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The Journal of Immunology, 2007, 179, 2349-2358
Copyright © 2007 by The American Association of Immunologists, Inc.
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Fimbrial Proteins of Porphyromonas gingivalis Mediate In Vivo Virulence and Exploit TLR2 and Complement Receptor 3 to Persist in Macrophages1

Min Wang*, Muhamad-Ali K. Shakhatreh*, Deanna James*, Shuang Liang*, So-ichiro Nishiyama{dagger}, Fuminobu Yoshimura{dagger}, Donald R. Demuth*,{ddagger} and George Hajishengallis2,*,{ddagger}

* Department of Periodontics/Oral Health and Systemic Disease, University of Louisville Health Sciences Center, Louisville, KY 40292; {dagger} Department of Microbiology, School of Dentistry, Aichi-Gakuin University, Nagoya, Japan; and {ddagger} Department of Microbiology and Immunology, University of Louisville Health Sciences Center, Louisville, KY 40292


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Porphyromonas gingivalis is an oral/systemic pathogen implicated in chronic conditions, although the mechanism(s) whereby it resists immune defenses and persists in the host is poorly understood. The virulence of this pathogen partially depends upon expression of fimbriae comprising polymerized fimbrillin (FimA) associated with quantitatively minor proteins (FimCDE). In this study, we show that isogenic mutants lacking FimCDE are dramatically less persistent and virulent in a mouse periodontitis model and express shorter fimbriae than the wild type. Strikingly, native fimbriae allowed P. gingivalis to exploit the TLR2/complement receptor 3 pathway for intracellular entry, inhibition of IL-12p70, and persistence in macrophages. This virulence mechanism also required FimCDE; indeed, mutant strains exhibited significantly reduced ability to inhibit IL-12p70, invade, and persist intracellularly, attributable to failure to interact with complement receptor 3, although not with TLR2. These results highlight a hitherto unknown mechanism of immune evasion by P. gingivalis that is surprisingly dependent upon minor constituents of its fimbriae, and support the concept that pathogens evolved to manipulate innate immunity for promoting adaptive fitness and thus their capacity to cause disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Periodontitis is an infection-driven chronic inflammatory disease characterized by complex host-microbe interactions that lead to resorption of tooth-supporting alveolar bone (1). Porphyromonas gingivalis is an obligate anaerobic pathogen that is strongly associated with periodontitis (2, 3) and has also been implicated as a contributory factor in atherosclerosis (4, 5, 6). The virulence of P. gingivalis has been attributed to a variety of factors, including hemagglutinins, cysteine proteinases, and fimbriae (7). P. gingivalis fimbriae are adhesive filamentous appendages that are required for virulence in rodent models of periodontitis or atherosclerosis (5, 8). In humans, fimbriated P. gingivalis is readily detected in periodontal pockets by immunogold labeling with specific anti-fimbrial Ab (9) and is more frequently found in deep periodontal pockets or in sites with severe periodontal attachment loss than nonfimbriated strains (10). These studies suggest a strong association between the expression of fimbriae and periodontal tissue destruction in humans. Although fimbriae function as a colonization factor (7), accumulating evidence from our laboratory suggests that their virulence role may extend to immunomodulation of the macrophage response to P. gingivalis, through coordinated interactions with pattern-recognition receptors (11, 12, 13, 14, 15).

The major structural component of P. gingivalis fimbriae is FimA, a multifunctional protein that interacts with extracellular matrix, other bacteria, and innate immune cells. Peptide-mapping studies of FimA have defined specific interactive domains involved in these interactions (reviewed in Ref. 16). However, several other P. gingivalis proteins, designated FimC, FimD, and FimE with molecular masses of 50, 80, and 60 kDa, respectively, have been identified as quantitatively minor components (≤1%) of native fimbriae (17, 18). These accessory proteins are encoded by genes that reside immediately downstream of fimA (18, 19, 20) (Fig. 1). Interestingly, inactivation of any of the accessory protein genes (i.e., fimC, fimD, or fimE) results in fimbriae that are devoid of all accessory proteins (henceforth referred to as DAP3 fimbriae) (18). For example, insertional inactivation of fimC abrogates expression of FimCDE due to concomitant polar effects on the downstream genes (fimD and fimE) of the operon. Moreover, although inactivation of fimE does not prevent the expression of fimC or fimD, their products are released into the culture medium and are not associated with fimbriae (18). This suggests that FimCDE may form a functional tripartite complex that associates with fimbriae.


Figure 1
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  		FIGURE 1. The P. gingivalis fimbrial gene cluster and strains used in this study. The P. gingivalis 33277 fimbrial gene cluster contains fimA, encoding the main fimbrillin subunit, and fimCDE encoding accessory proteins associated with fimbriae (the role of open reading frame 1 (ORF1) is unknown; however, the ORF1 product is not associated with fimbriae, and a mutant lacking ORF1 expresses wild-type fimbriae) (18 19 ). The direction of transcription for each ORF is shown with the TIGR designation (www.tigr.org) below the ORF. Strains OZ5001C and KO4 lacking fimC and fimE, respectively, express DAP, whereas lack of fimA abrogates expression of both FimA and the accessory proteins, resulting in a nonfimbriated state (18 ).

 
The biological significance of the fimbrial accessory proteins and their contributions to the function of fimbriae, in terms of their adhesive or immunomodulatory properties, has remained elusive. Only recently have functional distinctions been made between wild-type and DAP fimbriae. Specifically, we found that native fimbriae use either TLR1 or TLR6 for inducing cooperative TLR2 signaling, whereas DAP fimbriae display an absolute preference for TLR1 as a TLR2 signaling partner (12). This suggests that the innate immune system can differentially detect fimbriae that lack the accessory proteins. Furthermore, DAP fimbriae do not retain the ability to interact with Streptococcus oralis GAPDH or with fibronectin and type I collagen (18). This suggests that the fimbrial accessory proteins may contribute to colonization of the oral cavity by facilitating interactions of P. gingivalis with members of the dental biofilm (e.g., S. oralis) (7) or with extracellular matrix proteins.

In this study, we show for the first time that the fimbrial accessory proteins of P. gingivalis are essential for the assembly of mature fimbriae and for P. gingivalis virulence in experimental periodontitis. At the mechanistic level, we demonstrate that native fimbriae containing accessory proteins mediate P. gingivalis entry into macrophages in a way that promotes its intracellular persistence. This intriguing virulence property is attributable to exploitation of complement receptor 3 (CR3; CD11b/CD18) (3), and is not shared by isogenic mutants expressing DAP fimbriae. Whereas CR3 is hijacked by wild-type P. gingivalis for intracellular entry and inhibition of IL-12p70 production, TLR2 inside-out signaling is required for activating CR3 binding of native fimbriae. This study therefore establishes a critical role for the fimbrial accessory proteins in P. gingivalis virulence. The findings moreover suggest that exploitation of the host innate response mediated by fimbrial interactions with TLR2 and CR3 may contribute to virulence by facilitating entry and intracellular persistence of the organism in macrophages.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Bacteria

P. gingivalis 33277 and its isogenic mutants, OZ5001C, KO4, and JI-1 (Fig. 1), were grown anaerobically at 37°C in modified GAM medium (contains 5 µg/ml hemin and 1 µg/ml menadione) (Nissui Pharmaceutical). Fimbriae were purified from these strains, as previously described (21). The fimbrial preparations were free of any contaminating substances, as assayed using silver-stained SDS-PAGE, and tested negative for endotoxin (<6 EU/mg protein), according to quantitative Limulus amebocyte lysate assay (BioWhittaker). Aggregatibacter (Actinobacillus) actinomycetemcomitans LPS was provided by E. Haase (University of Buffalo, Buffalo, NY). Polyclonal Ab to P. gingivalis 33277 fimbriae was raised in rabbits, as previously described (21).

Cell culture

Thioglycolate-elicited macrophages were isolated from the peritoneal cavity of mice deficient in CR3 (CD11b–/–), TLR2 (TLR2–/–), or TLR4 (C3H/HeJ), or from wild-type controls (C57BL/6 or C3H/HeOuJ) (The Jackson Laboratory), as previously described (22). The animal procedures were conducted in compliance with established federal guidelines and institutional policies. Primary mouse macrophages as well as the macrophage cell line J774A.1 (American Type Culture Collection TIB-67) and human monocytic THP-1 cells (American Type Culture Collection TIB-202) were cultured at 37°C and 5% CO2 atmosphere, in RPMI 1640 (Invitrogen Life Technologies) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 10 mM HEPES, 100 U/ml penicillin G, 100 µg/ml streptomycin, and 0.05 mM 2-ME. THP-1 cells were differentiated with 10 ng/ml PMA for 3 days. Cell viability was monitored using the CellTiter-Blue assay kit (Promega). None of the experimental treatments affected cell viability compared with medium-only control treatments.

Fimbria-binding assay

The binding of purified fimbriae to macrophages was performed using a fluorescent cell-based assay in 96-well plates, as we previously described (15). Briefly, biotinylated fimbriae (1 µg/ml) were allowed to bind for 30 min at 37°C, and bound protein was probed with FITC-labeled streptavidin. Upon washing, binding was determined by measuring cell-associated fluorescence on a microplate fluorescence reader (FL600; Bio-Tek Instruments) with excitation/emission wavelength settings of 485/530 nm.

Flow cytometric adherence and internalization assays

Mouse macrophages were incubated at 37°C with FITC-labeled P. gingivalis at a multiplicity of infection (MOI) of 10:1 for various time points (5–60 min). To determine adherence, the macrophages were pretreated for 30 min with cytochalasin D (5 µg/ml) to block phagocytosis. The cells were washed, fixed with 1% paraformaldehyde, and analyzed by flow cytometry (see below). In macrophages that were not treated with cytochalasin D (to assess P. gingivalis internalization), phagocytosis was stopped at various time points by cooling the incubation tubes on ice. After cell washing to remove nonadherent bacteria, extracellular fluorescence (representing attached, but not internalized bacteria) was quenched with 0.2% trypan blue. The cells were washed again, fixed, and analyzed by flow cytometry (percentage of positive cells for FITC-P. gingivalis and mean fluorescence intensity) using the FACSCalibur and the CellQuest software (BD Biosciences). Control experiments indicated that cytochalasin D-pretreated macrophages incubated with FITC-P. gingivalis and subsequently exposed to trypan blue did not show significant fluorescence, thus confirming that cytochalasin D blocks internalization and that trypan blue quenches extracellular fluorescence. Adherence or internalization indices were calculated using the formula (percentage of positive cells x mean fluorescence intensity (MFI))/100. Adherent or internalized bacteria were visualized by confocal laser-scanning microscopy (Olympus FV500).

Antibiotic protection-based intracellular survival assay

To determine persistence of viable internalized bacteria, we used an antibiotic-protection phagocytosis assay (23). Mouse macrophages (primary or the J774A.1 cell line) or PMA-differentiated human THP-1 cells were incubated at 37°C with P. gingivalis for various time points up to 72 h. Extracellular nonadherent bacteria were removed by washing, whereas extracellular adherent bacteria were killed by addition of gentamicin (300 µg/ml) and metronidazole (200 µg/ml) for 1 h. After washing, internalized bacteria were released by lysis of the mammalian cells in sterile distilled water for 20 min (this treatment does not affect the viability of P. gingivalis). Serial dilutions of the lysates were plated on blood agar supplemented with hemin and menadione and cultured anaerobically for CFU enumeration.

Cell activation assays

Induction of cytokine release in culture supernatants of activated mouse macrophages was measured using ELISA kits (eBioscience). Activation of NF-{kappa}B was assessed using THP-1-Blue cells stably transfected with a NF-{kappa}B-inducible reporter system, following the manufacturer’s protocol (InvivoGen). Mouse rIFN-{gamma}, used to prime cells for IL-12p70 production, was purchased from R&D Systems.

Confocal microscopy

To demonstrate colocalization of P. gingivalis with CR3 or TLR2, mouse macrophages were grown on chamber slides (Lab-Tek) and were exposed to FITC-labeled P. gingivalis for 20 min. Immediately afterward, the cells were fixed with 2% paraformaldehyde and permeabilized with.05% Triton X-100. Cells were then stained with Texas Red-labeled anti-CD11b mAb (3A33; Abcam) or PE-labeled anti-TLR2 mAb (6C2; eBioscience) and mounted with coverslips before imaging on an Olympus FV500 confocal microscope.

Detection of P. gingivalis fimbriae

P. gingivalis cells were negatively stained with phosphotangstic acid (neutral pH), and surface structures were visualized using a Philips CM-10 Transmission Electron Microscope at the Analytical Microscopy Facility of the University of Louisville School of Medicine. The expression of fimbriae was examined by ELISA of P. gingivalis cells coated on microtiter plates using rabbit anti-fimbria Ab, followed by peroxidase-conjugated goat anti-rabbit IgG and addition of tetramethylbenzidine chromogenic substrate. The OD signal at 450 nm was read in a Bio-Tek microplate reader.

Mouse periodontitis model

Specific pathogen-free BALB/cByJ mice (10 wk old; purchased from The Jackson Laboratory) were orally infected with P. gingivalis American Type Culture Collection 33277 or isogenic mutants (OZ5001C, KO4, and JI-1) for induction of periodontal bone loss, according to the Baker model (24). Briefly, upon suppression of the normal oral flora with antibiotics, mice were orally infected five times at 2-day intervals with 109 CFU P. gingivalis suspended in 2% carboxymethylcellulose/PBS. Sham-infected controls received 2% carboxymethylcellulose/PBS alone. Six weeks after the final infection, mice were euthanized with CO2 inhalation. Assessment of periodontal bone loss in defleshed maxillae was performed under a dissecting microscope (x40) fitted with a video image marker measurement system (model VIA-170K; Fryer) standardized for measurements in millimeters (mm). Specifically, the distance from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) was measured on 14 predetermined points on the buccal surfaces of the maxillary molars (24). To calculate periodontal bone loss, the 14-site total CEJ-ABC distance for each mouse was subtracted from the mean CEJ-ABC distance from the group of sham-infected mice; results were expressed as mm change in bone, and negative values indicated bone loss compared with sham-infected controls (24). All animal procedures received Institutional Animal Care and Use Committee approval and were in accordance with federal guidelines for the care and use of laboratory animals.

Statistical analysis

Data were evaluated by ANOVA and the Dunnett multiple-comparison test using the InStat program (GraphPad). Where appropriate (comparison of two groups only), two-tailed t tests were also performed. Statistical differences were considered significant at the level of p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Phenotypic characterization of DAP fimbria-expressing P. gingivalis

Fimbriae isolated from strains OZ5001C (fimC-) and KO4 (fimE-) (Fig. 1) consist of FimA, but lack all three accessory proteins (FimCDE), which appear to exist as a tripartite complex in wild-type fimbriae (18). To determine whether the absence of the accessory proteins affects the structure of fimbriae in strains OZ5001C and KO4, we examined these organisms by transmission electron microscopy. Although OZ5001C and KO4 were indistinguishable from each other, both strains expressed relatively short fimbriae compared with the wild-type 33277 strain (Fig. 2A; strain JI-1 served as a nonfimbriated control). Although OZ5001C and KO4 exhibited relatively reduced reactivity with Abs against wild-type fimbriae (Fig. 2B), both strains reacted strongly compared with strain JI-1. In contrast, all wild-type and mutant strains (including JI-1) displayed comparable reactivities with polyclonal Ab against whole cells of P. gingivalis 33277 (Fig. 2C). Given that none of the accessory proteins are associated with fimbriae in OZ5001C and KO4 (18), these results suggest that these strains still express significant levels of FimA, but are incapable of assembling mature fimbriae. We next examined the virulence of strains OZ5001C and KO4 using a mouse model of periodontitis.


Figure 2
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  		FIGURE 2. Electron micrographs of P. gingivalis surface structures and reactivity with anti-fimbria Ab. A, Samples of wild-type P. gingivalis (33277) and isogenic mutants (OZ5001C, KO4, and JI-1) were prepared by negative staining with phosphotangstic acid (neutral pH), and surface structures were visualized by transmission electron microscopy. Strain 33277 expresses long fimbriae, whereas OZ5001C and KO4 express relatively short fimbrial structures. JI-1 is a FimA-deficient (nonfimbriated) mutant used for control purposes. B and C, The indicated P. gingivalis strains were coated on microtiter plates at the bacterial cell numbers shown, and expression of fimbriae was detected by ELISA using specific anti-fimbria polyclonal Ab (B). For control purposes, similarly coated wells were assayed using polyclonal Ab against P. gingivalis 33277 whole cells (C). Data are means ± SD (n = 3).

 
Diminished periodontal virulence of P. gingivalisexpressing DAP fimbriae

In the context of periodontal disease, the virulence of P. gingivalis can be estimated by its ability to induce alveolar bone resorption in animal models (25). Therefore, we compared the virulence capacity of P. gingivalis strains 32277, OZ5001C, KO4, and JI-1 with a validated model of mouse periodontitis that uses bone resorption as the measurable outcome (24). We found that wild-type P. gingivalis induced significantly greater bone loss compared with all mutant strains (p < 0.05), whereas OZ5001C and KO4 did not cause significant bone loss relative to sham infection (Fig. 3). The ability of the FimA-deficient JI-1 strain to cause bone loss was modest and intermediate to the wild-type strain and the OZ5001C and KO4 mutants (Fig. 3). At termination of the experiment, only the wild-type 33277 strain was recoverable by paper point sampling of the murine oral cavity and subsequent anaerobic culturing on blood agar (viable cell counts = 4170 ± 2502 CFU). This indicates that only the wild-type strain is capable of persisting in the mouse host. Together, these results suggest that the loss of fimbrial accessory proteins significantly reduces P. gingivalis virulence and persistence in the mouse oral cavity.


Figure 3
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  		FIGURE 3. Diminished induction of periodontal bone loss by P. gingivalis expressing DAP fimbriae. BALB/cByJ mice were orally infected or not with P. gingivalis 33277 (wild-type fimbriae), OZ5001C (DAP fimbriae), KO4 (DAP fimbriae), or JI-1 (FimA deficient). A, The mm distance from the CEJ to the ABC was measured at 14 predetermined sites in defleshed maxillae and was totaled for each mouse. Data are shown for each mouse, and horizontal lines indicate the mean value. B, The data from A were transformed to indicate bone loss, as outlined in Materials and Methods. Data are shown as means ± SD (n = 5). Asterisks indicate significant (p < 0.05) differences between infected and sham-infected mice. The sign "x" indicates significant difference between 33277 and JI-1.

 
Differential P. gingivalis-macrophage interactions determined by the fimbrial accessory proteins

Macrophages play a central role in the innate host response in periodontitis and other chronic infections (26, 27), and we previously showed that macrophages readily recognize and respond to P. gingivalis fimbriae (11, 12, 14). We thus investigated whether the reduction in virulence exhibited by the accessory protein-deficient strains could be attributed, at least in part, to differential interactions with macrophages.

We initially examined whether the fimbrial accessory proteins play a role in the ability of P. gingivalis to adhere to and become internalized by macrophages. The wild-type strain 33277 displayed significantly higher adherence to macrophages than OZ5001C, which in turn adhered better than the FimA-deficient strain JI-1 (p < 0.05; Fig. 4, A and D). These data suggest that wild-type fimbriae are required for optimal binding of P. gingivalis by macrophages. Consistent with this, the adherence of biotinylated DAP fimbriae was reduced by 30–40% relative to wild-type fimbriae (p < 0.05; Fig. 4B). The use of unlabeled wild-type or DAP fimbriae in 100-fold excess competitively inhibited the binding of the corresponding biotinylated fimbriae (data not shown), confirming that biotinylation did not alter their binding activity. Furthermore, wild-type P. gingivalis and mutants were differentially internalized by macrophages in the same order as seen with adherence, i.e., 33277 > OZ5001C > JI-1 (Fig. 4, C and E). Additional experiments confirmed that strain KO4 behaved similarly to OZ5001C (data not shown). Thus, the expression of FimA alone by P. gingivalis is sufficient for recognition and adherence by macrophages, although mature fimbriae that contain accessory proteins are necessary for maximal adhesion to and internalization by macrophages.


Figure 4
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  		FIGURE 4. Role of wild-type and DAP fimbriae in P. gingivalis adhesive and internalization interactions with macrophages. A, Following 30-min pretreatment with cytochalasin D (5 µg/ml), J774A.1 mouse macrophages were incubated for 30 min with the indicated FITC-labeled P. gingivalis strains, and adherence was determined by flow cytometry. The adherence index was calculated using the formula (percentage of positive cells x MFI)/100. B, Mouse macrophages were incubated for 30 min with the indicated concentrations of biotinylated wild-type or DAP fimbriae (from P. gingivalis OZ5001C or KO4), and binding was measured as cell-associated fluorescence (in relative fluorescence units (RFU)) upon staining with streptavidin-FITC. C, J774A.1 mouse macrophages were incubated for 30 min with the indicated FITC-labeled P. gingivalis strains, and internalization was determined by flow cytometry after quenching extracellular fluorescence. The phagocytic index was calculated using the formula (percentage of positive cells x MFI)/100. Adherent (D) or internalized (E) bacteria were visualized using confocal micrscopy. The third columns represent merge images of fluorescent bacteria (first columns) and macrophages (second columns). Numeric data are presented as means ± SD (n = 3) from one of three independent sets of experiments that yielded similar results. A and C, Asterisks denote significant (p < 0.05) differences between 33277 and both mutant strains, whereas • denote significant (p < 0.05) differences between OZ5001C and JI-1. B, Asterisks indicate statistically significant (p < 0.05) differences in binding between wild-type and DAP fimbriae.

 
We next followed the fate of internalized P. gingivalis in macrophages by monitoring the recovery of viable cells with time after infection using an antibiotic protection-based intracellular survival assay. Surprisingly, wild-type P. gingivalis persisted intracellularly in a viable state for at least 63 h and was recovered in significantly greater numbers (2–3 log10 U) than strains OZ5001C or KO4 at all time points assessed (p < 0.05; Fig. 5A). OZ5001C and KO4 displayed similar intracellular persistence kinetics, consistent with their phenotypic similarities in the previous experiments described above. Both strains were nondetectable at 63 h (Fig. 5A). Interestingly, the isogenic FimA-deficient mutant (JI-1) was recovered at significantly higher CFU numbers than OZ5001C or KO4, although at significantly lower levels relative to wild-type P. gingivalis (p < 0.05; Fig. 5A). Similar differences in persistence (i.e., 33277 > JI-1 > OZ5001C = KO4) were observed using differentiated (macrophage-like) human THP-1 cells and incubations up to 72 h (Fig. 5, B and C). In THP-1 cells, viable OZ5001C and KO4 were not detectable after 24 h (Fig. 5B). Strain JI-1 was not recovered at 72 h, but viable counts were obtained after 24 and 48 h, albeit at lower levels than wild-type P. gingivalis (Fig. 5C). As a control, macrophages were also infected with fimbriated and nonfimbriated A. actinomycetemcomitans, another periodontal pathogen (28). No viable A. actinomycetemcomitans cells were recovered after 90 min or overnight incubation with macrophages (data not shown). These results show for the first time that P. gingivalis resists killing by macrophages after internalization and can persist in viable form for greater than 72 h in these cells. Furthermore, this activity is dependent on the presence of wild-type fimbriae containing accessory proteins. Interestingly, the expression of FimA alone (i.e., DAP fimbriae) appeared to be more deleterious for P. gingivalis intracellular viability than the FimA-deficient state. However, these results reflect the differential in vivo virulence of the respective strains (see Fig. 3). It is intriguing that the data in Fig. 5 suggest that wild-type fimbriae may mediate P. gingivalis uptake through a pathway that allows the pathogen to resist killing, a possibility that is addressed below.


Figure 5
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  		FIGURE 5. Expression of wild-type fimbriae correlates with increased intracellular survival of P. gingivalis. Mouse J774A.1 macrophages (A) or differentiated human THP-1 cells (B and C) were incubated with the indicated P. gingivalis strains (MOI = 10:1) for the designated time points. The persistence of viable internalized bacteria was determined using an antibiotic protection-based survival assay. Data are means ± SD (n = 3) from typical experiments that were performed three (A and B) or two (C) times, yielding similar findings. Asterisks indicate significant (p < 0.05) differences between 33277 and all mutant strains, whereas • denote significant (p < 0.05) differences between JI-1 and the other mutants.

 
P. gingivalis expressing wild-type, but not DAP fimbriae exploit CR3 for intracellular entry and survival

We have previously shown that wild-type P. gingivalis uses CR3 (CD11b/CD18) for entry into mouse macrophages (13). To determine the role of the fimbrial accessory proteins in CR3-dependent entry of P. gingivalis, macrophage internalization of wild-type and OZ5001C strains was compared. Consistent with our findings above, OZ5001C was internalized less efficiently than 332277 by normal macrophages (Fig. 6, A and B). However, in comparative experiments using CD11b–/– macrophages, the uptake of OZ5001C was only slightly affected by CR3 deficiency, whereas uptake of strain 33277 was reduced by ~50% (Fig. 6, A and B). These findings indicate that OZ5001C does not efficiently use CR3 as a receptor for intracellular entry. Furthermore, confocal analysis showed that OZ5001C did not colocalize with CR3 in macrophages as did the wild-type organism (Fig. 6C), but both strains colocalized similarly with TLR2 (Fig. 6D).


Figure 6
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  		FIGURE 6. CR3-mediated internalization and persistence of P. gingivalis and inhibitory effect on IL-12 induction. A, Macrophages from normal or CR3-deficient (CD11b–/–) mice were incubated with P. gingivalis 33277 or OZ5001C (MOI = 10:1) for the indicated times at 37°C. Internalization was assessed using FITC-labeled bacteria and flow cytometry after quenching extracellular fluorescence, and was expressed as percentage of FITC-positive macrophages. The MFI at the 60-min time point is also shown (B) as a relative measure of the number of internalized bacteria. C and D, Mouse macrophages were exposed to FITC-labeled 33277 or OZ5001C for 20 min. After cell fixation and staining with Texas Red-labeled anti-CD11b mAb (C) or with PE-labeled anti-TLR2 mAb (D), the cells were examined by confocal microscopy. The merge images demonstrate colocalization of the 33277 strain with CR3 (C) or both 33277 and OZ5001C with TLR2 (D). E and F, The persistence of viable internalized bacteria in normal, CD11b–/–, or TLR2–/– macrophages was determined by an antibiotic protection-based survival assay. G, Normal mouse macrophages were monitored for IL-12p70 induction by ELISA, upon stimulation with IFN-{gamma} (1 µg/ml) and A. actinomycetemcomitans LPS (0.2 µg/ml), in the absence or presence of P. gingivalis 33277 or OZ5001C (MOI = 10:1), with or without macrophage treatment with cytochalasin D (5 µg/ml). Data are means ± SD (n = 3) from typical experiments that were repeated, yielding similar findings. Asterisks indicate significant (p < 0.05) differences between receptor-deficient and wild-type macrophages, whereas • denote significant (p < 0.05) differences between 33277 and OZ5001C (A and B, and E and F). G, Asterisks denote statistically significant (p < 0.05) inhibition of IL-12p70 production.

 
We next determined the significance of CR3-mediated uptake of P. gingivalis. Interestingly, the recovery of viable internalized P. gingivalis 33277 CFU from CR3-deficient macrophages was significantly reduced by ~2–3 log10 U after 1.5- and 15-h incubation, compared with its recovery from wild-type macrophages (p < 0.05; Fig. 6E). Thus, although CR3 deficiency reduced P. gingivalis 33277 internalization by ~2-fold (Fig. 6B), it had a dramatically more pronounced effect on the pathogen’s ability to persist within macrophages (Fig. 6E). This suggests that CR3 is associated with increased intracellular survival of wild-type P. gingivalis. In contrast, the limited ability of OZ5001C to persist intracellularly was not significantly affected by CR3 deficiency (Fig. 6E). Because the ability of P. gingivalis fimbriae to bind CR3 depends strictly upon TLR2 inside-out signaling, required for activating the ligand-binding capacity of CR3 (13, 15), we hypothesized that TLR2 deficiency would similarly diminish the ability of wild-type P. gingivalis to resist intracellular killing. Indeed, the intracellular survival of wild-type P. gingivalis was dramatically reduced by ~3 log10 U in TLR2-deficient macrophages relative to wild-type controls (p < 0.05; Fig. 6F). Although TLR2 deficiency had a relatively minor effect on the survival of OZ5001C (<1 log10 U difference compared with wild-type macrophages), the observed differences reached statistical significance (p < 0.05; Fig. 6F), suggesting an auxiliary TLR2-dependent, but CR3-independent evasion mechanism. These data collectively suggest that expression of wild-type fimbriae containing accessory proteins allows P. gingivalis to exploit the TLR2/CR3 pathway for intracellular entry and enhanced survival within macrophages.

Inhibition of IL-12p70 represents a microbial tactic to evade immunity (29). In addition to the observations that P. gingivalis uses CR3 to facilitate its internalization by macrophages, we previously showed that purified fimbriae down-regulate macrophage production of IL-12p70 in a CR3-dependent manner (11). To determine whether the fimbrial accessory proteins influence this activity, we used a model in which high levels of IL-12p70 are induced upon stimulation of macrophages with IFN-{gamma} and LPS from A. actinomycetemcomitans. In the presence of P. gingivalis 33277, but not OZ5001C, the induction of IL-12p70 in this system was significantly inhibited (p < 0.05; Fig. 6G), suggesting the importance of the fimbrial accessory proteins in this potential immune evasion mechanism. This inhibitory activity was not dependent upon P. gingivalis internalization because pretreating cells with cytochalasin D did not reverse it (Fig. 6F). However, the inhibition of IL-12p70 production was abrogated in CR3-deficient macrophages (data not shown). Taken together, these results indicate that the interaction of wild-type P. gingivalis with CR3 at the macrophage cell surface suppresses IL-12p70 production in response to other bacterial stimuli. In the context of the oral cavity, this is intriguing, because this mechanism may promote the survival of both P. gingivalis and cohabiting organisms in the subgingival pocket. The isogenic KO4 mutant behaved similarly to OZ5001C in the Fig. 6 assays (data not shown), consistent with the fact that they express phenotypically similar fimbriae lacking all accessory proteins.

Differential macrophage activation by wild-type P. gingivalis or fimbrial mutants

The observations that the DAP mutants were even less virulent than the FimA-deficient mutant both in vivo (Fig. 3) and in vitro (Fig. 5) are intriguing. One plausible explanation may be that the DAP fimbriae elicit a robust host response that clears the organism. In this regard, the DAP strains were found to be more potent inducers of NF-{kappa}B activation and TNF-{alpha} production than the FimA-deficient mutant (JI-1) or even wild-type P. gingivalis (33277) (Fig. 7, A and B). Interestingly, although wild-type fimbriae display higher binding to macrophages than DAP fimbriae (Fig. 4B), the latter were more proinflammatory than wild-type fimbriae (Fig. 7, C and D). However, both wild-type and DAP fimbriae required TLR2 for macrophage activation (Fig. 7C), consistent with the finding that both wild-type P. gingivalis and DAP mutants colocalize with TLR2 (Fig. 6D).


Figure 7
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  		FIGURE 7. Cell activation by wild-type P. gingivalis or fimbrial mutants. A and B, The indicated P. gingivalis strains (MOI = 10:1) were used to stimulate THP-1-Blue cells for NF-{kappa}B activation, determined colorimetrically by measuring the activity of NF-{kappa}B-inducible alkaline phosphatase secreted in the culture supernatants (A), or primary mouse macrophages for TNF-{alpha} induction (B). C and D, Mouse macrophages were stimulated for 16 h with purified fimbriae from the indicated P. gingivalis strains, and induction of TNF-{alpha} production was determined by ELISA. The indicated normal or TLR-deficient macrophages were used in C, in which fimbriae were used at 2 µg/ml. Normal C57BL/6 macrophages were used for a dose-response comparison in D. Data are means ± SD (n = 3) from a typical set of experiments that were repeated, yielding similar results. A, B, and D, Asterisks indicate significantly (p < 0.05) lower responses compared with whole cells or fimbriae from the DAP strains (OZ5001C and KO4). C, Asterisks denote significant (p < 0.05) differences between normal and TLR-deficient macrophages.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our findings show for the first time that the accessory proteins, which constitute <1% of the total fimbrial protein (17), are essential for the maintenance of fimbrial structure and virulence of P. gingivalis. In addition, bacterial mutants that cannot incorporate the accessory proteins into fimbriae were instrumental for elucidating the role of the accessory components in P. gingivalis interactions with macrophages. These studies indicate that P. gingivalis expressing wild-type fimbriae may exploit innate immune function by entering and persisting in macrophages and by reducing the production of IL-12p70, which may facilitate the survival of other microbes that cohabit its niche in the oral biofilm.

The reduced intracellular survival of the strains expressing fimbriae lacking the accessory components is attributable to their inability to exploit CR3, and cannot be explained by their reduced intracellular entry relative to wild-type P. gingivalis. For example, although the OZ5001C mutant was internalized ~2-fold less efficiently than wild-type strain 33277, its intracellular persistence was decreased by 2–3 log10 U compared with 33277. Moreover, the FimA-deficient JI-1 strain survived longer and at higher levels than OZ5001C, despite its lower internalization efficiency. Therefore, bacterial internalization efficiency did not directly determine intracellular bacterial persistence.

One explanation for these results is that P. gingivalis may be directed to different intracellular compartments depending on whether it presents wild-type, DAP fimbriae, or no fimbriae at all. In this context, expression of DAP fimbriae compromises the CR3-mediated uptake of P. gingivalis. Similarly, lack of FimA expression also abrogates CR3-dependent internalization (13). Interestingly, not all phagocytic receptors are created equal when considering their ability to mediate intracellular killing (30, 31). Indeed, CR3 is a preferred receptor used also by certain other pathogens for intracellular entry and persistence (32, 33, 34, 35), perhaps because CR3 is not linked to vigorous microbicidal mechanisms (30, 36, 37, 38, 39). Consistent with this notion, the in vivo phagocytic uptake of Bordetella pertussis via Fc{gamma}Rs facilitates its clearance, in contrast to CR3-mediated uptake (34). Our findings are consistent with these observations because CR3 deficiency results in enhanced intracellular killing of wild-type P. gingivalis. Indeed, in the absence of CR3, the wild-type strain is rendered equally susceptible to intracellular killing as is the DAP fimbria-expressing mutant. Therefore, our results are consistent with the hypothesis that CR3 is used by P. gingivalis for enhancing its survival, and that this exploitation of macrophages requires expression of wild-type fimbriae (see Fig. 8). In contrast, the DAP fimbria-expressing mutants, which do not interact with CR3, may be predominantly internalized via alternative phagocytic receptor(s) associated with increased intracellular killing (Fig. 8).


Figure 8
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  		FIGURE 8. TLR2/CR3-mediated entry and persistence of P. gingivalis. P. gingivalis enters macrophages by exploiting its fimbria-mediated interaction with CR3. This interaction requires prior activation of the CR3 binding affinity, dependent upon CD14/TLR2 inside-out signaling instigated by fimbriae (14 15 ). Expression of DAP fimbriae abrogates CR3-mediated entry of P. gingivalis, which is, however, still internalized by macrophages, apparently through alternative receptor(s). Because P. gingivalis expressing wild-type fimbriae displays enhanced intracellular persistence compared with the DAP fimbria-expressing mutant, it appears that CR3 constitutes a relatively safe portal of entry for P. gingivalis, consistent with the notion that CR3 is not linked to vigorous microbicidal mechanisms (30 32 36 37 38 39 ).

 
Interestingly, CR3 exploitation by P. gingivalis is initiated at the level of TLR2, because the interaction of P. gingivalis fimbriae with CR3 depends strictly upon fimbria-induced TLR2 inside-out signaling, which activates the ligand-binding capacity of CR3 (14, 15) (Fig. 8). In fact, although TLR2 is not a phagocytic receptor, TLR2 deficiency suppresses P. gingivalis internalization comparably to CR3 deficiency (13). Consistent with the notion that TLR2 plays a critical role in CR3 exploitation by P. gingivalis, we have now found that this pathogen loses the ability to resist intracellular killing in the absence of TLR2 expression. Moreover, our results are in line with a recent report that TLR2-deficient mice are more resistant to P. gingivalis-induced periodontal bone loss than wild-type controls (40).

The exploitability of macrophage CR3 by P. gingivalis is also suggested by our findings that this pathogen inhibits IL-12p70 production in a CR3-dependent way, as long as it expresses fimbriae containing the accessory components. Inhibition of IL-12p70 production may lead to suppression of efficient stimulation of CTLs and NK cells, because their activation is positively regulated by macrophage-derived IL-12p70 (41, 42). The implications of this potential immune subversion mechanism are currently uncertain, although the mechanism could conceivably promote the persistence of P. gingivalis in infected periodontal tissue. This is particularly relevant to oral disease because P. gingivalis readily takes intracellular refuge in permissive cells, such as epithelial cells (23) and endothelial cells (43), and a reduction in IL-12p70-dependent stimulation of CTLs or NK cells may compromise the killing of these P. gingivalis-infected cells. This is turn may allow P. gingivalis a window of opportunity to establish infection and create a niche that is suitable for its survival and growth, ultimately resulting in enhanced virulence and induction of inflammatory periodontal bone resorption.

The capacity of P. gingivalis for CR3-mediated uptake and persistence for at least 72 h in macrophages is also intriguing in the context of the link between severe periodontitis and systemic diseases, such as atherosclerosis (4, 5, 6). Interestingly, viable P. gingivalis has been recently demonstrated in atherosclerotic plaques (4), although it is unknown how the pathogen resists immune elimination and relocates there. Furthermore, the interaction of P. gingivalis with TLR2 not only promotes CR3-mediated internalization, but also stimulates the transmigratory activity of monocytes (14). Conceivably, the persistence of P. gingivalis in macrophages may be sufficient to co-opt the migration potential of these cells, facilitating relocation to systemic tissues and colonization or infection of more permissive cells (e.g., endothelial cells). Although monocytes/macrophages might be exploited as "Trojan horses" for P. gingivalis systemic dissemination, more studies are warranted to investigate this fascinating possibility.

Importantly, the notion that the fimbrial accessory proteins contribute to the virulence of P. gingivalis is corroborated by the findings from the mouse periodontitis model, in which maximal induction of periodontal bone loss by the pathogen required expression of wild-type fimbriae. Although these results are most likely partly attributable to the ability of wild-type P. gingivalis to exploit its fimbria-mediated interactions with CR3 in macrophages, additional mechanisms may be involved. In this regard, the contribution of the fimbrial accessory factors to the virulence function of fimbriae may be multifaceted. For instance, the enhanced ability of wild-type fimbriae to bind extracellular matrix proteins relative to DAP fimbriae (18) may confer a colonization advantage to the wild-type strain. Interestingly, the DAP mutants were even less virulent than the FimA-deficient mutant, which induced modest periodontal bone loss. It is possible that DAP fimbriae may elicit robust host responses that could eliminate the pathogen; indeed, the DAP strains were stronger inducers of NF-{kappa}B activation than the FimA-deficient mutant. In general, increased microbial immunostimulatory potential correlates with reduced microbial survival in the host, as exemplified by genetically modified Yersinia pestis expressing an immunostimulatory version of LPS (44). In contrast to DAP fimbriae, expression of wild-type fimbriae appears to offset this potential disadvantage through efficient exploitation of CR3 (and perhaps other receptors), and thereby undermining the innate host defense and promoting the persistence of the pathogen. In fact, wild-type P. gingivalis was the only strain that was recoverable from the oral cavity of infected mice in the experimental periodontitis study. Interestingly, although wild-type fimbriae exhibited higher binding to macrophages than DAP fimbriae, the latter were more immunostimulatory and similar differences applied to their respective P. gingivalis strains. It is possible, therefore, that wild-type fimbriae containing the accessory proteins may additionally interact with a receptor that limits cellular activation.

Although the P. gingivalis fimbrial accessory proteins are associated with adhesive functions, transmission electron microscopy clearly showed that they are also essential for maintenance of fimbrial structure. Indeed, their absence in strains OZ5001C or KO4 results in physically shorter fimbriae compared with the wild-type strain, suggesting a possible role in fimbrial assembly or polymerization. We cannot exclude the possibility that full-length DAP fimbriae do form, but are unstable in the absence of accessory proteins, and they are thus sheared off during processing of the cells. However, the reactivity of OZ5001C and KO4 with specific anti-fimbria Ab was not substantially less than that of the wild type, suggesting that FimA is present in significant amounts on the mutant cells. In conclusion, OZ5001C and KO4 appear to be incapable of assembling mature fimbriae. The specific control of fimbrial polymerization has precedence in the activity of CsgB, a minor component of an Escherichia coli fimbrial structure known as curli (45). Specifically, CsgB appears to facilitate the polymerization of the major fimbrial subunit, CsgA, and is moreover distributed along the length of the fimbrial structure (45). It is conceivable that P. gingivalis accessory proteins occur at regular intervals along the fimbrial length, perhaps including the tip, and serve both accessory roles in polymerization of fimA and adhesive interactions.

In summary, it is clear that the accessory proteins of P. gingivalis fimbriae are essential components of the fimbrial structure and function. Their absence results in a dramatic attenuation of P. gingivalis virulence in vivo, which may be explained in part through the outcomes of fimbrial interactions with macrophages. Despite the fact that these proteins are minor components of P. gingivalis fimbriae (17), they play major roles in P. gingivalis evasion of intracellular killing and induction of experimental periodontitis, and may moreover regulate the extent of P. gingivalis fimbriation. Our study supports the concept that pathogens evolved to manipulate innate immunity for promoting their adaptive fitness and, consequently, their capacity to cause disease (46).


   Disclosures
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 Abstract
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 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by U.S. Public Health Service Grants DE015254 and DE018292 (to G.H.), and DE14605 (to D.R.D.) from the National Institutes of Health; Grants-in-Aid for Scientific Research (15591957 to F.Y. and 17791318 to S.N.) from the Japan Society for the Promotion of Science; and the AGU High-Tech Research Center Project from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (to F.Y.). Back

2 Address correspondence and reprint requests to Dr. George Hajishengallis, University of Louisville Health Sciences Center, 501 South Preston Street, Room 206, Louisville, KY 40292. E-mail address: g0haji01{at}louisville.eduU Back

3 Abbreviations used in this paper: DAP, devoid of all accessory proteins; ABC, alveolar bone crest; CEJ, cementoenamel junction; CR3, complement receptor 3; MFI, mean fluorescence intensity; MOI, multiplicity of infection. Back

Received for publication April 26, 2007. Accepted for publication June 8, 2007.


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 Materials and Methods
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J. Immunol., June 1, 2009; 182(11): 6690 - 6696.
[Abstract] [Full Text] [PDF]

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 J. Immunol.Home page
S. Liang, K. B. Hosur, S. Lu, H. F. Nawar, B. R. Weber, R. I. Tapping, T. D. Connell, and G. Hajishengallis
Mapping of a Microbial Protein Domain Involved in Binding and Activation of the TLR2/TLR1 Heterodimer
J. Immunol., March 1, 2009; 182(5): 2978 - 2985.
[Abstract] [Full Text] [PDF]

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M. T.-H. Huang, D. J. Taxman, E. A. Holley-Guthrie, C. B. Moore, S. B. Willingham, V. Madden, R. K. Parsons, G. L. Featherstone, R. R. Arnold, B. P. O'Connor, et al.
Critical Role of Apoptotic Speck Protein Containing a Caspase Recruitment Domain (ASC) and NLRP3 in Causing Necrosis and ASC Speck Formation Induced by Porphyromonas gingivalis in Human Cells
J. Immunol., February 15, 2009; 182(4): 2395 - 2404.
[Abstract] [Full Text] [PDF]

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G. Hajishengallis, M. Wang, G. J. Bagby, and S. Nelson
Importance of TLR2 in Early Innate Immune Response to Acute Pulmonary Infection with Porphyromonas gingivalis in Mice
J. Immunol., September 15, 2008; 181(6): 4141 - 4149.
[Abstract] [Full Text] [PDF]

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G. Hajishengallis, M. Wang, S. Liang, M. Triantafilou, and K. Triantafilou
Pathogen induction of CXCR4/TLR2 cross-talk impairs host defense function
PNAS, September 9, 2008; 105(36): 13532 - 13537.
[Abstract] [Full Text] [PDF]

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 JDRHome page
S.L. Gaffen and G. Hajishengallis
A New Inflammatory Cytokine on the Block: Re-thinking Periodontal Disease and the Th1/Th2 Paradigm in the Context of Th17 Cells and IL-17
Journal of Dental Research, September 1, 2008; 87(9): 817 - 828.
[Abstract] [Full Text] [PDF]

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