The effects of immunization with invasive or noninvasive Porphyromonas (Bacteroides) gingivalis strains on the pathogenesis of infection in a mouse chamber ...
INFECTION AND IMMUNITY, Apr. 1992, p. 1447-1454
Vol. 60, No. 4
0019-9567/92/041447-08$02.00/0 Copyright © 1992, American Society for Microbiology
Influence of Immunization on Porphyromonas gingivalis Colonization and Invasion in the Mouse Chamber Model CAROLINE ATFARDO GENCO,lt* DARRELL R. KAPCZYNSKI,' CHRISTOPHER W. CUTLER,lt ROBERT J. ARKO,2 AND ROLAND R. ARNOLD'f Department of Oral Biology, Emory University, Atlanta, Georgia 30322,1 and Division of Sexually Transmitted Diseases Laboratory Research, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 303332 Received 19 August 1991/Accepted 10 January 1992
The effects of immunization with invasive or noninvasive Porphyromonas (Bacteroides) gingivalis strains on the pathogenesis of infection in a mouse chamber model were examined. BALB/c mice were immunized by a single injection of heat-killed P. gingivalis invasive strain A7436 or W83 or noninvasive strain 33277, HG405, or 381 directly into subcutaneous chambers. P. gingivalis-specific antibody was detected in chamber fluid 21 days postimmunization, and mice were subsequently challenged by injection of exponential-phase P. gingivalis into chambers. Immunization with A7436 or W83 followed by challenge with A7436 protected mice against secondary abscess formation and death; however, P. gingivalis persisted in chambers for up to 14 days postchallenge. Immunization with noninvasive strain 33277, HG405, or 381 followed by challenge with invasive strain A7436 or W83 protected mice against secondary lesion formation and death. P. gingivalis was cultured from 33277- or HG405-immunized and nonimmunized animals to day 14. All P. gingivalis strains induced an immunoglobulin G response, as measured by an enzyme-linked immunosorbent assay and Western immunoblotting of P. gingivalis whole-cell and outer membrane protein preparations. Western blot analyses indicated that sera from mice immunized with different invasive and noninvasive strains recognized common P. gingivalis antigens. In summary, immunization with invasive P. gingivalis A7436 and W83 or noninvasive P. gingivalis 33277, HG405, and 381 protected mice from secondary lesion formation and death after challenge with invasive P. gingivalis A7436 or W83. P. gingivalis-specific antibody did not, however, inhibit the colonization of P. gingivalis within chambers.
The anaerobic oral bacterium Porphyromonas (Bacteroides) gingivalis has been implicated as a major pathogen in adult periodontitis (46). P. gingivalis produces a number of potential virulence factors which are believed to be crucial to the ability of this organism to colonize and invade the periodontal pocket (46). These include fimbriae, hemagglutinin, capsule, lipopolysaccharide (LPS), outer membrane vesicles, and enzymatic activities that can perturb host defense mechanisms as well as initiate tissue destruction (31). The ability of P. gingivalis to invade gingival tissues in periodontal disease has been suggested to be an important component of the disease process (46, 48). We previously reported on the application of a mouse chamber model for the study of the virulence of and host response to P. gingivalis (19). On the basis of the pathology observed in mice following inoculation of P. gingivalis strains into subcutaneous chambers, strains of P. gingivalis have been classified as invasive or noninvasive; noninvasive strains produce a localized abscess at the chamber site, with chamber rejection, while invasive strains spread to distant sites and produce abdominal abscesses, septicemia, and death (19). The host response in human periodontal disease comprises a complex series of events involving activation of the acute inflammatory, cellular immune, and humoral immune systems (20). Studies with humans have suggested that
although the majority of adult periodontitis patients produce antibodies to P. gingivalis, these antibodies are apparently ineffective at limiting continued disease progression (15, 23, 34, 35, 41, 47, 49). Cutler et al. (8, 9) have suggested that this ineffectiveness of the antibody response may be due to the lack of a specific antibody necessary for the opsonization of P. gingivalis. To examine the consequences of the production of P. gingivalis-specific antibody in vivo, we performed a series of immunization experiments using the mouse subcutaneous (s.c.) chamber model (19). Previous studies by other investigators using a murine abscess model demonstrated protection in mice that were immunized with homologous invasive but not heterologous noninvasive P. gingivalis strains (3, 4). Using several different invasive and noninvasive strains for immunization and challenge, we demonstrated here protection induced against invasion but not colonization of P. gingivalis by immunization with either heterologous invasive or heterologous noninvasive P. gingivalis strains.
MATERIALS AND METHODS Bacterial strains. The P. gingivalis strains used in this study are listed in Table 1. P. gingivalis A7436 was originally isolated from a refractory periodontitis patient and was characterized in our laboratories. In mice, this P. gingivalis isolate is invasive and produces ulcerated lesions distant from the injection site, septicemia, and death (19). P. gingivalis W83 and HG405 were obtained from A. van Winkelhoff, Vrije Universiteit, Amsterdam, The Netherlands; strain 33277 was obtained from the American Type Culture Collection, Rockville, Md.; and strain 381 was obtained from
* Corresponding author. t Present address: Department of Microbiology and Immunology, Morehouse School of Medicine, Atlanta, GA 30310. : Present address: Dental Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7455.
1447
1448
INFEcr. IMMUN.
GENCO ET AL. TABLE 1. P. gingivalis strains
Strain
Serotype/ clonal typea
A7436 W83 HG405 33277 381
ND
Pathology mice" in
Invasive
B/2
Invasive
ND A/1 A/1
Noninvasive Noninvasive Noninvasive
a Serotypes are as described by Fisher et al. (18); clonal types are as described by Loos et al. (29). ND, not determined. b Determined as described by Genco et al. (19).
J. J. Zambon, School of Dental Medicine, State University of New York at Buffalo, Buffalo. P. gingivalis was typically grown on anaerobic blood agar plates (Remel, Lenexa, Kans.) in an anaerobic chamber (Coy Laboratory Products Inc., Ann Arbor, Mich.) with 85% N2-5%H2-10% CO2. After incubation at 37°C for 2 to 3 days, the bacterial cells were inoculated into Schaedler broth (Difco Laboratories, Detroit, Mich.) and grown for 1 to 2 days until the cultures reached an A660 of 1.2, as read on an LKB spectrophotometer. This absorbance corresponded to approximately 109 CFU/ml. Cultures were concentrated by centrifugation as previously described (19). Viable bacterial cell counts were determined immediately prior to inoculation by serial dilution. Antigen preparation. Antigens used for immunization were prepared from whole cells of P. gingivalis A7436, W83, HG405, 33277, or 381. Cultures were concentrated by centrifugation at 10,000 x g (10 min at room temperature) and resuspended in 1/10 the original volume of prereduced Schaedler broth. The bacterial cells were heated to 95°C for 10 min. Heat-treated preparations were plated on anaerobic blood agar and incubated for 7 days under anaerobic conditions to ensure effective killing of P. gingivalis. In all instances, the growth of P. gingivalis was not detected. Experimental animals. Female BALB/c mice approximately 8 weeks old and obtained from Charles River Laboratory, Wilmington, Mass., were used in this study. Coilshaped s.c. chambers were surgically implanted in the s.c. tissue of the dorsolumbar region of each mouse (19). Ten days after implantation, mice were immunized by direct injection into chambers of a suspension of heat-killed P. gingivalis (0.1 ml; 109 CFU). Control mice were injected with Schaedler broth only. Twenty-one days postimmunization, chamber fluid was removed with a hypodermic needle (26 gauge) and syringe, and immunoglobulin G (IgG) specific for P. gingivalis whole cells was quantitated by an enzymelinked immunosorbent assay (ELISA) as described below. Twenty-three days postimmunization, mice were challenged by injection of 0.1 ml of P. gingivalis A7436, W83, HG405, 33277, or 381 (109 to 1010 CFU) into chambers. Mice were examined daily for size and consistency of primary and/or secondary lesions and health status. Severe cachexia was defined as ruffled hair, hunched bodies, and weight loss. Chamber fluid was removed from each implanted chamber at 1 to 7, 14, and 28 days postchallenge for bacteriological culturing and microscopic examination. All surviving animals were sacrificed 30 days postinoculation, and sera were separated from blood obtained by cardiac puncture. Chamber fluid analysis. (i) Microbiology. Aliquots of fluid from each chamber were streaked for isolation onto anaerobic blood agar plates and cultured at 37°C for 7 days under anaerobic conditions as described above.
(ii) Host response. A combination of fluorescent dyes, cytocentrifugation, and fixation was used to examine the association of polymorphonuclear leukocytes with P. gingivalis in mouse chamber fluid as described previously (19). IgG specific for P. gingivalis whole cells was assayed by a modification of an ELISA described by Ebersole et al. (12), and results were read with a Vm,, kinetic spectrophotometer (Molecular Devices) at 450 nm. Western immunoblot analysis. Outer membrane proteins of P. gingivalis were extracted as follows. Cells were grown to the late exponential phase and harvested by centrifugation at 10,000 x g for 20 min. The resulting cell pellet was resuspended in 10 mM N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid (HEPES) (pH 7.4)-50 ,uM phenylmethylsulfonyl fluoride (PMSF)-2 mM N-a-p-tosyl-L-lysine chloromethyl ketone (TLCK). Cells were sonicated on ice for five 1-min periods with 1-min intervals. N-Lauryl sarcosyl (0.3%) was added to the sonicate, and the mixture was incubated at 37°C for 60 min. Whole cells and cell debris were removed by centrifugation at 8,000 x g for 20 min, and the supernatant was further centrifuged at 100,000 x g for 2 h. The resulting pellet (consisting of outer membranes) was suspended in 10 mM HEPES (pH 7.4), and the suspension was frozen at -20°C until used. Protein concentrations were determined by use of a bicinchoninic acid protein assay with bovine serum albumin (BSA) as the standard (Pierce, Rockford, Ill.). For whole-cell preparations, organisms were grown on anaerobic blood agar plates and cells were removed, washed, and resuspended in 10 mM HEPES (pH 7.4)-50 ,uM PMSF. Cells were lysed by being boiled in 2x sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) buffer for 10 min. Outer membrane proteins and whole cells were separated by SDS-PAGE by the method of Laemmli (26). Gels were stained with Coomassie brillant blue or silver stain (21). Following separation by SDS-PAGE, proteins were transferred to nitrocellulose by Western immunoblotting. The nitrocellulose filters were blocked with BSA and incubated with sera at 1:500 or 1:750 at room temperature for 2 h. Sera included those obtained from mice immunized and challenged with P. gingivalis and those obtained from nonimmunized, nonchallenged mice (control). Sera were pooled from individual mice in each group. Following incubation with the primary antibody, goat anti-mouse IgG conjugated to horseradish peroxidase was added, serving as the secondary antibody. Hydrogen peroxide and diaminobenzidine were added for color development of the immunoblot.
RESULTS Virulence of P. gingivalis in immunized mice. Nonimmunized animals challenged with P. gingivalis A7436 did not develop localized lesions but did develop secondary, ulcerated necrotic lesions on their abdomens. These mice exhibited severe cachexia, with ruffled hair, hunched bodies, and weight loss, and 9 of 10 died by day 3 (Table 2). In contrast, immunization with P. gingivalis A7436 whole cells followed by challenge with A7436 resulted in the protection of mice from secondary abscess formation and death (Table 2). However, these mice developed chamber lesions which gradually increased in size and resulted in chamber rejection on day 7. Nonimmunized mice challenged with P. gingivalis W83 were severely cachectic and developed ulcerated necrotic lesions on their abdomens. However, six of nine mice immunized with P. gingivalis A7436 and challenged with P.
VOL. 60, 1992
INFLUENCE OF IMMUNIZATION ON P. GINGIVALIS
1449
TABLE 2. Pathological course of P. gingivalis infection in immunized mice Lesionc
Cachexia
No.no.ofofdead micemice/total tested
A7436 A7436 A7436 A7436 A7436 A7436
Abdomen (9/10); CR (none) Abdomen (3/10); CR (4/10) CR (1/5) Chamber (7/10); CR (8/10) Chamber (5/10); CR (7/9) Chamber (5/7); CR (3/7)
Severe Mild to moderate Mild Mild Mild Mild
9/10 1/10 1/5 0/10 1/10 0/7
W83 W83 W83 W83 W83 W83
Abdomen (10/10); CR (none) Abdomen (6/9); CR (2/9) Abdomen (5/5); CR (2/5) Abdomen (1/10); CR (8/9) Chamber (10/10); CR (10/10) ND
Severe Moderate Moderate Mild Mild
9/10 3/9 2/5 1/10 0/10
Immunogena
Challengeb
None A7436 W83 HG405 33277 381 None A7436 W83 HG405 33277 381
a At 10 days after chamber implantation, mice were immunized by direct injection into chambers of a suspension of heat-killed P. gingivalis (0.1 ml). b At 23 days postimmunization, mice were challenged by injection into chambers of 0.1 ml of P. gingivalis (109 to 1010 CFU). Chamber lesion at injection site (chamber) or secondary lesion on the ventral abdomen (abdomen) (number of mice with lesions/total number of mice tested); CR, chamber rejection (number of mice which rejected chambers/total number of mice tested). ND, not determined.
gingivalis W83 developed ulcerated necrotic lesions on their abdomens and were moderately cachectic. Nine of 10 nonimmunized mice died, and 3 of 9 A7436-immunized mice died. We obtained similar results when we immunized mice with invasive P. gingivalis W83 and challenged them with this strain. All immunized mice developed ulcerated necrotic lesions on their abdomens; however, most mice resolved these lesions, and only two of five died (Table 2). W83immunized mice challenged with the homologous strain also developed localized lesions at the chamber site that resulted in the rejection of chambers by day 6. Thus, immunization with invasive strains resulted in protection following challenge with homologous or heterologous invasive P. gingivalis strains. Immunization with noninvasive P. gingivalis 33277 followed by challenge with P. gingivalis A7436 or W83 resulted in abscess formation at the injection site but no secondary abscess formation (Table 2). Mice were only mildly cachectic, and seven of nine rejected their chambers by day 14 following challenge with P. gingivalis A7436. We obtained similar results when we immunized mice with noninvasive P. gingivalis HG405 or 381 and challenged them with A7436 (Table 2). Mice immunized with noninvasive or invasive P. gingivalis strains responded similarly when challenged with noninvasive strains (data not shown). Analysis of chamber fluid for viable bacteria. Chamber fluid was cultured throughout the course of the experiments to correlate bacterial survival and growth within chambers with the pathology observed in mice. Immunization with invasive strain A7436 followed by challenge with strain A7436 did not inhibit the local survival or colonization of P. gingivalis. P. gingivalis was cultured from chamber fluid from all immunized and all nonimmunized mice up to day 14 (Table 3). Challenge of W83-immunized mice with strain W83 also did not inhibit the local survival or colonization of this invasive P. gingivalis strain, as assessed by growth in chamber fluid up to day 14. Quantitation of the number of viable cells indicated that P. gingivalis A7436 and W83 increased in numbers relative to the initial inoculum (108 to 1012 CFU) throughout the course of the experiments (data not shown). P. gingivalis was also cultured from chamber fluid obtained from mice immunized with invasive strain A7436 and challenged with noninvasive strain HG405 (data not shown).
Immunization of mice with noninvasive strains 33277, HG405, or 381 followed by challenge with invasive strain A7436 did not inhibit the survival or colonization of P. gingivalis within chambers. Analysis of chamber fluid from these mice revealed that viable P. gingivalis could be recovered up to day 14 from 33277-immunized mice and up to day 7 from HG405- or 381-immunized mice (Table 3) and that P. gingivalis A7436 increased in numbers relative to the initial inoculum (data not shown). Taken together, these results indicate that immunization with invasive or noninvasive P. gingivalis strains can limit the invasive capabilities of invasive strains but not the colonization of P. gingivalis within chamber fluid. They further suggest that immunization with the noninvasive strains chosen in this study may afford greater protection than homologous invasive immunogens. Serum IgG response to P. gingivalis. We measured the antibody response induced in mice by injection of P. gingivalis A7436, W83, 33277, and HG405 heat-killed whole cells into chambers. All strains induced a high IgG response, as TABLE 3. P. gingivalis cultured from chamber fluid Immu-
nogena
Challenge'
No. of mice from which P. gingivalis was cultured/ total no. of mice sampled on the following day postinoculation: 1
2
3
5/5 5/5 4/4 10/10
4/4 4/4 3/4 10/10 10/10
3/3
None A7436 W83 HG405 33277 381
A7436 A7436 A7436 A7436 A7436 A7436
10/10 7/7
5/5 5/5 4/4 10/10 10/10 7/7
None A7436 W83 HG405 33277 381
W83 W83 W83 W83 W83
5/5 5/5 5/5 10/10 10/10
5/5 4/4 5/5 9/10 10/10
W83
ND
NDC
5/5 3/3
8/8 10/10
5
7
2/2
2/2
5/5 3/4
5/5 3/4
3/3 2/3 3/3 5/8
1/1 0/2 3/3 5/5 6/6
14
28
2/2 0/1 5/5 0/2 1/1 0/1 6/10 4/4 0/1 8/10 8/10 2/5 0/2 7/7 7/7 0/4
ND
0/1 1/1 0/1 1/1 0/1 ND
a At 10 days after chamber implantation mice were immunized by direct injection into chambers of a suspension of heat-killed P. gingivalis (0.1 ml). bAt 23 days postimmunization, mice were challenged by injection into chambers of 0.1 ml of P. gingivalis (109 to 1010 CFU). c ND, not determined.
1450
INFECT. IMMUN.
GENCO ET AL.
TABLE 4. Systemic IgG response to P. gingivalis whole cellsa Immu-
nogen
Prechallenge
ELISA+ units SD)b (mean
Challenge
Postchallenge
ELISA units (mean t SD)b
None A7436 W83 HG405 33277
3.5 57.2 85.8 36.9
+ 0.03 ± 1.9 ± 13.98 ± 4.3 NDC
A7436 A7436 A7436 A7436 A7436
145.3 149.7 158.4 103.2 148.2
± 3.0
None A7436 W83 HG405 33277
2.4 63.5 67.9 36.9
± 0.6 ± 1.3
W83 W83 W83 W83 W83
131.7 133.0 131.7 114.9 117.2
± 0.8 + 19.0 ± 0.71 ± 23.8 ± 6.9
+ 1.4 ± 4.3 ND
± ± ± ±
16.8 2.4 7.16 44.5
a Mice were immunized by one injection of heat-killed P. gingivalis A7436, W83, HG405, or 33277 directly into subcutaneous chambers and challenged 23 days postimmunization with live P. gingivalis A7436, W83, HG405, or 33277. At 30 days postchallenge, serum was separated from blood obtained by cardiac puncture. h ELISA units = (sample Vmnx in OD/min)/(positive-pool Vmax in OD/min), where OD = optical density. Prechallenge antibody was determined from chamber fluid taken 21 days postimmunization and tested against the homologous immunizing strain. Postchallenge antibody was determined from sera taken 30 days postchallenge and tested against the homologous immunizing strain. c ND, not determined.
detected in chamber fluid 21 days postimmunization with heat-killed whole cells (Table 4). We also examined sera 30 days postchallenge for P. gingivalis-specific antibody. In all cases, challenge of A7436-, W83-, 33277-, or HG405-immunized mice with homologous or heterologous strains resulted in a high IgG response to P. gingivalis whole cells. All immunogens examined induced comparable quantitative levels of antibody, and this antibody response was much higher than that in nonimmunized mice. Analysis of chamber fluid by fluorescence microscopy. A combination of fluorescent dyes, cytocentrifugation, and fixation was used to examine host cells and P. gingivalis in mouse chamber fluid. Typically, when we examined chamber fluid from A7436- or W83-immunized mice, we observed a heavy polymorphonuclear leukocyte infiltrate 1 day postchallenge with strain W83 or A7436 (data not shown). Analysis of chamber fluid from all immunized mice following challenge with P. gingivalis also revealed massive inflammatory cell debris (data not shown). We believe that this cellular debris was indicative of the walling off of the infection, with the formation of a primary abscess at the chamber site and eventual chamber rejection. Cell debris was not typically observed in chamber fluid obtained from nonimmunized mice challenged with invasive or noninvasive P. gingivalis strains. Western blot analysis. Antisera raised to whole cells of P. gingivalis A7436, W83, 33277, HG405, and 381 (obtained from immunized and challenged mice) were reacted in immunoblotting with whole cells and outer membrane proteins of P. gingivalis A7436, W83, 33277, 381, and HG405 to determine the specificity of the P. gingivalis response in mice. As shown in Fig. 1, antisera from mice immunized and challenged with A7436, W83, 33277, HG405, or 381 reacted with many common antigens present in both whole-cell and outer membrane protein preparations from P. gingivalis A7436, 33277, HG405, and 381. Control antisera from nonimmunized, nonchallenged mice did not react with any antigens in whole-cell or outer membrane protein prepara-
tions (data not shown). A 70-kDa protein present in both whole cells and outer membrane proteins of P. gingivalis A7436, 381, and 33277 was a major antigen recognized by antisera from mice immunized and challenged with A7436, 381, HG405, and 33277. Antisera from mice immunized and challenged with W83 did not react with this 70-kDa band, suggesting that P. gingivalis W83 does not share this determinant. Although antisera raised to P. gingivalis HG405 reacted with this 70-kDa band, analysis of HG405 outer membrane protein preparations by SDS-PAGE indicated that this antigenic determinant migrated differently (Fig. 2). Mouse antisera raised to all strains of P. gingivalis also reacted with LPS present in both whole-cell and outer membrane protein preparations from P. gingivalis A7436, 381, HG405, and 33277. This result was most apparent in immunoblots reacted with 381 antisera (Fig. 1B). Mouse antisera raised to all five strains of P. gingivalis also reacted with a 43-kDa antigen present in both whole cells and outer membrane proteins of P. gingivalis A7436, 381, 33277, and HG405. The size of this immunoreactive antigen is consistent with that of the P. gingivalis fimbrillin subunit or the hemagglutinating adhesin (2, 33, 50). Other antigens which exhibited strong reactivity with all P. gingivalis antisera included an antigen that had an apparent molecular mass of 33 kDa and that was present in outer membrane protein preparations and a 29- to 31-kDa antigen that was present in whole-cell preparations (Fig. 1). Immunoreactivity was also observed with low-molecular-mass antigens (13 to 16 kDa) present in outer membrane protein and whole-cell preparations from P. gingivalis A7436, 381, 33277, and HG405 (Fig. 1). Interestingly, neither whole cells nor purified outer membrane proteins isolated from P. gingivalis W83 were strongly immunoreactive when probed with mouse antisera raised to P. gingivalis A7436, W83, HG405, 33277, or 381 (Fig. 1).
DISCUSSION In this study, we examined the protective role of antibody directed to P. gingivalis heat-killed whole cells. All strains of P. gingivalis examined induced a P. gingivalis antibody response, as assessed by an ELISA postimmunization as well as postchallenge and by Western blot analysis postimmunization and postchallenge. Our results indicate that P. gingivalis-specific antibody can modulate the virulence of P. gingivalis infection in a mouse chamber model by preventing the secondary spread of invasive P. gingivalis strains. Of particular importance was the protection induced by immunization with heterologous P. gingivalis strains. Immunization with invasive P. gingivalis A7436 or W83 or noninvasive P. gingivalis HG405, 33277, or 381 protected mice from secondary lesion formation and death after challenge with invasive P. gingivalis A7436 or W83. In contrast, challenge of A7436-immunized mice with noninvasive P. gingivalis HG405 did not modify the pathology observed in mice. We obtained similar results when we challenged 33277-immunized mice with the homologous noninvasive strain. In all instances, immunization followed by challenge with invasive P. gingivalis resulted in the rapid rejection of chambers. Immunization with P. gingivalis heat-killed cells did not prevent the local survival and colonization of P. gingivalis within s.c. chambers; P. gingivalis was cultured directly from s.c. chambers throughout the course of the infections from both immunized and nonimmunized mice. Taken together, these results indicate that the immune response
INFLUENCE OF IMMUNIZATION ON P. GINGIVALIS
VOL. 60, 1992
A
1
B
2 3 4 5 6 7 8 9 10
97.4 66.2-
45
-
31
-
1
2 3 4
1451
5 6 7 8 910
97.4 66.2 45 -
-.4
.-a -OK
aI
31
-
*1
]4
t....
P) .4 c
21.5 -
;. b
=:.
-
14.4-
14.4-
1
C
97.4,-
45
2
N
-
*I
3
5
6
8
9
1
7
8
9
10
*|*
of
D
--.
97 .4 66 2
-
45
-
...
-
-OK
X ... i. .
3ti
4
7..
-
_,
31
q
.
Wm,
A"
21.5-
Il"
14 4
A'A'"
ak
.0z.,
AM&&
-MNI.-,
::
n
14.4-
E
*-
::A
4
pii _ - -
!j.1
*fitj
-W
gkit.,
TIWII
*1412p:
A&-
io
40..
n
FIG. 1. Immunoblot analysis of mouse sera raised against P. gingivalis whole-cell and outer membrane protein preparations and obtained from immunized and challenged animals. (A) A7436 antisera; (B) 381 antisera; (C) W83 antisera; (D) 33277 antisera; (E) HG405 antisera. In each panel, the sizes of the molecular mass standards are indicated on the left in kilodaltons. Lanes: 1 and 2, A7436; 3 and 4, 381; 5 and 6, W83; 7 and 8, 33277; 9 and 10, HG405. For each P. gingivalis strain, whole-cell preparations (10 pug of protein per ml) were loaded in the odd-numbered lane and outer membrane protein preparations (10 ,ug of protein per ml) were loaded in the even-numbered lane. Arrowheads on the right indicate reactive bands common to all five antisera. Brackets indicate reactive bands common to A7436, 381, 33277, and HG405 antisera.
produced in mice to invasive or noninvasive P. gingivalis heat-killed whole cells was capable of inhibiting the invasion of invasive strains but not the colonization of invasive or noninvasive strains.
The inability to inhibit the colonization of P. gingivalis, as demonstrated here, is in contrast to a recent report by Dahlen and Slots (10). In that study, New Zealand White rabbits immunized with sonicated P. gingivalis 381 or W83
1452
GENCO ET AL.
1 .N.v:I!
3
2 I.~< ...... _
%:.::
97.46. 2
P"..".77T
-
z;"
"
4
.
INFECT. IMMUN.
5 IM
c;i ~:.-
_'K"m
.1
-'-
.aw
Aimiwam&
45
-
31
-
.s
21.5 -
14.4 -
FIG. 2. Outer membrane protein profiles of P. gingivalis. Ten micrograms of outer membrane protein was applied to each lane, and the membrane proteins were separated by SDS-PAGE and stained with silver stain (21). The sizes of the molecular mass standards are indicated on the left in kilodaltons. Arrowheads on the right indicate a 70-kDa protein band and a 43-kDa protein band with which P. gingivalis antisera reacted (Fig. 1). Lanes: 1, A7436; 2, 381; 3, W83; 4, 33277; 5, HG405.
and challenged with the homologous P. gingivalis strain showed complete elimination of bacteria from implanted tissue cages by day 3. Chen et al. (3) also examined the protective role of immunization using invasive P. gingivalis 53977 or AJW4 as an antigen in a murine model. Protection against secondary abscess formation following challenge with an invasive strain was observed in mice immunized three times by intraperitoneal injection with either live cells or a lithium diiodosalicylate extract preparation but not in mice immunized once with LPS by intravenous or intraperitoneal injection. In agreement with the results presented here, mice immunized with live cells or the lithium diiodosalicylate extract and challenged with virulent P. gingivalis also showed localization of P. gingivalis at the site of injection. In an earlier study, Chen et al. (4) did not observe protection against secondary abscess formation in mice immunized with noninvasive strain 381 and challenged with invasive strain A7A1-28. These mice developed both localized and secondary lesions from which P. gingivalis was cultured. These results are in contrast to the observations reported in the present study, in which we demonstrated protection against invasion of invasive P. gingivalis strains by immunization with heat-killed organisms from noninvasive strain HG405, 33277, or 381. The inability of P. gingivalis-specific antibody to inhibit P. gingivalis colonization has been observed in several other animal models. Okuda et al. (39) showed that peroral immunization with whole cells or s.c. immunization with purified P. gingivalis hemagglutinin could not eliminate the colonization of P. gingivalis in ligated hamsters. McArthur et al. (32) demonstrated that immunization of squirrel monkeys with P. gingivalis or Prevotella internedia (formerly Bacteroides intermedius [7]) was associated with a significant
reduction in the colonization of black-pigmented Bacteroides spp. Nisengard et al. (37) reported that a heightened humoral response in monkeys to Bacteroides macacae (the monkey equivalent of the human species P. gingivalis) as a result of immunization caused a reduction in the subgingival recolonization of B. macacae. In this study, we did not detect differences in the survival and colonization of P. gingivalis within s.c. chambers of immunized or nonimmunized mice, but differences were observed in invasiveness in mice that were immunized. It is important to note that our animal model, immunization protocol, and antigen preparation differed from those used by Dahlen and Slots (10), Chen et al. (3, 4), Okuda et al. (39), Nisengard et al. (37), and McArthur et al. (32). It could be argued that a sufficient immune response was not mounted by a single injection of heat-killed cells to inhibit the colonization and proliferation of P. gingivalis within chambers. However, in the report by Chen et al. (3), one injection of LPS did not protect against secondary lesion formation, colonization, or death, whereas in our study, immunization was sufficient to protect against secondary lesion formation and death. Since antibody produced to invasive or noninvasive P. gingivalis strains was found to protect against the secondary spread of invasive strains, these results would seem to suggest that a specific antibody that could prevent invasion was produced to a common heat-stable (95°C for 10 min) antigen(s) found on different invasive and noninvasive P. gingivalis strains. Virulence factors responsible for pathogenicity may be produced to some degree by all P. gingivalis strains, and differences in the amounts or levels of activity of these factors may differentiate virulent from avirulent organisms (22, 36). The invasive response may also be multifactorial, such that an immune response produced to one component may be capable of neutralizing the entire invasive process. The exact mechanism or type of immune response responsible for protection is unknown; however, both chamber fluid and serum antibodies were opsonic for P. gingivalis when used with human polymorphonuclear leukocytes in our in vitro phagocytosis assay (unpublished data). Although P. gingivalis produces a number of potential virulence factors (6, 22, 31, 46), potent proteolytic activity is thought to be particularly relevant in the disease process (46). A P. gingivalis trypsinlike protease and a collagenase were described previously (31). P. gingivalis is characteristically resistant to phagocytosis (8), and this resistance is likely due at least in part to the proteolytic digestion and inactivation of the serum opsonin IgG and C3 (42, 43). Virulent P. gingivalis W83 and A7436 proteolytically digest C3, whereas avirulent P. gingivalis 33277 does not (7a, 43). Our recent studies indicate that antibody can neutralize P. gingivalis proteolytic activity, allowing C3b, C3bi, and immunoglobulin accumulation on W83 and A7436 surfaces, and consequently facilitate phagocytosis (7a). Although proteolytic enzymes may play a role in tissue destruction and the evasion of the host response, the specific role of these enzymes in invasiveness is still speculative (36). The degree of encapsulation of P. gingivalis has also been suggested to play a role in the ability of P. gingivalis to invade host tissue (30). Immunization with a purified polysaccharide antigen from P. gingivalis has been shown to protect mice from subsequent challenge with the homologous P. gingivalis strain in a murine abscess model (44). However, the ability of purified polysaccharide antigens from different P. gingivalis strains to protect mice from challenge with heterologous P. gingivalis strains was not
VOL. 60, 1992
examined in this study. The polysaccharide antigen appears to exhibit strain differences (45), and Schifferle et al. (45) have suggested that this antigen could be a serotype antigen used to define bacterial serotypes. The outer membrane proteins of P. gingivalis exhibit significant species and strain specificities (1, 25). Currently, P. gingivalis isolates are characterized by biotyping or serotyping; two biotypes based on catalase activity have been reported, and three serogroups have been described (18, 40). The strains used in the current study included serogroups A (381 and 33277) and B (W83). Serogroup A strains appear to share a 76-kDa protein, whereas serogroup C strains share a 54-kDa antigen (18). Loos et al. (29) recently characterized isolates of P. gingivalis by genomic DNA fingerprinting and identified 29 distinct DNA fingerprints. Noninvasive serogroup A strains 381 and 33277 belong to clonal type 1, and invasive serogroup B strains belong to clonal type 2. Our results seem to suggest that antigens common to different P. gingivalis serogroups and clonal types are responsible for invasion in the mouse model. The antigenic specificity of the systemic responses to P. gingivalis is obviously an area of great interest. Serum IgG antibodies to P. gingivalis whole cells, capsular antigen, outer membrane proteins, fimbriae, LPS, and the trypsinlike protease have been observed in periodontitis patients (5, 13, 14, 17, 24, 27, 35, 41, 49). Local IgG antibodies to P. gingivalis fimbriae have also been observed in the gingival tissue of adult periodontitis patients (38). In most of these studies, the detection of P. gingivalis-specific antibody was assessed with only one strain of P. gingivalis as the source of antigens in Western blots. However, Ebersole et al. (13) examined several different P. gingivalis strains with which antisera from periodontitis patients reacted and found similarities in the reactive antigens among the different strains. In this study, mouse antisera produced following immunization with heat-killed whole cells and challenge with live cells reacted with a number of common antigens on different P. gingivalis strains. In addition, antisera raised to different P. gingivalis strains reacted with common P. gingivalis antigens. A 43-kDa immunodominant antigen was the major antigen recognized by mouse antisera raised to four of the five strains of P. gingivalis used in this study. This antigen may be either the hemagglutinating adhesin (HA-Ag2) (2) or the major structural subunit of the fimbriae (fimbrillin) (50). HA-Ag2 is an antigen common to different P. gingivalis strains (2). In SDS-PAGE, HA-Ag2 migrates very close to 43 kDa (33), the apparent molecular mass reported for fimbrillin by Yoshimura et al. (50). Interestingly, a 44-kDa protein produced by P. gingivalis (not fimbrillin) was shown to be strongly immunoreactive when reacted with sera from adult periodontitis patients (28). It has been noted that the immune response to oral colonization is not necessarily static but may fluctuate during periodontal disease (11). Ebersole et al. (15, 16) have noted that elevated serum antibodies to periodontopathic bacteria are reflective of subgingival colonization and exist as a response to a bacterial infection at active disease sites. P. gingivalis can invade the gingival tissues in periodontal disease, and this invasive capacity has been suggested to be an important component of the pathogenesis of periodontal disease. Our results would seem to challenge this notion and would support the ability of P. gingivalis to colonize as opposed to its ability to invade as an important component in the proliferation and persistence of P. gingivalis. An estimation of actual CFU within chambers indicated that P. gingivalis was multiplying within chambers, with an upper limit of
INFLUENCE OF IMMUNIZATION ON P. GINGIVALIS
1453
1012 CFU/ml detected in chamber fluid from A7436-challenged animals (data not shown). This study indicates that local exposure to a single injection of killed homologous or heterologous invasive or noninvasive strains of P. gingivalis influences the pathologic progression of challenge with invasive strains. This effect is manifested as an inhibition of invasion, secondary lesions, and death but not colonization and bacterial division. Both local and systemic antibody responses were observed. Whether this antibody response contributes wholly or partly to the protective immune response is the subject of a current investigation in our laboratories. ACKNOWLEDGMENTS We thank Kenneth Maloney for technical assistance. This study was supported by Public Health Service grants DE09368 and DE07808 from the National Institute of Dental Research. REFERENCES 1. Bowden, G. H., and N. Nolette. 1990. Study of the antigenic relationships between strains of Bacteroides intermedius, B. melaninogenicus, P. corporis, and B. denticola revealed by immunoblotting with rabbit antisera. Can. J. Microbiol. 36:637648. 2. Chandad, F., and C. Mouton. 1990. Molecular size variation of hemagglutinating adhesion HA-Ag2, a common antigen of Bacteroides gingivalis. Can. J. Microbiol. 36:690-696. 3. Chen, P. B., L. B. Davern, R. Schifferle, and J. J. Zambon. 1990. Protective immunization against experimental Bacteroides (Porphyromonas)gingivalis infection. Infect. Immun. 58:3394-3400. 4. Chen, P. B., M. E. Neiders, S. J. Millar, H. S. Reynolds, and J. J. Zambon. 1987. Effect of immunization on experimental Bacteroides gingivalis infection in a murine model. Infect. Immun. 55:2534-2537. 5. Chen, C.-K. C., A. DeNardin, D. W. Dyer, R. J. Genco, and M. E. Neiders. 1991. Human immunoglobulin G antibody response to iron-repressible and other membrane proteins of Porphyromonas (Bacteroides)gingivalis. Infect. Immun. 59:2427-2433. 6. Chu, L., T. E. Bramanti, J. L. Ebersole, and S. C. Holt. 1991. Hemolytic activity in the periodontopathogen Porphyromonas gingivalis: kinetics of enzyme release and localization. Infect. Immun. 59:1932-1940. 7. Clark, W. B., I. Magnusson, J. E. Beem, J. M. Jung, R. G. Marks, and W. P. McArthur. 1991. Immune modulation of Prevotella intermedia colonization in squirrel monkeys. Infect. Immun. 59:1927-1931. 7a.Cutler, C. W., et al. Submitted for publication. 8. Cutler, C. W., J. R. Kalmar, and R. R. Arnold. 1991. Phagocytosis of virulent Porphyromonas gingivalis by human polymorphonuclear leukocytes requires specific immunoglobulin G. Infect. Immun. 59:2097-2104. 9. Cutler, C. W., J. R. Kalmar, and R. R. Arnold. 1991. Antibodydependent alternate pathway of complement activation in opsonophagocytosis of Porphyromonasgingivalis. Infect. Immun. 59:2105-2109. 10. Dahlen, G., and J. G. Slots. 1989. Experimental infections by Bacteroidesgingivalis in nonimmunized and immunized rabbits. Oral Microbiol. Immunol. 4:6-11. 11. Ebersole, J. L. 1990. Systemic humoral immune responses in periodontal disease. Oral Biol. Med. 1:283-331. 12. Ebersole, J. L., D. E. Frey, M. A. Taubman, D. J. Smith, S. S. Socransky, and A. C. R. Tanner. 1984. Serological identification of oral Bacteroides spp. by enzyme-linked immunosorbent assay. J. Clin. Microbiol. 19:639-644. 13. Ebersole, J. L., M. J. Steffen, and S. C. Holt. 1989. Antigenic specificities of human IgG antibody to B. gingivalis, abstr. 4. J. Dent. Res. 68:222. 14. Ebersole, J. L., M. A. Taubman, D. J. Smith, and D. E. Frey. 1986. Human immune responses to oral microorganisms: patterns of systemic antibody levels to Bacteroides species. Infect.
1454
GENCO ET AL.
Immun. 51:507-513. 15. Ebersole, J. L., M. A. Taubman, D. J. Smith, D. E. Frey, A. D. Haffajee, and S. S. Socransky. 1987. Human serum antibody responses to oral microorganisms. IV. Correlation with homologous infection. Oral Microbiol. Immunol. 2:53-59. 16. Ebersole, J. L., M. A. Taubman, D. J. Smith, and A. D. Haffajee. 1985. Effect of subgingival scaling on systemic antibody responses to oral microorganisms. Infect. Immun. 48:534-539. 17. Farida, R., M. Wilson, and L. Ivanyi. 1986. Serum IgG antibodies to lipopolysaccharides in various forms of periodontal disease in man. Arch. Oral Biol. 31:711-715. 18. Fisher, J. G., J. J. Zambon, and R. J. Genco. 1987. Identification of serogroup specific antigens among Bacteroides gingivalis components, abstr. 927. J. Dent. Res. 66:222. 19. Genco, C. A., C. W. Cutler, D. R. Kapczynski, K. Maloney, and R. R. Arnold. 1991. A novel mouse model to study the virulence of and host response to Porphyromonas (Bacteroides) gingivalis. Infect. Immun. 59:1255-1263. 20. Genco, R. J., and J. Slot. 1984. Host responses in peridontal diseases. J. Dent. Res. 63:441-451. 21. Gorg, A., W. Postel, J. Wisser, H. W. Schivara, and W. H. Boesken. 1985. Horizontal SDS electrophoresis in ultrathin pore-gradient gels for the analysis of urinary proteins. Sci. Tools 32:5-9. 22. Grenier, D., and D. Mayrand. 1987. Selected characteristics of pathogenic and nonpathogenic strains of Bacteroides gingivalis. J. Clin. Microbiol. 25:738-740. 23. Gunsolley, J. C., J. G. Tew, C. Gooss, D. R. Marshall, J. A. Burmeister, and H. A. Schenkein. 1990. Serum antibodies to periodontal bacteria. J. Periodontol. 61:412-419. 24. Ismaiel, M. O., J. Greenman, and C. Scully. 1988. Serum antibodies against the trypsin-like protease of Bacteroides gingivalis in periodontitis. J. Periodontal Res. 23:193-198. 25. Kennell, W., and S. C. Holt. 1990. Comparative studies of the outer membranes of Bacteroides gingivalis strains ATCC 33277, W50, W83, 381. Oral Microbiol. Immunol. 5:121-130. 26. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London)
227:680-685. 27. Lamster, I. B., R. Celenti, and J. L. Ebersole. 1990. The relationship of serum IgG antibody titers to periodontal pathogens to indicators of the host response in crevicular fluid. J. Clin. Periodontol. 17:419-425. 28. Laosrisin, N., K. Nakashima, and I. Ishikawa. 1990. Detection of Bacteroides gingivalis antigenic proteins by immunoblotting analysis. J. Periodontal Res. 61:261-268. 29. Loos, B. G., D. Mayrand, R. J. Genco, and D. P. Dickinson. 1990. Genetic heterogeneity of Porphyromonas (Bacteroides) gingivalis by genomic DNA fingerprinting. J. Dent. Res. 69: 1488-1493. 30. Mansheim, B. J., C. A. Solstad, and D. L. Kasper. 1978. Identification of subspecies-specific capsular antigen from Bacteroides melaninogenicus subspecies asaccharolyticus by immunofluorescence and electron microscopy. J. Infect. Dis.
138:736-741. 31. Mayrand, D., and S. C. Holt. 1988. Biology of asaccharolytic black-pigmented Bacteroides species. Microbiol. Rev. 52:134152. 32. McArthur, W. P., I. Magnusson, R. G. Marks, and W. B. Clark. 1989. Modulation of colonization by black-pigmented Bacteroides species in squirrel monkeys by immunization with Bacteroides gingivalis. Infect. Immun. 57:2313-2317. 33. Mouton, C., N. E. D. Deslauriers, and L. Lamy. 1991. The hemagglutinating adhesin HA-Ag2 of Bacteroides gingivalis is distinct from fimbrillin. Oral Microbiol. Immunol. 6:6-11.
INFECT. IMMUN.
34. Mouton, C., P. G. Hammond, J. Slots, and R. J. Genco. 1981. Serum antibodies to oral Bacteroides asaccharolyticus (Bacteroides gingivalis): relationship to age and periodontal disease. Infect. Immun. 31:182-192. 35. Naito, Y., K. Okuda, and I. Takazoe. 1987. Detection of specific antibody in adult human periodontitis sera to surface antigens of Bacteroides gingivalis. Infect. Immun. 55:832-834. 36. Neiders, M. E., P. B. Chen, H. Suido, H. S. Reynolds, J. J. Zambon, M. Shlossman, and R. J. Genco. 1989. Heterogeneity of virulence among strains of Bacteroides gingivalis. J. Periodontal Res. 24:192-198. 37. Nisengard, R., D. Blann, L. Zelonis, K. McHenry, H. Reynolds, and J. Zambon. 1989. Effects of immunization with B. macacae on induced periodontitis-preliminary findings. Immunol. Invest. 18:225-237. 38. Ogawa, T., M. L. McGhee, Z. Moldoveanu, S. Hamada, J. Mestecky, J. R. McGhee, and H. Kiyono. 1989. Bacteroidesspecific IgG and IgA subclass antibody-secreting cells isolated from chronically inflamed gingival tissues. Clin. Exp. Immunol. 76:103-110. 39. Okuda, K., T. Kato, Y. Naito, I. Takazoe, Y. Kikuchi, T. Nakamura, T. Kiyoshige, and S. Sasaki. 1989. Protective efficacy of active and passive immunizations against experimental infection with Bacteroides gingivalis in ligated hamsters. J. Dent. Res. 67:807-811. 40. Parent, R., C. Mouton, L. Lamonde, and D. Bouchard. 1986. Human and animal serotypes of Bacteroides gingivalis defined by crossed immunoelectrophoresis. Infect. Immun. 51:909-918. 41. Schenck, K. 1985. IgG, IgA and IgM serum antibodies against lipopolysaccharide from Bacteroides gingivalis in periodontal health and disease. J. Periodontal Res. 20:368-377. 42. Schenkein, H. A. 1988. The effect of periodontal proteolytic Bacteroides species on proteins of the human complement system. J. Periodontal Res. 23:187-192. 43. Schenkein, H. A. 1989. Failure of Bacteroides gingivalis W83 to accumulate bound C3 following opsonization with serum. J. Periodontal Res. 24:20-27. 44. Schifferle, R. E., P. B. Chen, L. B. Davern, A. Aguirre, R. J. Genco, and M. J. Levine. 1990. Immunogenicity of polysaccharide from Bacteroidesgingivalis, abstr. 73. J. Dent. Res. 69:118. 45. Schifferle, R. E., R. S. Molakala, J. J. Zambon, R. J. Genco, and M. J. Levine. 1989. Characterization of polysaccharide antigen from Bacteroides gingivalis. J. Immunol. 143:3035-3042. 46. Slots, J., and R. J. Genco. 1984. Microbial pathogenicity of black-pigmented Bacteroides species, Capnocytophaga species and Actinobacillus actinomycetemcomitans in human periodontal disease: virulence factors in colonization, survival, and tissue destruction. J. Dent. Res. 63:412-421. 47. Turner, D. W., G. J. Dai, B. Merrell, and J. P. Robinson. 1989. Serum and gingival tissue antibody levels to oral microbial antigens in human chronic adult periodontitis. Microbios 60: 133-140. 48. Winkler, J. R., V. Matareset, C. I. Hoover, R. H. Kramer, and P. A. Murray. 1987. An in vitro model to study bacterial invasion of periodontal tissues. J. Periodontol. 59:40-45. 49. Yoshimura, F., T. Sugano, M. Kawanami, H. Kato, and T. Suziki. 1987. Detection of specific antibodies against fimbriae and membrane proteins from the oral anaerobe Bacteroides gingivalis in patients with periodontal disease. Microbiol. Immunol. 31:935-941. 50. Yoshimura, F., K. Takahashi, Y. Nodasaka, and T. Suzuki. 1984. Purification and characterization of a novel type of fimbriae from the oral anaerobe Bacteroides gingivalis. J. Bacteriol. 160:949-957.
