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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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Homotypic biofilm formation by P. gingivalis wild-type strain and mutants in PBS. P. gingivalis strains were stained with CFSE (green) and incubated in PBS for 24 hours. After washing, the biofilms that developed on the coverglass were observed with a CLSM equipped with a 40× objective. Optical sections were obtained along the z axis at 0.7-μm intervals, and images of the x-y and x-z planes were reconstructed with an imaging software as described in the text. Upper panels indicate z stacks of the x-y sections. Lower panels are x-z sections. P. gingivalis strains used in this assay are listed in Table 4. The experiment was repeated independently three times with each strain in triplicate. Representative images are shown.
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Figure 1: Homotypic biofilm formation by P. gingivalis wild-type strain and mutants in PBS. P. gingivalis strains were stained with CFSE (green) and incubated in PBS for 24 hours. After washing, the biofilms that developed on the coverglass were observed with a CLSM equipped with a 40× objective. Optical sections were obtained along the z axis at 0.7-μm intervals, and images of the x-y and x-z planes were reconstructed with an imaging software as described in the text. Upper panels indicate z stacks of the x-y sections. Lower panels are x-z sections. P. gingivalis strains used in this assay are listed in Table 4. The experiment was repeated independently three times with each strain in triplicate. Representative images are shown.

Mentions: First, we evaluated the roles of long/short fimbriae and gingipains in initial attachment and organization of biofilms which is a crucial event in the early phase of biofilm formation [19]. When cultured in TSB as free-living cells, wild type and all mutant strains showed the similar growth rates, as reported in previous study [20]. In contrast, when incubated in PBS for 24 h, wild type and mutants lacking long and/or short fimbriae formed distinct biofilms (Figure 1 and Table 1). Wild type strain 33277 formed biofilms with a dense basal monolayer and dispersed microcolonies. Compared with the wild type, the long fimbria mutant KDP150 formed patchy and sparser biofilms with a significantly greater distance between fewer peaks, although mean peak height was almost the same as that of the wild type strain. In contrast, the short fimbria mutant MPG67 developed cluster and channel-like biofilms consisting of significantly taller microcolonies compared to the wild type. Similar to MPG67, the mutant (MPG4167) lacking both types of fimbriae also formed thick biofilms with significantly taller microcolonies than the wild type. Viability of the cells in biofilms of each strain was tested by colony count and confirmed at 24 h (data not shown). These results suggest that the long fimbriae are involved in initial attachment and organization of biofilms by P. gingivalis, whereas the short fimbriae have a suppressive regulatory role for these steps.


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)

Homotypic biofilm formation by P. gingivalis wild-type strain and mutants in PBS. P. gingivalis strains were stained with CFSE (green) and incubated in PBS for 24 hours. After washing, the biofilms that developed on the coverglass were observed with a CLSM equipped with a 40× objective. Optical sections were obtained along the z axis at 0.7-μm intervals, and images of the x-y and x-z planes were reconstructed with an imaging software as described in the text. Upper panels indicate z stacks of the x-y sections. Lower panels are x-z sections. P. gingivalis strains used in this assay are listed in Table 4. The experiment was repeated independently three times with each strain in triplicate. Representative images are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Homotypic biofilm formation by P. gingivalis wild-type strain and mutants in PBS. P. gingivalis strains were stained with CFSE (green) and incubated in PBS for 24 hours. After washing, the biofilms that developed on the coverglass were observed with a CLSM equipped with a 40× objective. Optical sections were obtained along the z axis at 0.7-μm intervals, and images of the x-y and x-z planes were reconstructed with an imaging software as described in the text. Upper panels indicate z stacks of the x-y sections. Lower panels are x-z sections. P. gingivalis strains used in this assay are listed in Table 4. The experiment was repeated independently three times with each strain in triplicate. Representative images are shown.
Mentions: First, we evaluated the roles of long/short fimbriae and gingipains in initial attachment and organization of biofilms which is a crucial event in the early phase of biofilm formation [19]. When cultured in TSB as free-living cells, wild type and all mutant strains showed the similar growth rates, as reported in previous study [20]. In contrast, when incubated in PBS for 24 h, wild type and mutants lacking long and/or short fimbriae formed distinct biofilms (Figure 1 and Table 1). Wild type strain 33277 formed biofilms with a dense basal monolayer and dispersed microcolonies. Compared with the wild type, the long fimbria mutant KDP150 formed patchy and sparser biofilms with a significantly greater distance between fewer peaks, although mean peak height was almost the same as that of the wild type strain. In contrast, the short fimbria mutant MPG67 developed cluster and channel-like biofilms consisting of significantly taller microcolonies compared to the wild type. Similar to MPG67, the mutant (MPG4167) lacking both types of fimbriae also formed thick biofilms with significantly taller microcolonies than the wild type. Viability of the cells in biofilms of each strain was tested by colony count and confirmed at 24 h (data not shown). These results suggest that the long fimbriae are involved in initial attachment and organization of biofilms by P. gingivalis, whereas the short fimbriae have a suppressive regulatory role for these steps.

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