TEXT

Top Abstract Text References

Porphyromonas gingivalis, an anaerobic gram-negative organism, has been implicated in the pathogenesis of periodontal diseases (8, 22, 28). This organism possesses a variety of putative virulence factors, such as fimbriae, hemagglutinins, proteolytic enzymes, lipopolysaccharide, vesicles, and outer membrane proteins (16). Many strains of P. gingivalis are known to have fimbriae (18, 23), which are considered to play an important role in adherence of the organism to oral epithelial cells as the initial step in the progression of periodontitis (9). Affinity of the fimbriae to mammalian cells (7), bacterial cells (6, 12), or saliva-coated hydroxyapatite (13) has also been recognized. It has also been reported that vaccination with fimbrial proteins of P. gingivalis can protect experimental animals from periodontal tissue destruction (5).

The fimbriae of P. gingivalis were originally purified by Yoshimura et al. from the strain 381 (26), and these authors demonstrated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis that fimbrillin, the major subunit of the fimbriae (FimA₃₈₁), was a 43-kDa protein. Using a synthetic oligonucleotide probe designed on the basis of the amino acid sequence of the N-terminal domain of the protein, Dickinson et al. (4) then cloned and sequenced a gene (fimA₃₈₁) encoding fimbrillin on a 2.5-kb SacI segment.

To assess the roles played in the virulence of this organism, various genes have been cloned and expressed, mainly in the host Escherichia coli (1-4, 11, 15, 21). However, expression of these genes cloned in foreign species is not necessarily qualitatively or quantitatively equivalent to that in the original species. In fact, although the product of the cloned fimA gene of P. gingivalis in E. coli could be detected by using anti-fimbrial antibodies, no fibrous structures were observed by electron microscopy on the cell surface (4).

Previously, we developed a host-vector system for P. gingivalis consisting of the host strain, YH522, a restriction-deficient derivative of SU60, and a plasmid vector, pYH420, capable of replicating stably in P. gingivalis (25). In this study, a fragment containing the fimA₃₈₁ gene that encodes the fimbrillin of P. gingivalis 381 was subcloned in the vector pYH420, and the resulting recombinant plasmid, pYHF2, was electroporated into restriction-deficient P. gingivalis, YH522, which possesses a kind of fimbriae serotypically different from those of the strains ATCC 33277 and 381.

The plasmid pUC13Bg12.1 (4) was employed as the source of the fimA gene of P. gingivalis 381 (fimA₃₈₁). The EcoRI-digested linear fragment of this plasmid was inserted into the unique EcoRI site of the vector, pYH420, to construct a chimeric plasmid, pYHF1. To delete the redundant fragment containing the gene for ampicillin resistance, pYHF1 was then digested with SalI, and the larger of the two generated fragments was self-ligated and electroporated into YH522 (Fig. 1). The generated plasmid, pYHF2, with a size of 11.5 kb, contained fimA₃₈₁, rep, stb (25), and the erythromycin resistance gene used for selection of transformants. The YH522 cells containing pYHF2 were designated YH522/pYHF2.

View larger version (23K): [in this window] [in a new window]   FIG. 1.   Construction of pYHF2. The fimA gene of P. gingivalis 381 was cleaved out from pUC13Bg12.1 and ligated into the EcoRI site of the vector plasmid pYH420 to construct a chimeric plasmid, pYHF1. pYHF1 was then digested with SalI, and the larger of the two generated fragments was self-ligated to construct pYHF2. Apr, ampicillin resistance gene; Emr, erythromycin resistance gene.

To determine the presence of the subcloned fimA₃₈₁ gene in the transformants, Southern hybridization analysis was performed. EcoRI- or BamHI-digested whole DNA samples of YH522/pYHF2 were then electorophoretically separated in 0.7% agarose gels and transferred onto nylon membranes. Hybridization was performed with a 2.5-kb SacI fragment of pUC13Bg12.1 (fimA₃₈₁ probe) or the whole vector plasmid (pYH420 probe) labeled by using the enhanced chemiluminescence gene detection system. pYHF2 was cleaved into an 11.5-kb fragment with EcoRI and into 5.7-, 5.1-, and 0.7-kb fragments with BamHI. The fimA₃₈₁ probe hybridized with the 11.5-kb EcoRI fragment and with the 5.7-kb BamHI fragment, indicating that these fragments contained the fimA₃₈₁ gene (Fig. 2A). The pYH420 probe hybridized with the 11.5-kb EcoRI fragment and with the 5.7-, 5.1-, and 0.7-kb fragments of the BamHI digests (Fig. 2B). Plasmid DNA identical in size to pYHF2 was always detectable and could be recovered from YH522/pYHF2, strongly suggesting that the plasmid was autonomously maintained in the transformant cells.

View larger version (47K): [in this window] [in a new window]   FIG. 2.   Southern hybridization analysis for detection of pYHF2 in YH522/pYHF2 cells. Whole DNA from the P. gingivalis strains was digested with EcoRI or BamHI, electrophoresed in agarose gels, transferred onto membrane filters, and hybridized with fimA381 probe (A) or pYH420 probe (B). Lanes: 1, 4, 7, and 10, YH522; 2, 5, 8, and 11, YH522/pYHF2; and 3, 6, 9, and 12, purified pYHF2 DNA.

To detect a protein(s) produced by the transformed fimA₃₈₁ gene in YH522, whole-cell lysates of YH522 with and without pYHF2 were analyzed by SDS-polyacrylamide gel electrophoresis. As shown in Fig. 3A, YH522/pYHF2 exhibited a protein band with almost the same mobility as that of ATCC 33277. The sizes of the bands from these strains were estimated as ca. 41 kDa, similar to the molecular mass of 43 kDa reported by Yoshimura et al. (26). However, an antigenic difference between these bands was demonstrated by Western blotting. The band from YH522/pYHF2 reacted strongly with an antibody raised against fimbriae of ATCC 33277, which were shown to have the same antigenicity as fimbriae of strain 381, and weakly with that raised against SU60 native fimbriae (Fig. 3B and C). However, the band from YH522 without the plasmid reacted only with an anti-SU60 fimbrial antibody and not with that against 33277 fimbriae. These observations indicated that the products of the two strains with similar molecular masses were serologically different fimbrillins. The band observed in YH522/pYHF2 was thus confirmed to be a mixture of the products of the transformed fimA₃₈₁ gene and the host's resident fimA_SU60 gene.

View larger version (27K): [in this window] [in a new window]   FIG. 3.   Expression of FimA protein from YH522/pYHF2 and YH522 cells. (A) Coomassie blue-stained SDS-polyacrylamide gel; (B and C) Western blotting analysis with polyclonal antibody raised against the fimbriae of P. gingivalis 33277 and SU60 (parent strain of YH522), respectively. Lanes: 1, 5, and 9, YH522; 2, 6, and 10, YH522/pYHF2; 3, 7, and 11, purified fimbriae of YH522; and 4, 8, and 12, purified fimbriae of 33277.

Interestingly, considerably lower expression level of FimA_SU60, the YH522-specific fimbrial protein, was observed in YH522/pYHF2 (Fig. 3C, lane 10) than in YH522 lacking plasmid (Fig. 3C, lane 9). Expression of the fimA_SU60 gene seemed to be suppressed by the presence of the fimA₃₈₁ gene in the same cell. One possible explanation of this was that the native fimA_SU60 on the chromosome and the introduced fimA₃₈₁ on the plasmid are commonly regulated by the mechanism involved in diffusible mediators. The decreased expression of fimA_SU60 in the presence of fimA₃₈₁ might be explained by the difference in copy number between the two genes: fimA_SU60 gene is present as a single copy on the chromosome, and fimA₃₈₁ is present on the plasmid in multiple copies. Judging from the ease of purification of the plasmid DNA from the transformants, we assumed that the vector plasmid, pYH420, was present in more than 10 copies per cell in P. gingivalis.

The 20 amino-terminal residues of purified fimbriae from YH522/pYHF2 were analyzed. The proteins transferred onto polyvinylidene difluoride membranes were stained with Coomassie brilliant blue R-250, and the 41-kDa band was recovered for amino acid sequence analysis. Although there was contamination with the native fimbrial protein of YH522, it was present in much smaller amounts than the recombinant protein. The sequence of the major fimbrillin from YH522/pYHF2 was identical to that of strain 381 (data not shown).

The cell surface of YH522/pYHF2 was then investigated by negative staining and electron microscopy. YH522/pYHF2 cells showed characteristic thin and long fimbrial structures, 0.5 to 3.0 µm in length and 5 nm in width, over the entire cell surface (Fig. 4B). In contrast, fewer fimbriae with a shorter size were observed on YH522 cells lacking plasmid (Fig. 4A). The thin and long fimbriae, which comprised the majority of the fimbrial structures observed on YH522/pYHF2 cells, were then immunologically confirmed to be the ATCC 33277-type fimbriae and not those native to YH522, i.e., the SU60-type fimbriae. Immunogold electron microscopy by using an anti-33277 fimbrial antibody showed that gold particles bound to the majority, but not all, of the fimbriae on YH522/pYHF2 cells (Fig. 5). No gold particles were seen on YH522 cells without the plasmid (data not shown). The majority of the objects on YH522/pYHF2 cells detectable electron microscopically were thus considered to be fimbrial structures composed of the product of the transformed fimA₃₈₁ gene.

View larger version (101K): [in this window] [in a new window]   FIG. 4.   Surface configuration of YH522/pYHF2 and YH522 cells. Samples were prepared by negative staining with 2% uranyl acetate. (A) Each YH522 cell had short and sparse fimbriae on its surface. (B) Each YH522/pYHF2 cell had long and dense fimbrial structures. Bars, 200 nm.

View larger version (112K): [in this window] [in a new window]   FIG. 5.   Immunogold-labeling of a YH522/pYHF2 cell. The long fimbrial structures on the YH522/pYHF2 cell surface were labeled with the anti-33277 fimbrial antibody, while some shorter structures remained unlabeled. Bars, 200 nm.

These results suggested that introduction of the fragment containing the fimA gene into P. gingivalis was sufficient for construction of the fimbrial fibrous structures. Since this was not observed when the same gene was expressed in E. coli (4), it is possible that the polymerization process of fimbrillin leading to production of the fibrous structures employs the host's native mechanism. The maturation process of the fimbriae in P. gingivalis was considered to probably occur by a mechanism like cleavage of the leader peptide and polymerization of the subunits, as well as by supplementation by some minor fimbrial components (17, 19, 27). It was reported that fimbrillin precursor (prefimbrillin) was cleaved by trypsin-like protease activity of P. gingivalis (17, 19), resulting in maturation of fimbrillin. It is, therefore, natural to assume that prefimbrillin produced in YH522/pYHF2 is processed by the inherent protease, and the resulting mature fimbrillin molecules are then readily polymerized to generate fibrous structures. In fact, the amino-terminal amino acid sequence of the fimbrial protein isolated from YH522/pYHF2 cells was the same as that of the previously reported "mature" fimbrillin (14).

At present, the detailed fimbriation mechanism of P. gingivalis remains unclear. Five open reading frames encoding 63-, 15-, 50-, 80-, and 19-kDa polypeptides have been reported to exist in the fimA-flanking region (24, 27). Among these, the 50- and 80-kDa polypeptides have been confirmed to be minor structural components of the fimbriae because antibodies raised against them reacted with purified fimbriae, although their functions are unknown. In E. coli, 11 (10) and 8 (20) genes are known to be responsible for production, maturation, or regulation of P fimbriae and of type 1 fimbriae, respectively. Therefore, it is likely that several as yet unidentified genes also participate in some essential processes in fimbriation in P. gingivalis.

To gain further insight into the fimbriation system and the function of fimbriae in P. gingivalis, more precise genetic analyses are required. Experiments to elucidate in more detail the structure, biological significance, and mechanism of fimbrial expression by using the host-vector system which we have developed for this species are currently in progress.