INTRODUCTION

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Porphyromonas gingivalis is a gram-negative, anaerobic rod that is considered to be among the major pathogens associated with adult periodontitis (64). A possible mechanism of pathogenesis may be cellular invasion. P. gingivalis has been demonstrated to be internalized within gingival epithelial cells in vitro (19, 34, 53) and buccal epithelial cells in vivo (51). Recent epidemiological studies have demonstrated a strong relationship between periodontal disease and coronary heart disease (2, 3, 17, 38, 39). Oral bacteria have a direct route to the circulatory system in periodontitis patients due to transient bacteremias produced by flossing, mastication, and toothbrushing (11, 57, 62). P. gingivalis localizes to atherosclerotic plaques (12, 30) and is capable of invasion of coronary artery cells in vitro (16, 18). Therefore, invasion and intracellular parasitism of endothelial cells by P. gingivalis in vivo may exacerbate the inflammatory response of atherosclerosis.

Invasion of nonphagocytic cells is a common strategy of evading the immune system for many pathogens (23). Once within the cell, these pathogens have developed various mechanisms for survival (28, 40). Legionella pneumophila and virulent Brucella abortus gain access to and replicate in vacuoles that resemble autophagosomes and are associated with endoplasmic reticulum proteins (46, 47). Autophagosomes, multimembranous vacuoles formed from invaginations of ribosome-free regions of the rough endoplasmic reticulum (RER) (20), are the organelles of the autophagic process. Autophagy is a process whereby cytosol and organelles are sequestered for lysosome degradation in response to nutrient deprivation (20). Under normal conditions, the autophagosome matures into an autolysosome, where the contents are degraded. The autophagosome-like vacuoles containing these bacterial species do not acquire lysosomal hydrolases (46, 66). Bacterial trafficking to the autophagic pathway has been proposed to be a mechanism of increasing the concentration of free amino acids to be utilized by the bacteria for their biochemical pathways and/or to inhibit host cell protein synthesis in order to reduce the cellular response to the pathogen (63).

In a previous study, we demonstrated that the vacuoles containing P. gingivalis in human coronary artery endothelial (HCAE) cells morphologically resembled autophagosomes, similar to the vacuoles containing L. pneumophila in macrophages and virulent B. abortus in HeLa cells (18). The first goal of this study was to characterize and delineate the trafficking of P. gingivalis within endothelial cells using strain 381. The second goal was to determine whether the autophagosome-like vacuole was the intracellular niche for P. gingivalis in the HCAE cell or whether the HCAE cell utilized the autophagic pathway to rid itself of this intracellular intruder.

	MATERIALS AND METHODS

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Bacterial and cell culture conditions. P. gingivalis strain 381 was subcultured on tryptic soy agar (Difco Laboratories, Detroit, Mich.) supplemented with 5.0% sheep blood (Lampire Biological Laboratories, Pipersville, Pa.), 0.5% yeast extract (Difco), hemin (5 µg/ml), and vitamin K (5 µg/ml). Liquid cultures were grown in brain heart infusion broth (Difco) supplemented with 0.5% yeast extract, 0.1% cysteine (Sigma), hemin (5 µg/ml), and vitamin K (5 µg/ml) under anaerobic conditions. These strains were grown at 37°C in a Coy (Ann Arbor, Mich.) anaerobic chamber with an atmosphere of 5% CO₂, 10% H₂, and 85% N₂. Escherichia coli MC1061was subcultured at 37°C aerobically on Luria-Bertani (LB) plates consisting of Bacto Agar (15 g/liter; Difco), Bacto Tryptone (10 g/liter; Difco), yeast extract (5 g/liter), and sodium chloride (10 g/liter; Fisher Scientific, Springfield, N.J.) and was also grown in LB broth media. The HCAE cells are a primary cell culture line purchased from Clonetics Inc. (San Diego, Calif.), cryopreserved on third passage, and were passaged an additional two or three times before use. The HCAE cells were maintained in endothelial growth medium-2 (EGM-2), which consisted of endothelial basal medium-2 supplemented with fetal bovine serum, hydrocortisone, human recombinant fibroblast growth factor, vascular endothelial growth factor, recombinant insulin growth factor-1, ascorbic acid, human recombinant epidermal growth factor, gentamicin, and amphotericin (Clonetics).

Antibodies. Rabbit polyclonal anti-BiP (provided by S. Frost); rabbit polyclonal anti-cathepsin L (C. Gabel); rabbit polyclonal anti-HsGSA7p (human-specific Gsa7p); 61BG1.3, a mouse monoclonal anti-P. gingivalis hemagglutinin A (R. Gmür) (7); rabbit polyclonal antilysosomal glycoprotein 120 (LGP120) (22); rabbit polyclonal anti-mannose 6-phosphate receptor (MPR) (P. Nissley); rabbit polyclonal anti-Rab5 (Stressgen Biotechnologies Corp., Vancouver, British Columbia, Canada); rabbit polyclonal anti-Rap1 (Stressgen); and rabbit polyclonal anti-2,6 Gal 1,4-GlcNAc sialyltransferase (G. Hart) antibodies were used in this study. Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse antibody (Sigma Chemical Co., St. Louis, Mo.) and tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit antibody (Sigma) were used as secondary antibodies.

Persistence assay. The numbers of intracellular bacteria at various times and in the absence or presence of autophagy inhibitors were quantitated. Approximately 10⁵ HCAE cells per well in a 24-well tissue culture plate were washed three times with phosphate-buffered saline (PBS). For inhibition of autophagy, both 10 mM 3-methyladenine (Sigma) and 10 nM wortmannin (Sigma) in EGM-2 were preincubated with HCAE cells for 60 min prior to the start of the invasion assay. For wells not receiving the autophagy inhibitors, media were replaced with fresh antibiotic-free EGM-2. Following preincubation, broth-grown bacteria were centrifuged at low speed and resuspended in antibiotic-free medium to a concentration of 10⁷ cells/ml as determined spectrophotometrically (Shimadzu UV-1201; VWR, Marietta, Ga.). Then 1.0 ml of the bacterial suspension was added to each well and the HCAE cells plus bacteria were incubated at 37°C in 5% CO₂ for 90 min. The autophagy inhibitors were present during the time of infection in the appropriate wells. The media were removed from infected cells after 90 min, and the cells were washed three times with PBS. Medium containing gentamicin (300 µg/ml) and metronidazole (200 µg/ml) was then added to each well to kill any extracellular bacteria, and the plates were incubated for an additional 60 min in 5% CO₂ at 37°C. The autophagy inhibitors were also present during the antibiotic incubation in the appropriate wells. The media were removed, and the cells were washed three times with PBS. The HCAE cells from one set of wells were then lysed by a 20-min incubation with 1.0 ml of sterile distilled water at 37°C. This was the 2.5-h time point. For the wells to be assessed at 4, 6, and 8 h postinfection, the media were replaced with antibiotic-free EGM-2 without autophagy inhibitors and further incubated at 37°C in 5% CO₂. At the appropriate time points, the HCAE cells were lysed by the aforementioned protocol. Dilutions of the lysates of cells infected with P. gingivalis were plated in triplicate on tryptic soy agar (Difco) plates supplemented with 5.0% sheep blood, 0.5% yeast extract, hemin (5 µg/ml), and vitamin K (5 µg/ml) and were cultured anaerobically. The dilutions of the lysates of E. coli MC1061, the negative control, were cultured on LB plates at 37°C aerobically. The CFU of invasive bacteria were then enumerated. Each condition was performed in duplicate, and each assay was performed three times independently. All of the inhibitors were also tested at the appropriate concentration for adverse effects on the viability of the bacteria by plate count. The viability of the HCAE cells were tested by trypan blue exclusion and by examining the confluency of the monolayer.

Transmission electron microscopy. The bacteria were incubated with the HCAE cells for 30, 60, and 90 min and were subsequently fixed in 2% glutaraldehyde in PBS at room temperature for 1 h. For inhibition studies, the HCAE cells were preincubated with 10 nM wortmannin and 10 mM 3-methyladenine in EGM-2 for 1 h, and the wortmannin was also present during the infection. After the cells were centrifuged and the pellet was washed with PBS (pH 7.3), 3 drops of 3% low-gelling-point agarose was added, and the pellet was solidified at 4°C for 10 min. The agarose-embedded pellet was then washed twice for 10 min in PBS, postfixed in 1% osmium tetroxide for 1 h, and washed three times in distilled H₂O for 10 min. The specimens were then dehydrated in a graded series of ethanol and stained overnight en bloc in 2% uranyl acetate. Following the last ethanol treatment, the specimens were infiltrated and embedded in EM Bed-812 (Electron Microscopy Sciences, Fort Washington, Pa.). Thin sections were cut, poststained with uranyl acetate and lead citrate, and examined on a Hitachi 7000 transmission electron microscope.

Immunofluorescence microscopy. HCAE cells were grown on glass coverslips in a six-well tissue culture plate. The HCAE cells were then washed three times with PBS prior to infection with P. gingivalis 381 at different time points at a multiplicity of infection of approximately 100. The media were removed, and the HCAE cells were washed vigorously three times with PBS. Wortmannin (10 nM) was added 1 h before infection and was present during infection. The infected HCAE cells were then fixed in 4% paraformaldehyde in PBS for 30 min at room temperature. This was followed by washing twice in PBS and quenching in 50 mM NH₄Cl-0.3% Tween 20-PBS for 10 min at room temperature. After quenching, the HCAE cells were washed two times in PBS. The primary antibodies, 1/50 dilution in PBS-5% normal goat serum-0.3% Tween 20, were applied for 2 h at room temperature. The HCAE cells were then washed four times in PBS for 5 min each wash. In all cases, the bacteria were detected and organelle and host protein markers were detected with FITC-conjugated goat anti-mouse antibody and TRITC-conjugated goat anti-rabbit antibody, respectively, as the secondary antibodies. The secondary antibodies (1/200 dilution in PBS-5% normal goat serum-0.3% Tween 20) were applied for 1 h at room temperature. The HCAE cells were then washed twice with PBS before mounting with Fluoromount-G (Southern Biotechnology Associates, Inc., Birmingham, Ala.) onto glass microscope slides.

Statistical analysis. Student's t test (Microsoft Excel; Microsoft, Redmond, Wash.) was used to compare differences in colocalization data between untreated and wortmannin-treated time points. Due to similar comparisons over multiple time points, Bonferroni's correction was utilized, and statistical significance was achieved when P was <0.01 (0.05/5). The percentages of colocalization from each individual field of view were compared for this analysis.

	RESULTS

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Morphological analysis of vacuoles containing P. gingivalis. Internalized P. gingivalis was found within structures resembling autophagosomes at 90 min postinfection (Fig. 1A). These vacuoles were bound by one or two membranes and contained undegraded cytoplasm and vesicles of cytoplasmic origin (Fig. 1C and D). The presence of cytoplasmic components in these vacuoles suggests that the vacuoles arose via autophagic events. Profiles of RER were routinely observed in close association with these vacuoles. The morphological data suggest that the internalized P. gingivalis 381 cells reside in autophagosomes, which have been derived from the RER. We next examined the intracellular fate of P. gingivalis when 3-methyladenine or wortmannin suppresses autophagy. Internalized P. gingivalis was observed within vacuoles in wortmannin-treated HCAE cells at 30 min postinfection (Fig. 1B). However, these vacuoles were morphologically distinct from autophagosomes. They were bounded by only a single membrane and lacked undegraded cytoplasmic ground substance. These inhibitors appeared to have no effect on bacterial internalization. At later time points only what appeared to be bacterial fragments were observed in HCAE cells preincubated with 3-methyladenine and wortmannin.

View larger version (162K): [in this window] [in a new window]   FIG. 1.   Internalized P. gingivalis in HCAE cells. HCAE cells were infected with P. gingivalis in the presence (A, C, and D) and absence (B) of wortmannin. After 90 min of infection, P. gingivalis could be observed in vacuoles within the cytoplasm of HCAE cells (A). These vacuoles were bound by one or two membranes (arrows) and contained undegraded vesicles and cytoplasmic ground substance (C and D). After 30 min of infection of wortmannin-treated HCAE cells, P. gingivalis appears to be in the process of degradation and is within vacuoles that resemble lysosomes (B).

Trafficking through the autophagic pathway. Autophagy is responsible for the sequestration and delivery of cytoplasmic proteins and organelles to the autolysosome, where they are degraded. Our data suggest that internalized P. gingivalis enters the autophagic pathway. Therefore, we utilized protein markers of autophagy to investigate the intracellular trafficking of P. gingivalis in HCAE cells. We found that P. gingivalis resided in a vacuole that contained HsGsa7p but lacked cathepsin L (Fig. 2A and C). We next examined the trafficking of P. gingivalis in HCAE cells treated with 10 nM wortmannin, an inhibitor of autophagy. Under these conditions, the bacterial vacuole contained cathepsin L (Fig. 2D) but not HsGsa7p (Fig. 2B). We also observed that the cellular distribution of HsGsa7p differed in the absence and presence of wortmannin (Fig. 2). HsGsa7p resided in a Golgi-like vacuole when autophagy was suppressed by wortmannin (Fig 2B). During bacterial invasion in the absence of wortmannin, the HsGsa7p was found in small unknown organelles distributed throughout the cell. We believe that this alteration in the cellular distribution of HsGsa7p reflects the onset of autophagy. Indeed, we found HsGsa7p colocalized with Golgi markers in fed HuH7 cells when autophagy is suppressed but distributed to round organelles in starved HuH7 cells when autophagy is enhanced (data not shown).

View larger version (16K): [in this window] [in a new window]   FIG. 2.   Localization of HsGsa7p and cathepsin L to vacuoles containing P. gingivalis in HCAE cells using deconvolution microscopy. (A) P. gingivalis colocalized with HsGsa7p (arrow). (B) In the presence of wortmannin, P. gingivalis did not colocalize with HsGsa7p (arrowhead). HsGsa7p resided in a Golgi-like vacuole in the wortmannin-treated HCAE cells (arrowhead). (C) P. gingivalis did not colocalize with cathepsin L (arrowhead). (D) P. gingivalis colocalized with cathepsin L (arrow) in the presence of wortmannin. Abbreviations: Gsa7p, HsGsa7p; W, wortmannin; CathL, cathepsin L; Pg, P. gingivalis; N, nucleus. Bar, 15 µm.

View larger version (16K): [in this window] [in a new window]   FIG. 3.   Localization of BiP, LGP120, Rab5, and MPR to vacuoles containing P. gingivalis in HCAE cells. At 90 min postinfection, the P. gingivalis (Pg) vacuoles contained BiP (A) and LGP120 (B) (arrows). (C) At 35 min after infection, P. gingivalis colocalized with Rab5 (arrow). (D) MPR was absent from a majority of the vacuoles observed up to 2 h postinfection (arrowheads indicate colocalization). Bar, 15 µm.

View larger version (35K): [in this window] [in a new window]   FIG. 4.   Colocalization of P. gingivalis with protein markers of the autophagic pathway. HCAE cells were incubated with P. gingivalis 381 in the absence (solid bars) and presence (open bars) of 10 nM wortmannin for 15 to 120 min. The data represent the percentage of P. gingivalis vacuoles that contained BiP (A), HsGsa7p (B), LGP120 (C), and cathepsin L (D), expressed as the mean + the standard deviation (error bars). The data suggest that the P. gingivalis vacuole first acquires HsGsa7p and then BiP and LGP120; however, the vacuole fails to acquire cathepsin L. In the presence of wortmannin, the vacuole does not acquire HsGsa7p or BiP but rather acquires cathepsin L rapidly. The effects of wortmannin at each time point were evaluated by Student's t test utilizing Bonferroni's correction. Statistical significance (*) was achieved when P was <0.01.

Trafficking along the endocytic pathway. We have demonstrated that P. gingivalis is internalized and sequestered into a late autophagosome containing BiP and LGP120 but lacking cathepsin L. P. gingivalis must either promote its entry into the autophagic pathway or block its entry into the endocytic pathway, thereby channeling itself into the autophagic pathway. When autophagy was inhibited by wortmannin, the bacteria were found in a cathepsin L-positive, lysosome-like vacuole. Hence, we propose that P. gingivalis does not block the endocytic pathway but rather promotes its entry into the autophagosome. Therefore, we suggest that bacteria are trafficked through the endocytic pathway to lysosomes during autophagy inhibition. To test this hypothesis, we traced the trafficking of P. gingivalis through the endocytic pathway in the presence and absence of wortmannin using antibody markers to endosomal vacuoles.

View larger version (68K): [in this window] [in a new window]   FIG. 5.   Colocalization of P. gingivalis with protein markers of the endocytic pathway. HCAE cells were incubated with P. gingivalis 381 in the absence (solid bars) and presence (open bars) of 10 nM wortmannin for 15 to 120 min. The data represent the percentage of P. gingivalis vacuoles that contained Rab5 (A) and MPR (B), expressed as the mean + the standard deviation (error bars). The data suggest that the P. gingivalis vacuole acquires Rab5 early but does not acquire the late endocytic marker MPR. In the presence of wortmannin, a higher percentage of P. gingivalis vacuoles acquire MPR. The effects of wortmannin at each time point were evaluated by Student's t test utilizing Bonferroni's correction. Statistical significance (*) was achieved when P was <0.01.

Bacterial persistence Since autophagy is a degradative pathway, endothelial cells could use this pathway as a defensive mechanism against invading bacterial aggregates. However, if the autophagosome is the intracellular niche of P. gingivalis (at least initially), then inhibition of autophagy should result in a decrease in the number of bacteria able to survive. A modification of the antibiotic protection assay was used to test the effects of the autophagy inhibitors 3-methyladenine and wortmannin on the persistence of P. gingivalis within HCAE cells.

View larger version (77K): [in this window] [in a new window]   FIG. 6.   Persistence of P. gingivalis within HCAE cells over 8 h. HCAE cells were preincubated for 1 h at 37°C with either fresh antibiotic-free EGM-2 (solid bars), 10 mM 3-methyladenine (open bars), or 10 nM wortmannin (checkered bars) (n = 3). P. gingivalis 381 cells were incubated with EGM-2 with or without the appropriate inhibitor. The number of CFU of HCAE cells infected with P. gingivalis in the absence of autophagy inhibitors grew over the 8 h, whereas the number of CFU of HCAE cells infected with P. gingivalis in the presence of either autophagy inhibitor declined over the 8 h. The number of CFU for E. coli MC1061, the negative control, at 2.5 h postinfection was <200 (n = 3).

	DISCUSSION

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P. gingivalis is invasive of a variety of cells in vitro. The intracellular location of P. gingivalis has not been singularly defined within the variety of cells tested for invasion. Deshpande et al. have demonstrated that P. gingivalis resides within uncharacterized vacuoles, which may or may not be autophagosomes, in fetal bovine heart endothelial cells, bovine aortic endothelial cells, and human umbilical vein endothelial cells (16). P. gingivalis was reported free in the cytoplasm in gingival epithelial cells (33). Sandros et al. also found evidence of P. gingivalis free in the cytoplasm and within endosomes in KB cells, human pocket epithelium, and human junctional epithelium (44, 53, 54). The data suggest that the intracellular niche of P. gingivalis may be host cell specific. We have examined the trafficking of P. gingivalis in primary HCAE cells to better understand the proposed relationship between periodontal disease and coronary heart disease.

P. gingivalis traffics to the late autophagosome. The data presented here are consistent with P. gingivalis being targeted to autophagosomes shortly after invasion of HCAE cells. We have shown that P. gingivalis cells are internalized into Rab5-positive vacuoles that rapidly acquire HsGsa7p. This likely represents the first step in the autophagic sequestration of P. gingivalis. The bacteria eventually traffic to a vacuole that contains both the RER protein BiP and the lysosomal protein LGP120 but is devoid of cathepsin L. This vacuole has been previously defined as a late autophagosome that contains RER and lysosomal membrane proteins but lacks hydrolytic enzymes (22). Then the late autophagosome containing P. gingivalis does not appear to acquire cathepsin L and thereby become an autolysosome, at least during the 2-h time course. The data suggest that P. gingivalis is sequestered into an early autophagosome which then matures into a late autophagosome, where the bacteria presumably replicate (Fig. 7A). These observations are substantiated by our ultrastructural observations. We observed the bacteria in a double-membrane-bound vacuole containing undegraded ribosomes and cytoplasmic ground substance. Profiles of dividing P. gingivalis can be observed in these vacuoles.

View larger version (21K): [in this window] [in a new window]   FIG. 7.   Model of P. gingivalis trafficking in HCAE cells. P. gingivalis is initially found within an early endosome after internalization. (A) The bacteria promote their own entry into the autophagic pathway. The P. gingivalis 381-containing vacuole acquires BiP and later LGP120. However, this vacuole does not acquire cathepsin L. (B) Upon inhibition of autophagy with wortmannin, P. gingivalis enters the endocytic pathway. The vacuole matures into a late endosome and then a lysosome, as characterized by the presence of Rap1 and cathepsin L.

Comparison to L. pneumophila and virulent B. abortus. Studies of B. abortus and L. pneumophila trafficking demonstrate several similarities to P. gingivalis intracellular trafficking. All three bacterial species reside in vacuoles bound by multiple membranes surrounded by ribosomes (47, 66). The data are consistent with all three of these microorganisms evading the endocytic pathway and entering the autophagic pathway. Additionally, both virulent B. abortus- and L. pneumophila-containing vacuoles do not acquire Rab7, which is characteristic of late endosomes (46, 50). Both P. gingivalis and B. abortus are present in early endosome-like vacuoles as assessed by Rab5 and EEA1, respectively (46). There does exist a rapid convergence between autophagosomes and early endosomes (37, 69). However, L. pneumophila completely avoids the endocytic system and does not colocalize with Rab5 (13). Additionally, virulent B. abortus does not colocalize with MPR (46). Each of the three wild-type, bacterial species also fails to traffic to vesicles that fuse with lysosomal hydrolases, but vesicles containing attenuated B. abortus or growth mutants of L. pneumophila fuse with lysosomes (47, 72).

Bacterial survival. The specific genes of P. gingivalis required for its intracellular survival have not yet been identified. However, studies have indicated specific mechanisms by which the other two species survive intracellularly. For example, the functional secretion systems Icm-Dot and VirB in L. pneumophila and B. abortus, respectively, are necessary for their intracellular replication (8, 61). When the replication vacuole and host cell are depleted of amino acids, L. pneumophila converts to a virulent phenotype (29). The virulence traits, e.g., motility and cytotoxicity, allow the bacteria to escape the host cell and infect adjacent cells to start this cycle anew. Similar to P. gingivalis, L. pneumophila uses short peptides and amino acids as its carbon and energy source (24). We expect that P. gingivalis has evolved similar mechanisms to survive, which we are just beginning to elucidate.

Regulation of autophagy. Our data suggest that P. gingivalis promotes its entry into the autophagosome. At this time, we are uncertain whether this is directly mediated by the bacterium or a consequence of cellular signals transduced during invasion. A functional secretion system in both L. pneumophila and B. abortus is necessary for the establishment of an intracellular niche divergent from the endocytic pathway (14, 15). Once established within this replication vacuole, the Icm-Dot secretion system is no longer expressed (9). This suggests that a secreted protein interacts with the host cell to induce autophagy and is essential for the biogenesis of the replication vacuole. However, the precise mechanism for the sequestration of the bacterium into the autophagosome-like vacuole is unknown.