INFECTION AND IMMUNITY, JUlY 1991, p. 2427-2433 0019-9567/91/072427-07$02.00/0 Copyright © 1991, American Society for Microbiology
Vol. 59, No. 7
Human Immunoglobulin G Antibody Response to Iron-Repressible and Other Membrane Proteins of Porphyromonas (Bacteroides) gingivalis CHIH-KUANG CASEY CHEN,' ANN DENARDIN,1 DAVID W. DYER,2 ROBERT J. AND MIRDZA E. NEIDERS3*
GENCO,1
Departments of Oral Biology,' Microbiology,2 and Stomatology and Interdisciplinary Sciences,3 State University of New York at Buffalo, Buffalo, New York 14214 Received 17 December 1990/Accepted 26 April 1991
The human immunoglobulin G (IgG) immune response against Porphyromonas (Bacteroides) gingivalis A7A1-28 iron-repressible membrane proteins (IRMPs) and other membrane proteins was examined by immunoblot analysis. Thirty sera from patients with adult periodontitis and 30 sera from periodontally healthy subjects were included. Iron limitation of P. gingivalis was achieved by growing bacteria in brain heart infusion broth supplemented with protoporphyrin IX and 250 ,uM aL,a'-dypyridyl, a ferrous iron chelator. Ironsufficient growth was achieved by growing bacteria in the same medium without a,a'-dypyridyl. Human sera, in particular those from patients with periodontitis who exhibited high levels of IgG against whole cells of P. gingivalis A7A1-28 in serum in an enzyme-linked immunosorbent assay (ELISA), commonly reacted with five membrane proteins with apparent molecular masses of 80, 67.5, 51, 40.5, and 28 kDa and four IRMPs of 46, 43, 37.5, and 22 kDa. More than 80% of the sera from patients with periodontitis and high levels of IgG against strain A7A1-28 in serum by ELISA reacted with the 46-, 43-, and 37.5-kDa IRMPs, and 40% of these subjects expressed immunoreactivity against the 22-kDa IRMP. Sera from patients with periodontitis and low levels of IgG against strain A7A1-28 in serum by ELISA and sera from periodontally healthy subjects exhibited less immunoreactivity against IRMPs and the five membrane proteins of P. gingivalis. The present study indicates that P. gingivalis IRMPs are immunogenic and that these proteins are expressed in vivo. A successful pathogen must be able to enter a host, proliferate in the host tissue, and resist host defense mechanisms. Various strategies are utilized by microorganisms to adapt to the diverse environments encountered during infection (4, 17). Bacteria possess regulatory systems which sense and respond to environmental factors such as pH, temperature, and the availability of various nutrients (4, 17). Thus, the antigenic characteristics, structural properties, or virulence factors of bacteria in vivo are often different from those of bacteria grown in vitro. Iron is an essential growth factor for most, if not all, microorganisms. In human tissue and body fluids, free iron is not readily available to bacteria. Iron is stored intracellularly in the form of heme, ferritin, or hemosiderin. Extracellular iron is sequestered from bacteria by host iron-binding proteins such as transferrin and lactoferrin (12, 24). Withholding iron from bacteria plays an important role in host resistance to bacterial infections. Thus, it is not surprising that many virulence determinants are regulated by iron availability. For example, low iron concentrations increase toxin synthesis by Pseudomonas aeruginosa, Corynebacterium diphtheriae, Shigella dysenteriae, Clostridium perfringens, Clostridium tetani, Escherichia coli, and Serratia marcescens (3, 7). Virulent bacteria often have specialized iron uptake systems that remove iron from transferrin and lactoferrin; these iron uptake systems are important for the ability of bacteria to cause disease (5, 6, 23). Enteric bacteria, such as Salmonella and Escherichia species, acquire the metal from ironbinding proteins by secreting siderophores which bind iron at high affinity and remove iron from transferrin and lacto-
*
ferrin (19). Other bacterial species, such as Neisseria gonorrhoeae, acquire iron through cell surface receptors for transferrin and lactoferrin (2, 16). In both cases, certain outer membrane proteins (commonly referred to as ironrepressible membrane proteins [IRMPs]), whose synthesis is repressed by high iron concentrations, are expressed and may play a role in iron assimilation (19). Porphyromonas (Bacteroides) gingivalis is a putative periodontal pathogen frequently found subgingivally in patients with adult periodontitis (21). A recent study in our laboratory showed that iron is an essential growth factor for P. gingivalis (1). In that study, it was found that P. gingivalis expressed IRMPs with molecular masses of 43 and 22 kDa during iron-limited growth (1). These proteins may play a role in iron acquisition by P. gingivalis. Various studies have shown that iron-regulated proteins are immunogenic and may serve as targets of the host immune response against infections (10, 20, 22). The objectives of the present study were to determine whether IRMPs are expressed in vivo and whether patients with destructive periodontal disease respond immunologically to IRMPs. This was done by examining sera from patients with adult periodontitis and from periodontally healthy subjects for immunoglobulin G (IgG) antibodies to P. gingivalis IRMPs. MATERIALS AND METHODS
Subject groups. The characteristics of the subject groups listed in Table 1. Serum samples and clinical information from 30 patients with adult periodontitis (ages 30 to 53) and 30 periodontally healthy subjects (ages 20 to 50) were provided by the Periodontal Research Center at the State University of New York at Buffalo. The patients with are
Corresponding author. 2427
2428
CHEN ET AL.
INFECT. IMMUN.
TABLE 1. Subject groups Group
No. of subjects in high- and lowlevel subgroupsa
Mean ± SD level of IgG against A7A1-28 P. gingivalis (ELISA U) in serum
Periodontally healthy subjects
High, 2 Low, 28
42 ± 8.5 17.1 ± 4.7
Patients with adult periodontitis
High, 13 Low, 17
114.3 ± 76.9 21.9 ± 6.1
andgroups"
a There were 30 persons in each group. The high-level subgroup consisted of subjects with more than 34.7 ELISA U (mean number of ELISA units of the healthy group plus two standard deviations) of IgG against P. gingivalis A7A1-28 in serum. Subjects with less than 34.7 ELISA U were assigned to the low-level subgroup.
periodontitis exhibited at least two interproximal sites with probing attachment loss of 6 mm or greater and at least one site with a probing pocket depth of 5 mm or more. None of the periodontally healthy subjects exhibited probing attachment loss greater than 2 mm. IgG antibody levels against formalinized whole cells of P. gingivalis A7A1-28 in serum were determined by a modification of the procedures of Mouton et al. (18) and Ebersole et al. (9). A detailed description of the enzyme-linked immunosorbent assay (ELISA) procedure will be published elsewhere (8). ELISA reactivity was calculated by comparison to a reference pool of serum that was assigned an arbitrary value of 100 ELISA U/ml. Bacterial strain and growth conditions. P. gingivalis A7A1-28 (ATCC 53977) was chosen for the present study as it was demonstrated that this strain expressed two IRMPs of 43 and 22 kDa during iron-limited growth (1). The porphyrin requirement of this strain was satisfied by adding iron-free protoporphyrin IX rather than hemin to the medium (1). Under these conditions, P. gingivalis can be iron limited by adding the ferrous iron chelator ot,ot'-dypyridyl to the medium (1). Prior to use, strain A7A1-28 was grown in protoporphyrin IX-supplemented (hemin-free) medium for at least three passages. This regimen depleted internal heme stores so that the organism could be subsequently iron limited. To obtain exponential-phase bacteria grown in iron-sufficient medium, freshly thawed cells of strain A7A1-28 were inoculated into 10 ml of modified half-strength brain heart infusion broth (modified 0.5x BHI) containing 18.5 g of BHI (Difco Laboratories, Detroit, Mich.) per liter, 0.5% yeast extract (Difco), 2.3 ,ug of protoporphyrin IX disodium salt (Sigma Chemical Co., St. Louis, Mo.) per ml, and 1.0 ,ug of vitamin K1 (Sigma) per ml. The cultures were incubated at 37°C in an atmosphere of 85% N2, 10% H2, and 5% CO2 (1025/1029 Anaerobic System; Forma Scientific, Marietta, Ohio) for 24 h. This starter culture was diluted with fresh modified 0.5x BHI (3% inoculum) and incubated until the culture was in mid-exponential growth. To obtain iron-limited exponentialphase bacteria, the starter culture was inoculated into modified 0.5x BHI (5% inoculum) supplemented with 250 ,uM a,a'-dypyridyl (Sigma) and grown to the mid-exponential phase. This concentration of at,a'-dypyridyl inhibited bacterial growth by more than 50%. Growth was monitored by measuring the optical density at 660 nm, and bacterial purity was assessed by Gram's stain. The final immunoblot data (described below) were obtained by examining all sera with membranes obtained from one set of cultures; however,
similar patterns were seen with other batched cultures. The iron-sufficient culture was harvested after 20 h of growth and had an optical density at 660 nm of 0.35, and the iron-limited culture was harvested after 29 h of growth and had an optical density at 660 nm of 0.1. Bacterial membrane preparation. Bacterial cells were harvested by centrifugation at 15,000 x g for 1 h at 4°C. The cell pellet was suspended in 10 ml of distilled water and disrupted by one passage through a French pressure cell (SLM Instruments, Inc., Urbana, Ill.) at 32,000 lb/in2. Unbroken cells and large debris were removed by centrifugation at 7,000 x g for 10 min. The membrane fractions in the supernatant were collected by ultracentrifugation at 100,000 x g for 1 h at 4°C. Following ultracentrifugation, the membranes were suspended in distilled water and the protein concentration was determined by a modified Lowry assay (15). SDS-PAGE and immunoblot analysis. Sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis (PAGE) was performed by using a modified Laemmli gel system (14) with a gel 0.75 mm thick, with a stacking gel consisting of 3.8% acrylamide and 0.1% bisacrylamide and a separating gel consisting of 12% acrylamide and 0.32% bisacrylamide. The bacterial membrane preparations were diluted with an equal volume of 2x sample buffer (0.125 M Tris-Cl [pH 6.8], 8% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.006% bromophenol blue) and boiled at 100°C for 10 min before electrophoresis. Insoluble materials were removed by centrifugation for 3 min at 10,000 rpm in an Eppendorf 5415 C centrifuge (Brinkmann Instruments, Inc., Westbury, N.Y.). One hundred twenty micrograms of membrane proteins was applied to a trough of a preparative gel, and electrophoresis was carried out at a constant 150 V for 2 h and 20 min on a vertical slab gel apparatus (Tall Mighty Small Vertical Slab Unit; Hoefer Scientific Instruments, San Francisco, Calif.). After SDS-PAGE, the sample was electrotransferred at 180 mA (2.5 mA/cm2) for 30 min to a nitrocellulose membrane (Bio-Rad Laboratories, Richmond, Calif.) by using a PolyBlot Electrotransfer System (American Bionetics, Inc., Hayward, Calif.) and the buffer system of Kyshe-Anderson (13). After transfer, the gel was stained with Coomassie brilliant blue R250 to determine the transfer efficiency. A 1-cm strip of nitrocellulose paper containing the molecular weight standards and a portion of the bacterial antigens was cut from the blot and stained with AuroDye forte as recommended by the manufacturer (Amersham International plc., Amersham, United Kingdom). The remaining portion of the nitrocellulose membrane was blocked with buffer containing 0.2 M Tris-HCl (pH 7.5)-0.5 M NaCl (TBS) and 0.05% Tween 20 (Fisher Scientific, Fairlawn, N.J.) (TBS-T) for 30 min at room temperature. This was followed by overnight incubation with a 1:100 dilution of human serum in TBS-T using a PR 150 mini-DecaProbe system (Hoefer Scientific Instruments). Subsequently, the nitrocellulose membrane was washed three times with TBS-T and incubated with a 1:1,000 dilution of horseradish peroxidase-conjugated goat anti-human IgG (Southern Biotechnology Associates, Inc., Birmingham, Ala.) in TBS-T at room temperature on a rocking platform for 1 h. The nitrocellulose membrane was then washed twice with TBS-T and once with TBS and developed by addition of a freshly prepared mixture of 100 ml of TBS containing 1 ml of 3% hydrogen peroxide and 60 mg of 4-chloro-1-naphthol (Bio-Rad Laboratories) dissolved in 20 ml of cold methanol. After development, the membrane was rinsed with distilled water and matched with the strip stained with AuroDye forte. A permanent record was obtained by using a Polaroid MP-4 Land Camera (Polaroid,
VOL. 59, 1991
HUMAN ANTIBODY RESPONSE TO P. GINGIVALIS IRMPs
Cambridge, Mass.) with a high-speed print (Polaroid 667 Professional). The SDS-PAGE and electroblot procedures used to assess the total membrane protein profile of P. gingivalis A7A1-28 were the same as those described above, except that 25 ,ug of the membrane proteins was applied per lane in a gel with 10 wells. After electroblotting, the nitrocellulose membrane containing the bacterial proteins was stained with AuroDye forte. With the electroblotting conditions described above, a nearly complete transfer of membrane proteins to nitrocellulose was achieved. Six serum samples from patients with adult periodontitis were used in pilot studies to maximize immunoblot reactivity (data not shown). In testing different membranes for immunoblotting, we found that immunoperoxidase stains on Immobilon-P membrane (Millipore Corp., Bedford, Mass.) had a tendency to fade rapidly after development. As this did not happen with nitrocellulose, it was selected as the membrane for immunoblotting. Various combinations of Tween 20, nonfat dry milk, and bovine serum albumin of different concentrations were tried as blocking reagents. TBS-T maximally reduced the nonspecific background and did not interfere with specific binding of human IgG with membrane antigens. Other blocking reagents either gave high background activity on nitrocellulose membranes or interfered with specific binding of IgG to antigens. TBS alone was used for the final wash prior to development because Tween 20 interfered with color development. The appropriate dilutions of human sera and the peroxidase-conjugated second antibody were chosen after testing various dilutions of each (1:50 to 1:200 for human sera and 1:1,000 to 1:2,000 for the second antibody) in immunoblotting. Blots incubated overnight with human sera maximized immunoreactivity compared with incubations of 1 or 2 h. Data analysis. From the immunoblot analysis, we found a number of P. gingivalis A7A1-28 membrane proteins that reacted with 6 sera in a pilot study and 60 sera in the full study. The prevalence and intensity of immunoreactivity against nine of these membrane proteins were chosen for final analysis. Five of the membrane proteins were chosen on the basis of high frequency of reactivity with human sera. The remaining four membrane proteins were chosen because they were IRMPs and were frequently recognized by human sera as well. Two of these IRMPs, with molecular masses of 43 and 22 kDa, were described previously (1). Other membrane proteins reacted infrequently with IgG in human serum. After selection of these nine membrane proteins, the immunoreactivities of sera against individual membrane proteins by immunoblotting were scored independently by three investigators (barely visible, ±; visible, +; strong reaction, + +) on the basis of Polaroid photographs. A highly reactive serum was used as a positive control.
RESULTS Membrane proteins of P. gingivalis A7A1-28 grown in iron-sufficient and iron-limited conditions. Figure 1 shows the transblotted membrane proteins of strain A7A1-28 grown in iron-limited and iron-sufficient media stained with proteinspecific AuroDye forte. Both samples contained more than 15 protein bands, although certain bands were not easily identified (particularly protein bands migrating in the 30- to 14-kDa region). Among these membrane proteins, four IRMPs and five membrane proteins with which human sera
2429
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FIG. 1. Total membrane protein profiles of P. gingivalis A7A1-28 grown in iron-limited (lane 1) and iron-sufficient (lane 2) conditions. Twenty-five micrograms of membrane proteins was applied to each lane, and the membranes were separated by SDSPAGE, transferred to nitrocellulose, and stained with AuroDye forte. The apparent molecular masses of the membrane proteins selected for immunoblot analysis are indicated on the left. The IRMPs are indicated by arrows. The sizes of the molecular mass standards (Low Molecular Weight Calibration Kit; Phamacia LKB Biotechnology Inc., Piscataway, N.J.) in lane 3 are shown in kilodaltons on the right.
frequently reacted were selected for further data analysis. The molecular masses of these proteins are indicated in kilodaltons. Immunoblot analysis. Immunoblot analysis of human serum IgG against P. gingivalis A7A1-28 membrane proteins revealed that sera from patients with periodontitis generally showed reactivity with higher numbers of membrane proteins and also with higher intensity than did sera from periodontally healthy subjects. Of the 30 periodontally healthy subjects, 8 showed no detectable immunoreactivity against any of the nine selected membrane proteins of P. gingivalis A7A1-28 by immunoblotting (data not shown). Within the adult periodontitis group, sera from those individuals with high levels of IgG against strain A7A1-28 in serum by ELISA showed higher levels of immunoreactivity than those with low levels by ELISA. Negative controls, including blots probed with human sera without the peroxidase-conjugated goat anti-human IgG second antibody or blots probed with the second antibody without human sera, showed no reactivity (data not shown). Figures 2 and 3 show two sets of representative immunoblots of P. gingivalis A7A1-28 membranes probed with sera
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20.lb 1 2 3 4 5 6 7 8 FIG. 2. Panels A and B show a set of representative immunoblots of P. gingivalis A7A1-28 membranes reacted with sera from patients with adult periodontitis. Panel A contained membranes prepared from bacteria grown in the iron-sufficient condition, while panel B contained membranes from cells grown in the iron-limited condition. In each panel, lane 1 contained molecular size standards and lane 2 contained the membrane proteins; both were stained with AuroDye forte. Corresponding lanes in panels A and B were reacted with the same serum sample from five patients with adult periodontitis (lanes 3 to 7), and lane 8 was probed with the positive control. The sera used in lanes 3, 4, 6, and 7 were from patients with high levels of IgG against strain A7A1-28 in serum, and that in lane 5 was from a patient with a low level by ELISA. The representative staining intensities of +, +, and + + are indicated on the right. The IRMPs are indicated by arrowheads. The sizes of the molecular mass standards are given in kilodaltons next to lane 1. The nine selected P. gingivalis A7A1-28 membrane proteins (including IRMPs) are indicated to the left of each panel, and their molecular masses are shown.
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1 2 3 4 FIG. 3. Immunoblot analysis of human sera from patients with adult periodontitis against P. gingivalis A7A1-28 membrane proteins. Panel A contained membranes prepared from bacteria grown in the iron-sufficient condition, while panel B contained membranes from cells grown in the iron-limited condition. In each panel, lane 1 contained molecular mass standards and lane 2 contained the membrane proteins; both were stained with AuroDye forte. Lanes 3 to 6 of both panels were probed with sera from patients with low IgG levels against strain A7A1-28 by ELISA, lane 7 was reacted with serum from a patient with a high IgG level against the same strain by ELISA, and lane 8 was reacted with the positive control serum. The IRMPs are indicated by arrowheads. The sizes of the molecular mass standards are shown in kilodaltons next to lane 1. The nine selected P. gingivalis A7A1-28 membrane proteins (including IRMPs) are indicated to the left of each panel, and their molecular masses are shown.
VOL. 59, 1991
2431
HUMAN ANTIBODY RESPONSE TO P. GINGIVALIS IRMPs
from patients with periodontitis. There was wide variation among individual serum samples in the specific membrane proteins recognized and in the intensity of the reactivity. For example, in Fig. 2, some serum samples exhibited strong reactivity against a 51-kDa protein while others reacted with the 40.5-kDa protein. The serum sample tested in Fig. 2, lane 7, demonstrated reactivity against all nine selected membrane proteins, as well as other membrane proteins, in particular, those in the 30- and 20-kDa regions. Lanes 3, 4, 6, and 7 in Fig. 2 and lane 7 in Fig. 3 were tested with sera from patients with periodontitis and high levels of IgG against P. gingivalis A7A1-28 in serum by ELISA. Sera from this group of patients generally showed higher immunoreactivity against membrane proteins than did sera from patients with low levels of IgG against this strain in serum by ELISA (lanes 5 in Fig. 2 and 3 to 6 in Fig. 3). Figure 4 shows a set of representative immunoblots tested with sera from periodontally healthy subjects. These sera generally reacted with fewer membrane proteins, and the reactivity was generally at a lower level. IRMPs of P. gingivalis A7A1-28 were among the most commonly recognized membrane proteins in immunoblotting with sera from patients with periodontitis. For example, several sera used for Fig. 2 showed strong reactivity against the 46-, 43-, and 37.5-kDa IRMPs (Fig. 2B, lanes 3, 4, 6, and 7), and in Fig. 3, a serum sample (lane 5) showed distinct immunoreactivity against the 22-kDa IRMP. Patterns of serum reactivity against individual membrane proteins. The pattern and intensity of reactivity of each serum sample were estimated independently by three investigators. The tabulated data were organized into the following three subject categories: (i) patients with adult periodontitis and high levels of IgG against P. gingivalis A7A1-28 in serum by ELISA, (ii) patients with adult periodontitis and low levels of IgG against strain A7A1-28 in serum by ELISA, and (iii) periodontally healthy subjects. The percentages of serum samples in these three subject groups that reacted with the nine selected membrane proteins are tabulated graphically in Fig. 5. More than half of the serum samples from patients with adult periodontitis with high IgG levels by ELISA reacted with the nine selected membrane proteins. IRMPs were among the membrane proteins of P. gingivalis most frequently recognized by IgG in human sera. More than 80% of the sera from patients with periodontitis and high levels of IgG against strain A7A1-28 in serum by ELISA reacted with 46-, 43-, and 37.5-kDa IRMPs of strain A7A128, while 40% of the sera from the same group reacted with the 22-kDa IRMP. The P. gingivalis A7A1-28 membrane proteins were also recognized, but to a lesser degree, by sera from patients with periodontitis and low levels of IgG against strain A7A1-28 in serum by ELISA and sera from periodontally healthy subjects. DISCUSSION Our previous studies indicated that P. gingivalis A7A1-28 expressed two IRMPs of 43 and 22 kDa (1). In the present study, we observed that these proteins are immunogenic and were frequently recognized by sera from patients with periodontitis. We also identified two additional IRMPs (46 and 37.5 kDa) which were recognized by sera examined in the present study. Since IgG antibodies against IRMPs are found in sera from patients with periodontitis, many of whom may be infected with P. gingivalis, the data suggest that IRMPs are expressed in vivo. In addition, the present study showed that a significant portion of the human serum IgG immune
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2432
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ACKNOWLEDGMENTS This work was supported by USPHS grants DE 08240, DE 04898, DE 52559, and DE 07034. We thank Mary Bayers-Thering for assistance in immunoblot
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response against P. gingivalis in patients with adult periodontitis was directed against IRMPs. Studying the critical antigens expressed in ivivo, which response, is may serve as targets of an effective host immune response, IS important for understanding the pathogenic me chanisms of bacterial infections. Furthermore, development of vaccines against such antigens remains a distinct possibil ity. Various studies have demonstrated host immune respoi nses against iron-regulated proteins (10, 11, 20). This immu ne response may have important consequences for bacterial pathogenesis. Antibody against P. aeruginosa iron-regulzated protein has been shown to inhibit iron uptake by the o0 rganism and in mice confer protection in certain experimental infecti ions that P gingivalis (22). In the present study, we found A7A1-28 IRMPs were among the major target an tigens of the human humoral immune response. Although the significance of this immune response against P. gingivalis is unknown, it may protect patients with periodontitis from further periodontal destruction. Since IRMPs of strain A7 A1-28 were recognized at high frequencies by the sera exanr nined in this study, these proteins may be structurally coriserved and share antigenic cross-reactivity among serologic variants of P. gingivalis. If the human immune response aigainst ironrepressible proteins is protective, then this anti Lgenic crossreactivity may be advantageous in developin~ g a vaccine against P. gingivalis infections. In conclusion, we found nine P. gingivalis A7 A1-28 membrane proteins that consistently reacted with senum antibody from patients with adult periodontitis. Four oif these nine membrane proteins were regulated by iron avail lability. The in ViVO data suggest that P. gingivalis IRMPs were expre and that a significant portion of the human I.gG immune response to P. gingivalis is directed at ironi-repressible proteins.
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REFERENCES Barua, P. K., D. W. Dyer, and M. E. Neiders. 1990. Effect of iron limitation on Bacteroides gingivalis. Oral Microbiol. Immunol. 5:263-268. Blanton, K. J., G. D. Biswas, J. Tsai, J. Adams, D. W. Dyer, S. M. Davis, G. G. Koch, P. K. Sen, and P. F. Sparling. 1990. Genetic evidence that Neisseria gonorrhoeae produces specific receptors for transferrin and lactoferrin. J. Bacteriol. 172:52255235. Braun, V. 1985. Iron supply as a virulence factor, p. 139-170. In G. G. Jackson and H. Thomas (ed.), The pathogenesis of bacterial infections. Springer-Verlag KG, Berlin. Brown, M. R., and P. Williams. 1985. The influence of environment on envelope properties affecting survival of bacteria in infections. Annu. Rev. Microbiol. 39:527-556. Bullen, J. J. 1981. The significance of iron in infection. Rev. Infect. Dis. 3:1127-1138. Bullen, J. J., H. J. Rogers, and E. Griffiths. 1978. Role of iron in bacterial infection. Curr. Top. Microbiol. Immunol. 80:1-35. Crosa, J. H. 1987. Bacterial iron metabolism, plasmids and other virulence factors, p. 168-176. In J. J. Bullen and E. Griffiths (ed.), Iron and infection. John Wiley & Sons, Inc., New York. DeNardin, A., et al. Unpublished data. Ebersole, J. L., D. E. Frey, M. A. Taubman, and D. J. Smith. 1980. An ELISA for measuring serum antibodies to Actinobacillus actinomycetemcomitans. J. Periodont. Res. 15:621632. Fernandez-Beros, M. E., C. Gonzalez, M. A. McIntosh, and F. C. Cabello. 1989. Immune response to the iron-deprivationinduced proteins of Salmonella typhi in typhoid fever. Infect. Immun. 57:1271-1275. Fohn, M. J., T. A. Mietzner, T. W. Hubbard, S. A. Morse, and E. W. Hook. 1987. Human immunoglobulin G antibody response to the major gonococcal iron-regulated protein. Infect. Immun. 55:3065-3069. Griffiths, E. 1987. Iron in biological systems, p. 1-25. In J. J. Bullen and E. Griffiths (ed.), Iron and infection. John Wiley & Sons, Inc., New York. J. 1984. Electroblotting multiple gels: a Kyshe-Anderson, simple apparatus without buffer tank forofrapid transfer of proteins from polyacrylamide to nitrocellulose. J. Biochem. Biophys. Methods 10:203-209. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685.
15. Markwell, M. A. K., S. M. Haas, L. L. Bieber, and N. E.
16.
Tolbert. 1978. A modification of the Lowry procedure to simplify protein determination in membrane and lipopolysaccharide samples. Anal. Biochem. 87:206-210. McKenna, W. R., P. A. Mickelsen, P. F. Sparling, and D. W. Dyer. 1988. Iron uptake from lactoferrin and transferrin by
Neisseria gonorrhoeae. Infect. Immun. 56:785-791. 17. Miller, J. F., J. J. Mekalanos, and S. Falkow. 1989. Coordinate regulation and sensory transduction in the control of bacterial virulence. Science 243:916-922. 18. Mouton, C., P. G. Hammond, J. Slots, and R. J. k,enco. 1981. Serum antibodies to oral Bacteroides asaccharolyticus (Bacteroides gingivalis): relationship to age and periodontal disease. Infect. Immun. 31:182-192.
19. Neilands, J. B. 1982. Microbial envelope proteins related to iron. Annu. Rev. Microbiol. 36:285-309. 20. Shand, G. H., H. Anwar, J. Kadurugamuwa, M. R. W. Brown, S. H. Silverman, and J. Meiling. 1985. In vivo evidence that bacteria in urinary tract infection grow under iron-restricted
VOL. 59, 1991
HUMAN ANTIBODY RESPONSE TO P. GINGIVALIS IRMPs
conditions. Infect. Immun. 48:35-39. 21. Socransky, S. S., and A. D. Haffajee. 1990. Microbial risk factors for destructive periodontal diseases, p. 79-90. In J. D. Bader (ed.), Risk assessment in dentistry. University of North Carolina Dental Ecology, Chapel Hill, N.C. 22. Sokol, P. A., and D. E. Woods. 1986. Characterization of
2433
antibody to the ferripyochelin-binding protein of Pseudomonas aeruginosa. Infect. Immun. 51:896-900. 23. Weinberg, E. D. 1974. Iron and susceptibility to infectious disease. Science 184:952-956. 24. Weinberg, E. D. 1978. Iron and infection. Microbiol. Rev. 42:45-66.
Vol. 59, No. 7
Human Immunoglobulin G Antibody Response to Iron-Repressible and Other Membrane Proteins of Porphyromonas (Bacteroides) gingivalis CHIH-KUANG CASEY CHEN,' ANN DENARDIN,1 DAVID W. DYER,2 ROBERT J. AND MIRDZA E. NEIDERS3*
GENCO,1
Departments of Oral Biology,' Microbiology,2 and Stomatology and Interdisciplinary Sciences,3 State University of New York at Buffalo, Buffalo, New York 14214 Received 17 December 1990/Accepted 26 April 1991
The human immunoglobulin G (IgG) immune response against Porphyromonas (Bacteroides) gingivalis A7A1-28 iron-repressible membrane proteins (IRMPs) and other membrane proteins was examined by immunoblot analysis. Thirty sera from patients with adult periodontitis and 30 sera from periodontally healthy subjects were included. Iron limitation of P. gingivalis was achieved by growing bacteria in brain heart infusion broth supplemented with protoporphyrin IX and 250 ,uM aL,a'-dypyridyl, a ferrous iron chelator. Ironsufficient growth was achieved by growing bacteria in the same medium without a,a'-dypyridyl. Human sera, in particular those from patients with periodontitis who exhibited high levels of IgG against whole cells of P. gingivalis A7A1-28 in serum in an enzyme-linked immunosorbent assay (ELISA), commonly reacted with five membrane proteins with apparent molecular masses of 80, 67.5, 51, 40.5, and 28 kDa and four IRMPs of 46, 43, 37.5, and 22 kDa. More than 80% of the sera from patients with periodontitis and high levels of IgG against strain A7A1-28 in serum by ELISA reacted with the 46-, 43-, and 37.5-kDa IRMPs, and 40% of these subjects expressed immunoreactivity against the 22-kDa IRMP. Sera from patients with periodontitis and low levels of IgG against strain A7A1-28 in serum by ELISA and sera from periodontally healthy subjects exhibited less immunoreactivity against IRMPs and the five membrane proteins of P. gingivalis. The present study indicates that P. gingivalis IRMPs are immunogenic and that these proteins are expressed in vivo. A successful pathogen must be able to enter a host, proliferate in the host tissue, and resist host defense mechanisms. Various strategies are utilized by microorganisms to adapt to the diverse environments encountered during infection (4, 17). Bacteria possess regulatory systems which sense and respond to environmental factors such as pH, temperature, and the availability of various nutrients (4, 17). Thus, the antigenic characteristics, structural properties, or virulence factors of bacteria in vivo are often different from those of bacteria grown in vitro. Iron is an essential growth factor for most, if not all, microorganisms. In human tissue and body fluids, free iron is not readily available to bacteria. Iron is stored intracellularly in the form of heme, ferritin, or hemosiderin. Extracellular iron is sequestered from bacteria by host iron-binding proteins such as transferrin and lactoferrin (12, 24). Withholding iron from bacteria plays an important role in host resistance to bacterial infections. Thus, it is not surprising that many virulence determinants are regulated by iron availability. For example, low iron concentrations increase toxin synthesis by Pseudomonas aeruginosa, Corynebacterium diphtheriae, Shigella dysenteriae, Clostridium perfringens, Clostridium tetani, Escherichia coli, and Serratia marcescens (3, 7). Virulent bacteria often have specialized iron uptake systems that remove iron from transferrin and lactoferrin; these iron uptake systems are important for the ability of bacteria to cause disease (5, 6, 23). Enteric bacteria, such as Salmonella and Escherichia species, acquire the metal from ironbinding proteins by secreting siderophores which bind iron at high affinity and remove iron from transferrin and lacto-
*
ferrin (19). Other bacterial species, such as Neisseria gonorrhoeae, acquire iron through cell surface receptors for transferrin and lactoferrin (2, 16). In both cases, certain outer membrane proteins (commonly referred to as ironrepressible membrane proteins [IRMPs]), whose synthesis is repressed by high iron concentrations, are expressed and may play a role in iron assimilation (19). Porphyromonas (Bacteroides) gingivalis is a putative periodontal pathogen frequently found subgingivally in patients with adult periodontitis (21). A recent study in our laboratory showed that iron is an essential growth factor for P. gingivalis (1). In that study, it was found that P. gingivalis expressed IRMPs with molecular masses of 43 and 22 kDa during iron-limited growth (1). These proteins may play a role in iron acquisition by P. gingivalis. Various studies have shown that iron-regulated proteins are immunogenic and may serve as targets of the host immune response against infections (10, 20, 22). The objectives of the present study were to determine whether IRMPs are expressed in vivo and whether patients with destructive periodontal disease respond immunologically to IRMPs. This was done by examining sera from patients with adult periodontitis and from periodontally healthy subjects for immunoglobulin G (IgG) antibodies to P. gingivalis IRMPs. MATERIALS AND METHODS
Subject groups. The characteristics of the subject groups listed in Table 1. Serum samples and clinical information from 30 patients with adult periodontitis (ages 30 to 53) and 30 periodontally healthy subjects (ages 20 to 50) were provided by the Periodontal Research Center at the State University of New York at Buffalo. The patients with are
Corresponding author. 2427
2428
CHEN ET AL.
INFECT. IMMUN.
TABLE 1. Subject groups Group
No. of subjects in high- and lowlevel subgroupsa
Mean ± SD level of IgG against A7A1-28 P. gingivalis (ELISA U) in serum
Periodontally healthy subjects
High, 2 Low, 28
42 ± 8.5 17.1 ± 4.7
Patients with adult periodontitis
High, 13 Low, 17
114.3 ± 76.9 21.9 ± 6.1
andgroups"
a There were 30 persons in each group. The high-level subgroup consisted of subjects with more than 34.7 ELISA U (mean number of ELISA units of the healthy group plus two standard deviations) of IgG against P. gingivalis A7A1-28 in serum. Subjects with less than 34.7 ELISA U were assigned to the low-level subgroup.
periodontitis exhibited at least two interproximal sites with probing attachment loss of 6 mm or greater and at least one site with a probing pocket depth of 5 mm or more. None of the periodontally healthy subjects exhibited probing attachment loss greater than 2 mm. IgG antibody levels against formalinized whole cells of P. gingivalis A7A1-28 in serum were determined by a modification of the procedures of Mouton et al. (18) and Ebersole et al. (9). A detailed description of the enzyme-linked immunosorbent assay (ELISA) procedure will be published elsewhere (8). ELISA reactivity was calculated by comparison to a reference pool of serum that was assigned an arbitrary value of 100 ELISA U/ml. Bacterial strain and growth conditions. P. gingivalis A7A1-28 (ATCC 53977) was chosen for the present study as it was demonstrated that this strain expressed two IRMPs of 43 and 22 kDa during iron-limited growth (1). The porphyrin requirement of this strain was satisfied by adding iron-free protoporphyrin IX rather than hemin to the medium (1). Under these conditions, P. gingivalis can be iron limited by adding the ferrous iron chelator ot,ot'-dypyridyl to the medium (1). Prior to use, strain A7A1-28 was grown in protoporphyrin IX-supplemented (hemin-free) medium for at least three passages. This regimen depleted internal heme stores so that the organism could be subsequently iron limited. To obtain exponential-phase bacteria grown in iron-sufficient medium, freshly thawed cells of strain A7A1-28 were inoculated into 10 ml of modified half-strength brain heart infusion broth (modified 0.5x BHI) containing 18.5 g of BHI (Difco Laboratories, Detroit, Mich.) per liter, 0.5% yeast extract (Difco), 2.3 ,ug of protoporphyrin IX disodium salt (Sigma Chemical Co., St. Louis, Mo.) per ml, and 1.0 ,ug of vitamin K1 (Sigma) per ml. The cultures were incubated at 37°C in an atmosphere of 85% N2, 10% H2, and 5% CO2 (1025/1029 Anaerobic System; Forma Scientific, Marietta, Ohio) for 24 h. This starter culture was diluted with fresh modified 0.5x BHI (3% inoculum) and incubated until the culture was in mid-exponential growth. To obtain iron-limited exponentialphase bacteria, the starter culture was inoculated into modified 0.5x BHI (5% inoculum) supplemented with 250 ,uM a,a'-dypyridyl (Sigma) and grown to the mid-exponential phase. This concentration of at,a'-dypyridyl inhibited bacterial growth by more than 50%. Growth was monitored by measuring the optical density at 660 nm, and bacterial purity was assessed by Gram's stain. The final immunoblot data (described below) were obtained by examining all sera with membranes obtained from one set of cultures; however,
similar patterns were seen with other batched cultures. The iron-sufficient culture was harvested after 20 h of growth and had an optical density at 660 nm of 0.35, and the iron-limited culture was harvested after 29 h of growth and had an optical density at 660 nm of 0.1. Bacterial membrane preparation. Bacterial cells were harvested by centrifugation at 15,000 x g for 1 h at 4°C. The cell pellet was suspended in 10 ml of distilled water and disrupted by one passage through a French pressure cell (SLM Instruments, Inc., Urbana, Ill.) at 32,000 lb/in2. Unbroken cells and large debris were removed by centrifugation at 7,000 x g for 10 min. The membrane fractions in the supernatant were collected by ultracentrifugation at 100,000 x g for 1 h at 4°C. Following ultracentrifugation, the membranes were suspended in distilled water and the protein concentration was determined by a modified Lowry assay (15). SDS-PAGE and immunoblot analysis. Sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis (PAGE) was performed by using a modified Laemmli gel system (14) with a gel 0.75 mm thick, with a stacking gel consisting of 3.8% acrylamide and 0.1% bisacrylamide and a separating gel consisting of 12% acrylamide and 0.32% bisacrylamide. The bacterial membrane preparations were diluted with an equal volume of 2x sample buffer (0.125 M Tris-Cl [pH 6.8], 8% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.006% bromophenol blue) and boiled at 100°C for 10 min before electrophoresis. Insoluble materials were removed by centrifugation for 3 min at 10,000 rpm in an Eppendorf 5415 C centrifuge (Brinkmann Instruments, Inc., Westbury, N.Y.). One hundred twenty micrograms of membrane proteins was applied to a trough of a preparative gel, and electrophoresis was carried out at a constant 150 V for 2 h and 20 min on a vertical slab gel apparatus (Tall Mighty Small Vertical Slab Unit; Hoefer Scientific Instruments, San Francisco, Calif.). After SDS-PAGE, the sample was electrotransferred at 180 mA (2.5 mA/cm2) for 30 min to a nitrocellulose membrane (Bio-Rad Laboratories, Richmond, Calif.) by using a PolyBlot Electrotransfer System (American Bionetics, Inc., Hayward, Calif.) and the buffer system of Kyshe-Anderson (13). After transfer, the gel was stained with Coomassie brilliant blue R250 to determine the transfer efficiency. A 1-cm strip of nitrocellulose paper containing the molecular weight standards and a portion of the bacterial antigens was cut from the blot and stained with AuroDye forte as recommended by the manufacturer (Amersham International plc., Amersham, United Kingdom). The remaining portion of the nitrocellulose membrane was blocked with buffer containing 0.2 M Tris-HCl (pH 7.5)-0.5 M NaCl (TBS) and 0.05% Tween 20 (Fisher Scientific, Fairlawn, N.J.) (TBS-T) for 30 min at room temperature. This was followed by overnight incubation with a 1:100 dilution of human serum in TBS-T using a PR 150 mini-DecaProbe system (Hoefer Scientific Instruments). Subsequently, the nitrocellulose membrane was washed three times with TBS-T and incubated with a 1:1,000 dilution of horseradish peroxidase-conjugated goat anti-human IgG (Southern Biotechnology Associates, Inc., Birmingham, Ala.) in TBS-T at room temperature on a rocking platform for 1 h. The nitrocellulose membrane was then washed twice with TBS-T and once with TBS and developed by addition of a freshly prepared mixture of 100 ml of TBS containing 1 ml of 3% hydrogen peroxide and 60 mg of 4-chloro-1-naphthol (Bio-Rad Laboratories) dissolved in 20 ml of cold methanol. After development, the membrane was rinsed with distilled water and matched with the strip stained with AuroDye forte. A permanent record was obtained by using a Polaroid MP-4 Land Camera (Polaroid,
VOL. 59, 1991
HUMAN ANTIBODY RESPONSE TO P. GINGIVALIS IRMPs
Cambridge, Mass.) with a high-speed print (Polaroid 667 Professional). The SDS-PAGE and electroblot procedures used to assess the total membrane protein profile of P. gingivalis A7A1-28 were the same as those described above, except that 25 ,ug of the membrane proteins was applied per lane in a gel with 10 wells. After electroblotting, the nitrocellulose membrane containing the bacterial proteins was stained with AuroDye forte. With the electroblotting conditions described above, a nearly complete transfer of membrane proteins to nitrocellulose was achieved. Six serum samples from patients with adult periodontitis were used in pilot studies to maximize immunoblot reactivity (data not shown). In testing different membranes for immunoblotting, we found that immunoperoxidase stains on Immobilon-P membrane (Millipore Corp., Bedford, Mass.) had a tendency to fade rapidly after development. As this did not happen with nitrocellulose, it was selected as the membrane for immunoblotting. Various combinations of Tween 20, nonfat dry milk, and bovine serum albumin of different concentrations were tried as blocking reagents. TBS-T maximally reduced the nonspecific background and did not interfere with specific binding of human IgG with membrane antigens. Other blocking reagents either gave high background activity on nitrocellulose membranes or interfered with specific binding of IgG to antigens. TBS alone was used for the final wash prior to development because Tween 20 interfered with color development. The appropriate dilutions of human sera and the peroxidase-conjugated second antibody were chosen after testing various dilutions of each (1:50 to 1:200 for human sera and 1:1,000 to 1:2,000 for the second antibody) in immunoblotting. Blots incubated overnight with human sera maximized immunoreactivity compared with incubations of 1 or 2 h. Data analysis. From the immunoblot analysis, we found a number of P. gingivalis A7A1-28 membrane proteins that reacted with 6 sera in a pilot study and 60 sera in the full study. The prevalence and intensity of immunoreactivity against nine of these membrane proteins were chosen for final analysis. Five of the membrane proteins were chosen on the basis of high frequency of reactivity with human sera. The remaining four membrane proteins were chosen because they were IRMPs and were frequently recognized by human sera as well. Two of these IRMPs, with molecular masses of 43 and 22 kDa, were described previously (1). Other membrane proteins reacted infrequently with IgG in human serum. After selection of these nine membrane proteins, the immunoreactivities of sera against individual membrane proteins by immunoblotting were scored independently by three investigators (barely visible, ±; visible, +; strong reaction, + +) on the basis of Polaroid photographs. A highly reactive serum was used as a positive control.
RESULTS Membrane proteins of P. gingivalis A7A1-28 grown in iron-sufficient and iron-limited conditions. Figure 1 shows the transblotted membrane proteins of strain A7A1-28 grown in iron-limited and iron-sufficient media stained with proteinspecific AuroDye forte. Both samples contained more than 15 protein bands, although certain bands were not easily identified (particularly protein bands migrating in the 30- to 14-kDa region). Among these membrane proteins, four IRMPs and five membrane proteins with which human sera
2429
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FIG. 1. Total membrane protein profiles of P. gingivalis A7A1-28 grown in iron-limited (lane 1) and iron-sufficient (lane 2) conditions. Twenty-five micrograms of membrane proteins was applied to each lane, and the membranes were separated by SDSPAGE, transferred to nitrocellulose, and stained with AuroDye forte. The apparent molecular masses of the membrane proteins selected for immunoblot analysis are indicated on the left. The IRMPs are indicated by arrows. The sizes of the molecular mass standards (Low Molecular Weight Calibration Kit; Phamacia LKB Biotechnology Inc., Piscataway, N.J.) in lane 3 are shown in kilodaltons on the right.
frequently reacted were selected for further data analysis. The molecular masses of these proteins are indicated in kilodaltons. Immunoblot analysis. Immunoblot analysis of human serum IgG against P. gingivalis A7A1-28 membrane proteins revealed that sera from patients with periodontitis generally showed reactivity with higher numbers of membrane proteins and also with higher intensity than did sera from periodontally healthy subjects. Of the 30 periodontally healthy subjects, 8 showed no detectable immunoreactivity against any of the nine selected membrane proteins of P. gingivalis A7A1-28 by immunoblotting (data not shown). Within the adult periodontitis group, sera from those individuals with high levels of IgG against strain A7A1-28 in serum by ELISA showed higher levels of immunoreactivity than those with low levels by ELISA. Negative controls, including blots probed with human sera without the peroxidase-conjugated goat anti-human IgG second antibody or blots probed with the second antibody without human sera, showed no reactivity (data not shown). Figures 2 and 3 show two sets of representative immunoblots of P. gingivalis A7A1-28 membranes probed with sera
2430
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20.lb 1 2 3 4 5 6 7 8 FIG. 2. Panels A and B show a set of representative immunoblots of P. gingivalis A7A1-28 membranes reacted with sera from patients with adult periodontitis. Panel A contained membranes prepared from bacteria grown in the iron-sufficient condition, while panel B contained membranes from cells grown in the iron-limited condition. In each panel, lane 1 contained molecular size standards and lane 2 contained the membrane proteins; both were stained with AuroDye forte. Corresponding lanes in panels A and B were reacted with the same serum sample from five patients with adult periodontitis (lanes 3 to 7), and lane 8 was probed with the positive control. The sera used in lanes 3, 4, 6, and 7 were from patients with high levels of IgG against strain A7A1-28 in serum, and that in lane 5 was from a patient with a low level by ELISA. The representative staining intensities of +, +, and + + are indicated on the right. The IRMPs are indicated by arrowheads. The sizes of the molecular mass standards are given in kilodaltons next to lane 1. The nine selected P. gingivalis A7A1-28 membrane proteins (including IRMPs) are indicated to the left of each panel, and their molecular masses are shown.
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1 2 3 4 FIG. 3. Immunoblot analysis of human sera from patients with adult periodontitis against P. gingivalis A7A1-28 membrane proteins. Panel A contained membranes prepared from bacteria grown in the iron-sufficient condition, while panel B contained membranes from cells grown in the iron-limited condition. In each panel, lane 1 contained molecular mass standards and lane 2 contained the membrane proteins; both were stained with AuroDye forte. Lanes 3 to 6 of both panels were probed with sera from patients with low IgG levels against strain A7A1-28 by ELISA, lane 7 was reacted with serum from a patient with a high IgG level against the same strain by ELISA, and lane 8 was reacted with the positive control serum. The IRMPs are indicated by arrowheads. The sizes of the molecular mass standards are shown in kilodaltons next to lane 1. The nine selected P. gingivalis A7A1-28 membrane proteins (including IRMPs) are indicated to the left of each panel, and their molecular masses are shown.
VOL. 59, 1991
2431
HUMAN ANTIBODY RESPONSE TO P. GINGIVALIS IRMPs
from patients with periodontitis. There was wide variation among individual serum samples in the specific membrane proteins recognized and in the intensity of the reactivity. For example, in Fig. 2, some serum samples exhibited strong reactivity against a 51-kDa protein while others reacted with the 40.5-kDa protein. The serum sample tested in Fig. 2, lane 7, demonstrated reactivity against all nine selected membrane proteins, as well as other membrane proteins, in particular, those in the 30- and 20-kDa regions. Lanes 3, 4, 6, and 7 in Fig. 2 and lane 7 in Fig. 3 were tested with sera from patients with periodontitis and high levels of IgG against P. gingivalis A7A1-28 in serum by ELISA. Sera from this group of patients generally showed higher immunoreactivity against membrane proteins than did sera from patients with low levels of IgG against this strain in serum by ELISA (lanes 5 in Fig. 2 and 3 to 6 in Fig. 3). Figure 4 shows a set of representative immunoblots tested with sera from periodontally healthy subjects. These sera generally reacted with fewer membrane proteins, and the reactivity was generally at a lower level. IRMPs of P. gingivalis A7A1-28 were among the most commonly recognized membrane proteins in immunoblotting with sera from patients with periodontitis. For example, several sera used for Fig. 2 showed strong reactivity against the 46-, 43-, and 37.5-kDa IRMPs (Fig. 2B, lanes 3, 4, 6, and 7), and in Fig. 3, a serum sample (lane 5) showed distinct immunoreactivity against the 22-kDa IRMP. Patterns of serum reactivity against individual membrane proteins. The pattern and intensity of reactivity of each serum sample were estimated independently by three investigators. The tabulated data were organized into the following three subject categories: (i) patients with adult periodontitis and high levels of IgG against P. gingivalis A7A1-28 in serum by ELISA, (ii) patients with adult periodontitis and low levels of IgG against strain A7A1-28 in serum by ELISA, and (iii) periodontally healthy subjects. The percentages of serum samples in these three subject groups that reacted with the nine selected membrane proteins are tabulated graphically in Fig. 5. More than half of the serum samples from patients with adult periodontitis with high IgG levels by ELISA reacted with the nine selected membrane proteins. IRMPs were among the membrane proteins of P. gingivalis most frequently recognized by IgG in human sera. More than 80% of the sera from patients with periodontitis and high levels of IgG against strain A7A1-28 in serum by ELISA reacted with 46-, 43-, and 37.5-kDa IRMPs of strain A7A128, while 40% of the sera from the same group reacted with the 22-kDa IRMP. The P. gingivalis A7A1-28 membrane proteins were also recognized, but to a lesser degree, by sera from patients with periodontitis and low levels of IgG against strain A7A1-28 in serum by ELISA and sera from periodontally healthy subjects. DISCUSSION Our previous studies indicated that P. gingivalis A7A1-28 expressed two IRMPs of 43 and 22 kDa (1). In the present study, we observed that these proteins are immunogenic and were frequently recognized by sera from patients with periodontitis. We also identified two additional IRMPs (46 and 37.5 kDa) which were recognized by sera examined in the present study. Since IgG antibodies against IRMPs are found in sera from patients with periodontitis, many of whom may be infected with P. gingivalis, the data suggest that IRMPs are expressed in vivo. In addition, the present study showed that a significant portion of the human serum IgG immune
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2432
INFECT. IMMUN.
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ACKNOWLEDGMENTS This work was supported by USPHS grants DE 08240, DE 04898, DE 52559, and DE 07034. We thank Mary Bayers-Thering for assistance in immunoblot
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response against P. gingivalis in patients with adult periodontitis was directed against IRMPs. Studying the critical antigens expressed in ivivo, which response, is may serve as targets of an effective host immune response, IS important for understanding the pathogenic me chanisms of bacterial infections. Furthermore, development of vaccines against such antigens remains a distinct possibil ity. Various studies have demonstrated host immune respoi nses against iron-regulated proteins (10, 11, 20). This immu ne response may have important consequences for bacterial pathogenesis. Antibody against P. aeruginosa iron-regulzated protein has been shown to inhibit iron uptake by the o0 rganism and in mice confer protection in certain experimental infecti ions that P gingivalis (22). In the present study, we found A7A1-28 IRMPs were among the major target an tigens of the human humoral immune response. Although the significance of this immune response against P. gingivalis is unknown, it may protect patients with periodontitis from further periodontal destruction. Since IRMPs of strain A7 A1-28 were recognized at high frequencies by the sera exanr nined in this study, these proteins may be structurally coriserved and share antigenic cross-reactivity among serologic variants of P. gingivalis. If the human immune response aigainst ironrepressible proteins is protective, then this anti Lgenic crossreactivity may be advantageous in developin~ g a vaccine against P. gingivalis infections. In conclusion, we found nine P. gingivalis A7 A1-28 membrane proteins that consistently reacted with senum antibody from patients with adult periodontitis. Four oif these nine membrane proteins were regulated by iron avail lability. The in ViVO data suggest that P. gingivalis IRMPs were expre and that a significant portion of the human I.gG immune response to P. gingivalis is directed at ironi-repressible proteins.
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12. 13.
14.
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