| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Biological Chemistry, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan
2 Department of Restorative Dentistry and Endodontology, Osaka University, 1-8 Yamada-Oka, Suita 560-0871, Japan
Correspondence
Hiroyuki Azakami
azakami{at}yamaguchi-u.ac.jp
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). This bacterium can also be pathogenic, causing a variety of soft-tissue and wound infections (Chen & Wilson, 1992), such as endocarditis (Decker et al., 1986) and other opportunistic infections. Monoinfection of germ-free or gnotobiotic rats with E. corrodens causes periodontal disease with severe alveolar bone loss (Listgarten et al., 1978). Since E. corrodens is observed in the tooth-attached plaque area (Noiri et al., 2001), it is believed that this bacterium may participate in the early stages of biofilm formation by specific coaggregation with Gram-positive and -negative bacteria in human periodontal pockets.
We have previously found that E. corrodens 1073 expresses a cell-associated N-acetyl-D-galactosamine (GalNAc)-specific Eikenella corrodens lectin-like substance (EcLS) that mediates adherence to various host-tissue cell surfaces (Ebisu & Okada, 1983; Yamazaki et al., 1981, 1988; Miki et al., 1987). We have also reported that EcLS mediates the coaggregation of E. corrodens with some strains of Streptococcus sanguis and Actinomyces viscosus that are predominant during the early stages of dental plaque formation (Ebisu et al., 1988). Since all these bacteria are thought to be the early colonizers in dental plaque (Kolenbrander et al., 2002), E. corrodens might play a key role in dental plaque formation. Moreover, we have reported that EcLS stimulates the mitogenic activity of B lymphocytes (Nakae et al., 1994). Furthermore, we have reported that E. corrodens 1073 induces KB cells (a human oral epidermoid carcinoma cell line) to secrete and express IL-6 and IL-8; a direct contact of E. corrodens with KB cells is not necessarily required for the stimulation of IL-6 and IL-8 secretion and expression (Yumoto et al., 2001). Thus, it is thought that EcLS contributes to the pathogenicity and virulence of E. corrodens and that its expression can be estimated by its haemagglutination (HA) activity.
Like several Gram-negative pathogens, including Neisseria gonorrhoeae, Neisseria meningitidis and Moraxella bovis, E. corrodens exhibits a phase variation resulting from the altered synthesis of type IV pili, which is reflected in the changes in colony morphology (Villar et al., 1999, 2001). Most type IV pili are composed primarily of type IV pilin, a protein that is approximately 150 to 165 amino acids long and is synthesized as a precursor form (prepilin) that contains a basic leader sequence of variable length (4–25 residues), with one exception: in N. gonorrhoeae (Wolfgang et al., 2000). Following translation, prepilin is cleaved at an atypical site by a cognate peptidase that simultaneously methylates the resultant amino-terminal amino acid, typically a phenylalanine residue. Following processing, the mature pilin is exported to the cell surface by a specific transport mechanism and assembled into pili (Strom & Lory, 1993). Type IV pili are thought to be involved in bacterial adhesion and to function as antigenic determinants.
On a solid medium, E. corrodens 1073 forms large, non-corroding colonies, whereas other strains form small, corroding colonies due to twitching motility. Rao & Progulske-Fox (1993) and Tonjum et al. (1993) have cloned two type IV pilin genes, from E. corrodens 23834 and E. corrodens 31745, respectively. However, in our previous electron microscopic study of E. corrodens 1073, we did not observe any pilus-like structure (Ebisu et al., 1981), although other investigators have suggested the presence of pili (Hood & Hirschberg, 1995). Therefore, the molecular mechanism(s) of phase variation in E. corrodens, including the formation of type IV pili, remains to be determined.
Recently, we isolated the plasmid pMU1 from E. corrodens 1073 and discovered seven ORFs on it (Azakami et al., 2005a). We demonstrated that the transformation of a derivative of pMU1 into strain 23834 resulted in loss of pilus structure from the cell surface and a morphological change in the colonies, i.e. from a corroding to a non-corroding form. In this study, we showed that the gene encoding the pilin gene-specific recombinase piv on plasmid pMU1 is involved in these phenomena. Moreover, we have suggested that the genomic recombination of the pilin gene locus of E. corrodens 23834 increases GalNAc-specific lectin activity.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
) was obtained from the ATCC. E. corrodens cells were grown in tryptic soy broth containing 2 mg KNO3 ml–1 and 5 mg haemin ml–1 or on sheep blood agar plates at 37 °C. Esch. coli XL-1 Blue was routinely cultured in LB broth or agar. Bacteria harbouring plasmids were cultured on a medium supplemented with 50 µg carbenicillin ml–1.
Transformation of E. corrodens strains.
E. corrodens was electrotransformed with 5–10 µg of plasmid DNA using a Gene Pulser electroporator (Bio-Rad). In brief, a 100 ml volume of bacteria was grown for 12 h, washed three times in solution A (272 mM sucrose, 1 mM MgCl2, pH 7·4) and resuspended in 100 µl solution A. Next, a 39 µl volume of bacteria was mixed with 1 µl DNA and electroporated at 2·1 kV, 25 µF and 200 
. For transformations involving the broad-host-range shuttle vector pLES2 and its derivatives, cells were recovered by centrifugation after incubation for 12 h at 37 °C. After recovery, recombinant cells were cultured on sheep blood agar plates containing 50 µg carbenicillin ml–1 at 37 °C.
Curing experiment.
Acridine orange was added at a final concentration of 100 µg ml–1 to a medium inoculated with approximately ten exponential-phase cells per millilitre. After 24 h incubation at 37 °C, the culture was diluted with the medium and plated out on sheep blood agar plates. The colonies on the plates were used for further experiments. Cured colonies were identified as those that were sensitive to carbenicillin.
Transmission electron microscopy.
Drops of water (20 µl) were deposited on 24 h-old E. corrodens colonies on the sheep blood agar plates, and within 30 s, a 200 mesh Formvar-coated copper grid was floated on the surface of each drop for 30 s. The grids were removed, floated on a drop of 1 % phosphotungstic acid for 1·5 min, and the excess dye was then removed. Grids were dried and examined at 75 kV in a Hitachi H-7600 electron microscope.
Southern blot analysis.
After electrophoresis on agarose gels, DNA fragments were transferred onto a Hybond-N membrane (Amersham Bioscience). Hybridization of the membrane with a labelled probe and the detection of hybridized bands were conducted using a non-radioactive DNA labelling and detection kit (Amersham). The probe including the ecpAB region was amplified using genomic DNA of strain 23834 as a template and primers 5'-AAGCCTAGCCGGAAATGAAG-3' and 5'-TTCGGAGGACAATATGCAGC-3'.
HA activity assay.
The HA activity assay was performed as previously described (Ebisu & Okada, 1983). Erythrocytes that were obtained by the centrifugation of preserved rabbit blood were washed three times with saline before being suspended in PBS (pH 7·2) at a concentration of 2 %. The HA assay was performed in microtitre plates (Vdispo; Nalge Nunc International). Test preparations (50 µl) were serially diluted twofold in PBS and mixed for 2 min with equal volumes of the 2 % erythrocyte suspension. HA activity was examined after 1 h, and HA titres were expressed as the maximum dilution of the test preparation that exhibited HA.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
(A), seven ORFs, which have been designated ORF 1 to ORF 7, are found on the same strand of pMU1. Introduction of the fragments from ORF 4 to ORF 7 into strain 23834 causes irreversible changes in colony morphology (Azakami et al., 2005a). Although strain 23834 forms corroding colonies on a solid medium, all the transformants carrying pMU1 were altered to non-corroding colonies. To identify the gene responsible for this effect, we subcloned ORF 4 and ORFs 5–6 into the Esch. coli/E. corrodens shuttle vector pLES2 and introduced the resultant plasmids pMU4 and pMU5–6 into E. corrodens 23834. As shown in Fig. 2, all transformants with pMU4 formed non-corroding colonies on the selection medium (panel B), whereas the transformants with pMU5-6 or pLES2 vectors formed corroding colonies similar to those of strain 23834 (panel A). Even after the elimination of pMU4 by acridine orange treatment, the transformants showed the non-corroding colony morphology (data not shown). These results suggest that ORF 4 on plasmid pMU1 irreversibly altered the colony morphology of E. corrodens from corroding to non-corroding.
|
|
, 2001). Thus, it is believed that E. corrodens forms corroding colonies on solid medium due to mobility imparted by pili. As described above, we showed that ORF 4 on pMU1 was involved in morphologically changing the colony from corroding to non-corroding. To determine whether the morphological change induced by ORF 4 was caused by the loss of pili, we observed the cell surface structure of transformants with pMU4 by transmission electron microscopy. As shown in Fig. 3, although strain 23834 had the pilus structure on its cell surface (panel A), strain 1073 carrying pMU1 did not have any pilus (panel B). Pilus structure was not observed on the surface of strain 23834 (panel B), whereas strain 23834 (pMU5-6) and strain 23834 (pLES2) maintained the pili on the surface (panel A). These results suggest that ORF 4 is involved in the loss of pilus structure in strain 23834. Moreover, it is suggested that the morphological changes in the colonies due to ORF 4 might be caused by loss of pili.
|
have cloned two type IV pilin genes, ecpA and ecpB, from E. corrodens 23834 and have shown that they are located in tandem on its genome (Fig. 1B
). To determine whether ORF 4 causes genomic recombination of pilin genes, we performed genomic Southern hybridization using the ecpAB region as a probe. We cloned a 1·2 kb fragment, including ecpA and ecpB, from strain 23834. Chromosomal DNAs were isolated from strains 23834, 23834 (pLES2), 23834 (pMU4) and 1073. These were digested with KpnI and HindIII or with HindIII alone, and then hybridized to a 1·2 kb pilin gene fragment. As shown in Fig. 4(A), two bands of approximately 1·5 and 0·9 kb were detected in strains 23834 (lane 2) and 23834 (pLES2; lane 4). However, the hybridized band was shifted to a single band of 2·7 kb in 23834 (pMU4; lane 5). The shifted band of 23834 (pMU4) was of the same size as that of strain 1073 (lane 6). These results suggested that ORF 4 might cause the recombination of the pilin gene locus on the chromosome, including the ecpA and ecpB genes. As shown in Fig. 4(B), similar genomic recombination was observed for the HindIII-digested genomic DNA. This result confirmed that ORF 4 might be involved in genomic recombination with the pilin gene locus.
|
, 23834 (pMU4) exhibited strong HA activity (128-fold), whereas strains 23834 and 23834 (pLES2) exhibited weak activity (16-fold). The increased HA activity of strain 23834 (pMU4) was inhibited in the presence of GalNAc (data not shown). These results suggested that a GalNAc-specific lectin on the cell surface might be exposed by the loss of pilus structure. The increased activity of strain 23834 (pMU4) was equivalent to that of strain 1073.
|
). To investigate whether morphological changes in the colonies and loss of pili resulting from the presence of ORF 4 are caused by phase variation, we compared the properties of strain 23834 (pMU4) with those of phase variants from strain 23834. The phase variation of strain 23834 from corroding to non-corroding did not alter the pilus structure on the cell surface (Fig. 3) or the HA activity (Table 1). Moreover, in phase variants of strain 23834, no genomic recombination was observed in the ecpAB region (Fig. 4, lane 3). These results suggested that the increase in HA activity resulting from the presence of plasmid pMU1 might be due to genomic recombination of the pilin gene locus. Furthermore, it is suggested that the loss of pili might be a result of this recombination and might not contribute directly to the increase in HA activity.
Introduction of the piv gene improves the growth of E. corrodens
E. corrodens 23834 grows at a slower rate than strain 1073 (Fig. 5). Cultures of strain 23834 reached stationary phase at OD600=0·4, whereas strain 1073 grew until OD600=0·7. Moreover, the lag phase of strain 23834 was longer than that of strain 1073. We compared the growth curve of strain 23834 (pMU4) with that of strain 23834. Interestingly, the growth curve of strain 23834 (pMU4) was nearly the same as that of strain 1073.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). In this study, we have demonstrated that ORF 4 was responsible for both the changes (Figs 2 and 3). These results suggested that a product of ORF 4 might be related to the loss of pilus structure from the cell surface of E. corrodens. It has been reported that the pili on a cell surface are involved in the twitching motility of cells as well as colony morphology (McMichael, 1992; Villar et al., 2001). Thus, we considered that the morphological change in the colonies from corroding to non-corroding occurred due to a loss of pili. Since the introduction of pMU4 into strain 23834 increased GalNAc-specific HA activity (Table 1), we felt that a GalNAc-specific lectin was exposed due to the loss of the pilus structure from the cell surface. However, this hypothesis was refuted because phase variants from strain 23834 exhibited similar HA activity (Table 1), but formed non-corroding colonies and had lost their pili in a similar manner to strain 23834 (pMU4) (Figs 2 and 3). This was confirmed by the finding that no genomic recombination was observed in the phase variants (Fig. 4). Although most of the E. corrodens 23834 formed corroding colonies, non-corroding variants arose irreversibly from corroding variants at a considerable frequency. Therefore, we concluded that GalNAc-specific lectin activity was increased due to genomic recombination within the ecpAB locus. Since it is believed that GalNAc-specific lectin contributes very significantly to the pathogenicity and virulence of E. corrodens, these organisms may require plasmid pMU1 to survive in the oral environment. We have summarized the characteristics of the E. corrodens strains in Table 2.
|
). For example, the genes encoding the major pilin subunits of Mor. bovis are located on an invertible segment of DNA that allows alternate expression of tfpQ or typI (type IV pilin; Fulks et al., 1990). The piv gene, located immediately adjacent to the invertible segment, encodes the site-specific DNA invertase. The expression of the type IV pili in Mor. bovis is regulated by a site-specific DNA inversion system by Piv (Heinrich & Glasgow, 1997). Thus, we assumed that the loss of pili following transformation of ORF 4 might be due to genomic recombination in the pilin genes. In this study, it is suggested that transformation of ORF 4 results in genomic recombination within the ecpAB locus, although the recombination sites and the events following recombination are yet to be determined (Fig. 4). Moreover, the shifted band of strain 23834 with the introduced ORF 4 gene was of the same size as that of strain 1073 carrying pMU1. This finding suggests that strains 1073 and 23834 might be derived from the same origin and that strain 1073 might have evolved from strain 23834 by horizontal transfer of a plasmid. A homology search has revealed that gene clusters homologous to pMU1 are located in other bacterial genomes (Azakami et al., 2005a). Genes homologous to all the ORFs on pMU1 are found in the genome of N. meningitidis. Moreover, genes homologous to ORFs 1, 3, 6 and 7 of pMU1 are found on a plasmid, pJTPS1, of Ralstonia solanacearum. These gene clusters might also be transferred horizontally in the environment. Following the acquisition of pMU1, strain 1073 may have acquired the ability to adhere in an oral environment, and this may have contributed to its high periodontal pathogenicity.
As shown in Fig. 5, the growth of strain 23834 was improved by the introduction of pMU4. Although the reason for this effect is not yet clear, it is thought that this might occur as the result of the genomic recombination by ORF 4. Moreover, we have found that E. corrodens forms a biofilm on polystyrene surfaces and that the biofilm formation of strain 23834 is increased remarkably after the introduction of pMU4 (Azakami et al., 2005b). Although pili are required for the initial attachment process in most bacteria, it is thought that the lectin on the cell surface plays an important role in E. corrodens. Therefore, it is suggested that biofilm formation by E. corrodens depends on the lectin rather than pili. It is believed that biofilm formation is a process by which pathogenic bacteria attach to a solid surface and thereby enhance their pathogenicity (Kolenbrander et al., 2002). Therefore, along with an increase in growth, increased biofilm formation caused by pMU4 might also be one of the strategies by which E. corrodens survives in the oral cavity and enhances its pathogenicity and virulence.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Azakami, H., Nakashima, H., Akimichi, H., Noiri, Y., Ebisu, S. & Kato, A. (2005b). Involvement of N-acetyl-D-galactosamine-specific lectin in the biofilm formation by the periodontopathogenic bacterium, Eikenella corrodens. Biosci Biotechnol Biochem (in press).
Chen, C. K. & Wilson, M. E. (1992). Eikenella corrodens in human oral and non-oral infections: a review. J Periodontol 63, 941–953.[Medline]
Decker, M. D., Graham, B. S., Hunter, B. S. & Liebowitz, S. M. (1986). Endocarditis and infections of intravascular devices due to Eikenella corrodens. Am J Med Sci 292, 209–212.[Medline]
Ebisu, S. & Okada, H. (1983). Agglutination of human erythrocytes by Eikenella corrodens. FEMS Microbiol Lett 18, 153–156.[CrossRef]
Ebisu, S., Miki, Y., Fukuhara, H., Okada, H. & Nakao, M. (1981). An electron microscopic study on morphology of Eikenella corrodens. J Osaka Univ Dent Sch 21, 137–143.[Medline]
Ebisu, S., Nakae, H. & Okada, H. (1988). Coaggregation of Eikenella corrodens with oral bacteria mediated by bacterial lectin-like substance. Adv Dent Res 2, 323–327.
Fulks, K. A., Marrs, C. F., Stevens, S. P. & Green, M. R. (1990). Sequence analysis of the inversion region containing the pilin genes of Moraxella bovis. J Bacteriol 172, 310–316.
Heinrich, D. W. & Glasgow, A. C. (1997). Transcriptional regulation of type 4 pilin genes and the site-specific recombinase gene, piv, in Moraxella lacunata and Moraxella bovis. J Bacteriol 179, 7298–7305.
Hood, B. L. & Hirschberg, R. (1995). Purification and characterization of Eikenella corrodens type IV pilin. Infect Immun 63, 3693–3696.[Abstract]
Kolenbrander, P. E., Andersen, R. N., Blehert, D. S., Egland, P. G., Foster, J. S. & Palmer, R. J., Jr (2002). Communication among oral bacteria. Microbiol Mol Biol Rev 66, 486–505.
Listgarten, M. A., Johnson, D., Dowotny, A., Tanner, A. C. R. & Socransky, S. S. (1978). Histopathology of periodontal disease in gnotobiotic rats monoinfected with Eikenella corrodens. J Periodont Res 13, 134–148.[CrossRef][Medline]
McMichael, J. C. (1992). Bacterial differentiation within Moraxella bovis colonies growing at the interface of the agar medium with the Petri dish. J Gen Microbiol 138, 2687–2695.
Miki, Y., Ebisu, S. & Okada, H. (1987). The adherence of Eikenella corrodens to guinea pig macrophages in the absence and presence of anti-bacterial antibodies. J Periodont Res 22, 359–365.[CrossRef][Medline]
Nakae, H., Yumoto, H., Matsuo, T. & Ebisu, S. (1994). Mitogenic stimulation of murine B lymphocytes by the N-acetyl-D-galactosamine specific bacterial lectin-like substance from Eikenella corrodens. FEMS Microbiol Lett 116, 349–354.[Medline]
Noiri, Y., Li, L. & Ebisu, S. (2001). The localization of periodontal-disease-associated bacteria in human periodontal pockets. J Dent Res 80, 1930–1934.
Rao, V. K. & Progulske-Fox, A. (1993). Cloning and sequencing of two type 4 (N-methylphenylalanine) pilin genes from Eikenella corrodens. J Gen Microbiol 139, 651–660.
Stein, D. C., Silver, L. E., Clark, V. L. & Young, F. E. (1983). Construction and characterization of a new shuttle vector, pLES2, capable of functioning in Escherichia coli and Neisseria gonorrhoeae. Gene 25, 241–247.[Medline]
Strom, M. S. & Lory, S. (1993). Structure-function and biogenesis of the type IV pili. Annu Rev Microbiol 47, 565–596.[CrossRef][Medline]
Tanner, A. C., Haffer, C., Bratthall, G. T., Visconti, R. A. & Socransky, S. S. (1979). A study of the bacteria associated with advancing periodontitis in man. J Clin Periodontol 6, 278–307.[CrossRef][Medline]
Tonjum, T., Weir, S., Bovre, K., Progulske-Fox, A. & Marrs, C. F. (1993). Sequence divergence in two tandemly located pilin genes of Eikenella corrodens. Infect Immun 61, 1909–1916.
Villar, M. T., Helber, J. T., Hood, B., Schaefer, M. R. & Hirschberg, R. L. (1999). Eikenella corrodens phase variation involves a posttranslational event in pilus formation. J Bacteriol 181, 4154–4160.
Villar, M. T., Hirschberg, R. L. & Schaefer, M. R. (2001). Role of the Eikenella corrodens pilA locus in pilus function and phase variation. J Bacteriol 183, 55–62.
Wolfgang, M., van Putten, J. P., Hayes, S. F., Dorward, D. & Koomey, M. (2000). Components and dynamics of fiber formation define a ubiquitous biogenesis pathway for bacterial pili. EMBO J 19, 6408–6418.[CrossRef][Medline]
Yamazaki, Y., Ebisu, S. & Okada, H. (1981). Eikenella corrodens adherence to human buccal epithelial cells. Infect Immun 31, 21–27.
Yamazaki, Y., Ebisu, S. & Okada, H. (1988). Partial purification of a bacterial lectin-like substance from Eikenella corrodens. Infect Immun 56, 191–196.
Yumoto, H., Nakae, H., Yamada, M., Fujinaka, K., Shinohara, C., Ebisu, S. & Matsuo, T. (2001). Soluble products from Eikenella corrodens stimulate oral epithelial cells to induce inflammatory mediators. Oral Microbiol Immunol 16, 296–305.[CrossRef][Medline]
Received 5 September 2005;
revised 14 November 2005;
accepted 21 November 2005.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
), 23834 (corroding) (
), 23834 (pMU4) (
), and 23834 (non-corroding) (
). Bacterial growth was monitored spectrophotometrically by OD600.