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Infection and Immunity, January 2006, p. 734-739, Vol. 74, No. 1
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.1.734-739.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Association between Epithelial Cell Death and Invasion by Microspheres Conjugated to Porphyromonas gingivalis Vesicles with Different Types of Fimbriae

Hiroaki Inaba,1 Shinji Kawai,1 Takahiro Kato,1 Ichiro Nakagawa,2 and Atsuo Amano1*

Departments of Oral Frontier Biology,1 Oral and Molecular Microbiology, Osaka University Graduate School of Dentistry, Suita-Osaka 565-0871, Japan2

Received 14 July 2005/ Returned for modification 29 August 2005/ Accepted 9 October 2005


   ABSTRACT
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Microspheres (MS) conjugated to Porphyromonas gingivalis vesicles with type II fimbriae were the most efficient human epithelial cell invaders among the six types. Cell death was induced by MS, though becoming less frequent over time, with invasion efficiency partially related to cell death and gingipains likely the major cause.


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Porphyromonas gingivalis has been shown to be a major etiologic agent of destructive adult periodontitis (14). The organism reportedly invaded several human epithelial cell lines and persisted in the intracellular location in vitro, which may protect it from detection by the immune system, leading to further spreading of the bacteria to adjacent tissues (13, 19, 20, 29-31). P. gingivalis fimbriae, which play a critical role in the mediation of bacterial interactions with host tissues (1), are classified into six genotypes (I to V and Ib) on the basis of the diversity of the fimA genes encoding FimA (a subunit of fimbriae) (2). Previously, we reported that a majority of periodontitis patients harbored P. gingivalis with type II fimbriae (3, 15) and also found that microspheres (MS) conjugated to the recombinant protein of type II fimbriae adhered to human epithelial cells to a significantly greater degree than those of other types (16). However, those proteins were recombinantly engineered with Escherichia coli, while several other components of P. gingivalis, such as gingipains, minor fimbriae, and hemagglutinin, have been suggested to be involved in bacterial adherence to and invasion of host cells (4, 5, 7, 21, 23, 27). Therefore, we evaluated the invasive efficiency of the six types of fimbriae by use of native preparations containing other possible invasion-mediating factors of P. gingivalis. Further, P. gingivalis infection has been shown to inhibit apoptosis of gingival epithelial cells, despite the presence of large numbers of intracellular P. gingivalis organisms (8, 18, 29, 30), and it was also shown that gingival epithelial cells did not detach from the substratum and remained viable for 24 h after infection (29, 30). In contrast, following the degradation of cell adhesion molecules such as cadherins and integrins, gingipains were shown to induce apoptosis or death in 50% of epithelial cells for 24 h (22). Therefore, we also examined the relationship between cell death and the invasion mediated by P. gingivalis fimbriae.

The following P. gingivalis strains were used in this study: ATCC 33277 (type I fimbriae), HG1691 (type Ib), OMZ314 (type II), 6/26 (type III), HG564 (type IV), HNA99 (type V), a fimA-deficient mutant of ATCC33277 [{Delta}fimA (I)] (26), a fimA-deficient mutant of OMZ314 [{Delta}fimA (II)] (17), and a gingipain-deficient mutant of ATCC33277 [{Delta}rgpAB{Delta}kgp (I)] (23). Their vesicles were prepared as described previously (9). Bacterial hemagglutination activities were assayed as described previously (11). The phenotypic characterizations of P. gingivalis strains were performed with an API 20A system (BioMérieux Industry, Marcy l'Etoile, France) as described previously (24, 28). HeLa cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, gentamicin, and 1% fungizone (all from Invitrogen Co., Carlsbad, CA) at 37°C, and cell viability was assayed as described previously (16). Covalent coupling of vesicles or bovine serum albumin (BSA) (as a control) to fluorescent MS (Molecular Probes, Eugene, OR) (diameter, 1.0 µm) was performed as described previously (16). The amounts of bound proteins and lipopolysaccharide (LPS) as well as proteolytic activities of gingipains were measured as described previously (10, 25). Western blotting of fimbriae was performed as described previously (17). Fimbriae on vesicle-conjugated MS (vcMS) were stained with polyclonal rabbit anti-fimbriae antibodies (diluted 1:1,000) (17), followed by Alexa Fluor 488 goat anti-rabbit antibody (Molecular Probes). Confocal microscopic analyses of adhesion to and invasion of HeLa cells by vcMS were performed using a laser scanning confocal microscope (model LSM510; Carl Zeiss, Thornwood, NY) as described previously (16). The cells were incubated with vcMS (200 vcMS/cell) in Dulbecco's modified Eagle medium containing 10% fetal bovine serum and then fixed with 4% paraformaldehyde in phosphate-buffered saline, after which F-actin was stained with Oregon Green 488-conjugated phalloidin (Molecular Probes). The efficiency of the adhesion and/or invasion was expressed as the average area measured in images collected in 10 independent experiments. To analyze the distribution of the invaded vcMS, optical sections were obtained along the z axis at 0.15-µm intervals (60 sections, 9-µm thickness), and images of the x-z and y-z planes were reconstructed using LSM510 software. Cellular apoptosis was identified with a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay using a DeadEnd fluorometric TUNEL system (Promega, Madison, WI) according to the manufacturer's instructions. All data are expressed as the means ± standard deviations and were analyzed with an unpaired Student's t test.

The MS were equivalently conjugated to vesicles with different types of fimbriae (proteins, 17.13 ± 1.34 to 21.71 ± 1.58 µg/109 vcMS; LPS, 0.24 ± 0.011 to 0.30 ± 0.18 µg/109 vcMS). Adequate coupling of fimbriae to vcMS was confirmed with Western blotting (Fig. 1A), and the presence of type II fimbriae (Fig. 1B) as well as other types of fimbriae (data not shown) on the surface of vcMS was also confirmed with a confocal microscope. The interaction of epithelial cells with vcMS was examined as shown in Fig. 1C, while quantitative measurement of vcMS following invasion was performed as described above (Fig. 1D). Using this assay, the adhesion to and/or invasion of epithelial cells by the different types of vcMS was quantitatively evaluated. Type II fimbria vcMS significantly adhered to and invaded the cells at 6 h after incubation compared to the results seen with other types (Fig. 2A and B). Further, following prolonged incubation for 12 and 24 h, vcMS of types I and III invaded at levels similar to those seen with type II vcMS, with the vcMS of types Ib, IV, and V eventually showing invasion at 56% to 80% of the type II vcMS level (Fig. 2B). Although gingipains and their adhesion domains were suggested to be involved in the adhesion and invasion (5, 6), the fimA-deficient mutants showed negligible adhesion and invasion, which was similar to the result seen with the gingipain-deficient mutant [{Delta}rgpAB{Delta}kgp (I)] that lacked fimbriae on the surface (12; Koji Nakayama, personal communication). In addition, no positive correlation of gingipain activities to invasive efficiency was found (Fig. 3). P. gingivalis reportedly suppressed cellular apoptosis and maintained the viability of infected cells (18, 29, 31), while its gingipains were shown to induce cellular apoptosis and death (5, 6, 22). In the present study, approximately 30% of the cells had detached from the plates and lost their viability after 6 h (Fig. 4A). However, the number of floating cells did not increase with prolonged incubation, suggesting that vcMS suppressed further apoptotic changes. As shown in Fig. 4B and C, apoptotic changes of the attached cells were most frequently observed after 6 h, though the ratio was lower than 8% of all cells. Apoptosis was more frequently observed in association with vcMS invasion, especially with type II vcMS invasion, compared to noninvaded cell results. Further, the number of apoptotic cells gradually decreased as incubation time increased, even among cells invaded by type II-vcMS. The gingipain-deficient mutant caused very little cell death and apoptosis, suggesting that gingipains play a major role in cellular death and apoptosis.



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  		FIG. 1. Intracellular localization of type II fimbria vcMS. (A) Western blotting of fimbriae on vcMS. Lanes: 1, strain ATCC 33277 (I); 2, HG1691 (Ib); 3, OMZ314 (II); 4, 6/26 (III); 5, {Delta}fimA (I); 6, {Delta}fimA (II); 7, HG564 (IV); 8, HNA99 (V). (B) Presence of type II fimbriae on vcMS. Fimbriae on the surfaces of vcMS were visualized with antifimbria antibodies by use of a confocal microscope. Bar, 1 µm. (C) Epithelial cells were incubated with type II fimbria vcMS for 6 h. Images (red, type II vcMS; green, actin) were collected and analyzed with a confocal microscope. (D) The number of the cells invaded by type II vcMS increased with incubation time. To analyze the distribution of the vcMS-invaded cells, 60 optical sections were obtained along the z axis at 0.15-µm intervals, and images of the x-z and y-z planes were reconstructed with LSM510 software. Magnification, x63. Bar, 20 µm.

 


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  		FIG. 2. Quantitative analysis of adhesion to and/or invasion of epithelial cells by vcMS. (A) Confocal microscopic images of invaded vcMS (red) at 6 h after incubation. BSA-conjugated MS was used as a control. Magnification, x63. Bar, 20 µm. (B) Time course of adhesion to and invasion of epithelial cells by vcMS. The fimbria type of the strains used for vesicle preparation is shown in parentheses. •, adhered and invaded vcMS; {circ}, invaded vcMS; {square}, BSA-coated MS as a control. *, significant difference (P < 0.01) compared to the results seen with others at the same time point. Phenotypes of these strains including the mutants were found to be identical as follows: hemagglutination (+), proteolytic activity (+), catalase (–), indole production (–), urease (–), glucose (–), D-mannitol (–), lactose (–), sucrose (–), maltose (–), salicin (–), D-xylose (–), L-arabinose (–), ß-glucosidase (–), glycerin (–), D-cellobiose (–), D-mannose (–), D-melezitose (–), D-raffinose (–), D-sorbitol (–), L-rhamnose (–), and D-trehalose (–).

 


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  		FIG. 3. Correlation between vcMS invasive efficiency and gingipain activities. The correlation between gingipain activities (Arg-specific gingipain; Rgp- and Lys-specific gingipain; Kgp) and the area invaded by vcMS at 6 h was analyzed. No positive associations were observed.

 


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  		FIG. 4. Cell death-apoptosis induced by vcMS. (A) The ratios of epithelial cells attached to the culture plates during the experimental period are shown. The fimbria type of the strains used for vesicle preparation is shown in parentheses. Floating cell viability was found to be nearly lost. *, significant difference (P < 0.01) compared to vcMS of other fimbria types at the same time point. (B) Cellular apoptosis was detected by a TUNEL assay and visualized with green fluorescence at 6 h after starting incubation. Magnification, x63. Bar, 20 µm. (C) Time course of cellular apoptosis associated with invasion and noninvasion of vcMS. Ten fields were analyzed in each of the experiments, which were repeated 10 times. Data represent the ratios (%) of apoptotic cells to total adhered cells, vcMS-invaded cells, and noninvaded cells. *, significant difference (P < 0.01) compared to control (BSA) results at the same time point.

 
Vesicles of gram-negative bacteria retain a full complement of outer membrane constituents of the cell surface, including proteins, LPS, muramic acid, capsule, and fimbriae (32). The use of vcMS allowed us to examine a homogenous artificial intruder without bacterial multiplication for 24 h of incubation, and we were also able to distinguish invaded and noninvaded cells. Type II vcMS adhered quickly to the cells and then showed an invasion capability after 6 h greater than that seen with the other types. However, with prolonged incubation, some of the other types were able to adhere to and invade the cells to an extent similar to that seen with type II vcMS, suggesting that once P. gingivalis adheres to the cell surface, nearly all types can subsequently invade epithelial cells. In the present assay, vcMS interacted with the epithelial cells for up to 24 h, which likely permitted the delayed invasion by the other types. Thus, type II fimbriae are likely to contribute to an efficient invasion of gingival epithelial cells by P. gingivalis. Since cell death and apoptosis were suppressed with prolonged incubation, we speculated that P. gingivalis invades epithelial cells to acquire intracellular persistence and does not cause cell death. However, the invasion by type II vcMS caused greater apoptotic changes compared to the results seen with other types, which was considered to be due to a quick invasion prior to cellular antiapoptotic functions initiated by P. gingivalis components. Thus, the clonal variations of fimbriae may be related to bacterial cytotoxicity, though this issue requires additional study.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Oral Frontier Biology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita-Osaka 565-0871, Japan. Phone: 81-6-6879-2976. Fax: 81-6-6876-8455. E-mail: amanoa{at}dent.osaka-u.ac.jp. Back

Editor: J. D. Clements


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Infection and Immunity, January 2006, p. 734-739, Vol. 74, No. 1
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.1.734-739.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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