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1.  Isolation and characterization of fimbriae from a sparsely fimbriated strain of Porphyromonas gingivalis. 
Porphyromonas gingivalis W50 (ATCC 53978) possesses the gene for fimbriae; however, the surface-expressed fimbriae are sparse and have not been previously isolated and characterized. We purified fimbriae from strain W50 to homogeneity by ammonium sulfate precipitation and reverse-phase high-performance liquid chromatography [H. T. Sojar, N. Hamada, and R. J. Genco, Protein Expr. Purif. 9(1):49-52, 1997]. Negative staining of purified fimbriae viewed by electron microscopy revealed that the fimbriae were identical in diameter to fimbriae of other P. gingivalis strains, such as 2561, but were shorter in length. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, the apparent molecular weight of isolated fimbrillin from strain W50 was found to be identical to that of the fimbrillin molecule of strain 2561. Unlike 2561 fimbriae, W50 fimbriae, under reducing condition, exhibited a monomeric structure on SDS-PAGE at room temperature. However, under nonreduced conditions, even at 100 degrees C, no monomer was observed. In immunoblot analysis as well as immunogold labeling of isolated fimbriae, polyclonal antibodies against 2561 fimbriae, as well as antibodies against peptide I (V-V-M-A-N-T-G-A-M-E-V-G-K-T-L-A-E-V-K-Cys) and peptide J (A-L-T-T-E-L-T-A-E-N-Q-E-A-A-G-L-I-M-T-A-E-P-Cys), reacted. However, antifimbrial antibodies against strain 2561 reacted very weakly compared to anti-peptide I and anti-peptide J. Negative staining of whole W50 cells, as well as immunogold electron microscopy with anti-peptide I and anti-peptide J, showed fimbriae shorter in length and very few in number compared to those of strain 2561. Purified fimbriae showed no hemagglutinating activity. Amino acid composition was very similar to that of previously reported fimbriae of the 2561 strain.
PMCID: PMC168524  PMID: 9172351
2.  Salivary receptors for recombinant fimbrillin of Porphyromonas gingivalis. 
Infection and Immunity  1994;62(8):3372-3380.
Fimbriae are considered important in the adherence and colonization of Porphyromonas gingivalis in the oral cavity. It has been demonstrated that purified fimbriae bind to whole human saliva adsorbed to hydroxyapatite (HAP) beads, and the binding appears to be mediated by specific protein-protein interactions. Recently, we expressed the recombinant fimbrillin protein (r-Fim) of P. gingivalis corresponding to amino acid residues 10 to 337 of the native fimbrillin (A. Sharma, H.T. Sojar, J.-Y. Lee, and R.J. Genco, Infect. Immun. 61:3570-3573, 1993). We examined the ability of individual salivary components to promote the direct attachment of r-Fim to HAP beads. Purified r-Fim was radiolabeled with 125I and incubated with HAP beads which were coated with saliva or purified individual salivary components. Whole, parotid, and submandibular-sublingual salivas increased the binding of 125I-r-Fim to HAP beads. Submandibular-sublingual saliva was most effective in increasing the binding of 125I-r-Fim to HAP beads (1.8 times greater than that to uncoated HAP beads). The binding of 125I-r-Fim to HAP beads coated with acidic proline-rich protein 1 (PRP1) or statherin was four and two times greater, respectively, than that to uncoated HAP beads. PRP1 and statherin molecules were also found to bind 125I-r-Fim in an overlay assay. The binding of intact P. gingivalis cells to HAP beads coated with PRP1 or statherin was also enhanced, by 5.4 and 4.3 times, respectively, over that to uncoated HAP beads. The interactions of PRP1 and statherin with 125I-r-Fim were not inhibited by the addition of carbohydrates or amino acids. PRP1 and statherin in solution did not show inhibitory activity on 125I-r-Fim binding to HAP beads coated with PRP1 or statherin. These results suggest that P. gingivalis fimbriae bind strongly through protein-protein interactions to acidic proline-rich protein and statherin molecules which coat surfaces.
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PMCID: PMC302968  PMID: 8039907
3.  Oxidative and nonoxidative killing of Actinobacillus actinomycetemcomitans by human neutrophils. 
Infection and Immunity  1986;53(1):154-160.
Actinobacillus actinomycetemcomitans is a facultative gram-negative microorganism which has been implicated as an etiologic agent in localized juvenile periodontitis and in subacute bacterial endocarditis and abscesses. Although resistant to serum bactericidal action and to oxidant injury mediated by superoxide anion (O2-) and hydrogen peroxide (H2O2), this organism is sensitive to killing by the myeloperoxidase-hydrogen peroxide-chloride system (K.T. Miyasaki, M.E. Wilson, and R.J. Genco, Infect. Immun. 53:161-165, 1986). In this study, we examined the sensitivity of A. actinomycetemcomitans to killing by intact neutrophils under aerobic conditions, under anaerobic conditions, and under aerobic conditions in the presence of the heme-protein inhibitor sodium cyanide. Intact neutrophils killed opsonized A. actinomycetemcomitans under aerobic and anaerobic conditions, and the kinetics of these reactions indicated that both oxidative and nonoxidative mechanisms were operative. Oxidative mechanisms contributed significantly, and most of the killing attributable to oxidative mechanisms was inhibited by sodium cyanide, which suggested that the myeloperoxidase-hydrogen peroxide-chloride system participated in the oxidative process. We conclude that human neutrophils are capable of killing A. actinomycetemcomitans by both oxygen-dependent and oxygen-independent pathways, and that most oxygen-dependent killing requires myeloperoxidase activity.
PMCID: PMC260090  PMID: 3013778
4.  Blastogenic response of human lymphocytes to oral bacterial antigens: characterization of bacterial sonicates. 
Infection and Immunity  1976;14(5):1202-1212.
Soluble sonicate supernatant preparations were made from Actinomyces viscosus (ATCC 19246), A. naeslundii (ATCC 12104), two strains of Veillonella alcalescens (strain HV-1 and a human oral isolate), Streptococcus sanguis (ATCC 10556), S. mutans (strain 6715-T2), Bacteroides melaninogenicus (strain K110), and Leptotrichia buccalis (isolated from human dental plaque). These supernatants were characterized with reference to their chemical and antigenic components and their biological activity determined by using in vitro lymphocyte blastogenesis as a measure of the host's cellular immune response. The sonicate supernatant of each bacterium contained protein, neutral sugars, methylpentose, and nucleic acids. Protein was the major component in all except L. buccalis, in which neutral sugars predominated. The antigenic components in each supernatant were detected by using rabbit antisera prepared against the whole bacteria and the sonicate supernatant. The supernatants showed a complex antigenic distribution on immunoelectrophoretic analysis. The supernatants were shown to be antigenic and not mitogenic in nature, since neither cord blood lymphocytes nor all adult lymphocytes were stimulated. The supernatant antigen preparations showed a reproducible, dose-dependent, and kinetic response in vitro, which was similar to that seen with the antigen preparation streptokinase-streptodornase.
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PMCID: PMC415515  PMID: 977126
5.  A role for fimbriae in Porphyromonas gingivalis invasion of oral epithelial cells. 
Infection and Immunity  1997;65(5):1980-1984.
Isogenic mutants of Porphyromonas gingivalis which differ in the expression of fimbriae were used to examine the contribution of fimbriae in invasion of a human oral epithelial cell line (KB). At a multiplicity of infection of 100, the wild-type P. gingivalis strains 33277, 381, and A7436 exhibited adherence efficiencies of 5.5, 0.11, and 5.0%, respectively, and invasion efficiencies of 0.15, 0.03, and 0.10%, respectively. However, adherence to and invasion of KB cells was not detected with the P. gingivalis fimA mutants, DPG3 and MPG1. Adherence of P. gingivalis wild-type strains to KB cells was completely inhibited by the addition of hyperimmune sera raised to the major fimbriae. Examination by electron microscopy of invasion of epithelial cells by the P. gingivalis wild-type strain 381 revealed microvillus-like extensions around adherent bacteria; this was not observed with P. gingivalis fim mutants. Taken together, these results indicate that the P. gingivalis major fimbriae are required for adherence to and invasion of oral epithelial cells.
PMCID: PMC175257  PMID: 9125593
6.  Role of the carboxyl-terminal region of Porphyromonas gingivalis fimbrillin in binding to salivary proteins. 
Infection and Immunity  1997;65(2):422-427.
Porphyromonas gingivalis fimbriae are considered to play an important role in the adherence and colonization of the bacteria in the oral cavity. In this study, we generated and purified three carboxyl-terminal variants of recombinant fimbrillin (r-FimA 224-337, r-FimA 266-337, and r-FimA 287-337, corresponding to amino acid residues 224 to 337, 266 to 337, and 287 to 337, respectively, of the 43-kDa fimbrillin of P. gingivalis 2561). They were used as inhibitors of P. gingivalis cell binding to human salivary protein-coated hydroxyapatite (HAP) beads. All of the carboxyl-terminal region polypeptides inhibited binding in a dose-dependent manner; however, the inhibitory effect of r-FimA 287-337 was less than that of the other two polypeptides when HAP beads were coated with whole saliva or purified salivary proline-rich protein 1 (PRP1). Assays of binding of a synthetic peptide corresponding to amino acid residues 266 to 286 of P. gingivalis 2561 fimbrillin to salivary proteins showed that this peptide bound strongly to whole saliva or PRP1 but only weakly to statherin. These results suggest that the carboxyl-terminal region corresponding to amino acid residues 266 to 337 of P. gingivalis fimbrillin plays an important role in binding to salivary proteins and that the domain corresponding to amino acids 266 to 286 is likely a major binding site for PRP1s and the domain corresponding to amino acids 287 to 337 is likely a major binding site for statherin.
PMCID: PMC174611  PMID: 9009291
7.  Expression of functional Porphyromonas gingivalis fimbrillin polypeptide domains on the surface of Streptococcus gordonii. 
Applied and Environmental Microbiology  1996;62(11):3933-3938.
Genetically engineering bacteria to express surface proteins which can antagonize the colonization of other microorganisms is a promising strategy for altering bacterial environments. The fimbriae of Porphyromonas gingivalis play an important role in the pathogenesis of periodontal diseases. A structural subunit of the P. gingivalis fimbriae, fimbrillin, has been shown to be an important virulence factor, which likely promotes adherence of the bacterium to saliva-coated oral surfaces and induces host responses. Immunization of gnotobiotic rats with synthetic peptides based on the predicted amino acid sequence of fimbrillin has also been shown to elicit a specific immune response and protection against P. gingivalis-associated periodontal destruction. In this study we engineered the human oral commensal organism Streptococcus gordonii to surface express subdomains of the fimbrillin polypeptide fused to the anchor region of streptococcal M6 protein. The resulting recombinant S. gordonii strains expressing P. gingivalis fimbrillin bound saliva-coated hydroxyapatite in a concentration-dependent manner and inhibited binding of P. gingivalis to saliva-coated hydroxyapatite. Moreover, the recombinant S. gordonii strains were capable of eliciting a P. gingivalis fimbrillin-specific immune response in rabbits. These results show that functional and immunologically reactive P. gingivalis fimbrillin polypeptides can be expressed on the surface of S. gordonii. The recombinant fimbrillin-expressing S. gordonii strains may provide an effective vaccine or a vehicle for replacement therapy against P. gingivalis. These experiments demonstrated the feasibility of expressing biologically active agents (antigens or adhesin molecules) by genetically engineered streptococci. Such genetically engineered organisms can be utilized to modulate the microenvironment of the oral cavity.
PMCID: PMC168210  PMID: 8899979
8.  Isolation and characterization of a minor fimbria from Porphyromonas gingivalis. 
Infection and Immunity  1996;64(11):4788-4794.
We have discovered two distinctly different fimbriae expressed by the same Porphyromonas gingivalis strain. The construction of a fimA mutant of P. gingivalis ATCC 33277 has previously been reported by N. Hamada et al. (Infect. Immun. 62:1696-1704, 1994). Expression of fimbriae on the surface of the fimA mutant and the wild-type strain, ATCC 33277, were investigated by electron microscopy. The wild-type strain produced long fimbrial structures extending from the cell surface, whereas those structures were not observed on the fimA mutant. However, short fimbrial structures were seen on the surface of the fimA mutant. The short fimbrial protein was purified from the fimA mutant by selective protein precipitation and chromatography on DEAE Sepharose CL-6B. We have found that the second fimbrial structure of P. gingivalis ATCC 33277 is distinct from the 41-kDa (43-kDa) major fimbrial protein (FimA). We provisionally call this protein minor fimbriae. The molecular mass of the minor fimbriae is 67 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions after boiling at 100 degrees C. The component shows a ladder-like pattern at 80 degrees C under nonreducing conditions, suggesting a tendency to aggregate or polymerize. In immunoblotting analysis, anti-minor fimbria serum reacted with both the 100 degrees C- and the 80 degrees C-treated minor fimbriae. The anti-minor fimbria serum also reacts with the same-molecular-size fimbrial preparation from the wild-type strain. Immunogold electron microscopy showed that the anti-minor fimbria serum bound to the minor fimbria on the cell surface of the wild-type strain. This is the first report on the identification of the minor fimbria produced by P. gingivalis. These results suggest that the minor fimbriae appearing on the fimA mutant strain are produced together with numerous long major fimbriae on the wild-type strain. Moreover, the minor fimbriae are different in size and antigenicity from the earlier-reported FimA, a major 41-kDa fimbrial component of P. gingivalis.
PMCID: PMC174446  PMID: 8890240
9.  Binding sites of salivary statherin for Porphyromonas gingivalis recombinant fimbrillin. 
Infection and Immunity  1996;64(10):4249-4254.
We investigated the binding sites of salivary statherin involved in the interaction with Porphyromonas gingivalis recombinant fimbrillin (r-Fim). Synthetic peptides representing statherin analogs were used to localize the binding domains of statherin. Peptide F4 (residues 29 to 43) significantly bound to r-Fim and inhibited r-Fim binding to statherin-coated hydroxyapatite beads. Successive peptides in which pairs of amino acid residues were deleted starting at the N terminus of peptide F4 were synthesized. Peptide N1 without Leu-29-Tyr-30 had significantly reduced direct binding and inhibition ability. The deletions of residues 31 to 40 had little effect on interaction with r-Fim. The tripeptide N6 representing Tyr-41-Thr-42-Phe-43 retained significant binding to r-Fim. Another set of peptides was synthesized by deleting individual amino acid residues from the C and N termini of peptide F4 to identify functional residues among the five putative functional residues 29, 30, and 41 to 43. Peptide C1 missing Phe-43 lost over 50% of its binding ability. Binding ability was gradually reduced with deletions from the peptides. Peptide C5 (amino acids 31 to 40) weakly affected direct binding and inhibition. Collectively, the results of this study suggests that Leu-29-Tyr-30 and Tyr-41-Thr-42-Phe-43 are important binding regions that mediate the binding of statherin to P. gingivalis fimbrillin.
PMCID: PMC174364  PMID: 8926096
10.  Structural domains of Porphyromonas gingivalis recombinant fimbrillin that mediate binding to salivary proline-rich protein and statherin. 
Infection and Immunity  1996;64(5):1631-1637.
Fimbriae (the oligomeric form of fimbrillin) are considered important in the adherence and colonization of Porphyromonas gingivalis in the oral cavity. In the present study, we have identified the structural domains of P. gingivalis fimbrillin that mediate the binding to salivary proline-rich protein 1 (PRP1) and statherin. A series of synthetic fimbrillin peptides were used to localize the active fimbrillin domains involved in the binding to PRP1 and statherin. The binding of 125I-labeled 41-r-Fim (whole-length recombinant fimbrillin, amino acid [aa] residues 1 to 337) to PRP1-coated hydroxyapatite beads (HAP) was strongly inhibited by the fimbrillin C-terminal peptides corresponding to aa residues 266 to 286 and 318 to 337 (peptides 266-286, and 318-337, respectively), while the binding to statherin was inhibited by C-terminal peptides 266-286, 293-306 and 307-326. Peptide 126-146 also showed a weak inhibitory effect, about half that of other active peptides, on the binding to both PRP1 and statherin. P. gingivalis whole-cell binding to PRP1- or statherin-coated HAP was inhibited by more than 80% by the same active peptides. To confirm that the C-terminal portion of fimbrillin includes domains responsible for the binding, two C-terminally truncated variants of recombinant fimbrillin were generated and purified. These were designated 34.5-r-Fim, corresponding to aa residues 1 to 286, and 32-r-Fim, corresponding to aa residues 1 to 265. 125I-34.5-r-Fim revealed 35 and 34% loss of binding ability to PRP1 and statherin, respectively. 125I-32-r-Fim had significantly less binding ability to PRP1 and statherin than 125I-34.5-r-Fim, which was reduced 78 and 73%, respectively. Whole-cell binding to PRP1-, statherin-, or whole saliva-coated HAP was inhibited up to 100% by 41-r-Fim, while 32-r-Fim also showed considerable inhibition, possibly due to the region of aa 126 to 146. Collectively, these results suggest that there are separate and multiple binding sites for PRP1 and statherin in the P. gingivalis fimbrillin, and the combination of all of these binding sites may be indispensable in establishing stable bacterial adherence to saliva-coated surfaces in the oral cavity.
PMCID: PMC173972  PMID: 8613371
11.  Inactivation of the Porphyromonas gingivalis fimA gene blocks periodontal damage in gnotobiotic rats. 
Journal of Bacteriology  1994;176(4):1052-1059.
Fimbrial production by Porphyromonas gingivalis was inactivated by insertion-duplication mutagenesis, using the cloned gene for the P. gingivalis major fimbrial subunit protein, fimA. by several criteria, this insertion mutation rendered P. gingivalis unable to produce fimbrilin or an intact fimbrial structure. A nonfimbriated mutant, DPG3, hemagglutinated sheep erythrocytes normally and was unimpaired in the ability to coaggregate with Streptococcus gordonii G9B. The cell surface hydrophobicity of DPG3 was also unaffected by the loss of fimbriae. However, DPG3 was significantly less able to bind to saliva-coated hydroxyapatite than wild-type P. gingivalis 381. This suggested that P. gingivalis fimbriae are important for adherence of the organism to saliva-coated oral surfaces. Further, DPG3 was significantly less able to cause periodontal bone loss in a gnotobiotic rat model of periodontal disease. These observations are consistent with other data suggesting that P. gingivalis fimbriae play an important role in the pathogenesis of human periodontal disease.
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PMCID: PMC205156  PMID: 8106316
12.  Immunization with Porphyromonas (Bacteroides) gingivalis fimbriae protects against periodontal destruction. 
Infection and Immunity  1992;60(7):2926-2935.
Adhesive fimbriae from Porphyromonas gingivalis are cell surface structures which may be important in the virulence of this oral pathogen and thus may serve as a critical or target antigen. Immunization with highly purified 43-kDa fimbrial protein protected against periodontal tissue destruction when tested in the P. gingivalis-infected gnotobiotic rat model. A similarly highly purified 75-kDa cell surface component did not provide protection. Heat-killed whole-cell and sonicated cell surface extracts which contain the 43-kDa protein as well as the 75-kDa component were protective also. This study indicates that the fimbrial protein may serve as a model for the development of effective vaccines against periodontitis, a major human oral disease.
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PMCID: PMC257256  PMID: 1351883
13.  Serological studies of oral Bacteroides intermedius. 
Infection and Immunity  1988;56(6):1647-1651.
Bacteroides intermedius is a gram negative, anaerobic microorganism associated with certain forms of human periodontal disease, including adult periodontitis and acute necrotizing ulcerative gingivitis. Previous studies have indicated the presence of two DNA homology groups which could be distinguished by analysis of protein patterns on polyacrylamide gel electrophoresis, as well as at least two serogroups within B. intermedius. The present study examined the serology of B. intermedius and determined the distribution of B. intermedius serogroups in clinical isolates and patient plaque samples. Serological reactions with unabsorbed rabbit antisera and antisera immunoabsorbed with B. intermedius strains demonstrated a previously unreported antigenic group within B. intermedius, serogroup C, in both immunodiffusion and immunofluorescence assays. Of 79 B. intermedius isolates from 68 subjects examined with specific antisera, 55% of the isolates and 52% of the subjects were categorized in serogroup C, 40% of the isolates and 46% of the subjects were in serogroup B, and 5% of the isolates and 6% of the subjects were in serogroup A. In 31 samples of subgingival dental plaque from adolescents known to harbor B. intermedius, 81% demonstrated serogroup B, 16% had serogroup A, and 3% had serogroup C.
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PMCID: PMC259450  PMID: 3131249
14.  Resistance of Actinobacillus actinomycetemcomitans and differential susceptibility of oral Haemophilus species to the bactericidal effects of hydrogen peroxide. 
Infection and Immunity  1984;46(3):644-648.
We compared the sensitivities of oral and nonoral isolates of Actinobacillus actinomycetemcomitans, Haemophilus segnis, H. aphrophilus, and H. paraphrophilus to the bactericidal action of reagent hydrogen peroxide (H2O2). Susceptibility to a range of H2O2 concentrations (10(-6) to 10(-3) M) was assessed by incubating bacterial suspensions for 1 h at 37 degrees C in the presence of H2O2 and plating on chocolate agar to determine the concentration of H2O2 that would produce a 50% reduction in CFU (50% lethal dose). As a group, A. actinomycetemcomitans was more resistant to H2O2 than the oral haemophili, and H. aphrophilus was much more sensitive than all other organisms tested. The range of 50% lethal dose values for A. actinomycetemcomitans was between 8.5 X 10(-5) and 10(-3) M H2O2 or above. In contrast, H. aphrophilus exhibited 50% lethal dose values from below 1 X 10(-6) to 3.4 X 10(-4) M H2O2. The resistance of A. actinomycetemcomitans to H2O2 may be sufficient to protect these organisms from direct H2O2-mediated killing by host phagocytes.
PMCID: PMC261590  PMID: 6500706
15.  Use of combined immunization routes in induction of a salivary immunoglobulin A response to Streptococcus mutans in Macaca fascicularis monkeys. 
Infection and Immunity  1981;31(1):345-351.
Various combinations of immunization routes were examined for ability to elicit or prolong (or both) a salivary secretory immunoglobulin A response to Streptococcus mutans strain Ingbritt (serotype c) in Macaca fascicularis monkeys. Intraductal (i.d.), per os (p.o.), and subcutaneous (s.c.) routes were utilized. Four groups of three to five monkeys each were immunized by the following schedules: group I--p.o., s.c., i.d.; group II--i.d., p.o., i.d.; group III--s.c., p.o., i.d.; and group IV--control. Immune responses in the serum and parotid fluid were quantitated by using passive hemagglutination assays with purified serotype-specific polysaccharide and by indirect immunofluorescent staining assays. Both s.c. and i.d., but not p.o., routes resulted in detectable serum antibody responses. Only i.d. immunization resulted in a measurable salivary response. Indirect immunofluorescent staining revealed specific secretory immunoglobulin A antibodies in the parotid fluid which correlated with passive hemagglutination titers. The p.o. procedures used in this study did not result either in a prolonged immune response or in measurable tolerance related to the humoral or secretory immune system.
PMCID: PMC351789  PMID: 7216449
16.  Neutrophil Chemotaxis Dysfunction in Human Periodontitis 
Infection and Immunity  1980;27(1):124-132.
Polymorphonuclear leukocyte (PMNL) chemotaxis studies of 32 patients with localized juvenile periodontitis (periodontosis or LJP), 10 adult patients with a history of LJP (post-LJP), 8 patients with generalized juvenile periodontitis (GJP), and 23 adults with moderate to severe periodontitis were performed: (i) to determine the prevalence of a PMNL chemotaxis defect in a large group of LJP patients; (ii) to study PMNL chemotaxis in patients with other forms of severe periodontal disease; and (iii) to determine if the PMNL chemotaxis defect seen in LJP patients is a cell-associated defect or is mediated by humoral factors. The effect of periodontal treatment on PMNL chemotaxis was studied in nine LJP patients. The chemotactic response was measured with the Boyden chamber procedure, and patient's peripheral PMNL were compared with those of control subjects, using endotoxin-activated serum, bacterial factor, N-formylmethionyl-leucylphenylalanine, and leukocyte-derived chemotactic factor as the standard chemoattractants. Based upon statistical analysis of chemotaxis assays, most carried out on at least two and often three or more separate occasions, 26 of 32 LJP patients, 7 of 10 post-LJP patients, and 5 of 8 GJP patients exhibited cellular defects of chemotaxis, whereas only 2 of 23 of the patients with adult periodontitis exhibited depressed chemotaxis. Elevated PMNL chemotaxis was occasionally found in subjects with juvenile periodontitis (2 of 32 with LJP and two of eight with GJP); however, it was found in a significant number (10 of 23) of patients with adult periodontitis. In eight of nine LJP patients, depressed PMNL chemotaxis was observed before and after periodontal therapy. The results indicate that the PMNL chemotaxis defect observed in juvenile periodontitis is due to a cell-associated defect of long duration. These studies suggest that the PMNL plays a major protective role against periodontal infection and that the cellular chemotactic defects and may predispose subjects to LJP.
PMCID: PMC550734  PMID: 7358424
17.  Prevention of Streptococcus mutans infection of tooth surfaces by salivary antibody in Irus monkeys (Macaca fascicularis). 
Infection and Immunity  1975;12(2):293-302.
Four irus monkeys (Macaca fascicularis) were immunized with Streptococcus mutans 6715 killed cells and cell products by injection in the vicinity of the major salivary glands and by instillation into the parotid glands via the ducts. After immune group and a sham-immunized control group of monkeys were infected orally with viable strain 6715 organisms. Dental plaque samples were taken at intervals from the buccal and lingual grooves of the first permanent molars. These samples were evaluated for recovery of strain 6715 by cultural methods. In addition, individual samples were taken from 10 representative tooth surfaces and were evaluated by indirect immunofluorescent staining for strain 6715. Results showed that immune monkeys had fewer infected surfaces and fewer organisms on the infected surfaces than the control animals. These studies indicate that salivary antibody to cariogenic streptococci inhibits implantation of these organisms in dental plaque and may be protective against dental caries.
PMCID: PMC415283  PMID: 1097337
18.  Antibody response in the parotid fluid and serum of Irus monkeys (Macaca fascicularis) after local immunization with Streptococcus mutans. 
Infection and Immunity  1975;12(2):281-292.
The antibody response of Macaca fascicularis in parotid saliva and serum to local immunization by two routes with Streptococcus mutans was studied and compared over 1 year. Antibodies were titrated and classified by indirect immunofluorescent staining using specific antiglobulin conjugates. Antiglucosyltransferase activity was assayed by an enzyme inhibition test. Animals were immunized first by injecting formalin-killed bacterial cells and cell products subcutaneously into the vicinity of the four major salivary glands. The monkeys were next immunized by retrograde instillation of antigen into the parotid duct. Extensive subcutaneous local immunization gave a serum response only. After parotid duct immunization, high titers of immunoglobulin A (IgA) antibody, along with traces of immunoglobulin G (IgG) immunoglobulin M (IgM) antibody, appeared in the parotid saliva, and in the serum high titers of IgG antibody were present along with lower titers of IgA and IgM. IgA antibodies in parotid fluid were shown by double immunofluorescent staining to be associated with antigenic determinants which cross-reacted with an antiserum directed to human secretory component. Titers in parotid fluids and sera fell sharply when immunization was stopped. This response pattern was reproducible. High concentrations of antibody capable of inhibiting glucosyltransferase prepared from S. mutans were found in the sera, but relatively little was detected in the parotid fluids. Extensive immunization via the parotid duct resulted in transient functional impairment of the gland, as evidenced by diminished salivary flow rates. We conclude that parotid ductal immunization can be an effective method for stimulating a salivary secretory IgA antibacterial antibody response.
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PMCID: PMC415282  PMID: 50286
19.  Inhibition of Glucosyltransferase Activity by Antisera to Known Serotypes of Streptococcus mutans 
Infection and Immunity  1973;7(2):237-241.
Using a recently developed assay for glucosyltransferase activity based on 14C-glucose incorporation into an alcohol-insoluble polysaccharide, we were able to study inhibition by antibody of this enzyme activity. Rabbit antibody was relatively specific for the strain of Streptoccus mutans from which the enzyme was obtained. Absorption studies showed that neither removal of antibodies directed to dextran nor absorption with intact bacteria offset the enzyme-inhibitory capacity of these sera, whereas absorption with partially purified enzyme did result in removal of the inhibitory capacity.
PMCID: PMC422666  PMID: 4735375
20.  Effects of temperature stress on expression of fimbriae and superoxide dismutase by Porphyromonas gingivalis. 
Infection and Immunity  1994;62(10):4682-4685.
We examined the biosynthesis of fimbriae and superoxide dismutase (SOD) produced by the periodontopathic bacterium Porphyromonas gingivalis in response to elevated temperature. P. gingivalis 2561, grown at 37 degrees C to mid-logarithmic phase, was subsequently incubated at 39, 41, and 43 degrees C, respectively, to stationary phase. There was no difference in the growth of cells at 37 and 39 degrees C. However, at 39 degrees C there was a 54% reduction in the amount of fimbrillin (fimbriae) as well as decreased expression of mRNA for fimA. On the other hand, under the same conditions, a more than twofold increase in the amount of SOD activity, as well as in the levels of SOD mRNA, was observed. Moreover, cells cultured for 20 h at 39 degrees C showed an 86% decrease of fimbrillin protein and a threefold increase in SOD activity. These observations suggest that P. gingivalis may undergo alterations in its virulence and susceptibility to host immune responses as a result of the elevated temperatures found in inflamed periodontal pockets.
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PMCID: PMC303167  PMID: 7927742
21.  Structural and functional characterization of the human formyl peptide receptor ligand-binding region. 
Infection and Immunity  1994;62(5):1726-1732.
The formyl peptide (N-formyl-1-methionyl-1-leucyl-1-phenylalanine [FMLP]) receptor is involved in the activation of neutrophils and their subsequent response to chemotactic N-formylated peptides. Recently, we found that the first extracellular loop closest to the N-terminal end of the FMLP receptor exhibited the strongest ligand binding compared with that shown by other extracellular regions. By constructing amino acid substitutional variants of this domain, we have determined that residues Arg-84 and Lys-85 on this loop play major roles in ligand-binding activity. Furthermore, random rearrangement of the residues of this receptor region demonstrated that the position of these charged amino acids did not affect their involvement in ligand binding, although their presence was essential for this binding to occur. We propose that the portion of the first N-terminal extracellular loop of the FMLP receptor containing residues Arg-84 and Lys-85 contributes significantly to the active site in ligand-receptor binding. We further propose that this binding is not dependent on defined structure but rather that these charged moieties may function as important "contacts" in receptor-ligand interactions.
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PMCID: PMC186394  PMID: 8168934
22.  Differentiation of strains of Actinobacillus actinomycetemcomitans by arbitrarily primed polymerase chain reaction. 
Journal of Clinical Microbiology  1993;31(10):2773-2776.
The present study describes a method for amplifying DNA in Actinobacillus actinomycetemcomitans by using short, synthetic oligonucleotides of random sequence as primers in the polymerase chain reaction. Genomic DNA from each of 20 human isolates of A. actinomycetemcomitans was successfully amplified in a thermal cycler with a single synthetic primer (GGGTAACGCC) and reproducibly produced 14 different DNA amplification profiles (amplitypes). A. actinomycetemcomitans isolates from the same subject revealed the same amplitype. The arbitrarily primed polymerase chain reaction appears to be useful in characterizing human isolates of A. actinomycetemcomitans for studies of epidemiology and bacterial transmission.
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PMCID: PMC266011  PMID: 8253980
23.  Expression of a functional Porphyromonas gingivalis fimbrillin polypeptide in Escherichia coli: purification, physicochemical and immunochemical characterization, and binding characteristics. 
Infection and Immunity  1993;61(8):3570-3573.
Fimbriae have been reported to play an important role in the adherence of Porphyromonas gingivalis to oral surfaces and possibly in triggering host responses. A structural subunit of the fimbriae, fimbrillin, has been shown to be important in binding of the bacterium to saliva-coated oral surfaces. In the present study, a coding region of the fimbrillin gene from P. gingivalis 2561 was amplified by the polymerase chain reaction and cloned into the pET-11d vector. The recombinant plasmid was transformed into Escherichia coli BL21, and protein expression was induced with isopropyl-beta-D-thiogalactopyranoside. The expressed protein was purified from insoluble inclusion bodies after solubilization with urea and gel filtration chromatography. The purified recombinant fimbrillin polypeptide, r-fim 10-337, corresponded to amino acid residues 10 to 337 of the deduced amino acid sequence of fimbrillin. In immunoblot analysis, r-fim 10-337 reacted with antibodies to fimbrillin purified from P. gingivalis, as well as with antibodies to synthetic peptides corresponding to the amino acid sequence of fimbrillin. The apparent molecular mass of r-fim 10-337 was estimated to be 41 kDa on sodium dodecyl sulfate-polyacrylamide gels. The r-fim 10-337 polypeptide was capable of inhibiting the binding of P. gingivalis 2561 to saliva-coated hydroxyapatite beads. These results suggest that the fimbrillin subunit polypeptide plays an important role in binding of P. gingivalis cells to saliva-coated surfaces. We describe here the successful expression and purification of a functionally and immunologically reactive recombinant P. gingivalis fimbrillin subunit from E. coli.
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PMCID: PMC281044  PMID: 8392975
24.  Purification and characterization of a protease from Porphyromonas gingivalis capable of degrading salt-solubilized collagen. 
Infection and Immunity  1993;61(6):2369-2376.
An enzyme capable of hydrolyzing the substrate 4-phenylazobenzyloxycarbonyl-L-prolyl-leucyl-glycyl-prolyl-D-ar gin ine (pZ-peptide), pZ-peptidase, was purified from the oral bacterium Porphyromonas gingivalis. pZ-peptidase hydrolyzed salt-solubilized type I collagen from rat skin, rat plasma low-molecular-weight kininogen, and transferrin at room temperature in the presence of calcium and dithiothreitol. pZ-peptidase did not cleave acid-soluble type I calf skin collagen, type V placental collagen, lysozyme, albumin, or human plasma fibrinogen. Furthermore, the purified enzyme did not hydrolyze N-alpha-benzoyl-DL-Arg-p-nitroanilide, Gly-Pro-p-nitroanilide, N-p-tosyl-Gly-Pro-Arg-p-nitroanilide, N-p-tosyl-Gly-Pro-Lys-p-nitroanilide, azoalbumin, or azocasein. Under reducing conditions, the native enzyme migrated as a single band at 120 kDa on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. However, when heated to 100 degrees C for 10 min in SDS under reducing conditions, the enzyme migrated as a major band at 50 kDa and a minor band at 60 kDa on SDS-polyacrylamide gel electrophoresis. Zymography using calf skin gelatin revealed the gelatin-cleaving activity of the enzyme as evidenced by a diffuse band in the range of 120 to 300 kDa under reducing conditions at room temperature, suggesting that this is the native form of the enzyme. However, incubation at 50 degrees C for 10 min under reducing conditions showed gelatin-cleaving activity at a distinct band of 60 kDa. A minimum temperature of 50 degrees C was required to dissociate the 60-kDa chain from the native complex in active form on gelatin zymography. The ability of the enzyme to cleave other proteins, including kininogen and transferrin, suggests that it has specificity for the Pro-X-Gly sequence found in several proteins, including collagen.
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PMCID: PMC280857  PMID: 8388862
25.  Synthetic peptides analogous to the fimbrillin sequence inhibit adherence of Porphyromonas gingivalis. 
Infection and Immunity  1992;60(4):1662-1670.
Fimbriae are important in the adherence of many bacterial species to the surfaces they eventually colonize. Porphyromonas (Bacteroides) gingivalis fimbriae appear to mediate adherence to oral epithelial cells and the pellicle-coated tooth surface. The role and contribution of fimbriae in the binding of P. gingivalis to hydroxyapatite (HAP) coated with saliva as a model for the pellicle-coated tooth surface were investigated. 3H-labeled P. gingivalis or the radioiodinated purified fimbriae were incubated with 2 mg of HAP beads coated with whole human saliva (sHAP) and layered on 100% Percoll to separate unbound from sHAP-bound components. The radioactivity of the washed beads was a measure of the bound components. The binding of P. gingivalis 2561 (381) cells and that of purified fimbriae were concentration dependent and saturable at approximately 10(8) cells and 40 micrograms of fimbriae added, respectively. The addition of fimbriae inhibited binding of P. gingivalis to sHAP beads by 65%, while the 75-kDa protein, which is another major surface component of P. gingivalis 2561, did not show significant inhibition, suggesting that the fimbriae are important in adherence. Encapsulated and sparsely fimbriated P. gingivalis W50 did not bind to sHAP beads. On the basis of the predicted sequence of the fimbrillin, a structural subunit of fimbriae, a series of peptides were synthesized and used to localize the active fimbrillin domains involved in P. gingivalis adherence to sHAP beads. Peptides from the carboxyl-terminal one-third of the fimbrillin strongly inhibited P. gingivalis binding to sHAP beads. Active residues within the sequence of inhibitory peptide 226-245 (peptide containing residues 226 to 245) and peptide 293-306 were identified by using smaller fragments prepared either by trypsin cleavage of the peptide 226-245 or by synthesis of smaller segments of peptide 293-306. Hemagglutinin activity, lectinlike binding, and ionic interaction did not seem to be involved in this binding since lysine, arginine, carbohydrates, and calcium ions failed to affect the binding of P. gingivalis. The observation that poly-L-lysine, bovine serum albumin, and defatted bovine serum albumin, even at high concentrations, only partially blocked the binding of P. gingivalis indicates that hydrophobic interactions are not the major forces involved in P. gingivalis binding to sHAP beads. Protease inhibitors such as EDTA, leupeptin, pepstatin, 1,10-phenanthroline, and phenylmethylsulfonyl fluoride did not interfere with the binding of P. gingivalis. However, the binding of P. gingivalis to trypsin- or chymotrypsin-pretreated sHAP beads was reduced.(ABSTRACT TRUNCATED AT 400 WORDS)
PMCID: PMC257044  PMID: 1347762

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