A Major Fimbrilin Variant of Mfa1 Fimbriae in Porphyromonas gingivalis

Abstract

The periodontal pathogen Porphyromonas gingivalis is known to express 2 distinct types of fimbriae: FimA and Mfa1 fimbriae. However, we previously reported that fimbria-like structures were found in a P. gingivalis strain in which neither FimA nor Mfa1 fimbriae were detected. In this study, we identified a major protein in the bacterial lysates of the strain, which has been reported as the 53-kDa major outer membrane protein of P. gingivalis (53K protein) and subsequently reported as a major fimbrilin of a novel-type fimbria. Sequencing of the chromosomal DNA of the strain showed that the 53k gene (encoding the 53K protein) was located at a locus corresponding to the mfa1 gene (encoding the Mfa1 protein, which is a major fimbrilin of Mfa1 fimbriae) of the ATCC 33277 type strain. However, the 53K and Mfa1 proteins showed a low amino acid sequence homology and different antigenicity. The 53K protein was detected in 34 of 84 (41%) P. gingivalis strains, while the Mfa1 protein was detected in 44% of the strains. No strain expressed both 53K and Mfa1 proteins. Additionally, fimbriae were normally expressed in mutants in which the 53k and mfa1 genes were interchanged. These results indicate that the 53K protein is another major fimbrilin of Mfa1 fimbriae in P. gingivalis.

Keywords

bacteria bacterial virulence genetics adhesives biofilm(s)periodontal disease(s)/periodontitis

Introduction

Periodontal disease is a chronic, gingival inflammatory disease, which is caused by multispecies biofilm in the gingival crevice (Socransky and Haffajee 2002). Porphyromonas gingivalis, a gram-negative, anaerobic bacterium, is thought to be responsible for the initiation and progression of the disease (Socransky and Haffajee 2002; Hajishengallis 2015). It is well known that P. gingivalis generally expresses 2 distinct types of fimbriae, FimA and Mfa1 fimbriae, which regulate the adherent activity of the bacteria, such as that involved in biofilm formation (Amano 2010). There is a genetic variation in the fimA gene (encoding the FimA protein, which is a major fimbrilin of FimA fimbriae) (Amano et al. 2004), and different fimA genotypes produce antigenically different FimA fimbriae (Nagano et al. 2012; Nagano et al. 2013). However, to our knowledge, there is no report on a variety in Mfa1 fimbriae.

Mfa1 fimbriae are sometimes referred to as minor fimbriae. The designation, Mfa, is indeed derived from “minor fimbriae” (Hamada et al. 1996; Lamont et al. 2002). However, the expression level of the Mfa1 protein (major fimbrilin of Mfa1 fimbriae) is not necessarily lower than that of the FimA protein (unpublished data). Mfa1 fimbriae have also been called short fimbriae because a well-used P. gingivalis strain ATCC 33277 produces extremely long FimA fimbriae (several micrometers in length) (Yoshimura et al. 1984), whereas Mfa1 fimbriae are about 100 nm (Park et al. 2005). However, this strain has a nonsense mutation in the fimB gene, encoding the FimB protein that regulates the length of FimA fimbriae by terminating the fimbrial elongation, and consequently expresses extraordinarily long FimA fimbriae (Nagano et al. 2010). Thus, the description that Mfa1 fimbriae are minor and short in comparison with FimA fimbriae is not appropriate.

Mfa1 fimbriae are primarily composed of polymers of the Mfa1 protein, encoded by the mfa1 gene (Fig. 1). The Mfa2 protein (encoded by mfa2) localizes in the outer membrane and plays a role in anchoring and length regulation of Mfa1 fimbriae (Hasegawa et al. 2009). Mfa3, Mfa4, and Mfa5 (encoded by mfa3, mfa4, and mfa5, respectively) were incorporated into the fimbriae as minor accessory components (Hasegawa et al. 2009). Additionally, it was shown that Mfa3 was located at a tip of the fimbriae (Hasegawa et al. 2013). The mfa1 through mfa5 genes are arranged in tandem and form a cluster on the chromosome of a type strain of P. gingivalis ATCC 33277 (Naito et al. 2008) (Fig. 1).

In our previous study, we observed fimbria-like structures on the cell surface of a P. gingivalis strain in which neither FimA nor Mfa1 fimbriae were detected (Nagano et al. 2013). In this study, we detected a major protein in the bacterial lysate, which has been identified as the 53-kDa major outer membrane protein of P. gingivalis (53K protein) (Kokeguchi et al. 1990). A homologous gene encoding the 53K protein (53k gene) does not exist in the published, complete genomic sequence of P. gingivalis strains ATCC 33277 (Naito et al. 2008), TDC60 (Watanabe et al. 2011), and W83 (Nelson et al. 2003). The 53K protein has been also reported as a major fimbrilin of a novel-type fimbria in P. gingivalis (Arai et al. 2000). We further ascertained that the 53K protein was a major fimbrilin in this study. Furthermore, interestingly, we found that the 53K protein was a major fimbrilin variant of Mfa1 fimbriae in P. gingivalis.

Materials and Methods

P. gingivalis Strains and Culture Conditions

We used the Ando strain, which expresses only 53K fimbriae, and the ATCC 33277 type strain as a reference, which expresses both FimA and Mfa1 fimbriae. Here, we redesignated these strains and their derivative mutants (Table 1). We also used other P. gingivalis strains (see Appendix). All P. gingivalis strains were cultivated on Brucella HK Agar (Kyokuto Pharmaceutical Industrial Co. Ltd., Tokyo, Japan) supplemented with 5% laked rabbit blood and in Modified GAM broth (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) at 37°C under anaerobic conditions. When needed, 20 μg/mL erythromycin, 10 μg/mL chloramphenicol, and 1 μg/mL tetracycline were added to the media.

Table 1.

Porphyromonas gingivalis Strains Mainly Used in This Study.

Name		Description	Fimbrial Expression	Reference
This Study	Original	Description	Fimbrial Expression	Reference
A1	ATCC 33277	A type strain, sequenced genome	FimA, Mfa1	Naito et al. (2008)
A2	SMF1	A1 with disrupted mfa1 by insertion of an erythromycin-resistant cassette	FimA	Park et al. (2005)
A3	JI-1	A1 deleted fimA by replacing with cat	Mfa1	Hasegawa et al. (2009)
A4	SMF-fimA	A2 deleted fimA by replacing with cat	None	Hasegawa et al. (2013)
A5		A4 carrying a plasmid expressing mfa1 (cloned from A1)	Mfa1	This study
A6		A3 deleted mfa1 by replacing with 53k (cloned from B1) fused with tetQ	53K	This study
B1	Ando	Wild type	53K	Nagano et al. (2013)
B2		B1 deleted 53k by replacing with cat	None	This study
B3		B2 carrying a plasmid expressing mfa1 (cloned from A1)	Mfa1	This study
B4		B2 complemented 53k by replacing cat with 53k (cloned from B1) fused with tetQ	53K	This study

The cat and tetQ genes provide chloramphenicol and tetracycline resistances, respectively.

SDS-PAGE and Western Blot Analysis

Bacterial lysates were prepared by solubilization in BugBuster HT (EMD Millipore Co., Billerica, MA, USA). Pure fimbriae were prepared as previously described (Yoshimura et al. 1984) with modifications. Briefly, bacterial cells, in 50 mM Tris-HCl, pH 7.5, supplemented with protease inhibitors (1 mM phenylmethylsulfonyl fluoride and 0.1 mM Nα-p-tosyl-L-lysine chloromethyl ketone) were disrupted using a French press and mixed with ammonium sulfate at 50% saturation to precipitate a fraction containing fimbriae. Pure fimbriae were then obtained by fractionating twice by using DEAE Sepharose Fast Flow chromatography (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) with a linear gradient of NaCl (0 to 0.5 M). Sample protein concentration (bacterial lysates and pure fimbriae) was determined by a Pierce BCA Protein Assay kit (Thermo Scientific, Rockford, IL, USA). The samples were mixed with sodium dodecyl sulfate (SDS) and 2-mercaptoethanol and denatured by heating at 100°C for 10 min. After electrophoresis, gels were subjected to Coomassie Brilliant Blue (CBB) staining or to Western blot analysis. In Western blotting, we used antisera to 53K (see below), Mfa1, Mfa2, Mfa3, Mfa4, and Mfa5 (Hasegawa et al. 2009; Hasegawa et al. 2013) as primary antibodies. After reaction with horseradish peroxidase–labeled secondary antibodies, reacted proteins were developed by using a chemiluminescence substrate.

Protein Identification

Protein bands in the CBB-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) (Abiko et al. 2014a, 2014b). After in-gel tryptic digestion, the peptides were extracted, desalted, and analyzed using the 4800 MALDI TOF/TOF Analyzer (AB Sciex, Framingham, MA, USA). The identity of the proteins was deduced from the MS peaks by a comparative analysis of the mass with that in the Mascot database (http://www.matrixscience.com/). Homology searches were also performed by using BLAST (http://blast.ncbi.nlm.nih.gov/).

Transmission Electron Microscopy

P. gingivalis cells were placed on the Elastic Carbon support film grid (Okenshoji Co. Ltd., Tokyo, Japan), negatively stained with 0.1 to 0.5 M ammonium molybdate, pH 7.5, and observed by using the JEM1400 Plus Electron Microscope (JEOL, Tokyo, Japan). Fimbrial length was measured on electron microphotographs.

DNA Sequence Analysis

The draft genome sequence of Ando was analyzed at Filgen Inc. (Nagoya, Japan), and 100-bp paired-end sequences generated by HiSeq 2000 (Illumina Inc., San Diego, CA, USA) were mapped to the genomic sequence of ATCC 33277. Based on the mapped sequence and the 53k DNA sequence published in GenBank (accession number D31835), we determined an unread sequence flanking the 53k gene using a dye terminator method.

Construction of the 53k Gene Deletion Mutant of P. gingivalis

The 53k gene deletion mutant was constructed by replacing the 53k gene with the chloramphenicol acetyltransferase (cat) gene, which provides chloramphenicol resistance. Briefly, the DNA construct was prepared using a polymerase chain reaction (PCR)–based overlap extension method (Horton et al. 1993; Nagano et al. 2005). Primers used in this study are listed in Appendix Table 1. The upstream region (ending immediately before the translation initiation codon; primers 53Del-Up-F/53Del-Up-R-cat) and downstream region (beginning immediately after the terminating nonsense codon; primers 53Del-Lo-F-cat/53Del-Lo-R) of the 53k gene were amplified by PCR from the chromosomal DNA of Ando, and the open reading frame of cat (primers Cat-F/Cat-R) was amplified from pACYC184. These 3 amplicons were fused into a piece through PCR by using the primers 53Del-Up-F and 53Del-Lo-R. The final PCR product was cloned into a plasmid vector and verified the authenticity of the DNA sequence. The plasmid construct was linearized by digestion with appropriate restriction enzymes and then electroporated into P. gingivalis Ando. Possible transformants were selected on the solid medium containing 10 μg/mL chloramphenicol. Genetic replacement was confirmed by PCR and Western blotting (data not shown).

Introduction of the mfa1 and 53k Genes

We used the pT-COW plasmid carrying the mfa1 gene (Park et al. 2005) to introduce the mfa1 gene. The plasmid was introduced into P. gingivalis A4 and B2 by conjugation as described previously (Nagano et al. 2012).

The 53k gene was introduced into chromosomes because we could not clone the full length of the 53k gene in an Escherichia coli strain possibly due to the harmful effect of the gene product on E. coli, although the partial DNA fragment of the gene could be cloned as described below. A DNA fragment for introduction of the 53k gene was constructed by the PCR-based overlap extension method. The tetQ gene (conferring tetracycline resistance) was used as a selection marker. The primers are listed in Appendix Table 1. The DNA fragments containing the 598-bp upstream region through the termination codon of 53k (primers 53KC-Up-F/53KC-Up-R-tetQ) and the downstream region of 53k (primers 53KC-Lo-F-tetQ/53KC-Lo-R) were amplified by PCR from the chromosomal DNA of Ando, and the tetQ gene (primers Tet-F/Tet-R) was amplified from pT-COW. These 3 amplicons were fused into a piece through PCR by using the primers 53KC-Up-F and 53KC-Lo-R. The final PCR product was directly electroporated into P. gingivalis A4 and B2. Possible transformants were selected on the solid medium containing 1 μg/mL tetracycline. DNA sequencing confirmed the absence of mutation in the 53k gene.

Reverse Transcription (RT)–PCR

RT-PCR was performed as described previously (Abiko et al. 2014a, 2014b). Briefly, purified RNA (100 ng) was used to generate complementary DNA (cDNA) with a PrimeScript RT-PCR Kit and Random 6 mers (Takara Bio Inc., Kyoto, Japan). The resultant cDNA was used as a template for subsequent PCR, with primers listed in Appendix Table 1. The transcript was examined after agarose electrophoresis and ethidium bromide staining of the gel.

Preparation of Antiserum to the 53K Protein

The N-terminal of 50 amino acids of the 53K protein is removed in the maturation process as a leader peptide (Hongyo et al. 1997). We therefore cloned the DNA fragment encoding the mature 53K protein from Ando into the pET28(b) plasmid (Novagen, Darmstadt, Germany) to incorporate a hexa-histidine tag. The His-tagged protein was purified by a cobalt affinity column, emulsified with complete Freund’s adjuvant, and injected into a rabbit to obtain anti-53K antiserum. The protocol was approved by the Animal Research Committee of the Aichi Gakuin University School of Dentistry.

Results

Identification of the 53K Fimbrilin in P. gingivalis Ando

We previously reported that fimbria-like structures were observed on the cell surface of P. gingivalis Ando in which neither FimA nor Mfa1 fimbriae were detected (Nagano et al. 2013). In this study, we analyzed major proteins in the bacterial lysate by MS. An MS search engine (Mascot) indicated a statistically significant probability that a heavy band, indicated by an arrowhead in Figure 2, was the 53-kDa major outer membrane protein of P. gingivalis (53K protein) that had been reported by Kokeguchi et al. (1990). Because the 53K protein has also been reported as a major fimbrilin of a novel-type fimbria in P. gingivalis (Arai et al. 2000), we examined if the 53K protein was responsible for the fimbrial expression. Deletion of the 53k gene (encoding the 53K protein) resulted in afimbrial bacteria, and complementation of the gene restored the fimbrial structures (panels B1, B2, and B4 in Fig. 3). Western blotting confirmed the disappearance and restoration of the 53K protein in the 53k-deleted and -complemented mutants, respectively (Fig. 4). Thus, we concluded that P. gingivalis Ando expressed fimbriae composed of the 53K protein encoded by the 53k gene.

DNA Sequence of Flanking Regions of the 53k Gene in Ando

We obtained a draft genome sequence of Ando and mapped it to the genome sequence of ATCC 33277. Although the 53k gene was not mapped to the genome of ATCC 33277 as expected, the upper sequence of 53k was nearly identical to that of mfa1 of ATCC 33277. Additionally, a homologous sequence from mfa3 through mfa5 was found in the draft sequence. Using these sequence data, we determined flanking sequences of the 53k gene by dye terminator sequencing (accession number AB999995). The determined DNA sequence showed 99% identity to the 53k gene, which has been deposited in GenBank (accession number D31835) (Kokeguchi et al. 1990; Hongyo et al. 1997) (Appendix Fig. 1). Ando had the homologous sequence containing mfa2 through mfa5 in the immediate downstream of 53k, which was consistent with the mfa gene cluster of ATCC 33277 (Fig. 1B). RT-PCR showed that the genes from 53k through mfa5 could be co-transcribed as an operon (data not shown).

Proportion of P. gingivalis Strains Expressing 53K Fimbriae

We investigated the expression of 53K and Mfa1 proteins in 84 strains of P. gingivalis by Western blotting (summarized in Appendix Table 2). The 53K protein was detected in 34 strains (41%), while the Mfa1 protein was detected in 37 strains (44%). No strain expressed both the 53K and Mfa1 proteins. Taken together with the results of DNA sequencing, our findings indicated that P. gingivalis generally possesses either the 53k or mfa1 gene and expresses fimbriae consisting of either the 53K or Mfa1 fimbrilin. Additionally, we noticed the difference in antigenicity between the 53K and Mfa1 proteins.

Interchangeability of the 53K and Mfa1 Fimbrilins

As shown in Figure 3, fimbriae 200 to 300 nm in length were observed in fimA-deleted ATCC 33277 (A3) and Ando (B1), whereas no fimbrial structure was detected in mfa1-inactivated A3 (A4) and 53k-deleted B1 (B2). Introduction of the exogenous mfa1 gene into B2 induced an abundant fimbrial expression, which was consistent with the mfa1 gene–complemented ATCC 33277 derivative (panels B3 and A5 in Fig. 3, respectively). Conversely, exogenous 53K fimbriae were expressed in the ATCC 33277 derivative, which was consistent with 53k gene–complemented B2 (panels A6 and B4 in Fig. 3, respectively). The fimbriae purified from mutants interchanged between the 53k and mfa1 genes were concomitantly fractionated with the accessory components of Mfa3, Mfa4, and Mfa5, which were consistent with fimbriae purified from the parental and complemented strains (Fig. 4), indicating that the fimbriae of the gene-interchanged mutants were normally assembled and integrated with the accessories.

Discussion

Arai et al. (2000) showed that a 53-kDa protein purified from a P. gingivalis strain in which the fimA gene was disrupted showed a filamentous structure and that the antibody to the 53-kDa protein labeled fimbriae of the strain. They therefore concluded that the 53-kDa protein formed a novel fimbria (Arai et al. 2000). In this study, we further ascertained their conclusion using deleted and complemented mutants of the 53k gene. DNA sequence analysis showed that the 53k gene was located at the locus corresponding to the mfa1 gene of ATCC 33277, that is, the mfa2 through mfa5 genes arranged in tandem immediately downstream from the 53k gene. Furthermore, the 53k and mfa1 genes were interchangeable. Thus, we conclude that the 53K protein is another major fimbrilin of Mfa1 fimbriae in P. gingivalis. Additionally, this study is the first report about a variety in Mfa1 fimbriae. However, molecular weight was significantly different (Mfa1, 75 kDa; 53K, 53 kDa) (Fig. 4), and homologies between the DNA/amino acid sequences of 53k/53K and mfa1/Mfa1, respectively, were not high (Appendix Fig. 1). Thus, it is difficult to classify 53K/Mfa1 fimbriae into a genotype such as the fimA genotype.

Kokeguchi et al. (1990) reported that sera from periodontal patients reacted to 53K and/or to 67-kDa proteins (likely to be Mfa1 proteins), but whether P. gingivalis expressed either 53K or Mfa1 or both fimbriae remained unclear. Here, we showed that P. gingivalis strains express only the 53K or Mfa1 protein in almost equal proportion (about 40%) by analyzing 84 strains of P. gingivalis. Taken together with the sequence analysis, our results indicate that P. gingivalis expresses fimbriae consisting of either the 53K or Mfa1 fimbrilin. With regard to FimA fimbriae, all 84 strains presented the fimA gene, and FimA protein expression was confirmed in 73 strains (87%) (Nagano et al. 2013) (Appendix Table 2). Therefore, P. gingivalis generally expresses either 53K or Mfa1 fimbriae in addition to FimA fimbriae. Additionally, strains with fimA genotypes III and V expressed 53K fimbriae almost exclusively, whereas genotypes I and II tended to express Mfa1 fimbriae (Appendix Table 2). This suggests a genetic association between the fimA genotype and the presence of either the 53k or mfa1 gene.

P. gingivalis FimA fimbriae function in biofilm formation and adherence to host cells (Amano 2010). Mfa1 fimbriae were also reported to be essential for aggregation of P. gingivalis, eventually for biofilm formation (Lin et al. 2006). However, Kuboniwa et al. (2009) showed that Mfa1 fimbriae played a suppressive regulatory role for biofilm formation. We also previously reported that deletion of the mfa1 gene increased biofilm volume (Hasegawa et al. 2013). Furthermore, Umemoto and Hamada (2003) reported that disruption of the mfa1 gene increased the bacterial adherence to epithelial cells. However, in this study, we could not examine the impact of 53K fimbriae in biofilm formation because P. gingivalis Ando barely forms a biofilm on a polystyrene plate (Nagano et al. 2013). It is necessary to use 53K fimbriae–expressing strains that are able to form a biofilm to examine a role of 53K fimbriae in biofilm formation.

It was reported that Mfa1 fimbriae adhered to the SspB polypeptide on the surface of Streptococcus gordonii (Lamont et al. 2002; Park et al. 2005). The accessory components of Mfa3, Mfa4, and Mfa5 were predicted to be located at the tip of Mfa1 fimbriae and interact directly with external molecules such as SspB (Hasegawa et al. 2013). The 53K fimbriae were also associated with the accessories and, therefore, may also interact with SspB. However, we did not succeed in elucidating the interaction between the fimbriae and SspB. Additionally, it has been reported that P. gingivalis interacts with host cells through Mfa1 fimbriae to elicit inflammatory responses (Hiramine et al. 2003; Takahashi et al. 2006; Zeituni et al. 2009). Interaction of 53K fimbriae with host cells should be examined.

Author Contributions

K. Nagano, contributed to conception, design, data acquisition, analysis, and interpretation, drafted and critically revised the manuscript; Y. Hasegawa, contributed to design and data interpretation, drafted and critically revised the manuscript; Y. Yoshida, F. Yoshimura, contributed to conception and data interpretation, drafted and critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.

Footnotes

Acknowledgements

We thank Mikie Sato and Yuki Abiko for their contribution to MS and RT-PCR analyses.

This work was supported by JSPS KAKENHI Grant Number 25462880 (K.N.).

The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

A supplemental appendix to this article is published electronically only at .

References

Abiko

Nagano

Yoshida

Yoshimura

. 2014a. Characterization of Treponema denticola mutants defective in the major antigenic proteins, Msp and TmpC. PLoS One. 9(11):e113565.

Abiko

Nagano

Yoshida

Yoshimura

. 2014b. Major membrane protein TDE2508 regulates adhesive potency in Treponema denticola. PLoS One. 9(2):e89051.

Amano

. 2010. Bacterial adhesins to host components in periodontitis. Periodontol 2000. 52(1):12–37.

Amano

Nakagawa

Okahashi

Hamada

. 2004. Variations of Porphyromonas gingivalis fimbriae in relation to microbial pathogenesis. J Periodontal Res. 39(2):136–142.

Arai

Hamada

Umemoto

. 2000. Purification and characterization of a novel secondary fimbrial protein from Porphyromonas gingivalis strain 381. FEMS Microbiol Lett. 193(1):75–81.

Hajishengallis

. 2015. Periodontitis: from microbial immune subversion to systemic inflammation. Nat Rev Immunol. 15(1):30–44.

Hamada

Sojar

Cho

Genco

. 1996. Isolation and characterization of a minor fimbria from Porphyromonas gingivalis. Infect Immun. 64(11):4788–4794.

Hasegawa

Iwami

Sato

Park

Nishikawa

Atsumi

Moriguchi

Murakami

Lamont

Nakamura

. 2009. Anchoring and length regulation of Porphyromonas gingivalis Mfa1 fimbriae by the downstream gene product Mfa2. Microbiology. 155(Pt 10):3333–3347.

Hasegawa

Nagano

Ikai

Izumigawa

Yoshida

Kitai

Lamont

Murakami

Yoshimura

. 2013. Localization and function of the accessory protein Mfa3 in Porphyromonas gingivalis Mfa1 fimbriae. Mol Oral Microbiol. 28(6):467–480.

10.

Hiramine

Watanabe

Hamada

Umemoto

. 2003. Porphyromonas gingivalis 67-kDa fimbriae induced cytokine production and osteoclast differentiation utilizing TLR2. FEMS Microbiol Lett. 229(1):49–55.

11.

Hongyo

Kurihara

Kokeguchi

Miyamoto

Maeda

Hayakawa

Abiko

Takashiba

Murayama

. 1997. Molecular cloning and characterization of the gene encoding 53 kD outer membrane protein of Porphyromonas gingivalis. Microbios. 92(370):47–57.

12.

Horton

Pullen

Hunt

Cai

Pease

. 1993. Gene splicing by overlap extension. Methods Enzymol. 217:270–279.

13.

Kokeguchi

Kato

Kurihara

Nishimura

Murayama

. 1990. Purification and characterization of two major outer membrane proteins from Porphyromonas gingivalis. Dent Jpn (Tokyo). 27(1):29–34.

14.

Kuboniwa

Amano

Hashino

Yamamoto

Inaba

Hamada

Nakayama

Tribble

Lamont

Shizukuishi

. 2009. Distinct roles of long/short fimbriae and gingipains in homotypic biofilm development by Porphyromonas gingivalis. BMC Microbiol. 9:105.

15.

Lamont

El-Sabaeny

Park

Cook

Costerton

Demuth

. 2002. Role of the Streptococcus gordonii SspB protein in the development of Porphyromonas gingivalis biofilms on streptococcal substrates. Microbiology. 148(Pt 6):1627–1636.

16.

Lin

Xie

. 2006. Porphyromonas gingivalis minor fimbriae are required for cell-cell interactions. Infect Immun. 74(10):6011–6015.

17.

Nagano

Abiko

Yoshida

Yoshimura

. 2013. Genetic and antigenic analyses of Porphyromonas gingivalis FimA fimbriae. Mol Oral Microbiol. 28(5):392–403.

18.

Nagano

Hasegawa

Abiko

Yoshida

Murakami

Yoshimura

. 2012. Porphyromonas gingivalis FimA fimbriae: fimbrial assembly by fimA alone in the fim gene cluster and differential antigenicity among fimA genotypes. PLoS One. 7(9):e43722.

19.

Nagano

Hasegawa

Murakami

Nishiyama

Yoshimura

. 2010. FimB regulates FimA fimbriation in Porphyromonas gingivalis. J Dent Res. 89(9):903–908.

20.

Nagano

Read

Murakami

Masuda

Noguchi

Yoshimura

. 2005. Trimeric structure of major outer membrane proteins homologous to OmpA in Porphyromonas gingivalis. J Bacteriol. 187(3):902–911.

21.

Naito

Hirakawa

Yamashita

Ohara

Shoji

Yukitake

Nakayama

Toh

Yoshimura

Kuhara

. 2008. Determination of the genome sequence of Porphyromonas gingivalis strain ATCC 33277 and genomic comparison with strain W83 revealed extensive genome rearrangements in P. gingivalis. DNA Res. 15(4):215–225.

22.

Nelson

Fleischmann

DeBoy

Paulsen

Fouts

Eisen

Daugherty

Dodson

Durkin

Gwinn

. 2003. Complete genome sequence of the oral pathogenic bacterium Porphyromonas gingivalis strain W83. J Bacteriol. 185(18):5591–5601.

23.

Park

Simionato

Sekiya

Murakami

James

Chen

Hackett

Yoshimura

Demuth

Lamont

. 2005. Short fimbriae of Porphyromonas gingivalis and their role in coadhesion with Streptococcus gordonii. Infect Immun. 73(7):3983–3989.

24.

Socransky

Haffajee

. 2002. Dental biofilms: difficult therapeutic targets. Periodontol 2000. 28:12–55.

25.

Takahashi

Davey

Yumoto

Gibson

3rd Genco

. 2006. Fimbria-dependent activation of pro-inflammatory molecules in Porphyromonas gingivalis infected human aortic endothelial cells. Cell Microbiol. 8(5):738–757.

26.

Umemoto

Hamada

. 2003. Characterization of biologically active cell surface components of a periodontal pathogen: the roles of major and minor fimbriae of Porphyromonas gingivalis. J Periodontol. 74(1):119–122.

27.

Watanabe

Maruyama

Nozawa

Aoki

Okano

Shibata

Oshima

Kurokawa

Hattori

Nakagawa

. 2011. Complete genome sequence of the bacterium Porphyromonas gingivalis TDC60, which causes periodontal disease. J Bacteriol. 193(16):4259–4260.

28.

Yoshimura

Takahashi

Nodasaka

Suzuki

. 1984. Purification and characterization of a novel type of fimbriae from the oral anaerobe Bacteroides gingivalis. J Bacteriol. 160(3):949–957.

29.

Zeituni

Jotwani

Carrion

Cutler

. 2009. Targeting of DC-SIGN on human dendritic cells by minor fimbriated Porphyromonas gingivalis strains elicits a distinct effector T cell response. J Immunol. 183(9):5694–5704.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.77 MB