Young

Kuboniwa, Masae

Lamont, Richard J.

Park, Yoonsuk

Tribble, Gena D.

PubMed

The oral pathogen Porphyromonas gingivalis expresses a homolog of the internalin family protein InlJ. Inactivation of inlJ reduced monospecies biofilm formation by P. gingivalis. In contrast, heterotypic P. gingivalis-Streptococcus gordonii biofilm formation was enhanced in the InlJ-deficient mutant. The results indicate a nuanced role for InlJ in regulating biofilm accumulations of P. gingivalis.ope

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Yonsei University Medical Library Open Access Repository

  10.1128/IAI.74.5.3002-3005.2006. 2006, 74(5):3002. DOI:Infect. Immun. Yoonsuk Park, Gena D. Tribble and Richard J. LamontCindy A. Capestany, Masae Kuboniwa, Il-Young Jung, Biofilm DevelopmentProtein in Homotypic and Heterotypic  InlJPorphyromonas gingivalisRole of the http://iai.asm.org/content/74/5/3002Updated information and services can be found at: These include:REFERENCEShttp://iai.asm.org/content/74/5/3002#ref-list-1This article cites 18 articles, 7 of which can be accessed free at:CONTENT ALERTS more»articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when newhttp://journals.asm.org/site/misc/reprints.xhtmlInformation about commercial reprint orders: http://journals.asm.org/site/subscriptions/To subscribe to to another ASM Journal go to:  on June 18, 2014 by YONSEI UNIV MED LIBRARYhttp://iai.asm.org/Downloaded from  on June 18, 2014 by YONSEI UNIV MED LIBRARYhttp://iai.asm.org/Downloaded from INFECTION AND IMMUNITY, May 2006, p. 3002–3005 Vol. 74, No. 50019-9567/06/$08.000 doi:10.1128/IAI.74.5.3002–3005.2006Copyright © 2006, American Society for Microbiology. All Rights Reserved.NOTESRole of the Porphyromonas gingivalis InlJ Protein in Homotypicand Heterotypic Biofilm DevelopmentCindy A. Capestany, Masae Kuboniwa, Il-Young Jung,† Yoonsuk Park, Gena D. Tribble,and Richard J. Lamont*Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, Florida 32607Received 9 December 2005/Returned for modification 16 January 2006/Accepted 6 February 2006The oral pathogen Porphyromonas gingivalis expresses a homolog of the internalin family protein InlJ. In-activation of inlJ reduced monospecies biofilm formation by P. gingivalis. In contrast, heterotypic P. gingivalis-Streptococcus gordonii biofilm formation was enhanced in the InlJ-deficient mutant. The results indicate anuanced role for InlJ in regulating biofilm accumulations of P. gingivalis.The internalin protein family was originally identified inListeria monocytogenes and is characterized by the presence ofan N-terminal leucine-rich repeat (LRR) domain (1). LRRsare involved in protein-protein interactions including ligand-receptor binding (5). Indeed, internalin A (InlA) and InlB ofL. monocytogenes are involved in adherence and invasion ofthe organism (1, 3). Recently, a novel internalin, InlJ, wasidentified in L. monocytogenes (12). InlJ defines a new subclassfamily of cysteine-containing LRR proteins. An InlJ mutant ofListeria was found to be significantly attenuated in virulence inmice (12). InlJ type proteins are currently identified in only sixbacterial species, one of which is the gram-negative oralanaerobe Porphyromonas gingivalis (12). Expression of InlJ(PG0350) (http://www.lanl.gov) in P. gingivalis was originallydetected when the organism was in contact with host epithelialcells (2). However, an InlJ mutant of P. gingivalis did notexhibit an invasion-related phenotype (18), as is also the casefor the Listeria InlJ mutant (12). In addition to an intracellularlocation, a significant component of the P. gingivalis lifestyle iswithin the complex multispecies biofilm (dental plaque) thatdevelops on tooth surfaces. In this study, we investigated therole of the P. gingivalis InlJ protein in single-species and mul-tispecies biofilm formation by the organism. Interestingly, anInlJ-null mutant exhibited reduced monospecies biofilm devel-opment but enhanced heterotypic biofilm formation with Strep-tococcus gordonii.An InlJ-deficient mutant was generated by insertional inacti-vation in P. gingivalis strain 33277. A central 847-bp fragment ofinlJ (PG0350) was amplified by PCR using primers 5-TCTTCTGCAGGGGACTATGG-3 and 5-TTTCCACGTGTTCGGTTGTA-3 and subcloned into the suicide plasmid pVA3000. Therecombinant plasmid was introduced into P. gingivalis 33277 byconjugation as described previously (10). The absence of inlJtranscript was confirmed by reverse transcription-PCR. As theinlJ gene is located between two genes transcribed in the op-posite direction, the potential for pleiotropic effects of thismutation are diminished. The adjacent genes are PG0349, aputative hydrolase of the haloacid dehalogenase-like family,and PG0351, a hypothetical protein, neither of which has adocumented role in biofilm biogenesis. Homotypic biofilm for-mation was first tested in the microtiter plate assay describedpreviously by O’Toole and Kolter (8). Parental and mutantstrains were suspended in prereduced phosphate-buffered sa-line, and 5  107 cells were incubated at 37°C anaerobically inindividual wells of 96-well plates. The resulting biofilms werewashed, stained with 1% crystal violet, and destained with 95%* Corresponding author. Mailing address: Department of Oral Bi-ology, College of Dentistry, University of Florida, Gainesville, FL32610-0424. Phone: (352) 392-5067. Fax: (352) 392-2361. E-mail: rlamont@dental.ufl.edu.† Present address: Department of Conservative Dentistry, Collegeof Dentistry, Yonsei University, Sinchon-dong, Seodaemun-gu, Seoul120-752, Korea.FIG. 1. Microtiter plate monospecies biofilm production by P. gin-givalis 33277 and the InlJ mutant at 24 h and 48 h. Asterisks indicatea significant difference (P 0.05, t test; n 3) between the mutant andparental strains. OD, optical density.3002 on June 18, 2014 by YONSEI UNIV MED LIBRARYhttp://iai.asm.org/Downloaded from ethanol. Absorbance at 595 nm was determined using a Bench-mark microplate reader. Figure 1 shows that monospecies bio-film formation was reduced in the InlJ mutant by 13.6% after24 h and 56.1% after 48 h. For visualization and quantificationof biofilm structure, biofilms were generated in a 16-well Cul-ture Well chambered coverglass system, stained with 5 (and6)-carboxyfluorescein succinimidyl ester (fluorescein isothio-cyanate [FITC], 4 g ml1; Molecular Probes), and examinedby confocal microscopy (Bio-Rad MRC600 confocal scanninglaser microscope [Kr/Ar] system with an MS plan 60 1.4-numerical-aperture objective). Biofilms were observed with thereflected laser light of combined 488-, 546-, and 647-nm wave-lengths. The images were analyzed with Image J 1.35c andAdobe Photoshop 6.0 software. MCID-M5 5.1 software wasused to determine the total grain area. Biofilm formation bythe InlJ mutant was visibly more sparse than that by the parentstrain (Fig. 2A), and total accumulation was reduced by 46.2%(Fig. 2B). In addition, the average height across three randomFIG. 2. (A) Confocal laser scanning microscopy projections of monospecies biofilm formation by P. gingivalis strains 33277 and the InlJ mutantafter 24 h. Magnification, 40. (B) Total grain area analysis of a 268.6- by 268.6-m x-y section. (C) Average biofilm height of P. gingivalisaccumulation across three random x-z sections. Asterisks indicate a significant difference (P  0.05, t test; n  3) between the mutant and parentalstrains.VOL. 74, 2006 NOTES 3003 on June 18, 2014 by YONSEI UNIV MED LIBRARYhttp://iai.asm.org/Downloaded from x-z sections of the mutant biofilm was reduced by 36.5% com-pared to the parent strain (Fig. 2C). These experiments dem-onstrate that InlJ is required for optimal homotypic biofilmformation by P. gingivalis.On the tooth surfaces, P. gingivalis will be in contact with thediverse species that comprise the plaque biofilm. Thus, to be-gin to assess the role of InlJ in heterotypic biofilms, mixed S.gordonii-P. gingivalis biofilms were examined. S. gordonii is acommon component of dental plaque (11, 13, 14) and is en-countered by P. gingivalis upon initial colonization. S. gordoniicells were cultured for 24 h on chambered coverglass andstained with hexidium iodide (15 g ml1; Molecular Probes).P. gingivalis cells were stained with FITC as described aboveand reacted anaerobically with the S. gordonii biofilm for 24 hat 37°C in prereduced phosphate-buffered saline. After wash-ing, accumulations of heterotypic biofilms were observed byconfocal microscopy as described above. In contrast to themonospecies biofilm, the InlJ mutant formed more abundantaccumulations within the mixed P. gingivalis-S. gordonii biofilm(Fig. 3A). This observation was supported by the total grainFIG. 3. (A) Confocal laser scanning microscopy projections of mixed biofilms of S. gordonii DL1 with P. gingivalis strains 33277 and the InlJmutant after 24 h. S. gordonii was prestained with hexidium iodide (red), and P. gingivalis was prestained with FITC (green). Magnification, 40.(B) Total grain area analysis of a 268.6-  268.6-m x-y section. (C) Average biofilm height of P. gingivalis accumulation across three random x-zsections. Asterisks indicate a significant difference (P  0.05, t test; n  3) between the mutant and wild-type strains.3004 NOTES INFECT. IMMUN. on June 18, 2014 by YONSEI UNIV MED LIBRARYhttp://iai.asm.org/Downloaded from analysis of P. gingivalis accumulation (Fig. 3B). In addition,measurement of average biofilm height across three randomx-z sections showed higher vertical accretion of the InlJ mutantthan the parental strain (Fig. 3C). Hence, in the absence ofInlJ, more luxuriant heterotypic biofilms are formed by P.gingivalis.Biofilm accumulation proceeds through a series of develop-mental steps involving attachment of bacterial cells to a sur-face; accumulation by the recruitment of additional cells andproliferation; and, in certain cases, inclusion of additional spe-cies. Defined genetic profiles are considered important fordistinct phases of biofilm development (7, 15). Regulation ofbiofilm development can be hypothesized to involve mecha-nisms that both stimulate an increase in biomass and limit orstabilize accumulation according to environmental constraints.For example, in Pseudomonas aeruginosa, biofilm depth is re-duced by the transcription factor RpoS (4, 17). However, RpoSmutants of P. aeruginosa form biofilms of greater depth underflowing conditions (17). RpoS production is regulated at mul-tiple levels, including transcription, translation, and proteoly-sis, in response to different stress conditions such as nutrientlimitation (16). The results of the current study indicate thatthe InlJ protein of P. gingivalis is exploited to perform rolesboth in the stimulation of biofilm accumulation on abioticsurfaces and in biofilm control in the more complex and invivo-relevant situation where other organisms are present.Such a multifunctional role is not inconsistent with the struc-ture and properties of LRR proteins. Internalins of Listeria areinvolved in adherence, and adhesive activity mediated by InlJmay be important for biofilm initiation on abiotic surfaces. InlJdoes possess a signal peptide and is therefore likely to bepresent on the surface of P. gingivalis. Adherence of P. gingi-valis to S. gordonii, however, is mediated through the long andshort fimbriae (6, 9) and thus may not require the presence ofInlJ. LRRs can also be involved in signal transduction throughtheir capacity to provide a versatile structural framework forthe formation of protein-protein interactions. Such a role forInlJ may be important in constraining biofilm growth, possiblyto avoid excessive exposure to oxygen in the oral cavity. As astrict anaerobe, P. gingivalis is likely to favor an existence deepwithin the plaque biofilm. Alternatively, restriction of biofilmdevelopment may be important to maintain the integrity ofchannels that allow nutrient penetration into the biofilm.This work was supported by NIDCR DE12505 and DE11111.REFERENCES1. Cabanes, D., P. Dehoux, O. Dussurget, L. Frangeul, and P. Cossart. 2002.Surface proteins and the pathogenic potential of Listeria monocytogenes.Trends Microbiol. 10:238–245.2. Chen, W., K. E. Laidig, Y. Park, K. Park, J. R. Yates III, R. J. Lamont, andM. Hackett. 2001. Searching the Porphyromonas gingivalis genome with pep-tide fragmentation mass spectra. Analyst 126:52–57.3. Cossart, P., J. Pizarro-Cerda, and M. Lecuit. 2003. Invasion of mammaliancells by Listeria monocytogenes: functional mimicry to subvert cellular func-tions. Trends Cell Biol. 13:23–31.4. Heydorn, A., B. Ersboll, J. Kato, M. Hentzer, M. R. Parsek, T. Tolker-Nielsen, M. Givskov, and S. Molin. 2002. Statistical analysis of Pseudomonasaeruginosa biofilm development: impact of mutations in genes involved intwitching motility, cell-to-cell signaling, and stationary-phase sigma factorexpression. Appl. Environ. Microbiol. 68:2008–2017.5. Kajava, A. V. 1998. Structural diversity of leucine-rich repeat proteins. J.Mol. Biol. 277:519–527.6. Lamont, R. J., A. El-Sabaeny, Y. Park, G. S. Cook, J. W. Costerton, and D. R.Demuth. 2002. Role of the Streptococcus gordonii SspB protein in the devel-opment of Porphyromonas gingivalis biofilms on streptococcal substrates.Microbiology 148:1627–1636.7. Moorthy, S., and P. I. Watnick. 2005. Identification of novel stage-specificgenetic requirements through whole genome transcription profiling of Vibriocholerae biofilm development. Mol. Microbiol. 57:1623–1635.8. O’Toole, G. A., and R. Kolter. 1998. Initiation of biofilm formation inPseudomonas fluorescens WCS365 proceeds via multiple, convergent signal-ling pathways: a genetic analysis. Mol. Microbiol. 28:449–461.9. Park, Y., R. Simionato, K. Sekiya, Y. Murakami, D. James, W. Chen, M.Hackett, F. Yoshimura, D. R. Demuth, and R. J. Lamont. 2005. Shortfimbriae of Porphyromonas gingivalis and their role in coadhesion with Strep-tococcus gordonii. Infect. Immun. 73:3983–3989.10. Park, Y., O. Yilmaz, I. Y. Jung, and R. J. Lamont. 2004. Identification ofPorphyromonas gingivalis genes specifically expressed in human gingival ep-ithelial cells by using differential display reverse transcription-PCR. Infect.Immun. 72:3752–3758.11. Quirynen, M., R. Vogels, M. Pauwels, A. D. Haffajee, S. S. Socransky, N. 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Teitzel,S. Lory, and E. P. Greenberg. 2001. Gene expression in Pseudomonas aerugi-nosa biofilms. Nature 413:860–864.18. Zhang, Y., T. Wang, W. Chen, O. Yilmaz, Y. Park, I. Y. Jung, M. Hackett,and R. J. Lamont. 2005. Differential protein expression by Porphyromonasgingivalis in response to secreted epithelial cell components. Proteomics5:198–211.Editor: V. J. DiRitaVOL. 74, 2006 NOTES 3005 on June 18, 2014 by YONSEI UNIV MED LIBRARYhttp://iai.asm.org/Downloaded from 

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Role of the Porphyromonas gingivalis InlJ protein in homotypic and heterotypic biofilm development

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Role of the Porphyromonas gingivalis InlJ Protein in Homotypic and Heterotypic Biofilm Development

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