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Distinct roles of long/short fimbriae and gingipains in homotypic biofilm development by Porphyromonas gingivalis.

Kuboniwa M, Amano A, Hashino E, Yamamoto Y, Inaba H, Hamada N, Nakayama K, Tribble GD, Lamont RJ, Shizukuishi S - BMC Microbiol. (2009)

Bottom Line: In addition, deletion of FimA reduced the autoaggregation efficiency, whereas autoaggregation was significantly increased in the Kgp and Mfa mutants, with a clear association with alteration of biofilm structures under the non-proliferation condition.These results suggested that the FimA fimbriae promote initial biofilm formation but exert a restraining regulation on biofilm maturation, whereas Mfa and Kgp have suppressive and regulatory roles during biofilm development.Collectively, these molecules seem to act coordinately to regulate the development of mature P. gingivalis biofilms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Preventive Dentistry, Osaka University Graduate School of Dentistry, Suita-Osaka, Japan. kuboniwa@dent.osaka-u.ac.jp

ABSTRACT

Background: Porphyromonas gingivalis, a periodontal pathogen, expresses a number of virulence factors, including long (FimA) and short (Mfa) fimbriae as well as gingipains comprised of arginine-specific (Rgp) and lysine-specific (Kgp) cysteine proteinases. The aim of this study was to examine the roles of these components in homotypic biofilm development by P. gingivalis, as well as in accumulation of exopolysaccharide in biofilms.

Results: Biofilms were formed on saliva-coated glass surfaces in PBS or diluted trypticase soy broth (dTSB). Microscopic observation showed that the wild type strain formed biofilms with a dense basal monolayer and dispersed microcolonies in both PBS and dTSB. A FimA deficient mutant formed patchy and small microcolonies in PBS, but the organisms proliferated and formed a cohesive biofilm with dense exopolysaccharides in dTSB. A Mfa mutant developed tall and large microcolonies in PBS as well as dTSB. A Kgp mutant formed markedly thick biofilms filled with large clumped colonies under both conditions. A RgpA/B double mutant developed channel-like biofilms with fibrillar and tall microcolonies in PBS. When this mutant was studied in dTSB, there was an increase in the number of peaks and the morphology changed to taller and loosely packed biofilms. In addition, deletion of FimA reduced the autoaggregation efficiency, whereas autoaggregation was significantly increased in the Kgp and Mfa mutants, with a clear association with alteration of biofilm structures under the non-proliferation condition. In contrast, this association was not observed in the Rgp- mutants.

Conclusion: These results suggested that the FimA fimbriae promote initial biofilm formation but exert a restraining regulation on biofilm maturation, whereas Mfa and Kgp have suppressive and regulatory roles during biofilm development. Rgp controlled microcolony morphology and biovolume. Collectively, these molecules seem to act coordinately to regulate the development of mature P. gingivalis biofilms.

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Exopolysaccharide production by P. gingivalis wild-type strain and mutants in dTSB. A) Visualization of exopolysaccharide production in biofilms formed by P. gingivalis strains after staining with FITC-labelled concanavalin A and wheat germ agglutinin (green). Bacteria were stained with DAPI (blue). Fluorescent images were obtained using a CLSM. The z stack of the x-y sections was converted to composite images with the "Volume" function using Imaris software, after which a y stack of the x-z sections was created and is presented here. B) Fluorescent images were quantified using Imaris software and average of total exopolysaccharide biovolume per field was calculated. C) Exopolysaccharide levels are expressed as the ratio of exopolysaccharide/cells (FITC/DAPI) fluorescence. The experiment was repeated independently three times. Data are presented as averages of 8 fields per sample with standard errors of the means. Statistical analysis was performed using a Scheffe test. *p < 0.05 and **p < 0.01 in comparison to the wild-type strain.
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Figure 5: Exopolysaccharide production by P. gingivalis wild-type strain and mutants in dTSB. A) Visualization of exopolysaccharide production in biofilms formed by P. gingivalis strains after staining with FITC-labelled concanavalin A and wheat germ agglutinin (green). Bacteria were stained with DAPI (blue). Fluorescent images were obtained using a CLSM. The z stack of the x-y sections was converted to composite images with the "Volume" function using Imaris software, after which a y stack of the x-z sections was created and is presented here. B) Fluorescent images were quantified using Imaris software and average of total exopolysaccharide biovolume per field was calculated. C) Exopolysaccharide levels are expressed as the ratio of exopolysaccharide/cells (FITC/DAPI) fluorescence. The experiment was repeated independently three times. Data are presented as averages of 8 fields per sample with standard errors of the means. Statistical analysis was performed using a Scheffe test. *p < 0.05 and **p < 0.01 in comparison to the wild-type strain.

Mentions: As extracellular polysaccharide is important for the development of biofilm communities, we examined the influences of fimbriae and gingipains on the accumulation of exopolysaccharide in P. gingivalis biofilms. To visualize and quantify exopolysaccharide accumulation in biofilms under the proliferation condition, 4',6-diamino-2-phenylindole (DAPI)-labeled P. gingivalis cells and fluorescein isothiocyanate (FITC)-labeled exopolysaccharide were examined by confocal microscopy with digitally reconstructed image analysis. In all of the tested strains, DAPI-labeled cells exhibited the same microstructures of biofilms composed of 5-(and-6)-carboxyfluorescein succinimidyl ester (CFSE)-labeled cells, as shown in Figure 3, thus validating the use of these live-staining methods (data not shown). Exopolysaccharide visualization enabled us to assess the accumulation pattern (Figure 5A) and exopolysaccharide biovolume per base area (Figure 5B). Furthermore, the exopolysaccharide production was normalized to the levels of DAPI-labeled P. gingivalis cells in the biofilms and expressed as the exopolysaccharide/cell ratio (Figure 5C). Interestingly, a unique pattern of exopolysaccharide accumulation was observed in the Rgp mutant KDP133 in vertical sections (x-z plane) of biofilms (Figure 5A). In contrast to the other strains, exopolysaccharide accumulated in the middle layer, and the biofilm surface was not covered with exopolysaccharide. It was also notable that the long fimbria mutant KDP150 developed a biofilm enriched with exopolysaccharide (Figure 5A), reflecting a significantly higher exopolysaccharide/cell ratio (Figure 5C). The gingipain mutant KDP136 produced the most abundant exopolysaccharide per unit base area (Figure 5B). The minor fimbria mutant MPG67, long/short fimbriae mutant MPG4167 and Rgp mutant KDP133 also accumulated significantly larger amounts of exopolysaccharide than wild type; however, exopolysaccharide/cell ratio in KDP133 and MPG4167 was significantly lower than wild type because biofilms of these strains consisted of larger numbers of cells (Figure 5C).


Distinct roles of long/short fimbriae and gingipains in homotypic biofilm development by Porphyromonas gingivalis.

Kuboniwa M, Amano A, Hashino E, Yamamoto Y, Inaba H, Hamada N, Nakayama K, Tribble GD, Lamont RJ, Shizukuishi S - BMC Microbiol. (2009)

Exopolysaccharide production by P. gingivalis wild-type strain and mutants in dTSB. A) Visualization of exopolysaccharide production in biofilms formed by P. gingivalis strains after staining with FITC-labelled concanavalin A and wheat germ agglutinin (green). Bacteria were stained with DAPI (blue). Fluorescent images were obtained using a CLSM. The z stack of the x-y sections was converted to composite images with the "Volume" function using Imaris software, after which a y stack of the x-z sections was created and is presented here. B) Fluorescent images were quantified using Imaris software and average of total exopolysaccharide biovolume per field was calculated. C) Exopolysaccharide levels are expressed as the ratio of exopolysaccharide/cells (FITC/DAPI) fluorescence. The experiment was repeated independently three times. Data are presented as averages of 8 fields per sample with standard errors of the means. Statistical analysis was performed using a Scheffe test. *p < 0.05 and **p < 0.01 in comparison to the wild-type strain.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2697998&req=5

Figure 5: Exopolysaccharide production by P. gingivalis wild-type strain and mutants in dTSB. A) Visualization of exopolysaccharide production in biofilms formed by P. gingivalis strains after staining with FITC-labelled concanavalin A and wheat germ agglutinin (green). Bacteria were stained with DAPI (blue). Fluorescent images were obtained using a CLSM. The z stack of the x-y sections was converted to composite images with the "Volume" function using Imaris software, after which a y stack of the x-z sections was created and is presented here. B) Fluorescent images were quantified using Imaris software and average of total exopolysaccharide biovolume per field was calculated. C) Exopolysaccharide levels are expressed as the ratio of exopolysaccharide/cells (FITC/DAPI) fluorescence. The experiment was repeated independently three times. Data are presented as averages of 8 fields per sample with standard errors of the means. Statistical analysis was performed using a Scheffe test. *p < 0.05 and **p < 0.01 in comparison to the wild-type strain.
Mentions: As extracellular polysaccharide is important for the development of biofilm communities, we examined the influences of fimbriae and gingipains on the accumulation of exopolysaccharide in P. gingivalis biofilms. To visualize and quantify exopolysaccharide accumulation in biofilms under the proliferation condition, 4',6-diamino-2-phenylindole (DAPI)-labeled P. gingivalis cells and fluorescein isothiocyanate (FITC)-labeled exopolysaccharide were examined by confocal microscopy with digitally reconstructed image analysis. In all of the tested strains, DAPI-labeled cells exhibited the same microstructures of biofilms composed of 5-(and-6)-carboxyfluorescein succinimidyl ester (CFSE)-labeled cells, as shown in Figure 3, thus validating the use of these live-staining methods (data not shown). Exopolysaccharide visualization enabled us to assess the accumulation pattern (Figure 5A) and exopolysaccharide biovolume per base area (Figure 5B). Furthermore, the exopolysaccharide production was normalized to the levels of DAPI-labeled P. gingivalis cells in the biofilms and expressed as the exopolysaccharide/cell ratio (Figure 5C). Interestingly, a unique pattern of exopolysaccharide accumulation was observed in the Rgp mutant KDP133 in vertical sections (x-z plane) of biofilms (Figure 5A). In contrast to the other strains, exopolysaccharide accumulated in the middle layer, and the biofilm surface was not covered with exopolysaccharide. It was also notable that the long fimbria mutant KDP150 developed a biofilm enriched with exopolysaccharide (Figure 5A), reflecting a significantly higher exopolysaccharide/cell ratio (Figure 5C). The gingipain mutant KDP136 produced the most abundant exopolysaccharide per unit base area (Figure 5B). The minor fimbria mutant MPG67, long/short fimbriae mutant MPG4167 and Rgp mutant KDP133 also accumulated significantly larger amounts of exopolysaccharide than wild type; however, exopolysaccharide/cell ratio in KDP133 and MPG4167 was significantly lower than wild type because biofilms of these strains consisted of larger numbers of cells (Figure 5C).

Bottom Line: In addition, deletion of FimA reduced the autoaggregation efficiency, whereas autoaggregation was significantly increased in the Kgp and Mfa mutants, with a clear association with alteration of biofilm structures under the non-proliferation condition.These results suggested that the FimA fimbriae promote initial biofilm formation but exert a restraining regulation on biofilm maturation, whereas Mfa and Kgp have suppressive and regulatory roles during biofilm development.Collectively, these molecules seem to act coordinately to regulate the development of mature P. gingivalis biofilms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Preventive Dentistry, Osaka University Graduate School of Dentistry, Suita-Osaka, Japan. kuboniwa@dent.osaka-u.ac.jp

ABSTRACT

Background: Porphyromonas gingivalis, a periodontal pathogen, expresses a number of virulence factors, including long (FimA) and short (Mfa) fimbriae as well as gingipains comprised of arginine-specific (Rgp) and lysine-specific (Kgp) cysteine proteinases. The aim of this study was to examine the roles of these components in homotypic biofilm development by P. gingivalis, as well as in accumulation of exopolysaccharide in biofilms.

Results: Biofilms were formed on saliva-coated glass surfaces in PBS or diluted trypticase soy broth (dTSB). Microscopic observation showed that the wild type strain formed biofilms with a dense basal monolayer and dispersed microcolonies in both PBS and dTSB. A FimA deficient mutant formed patchy and small microcolonies in PBS, but the organisms proliferated and formed a cohesive biofilm with dense exopolysaccharides in dTSB. A Mfa mutant developed tall and large microcolonies in PBS as well as dTSB. A Kgp mutant formed markedly thick biofilms filled with large clumped colonies under both conditions. A RgpA/B double mutant developed channel-like biofilms with fibrillar and tall microcolonies in PBS. When this mutant was studied in dTSB, there was an increase in the number of peaks and the morphology changed to taller and loosely packed biofilms. In addition, deletion of FimA reduced the autoaggregation efficiency, whereas autoaggregation was significantly increased in the Kgp and Mfa mutants, with a clear association with alteration of biofilm structures under the non-proliferation condition. In contrast, this association was not observed in the Rgp- mutants.

Conclusion: These results suggested that the FimA fimbriae promote initial biofilm formation but exert a restraining regulation on biofilm maturation, whereas Mfa and Kgp have suppressive and regulatory roles during biofilm development. Rgp controlled microcolony morphology and biovolume. Collectively, these molecules seem to act coordinately to regulate the development of mature P. gingivalis biofilms.

Show MeSH
Related in: MedlinePlus