Antibodies to periodontal pathogens and coronary artery calcification in type 1 diabetic and nondiabetic subjects.

BACKGROUND AND OBJECTIVE: The aim of this study was to examine whether serum immunoglobulin G (IgG) levels to Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans are higher in type 1 diabetic patients than in controls and are associated with coronary artery calcification, a measure of atherosclerosis. MATERIAL AND METHODS: One-hundred and ninety nine type 1 diabetic patients (mean age 38 +/- 4 years) and 201 age- and gender-matched nondiabetic subjects had coronary artery calcification, as measured by electron beam computed tomography. Serum IgG levels to P. gingivalis W50 and to A. actinomycetemcomitans HK1651 whole cells were measured by enzyme-linked immunosorbent assay. RESULTS: A similar proportion of diabetic patients (29%) and controls (31%, p = 0.7) had elevated serum IgG to periodontal bacteria, defined as being above the median antibody level for both microorganisms. Elevated antibody levels were associated with higher systolic blood pressure (p = 0.02) and an increased odds of coronary artery calcification in all subjects combined (odds ratio = 1.7, p = 0.047) and in diabetic subjects examined separately (odds ratio = 2.01, p = 0.027). Association of serum IgG levels with coronary artery calcification was independent of social class, lipids and antibody levels to other microorganisms, but not systolic blood pressure (odds ratio = 1.4, p = 0.1 on adjustment for blood pressure). There was no association between serum IgG level and vascular endothelial function. CONCLUSION: Elevated levels of serum IgG to P. gingivalis and A. actinomycetemcomitans are associated with coronary artery atherosclerosis. This may reflect a direct role for periodontal infection or a role for the host response to infection in coronary atherosclerosis, particularly in patients with type 1 diabetes.

Platelet activation by Protease I of Porphyromonas gingivalis W83.

Porphyromonas gingivalis produces a trypsin-like enzyme, Protease I, which is thought to be an important virulence determinant of the organism in adult periodontal disease. Protease I is transiently inhibited by physiological inhibitors of human thrombin. The aim of the present work was to establish whether Protease I was able to mimic thrombin by activation of the thrombin receptor on human platelets. Protease I caused true platelet activation at concentrations comparable to thrombin as measured by aggregometry, morphology and fluorescence flow cytometric analysis of CD63 expression. The effect was blocked by protease inhibitors but not by anti-thrombin receptor antibodies which, by contrast, blocked platelet activation by thrombin. We conclude that the activation of platelets by P. gingivalis Protease I involves proteolysis, but not scission of the thrombin cleavage site of the thrombin receptor.

Interaction of a trypsin-like enzyme of Porphyromonas gingivalis W83 with antithrombin III.

We have previously observed that trypsin-like activity in Porphyromonas gingivalis culture supernatants is inhibitable by the plasma arg-serpin antithrombin III (ATIII). This report demonstrates that a partially purified P. gingivalis trypsin-like enzyme (M(r) 47,000) is inhibited by ATIII with an association rate constant (k(ass)) of 5.65 x 10(4) M-1 s-1 but does not form SDS-stable complexes. Heparin enhances the k(ass) and stabilizes the complexes but in either case such inhibition is temporary and results in ATIII inactivation by reactive centre proteolysis between R393-S394. In the absence of heparin this is accompanied by N-terminal cleavage between K39-I40.

Production and characterisation of monoclonal antibodies to the principle sonicate antigens of Porphyromonas gingivalis w50.

Protein antigens from whole cell sonicates of Porphyromonas gingivalis W50, previously shown to be discriminatory antigens for patients with adult periodontitis, were purified using SDS-PAGE. Electroeluted proteins were used to immunize mice for the production of monoclonal antibodies (mAbs). A combination of enzyme-linked immunosorbent assay (ELISA) and Western blotting were used to screen hybridoma supernatants for mAbs. MAbs were successfully raised against M(r) 115,000, M(r) 55,000 and M(r) 47,000 antigens together with a second M(r) 55,000 polypeptide which was a contaminant of the M(r) 55,000 antigen. No immunological cross-reactivity was found between these four proteins. The mAbs were used to examine the distribution of these antigens among fifteen P. gingivalis strains together with related oral bacteria using immunostaining of dot blots and Western blots. The antigens were confined to P. gingivalis with the M(r) 115,000 and M(r) 47,000 antigens being present in all strains tested. The distribution of the M(r) 55,000 antigens were slightly more restricted: one M(r) 55,000 (outer membrane location) was present in nine of the fifteen P. gingivalis strains tested, while the other M(r) 55,000 (location unknown) was only absent from one strain. Whole cell ELISA demonstrated that the M(r) 115,000 and the outer membrane M(r) 55,000 antigen possess epitopes which are located on the surface of the bacterium.

Critical pathways in microbial virulence.

The virulence of a microbe represents a combination of complex factors including the agent's transmissibility and the severity of the disease associated with infection and is also significantly influenced by the susceptibility of the colonized host. Virulence factors may be defined as those products of the organism which are required to complete the various stages of the life cycle leading to pathology in the host. In this review, we examine some of the approaches which have been adopted in other fields of infectious disease in order to categorically identify virulence factors using a classical genetics approach with relevant models or human subjects. The absence of an accurate experimental model for periodontal disease means that our understanding of the microbial virulence determinants and pathways in this disease remains hypothetical and based largely on observations in vitro. However, factors which enable the organism to persist in spite of the elevated immune and inflammatory pressure at sites of disease are liable to be critical. Periodontal bacterial genomics is liable to make a significant impact on the field through an increased appreciation of the role of gene acquisition and gene loss in the evolution of periodontal bacteria and of the consequences of strain variation in gene content on virulence potential.

Characterization of the trypsin-like enzymes of Porphyromonas gingivalis W83 using a radiolabelled active-site-directed inhibitor.

The trypsin-like enzyme activity of Porphyromonas gingivalis is an important virulence determinant of this organism in destructive periodontitis. An active-site-directed inhibitor, tyrosyl-alanyl-lysyl-arginine chloromethyl ketone (YAKR-CK) was radio-iodinated and used with SDS-PAGE and autoradiography to determine the number and molecular masses of enzymes with trypsin-like specificity produced by P. gingivalis W83. Two forms (I + II) were detected in both crude culture supernatant and whole cell sonicates. Protease I was a sharp band (47 kDa) on reducing SDS-PAGE; Protease II electrophoresed as a diffuse band in the range 70-90 kDa. The specificity with which the inhibitor bound to Protease I was established in competition experiments using other active-site-directed agents. YAKR-CK inhibited P. gingivalis whole cell haemagglutination, supporting the possible role of trypsin-like proteases of this organism in adhesion mechanisms.

Circulating immune complexes and symptoms in Hodgkin's disease.

Increased quantities of the third component of complement (C3) were found in the macromolecular fractions of plasma from patients with untreated Hodgkin's disease (H.D.) These changes provide indirect evidence that immune complexes are present in the plasma of patients with this disease; their presence is closely correlated with the symptoms of night sweats and fever which are associated with a poor prognosis. It is suggested that the detection of circulating complexes may help in assessing the severity of H.D.

Characterization, genetic analysis, and expression of a protease antigen (PrpRI) of Porphyromonas gingivalis W50.

Previous studies of the serum immunoglobulin G antibody response of periodontal patients have demonstrated significant reactivity to a cell surface or extracellular arginine-specific protease of Porphyromonas gingivalis which migrates as an approximately 50-kDa band on sodium dodecyl sulfate-polyacrylamide gels. In the present report, two forms of the enzyme (ArgI and ArgIA) with this electrophoretic behavior were isolated. ArgI is a heterodimer of alpha and beta subunits, and ArgIA is a monomer composed of the catalytically active alpha component alone. The gene encoding ArgI (prpR1 encoding protease polyprotein ArgI) was cloned from Sau3AI digests of P. gingivalis W50 DNA into pUC18. Sequence analysis demonstrated that the alpha and beta components are contiguous on the initial translation product and are flanked by large N- and C-terminal extensions. prpR1 is 97.5% identical to the rgp-1 gene from P. gingivalis H66. prpR1 expression in Escherichia coli demonstrated the presence of an internal transcription-translation initiation site which could permit independent expression of different regions of the polyprotein. Immunochemical analysis of P. gingivalis mid-logarithmic-phase cultures suggested that the processing of PrpRI may be closely coupled to its synthesis, with only the final stages taking place at the cell surface. Southern hybridization studies demonstrated that the prpR1 gene is widely distributed in other P. gingivalis strains and that a second homologous locus to the alpha component and at least two other homologous loci to the beta component are present on the P. gingivalis chromosome. These data indicate that the ArgI protease of P. gingivalis is a member of a family of sequence-related gene products which may share both functional and antigenic properties.

The prpR1 and prR2 arginine-specific protease genes of Porphyromonas gingivalis W50 produce five biochemically distinct enzymes.

The arginine-specific protease activity of Porphyromonas gingivalis is considered to be an important factor in the pathogenic potential of this organism in destructive periodontal disease. Multiple forms of closely related Arg-x proteases are present in the culture supernatants of P. gingivalis W50. RI is a heterodimer (alpha/beta) in which the catalytic alpha chain is associated with a second beta chain which functions as a haemagglutinin. RIA is a single-chain enzyme (alpha) and RIB is a highly post-translationally lipid-modified enzyme (LPS-alpha) with reduced solubility compared to the other two forms. The N-terminal sequence of the alpha chain of all three forms is identical, suggesting that all these enzymes may arise by differential processing of the prpR1 (protease polyprotein for RI). In the present study we constructed a prpR1- strain of P. gingivalis W50 by insertional gene inactivation and characterized the residual extracellular Arg-x protease activity of the resulting mutant. Loss of prpR1 expression led to the abolition of RI, RIA and RIB but the total Arg-x activity in the supernatant of this strain was reduced by only c. 66%. The remaining activity was composed of two novel forms of Arg-x protease (RIIA and RIIB) which appeared to be structurally and kinetically almost identical to RIA and RIB, respectively, except for two amino acid differences in the N-terminus at position 8 (Q-->E) and position 17 (A-->P) and with respect to their stability to high pH. Confirmation that RIIA and RIIB are the products of a homologous locus (prR2) was obtained by cloning and sequencing the prR2 which showed the predicted substitutions in the deduced translation. These data indicate that RI, RIA and RIB are produced by prpR1 expression and a maturation pathway which can give rise to a dimer and an unmodified- or LPS-modified catalytic monomer. Furthermore, RIIA and RIIB, the products of prR2, are exported into the culture supernatant in the absence of prpR1 expression and these forms may also contribute to the pathogenic potential of this organism in destructive disease.

Immunoconglutinins and C3 in human saliva.

High titres of immunoconglutinin activity (antibody to bound complement components) have been found in the parotid, sublingual and submandibular saliva of most healthy subjects. The immunoconglutinin (IK) titre in mixed saliva was substantially lower than in the other samples of saliva. C3 was detectable in only three of 164 samples of parotid, submandibular and sublingual saliva but was present in forty-seven of 117 mixed saliva samples. It is suggested that crevicular fluid is the major source of C3 in mixed saliva. A negative correlation was found in mixed saliva between the C3 concentration and the IK titre, and this suggested that C3 was the inhibitor of IK in mixed saliva. Binding of C3 to IK has been demonstrated in mixed saliva by using highly purified salivary IK and C3. Purified C3, C3i, C3c and C3d inhibited the activity of purified IK. It is suggested that salivary IK represents the secretion of a B-lymphocyte population which has evaded the mechanism responsible for inducing B-cell tolerance to autologous serum proteins. The reason for its persistence in the salivary glands, however, is not known at present.

Degradation of plasma proteins by the trypsin-like enzyme of Porphyromonas gingivalis and inhibition of protease activity by a serine protease inhibitor of human plasma.

The interaction between Porphyromonas gingivalis culture supernatant and human serum was examined. Hydrolysis of the major serum proteins was thiol-dependent and correlated with the trypsin-like activity of the sample. Transferrin and IgG light chains were less susceptible to degradation than albumin and IgG heavy chains and partially degraded IgG retained antigen-binding capability. Serum inhibited the trypsin-like activity in a fluorimetric assay. The inhibition was shown to be independent of the level of IgG antibody reactive with whole cells of P. gingivalis. Purified preparations of antithrombin III, a serine protease inhibitor, but not alpha 1-antitrypsin nor alpha 2-macroglobulin inhibited the trypsin-like activity in the fluorometric assay.

Identification of the major surface protein antigens of Porphyromonas gingivalis using IgG antibody reactivity of periodontal case-control serum.

The identity of the major surface antigens of Porphyromonas gingivalis was investigated. Outer membranes of P. gingivalis strains W83, W50, 381 and NCTC 11843 were prepared following inactivation of the trypsin-like enzyme activity. Three proteins, molecular weight 115, 55 and 40 kDa, were major components of the outer membranes of strains W83 and W50 and were also present in strains 381 and NCTC 11834. Two proteins, 55 and 47 kDa, were released from the cells during the sonication step of the outer membrane preparation procedure. Immunoblots using preparations of P. gingivalis W83 and serum from a case-control study of adult periodontitis demonstrated higher mean antibody reactivity in the case population to all the major proteins except for the 115 kDa outer membrane protein, which was recognized equally well by both populations. We conclude that the 55, 47 and 40 kDa proteins are important surface antigens of P. gingivalis. Characterization of the structure and function of these components should lead to an improved understanding of the host-parasite interactions in adult periodontitis.

The Tla protein of Porphyromonas gingivalis W50: a homolog of the RI protease precursor (PrpRI) is an outer membrane receptor required for growth on low levels of hemin.

The prpR1 gene of Porphyromonas gingivalis W50 encodes the polyprotein precursor (PrpRI) of an extracellular arginine-specific protease. PrpRI is organized into four distinct domains (pro, alpha, beta, and gamma) and is processed to a heterodimeric protease (RI) which comprises the alpha and beta components in a noncovalent association. The alpha component contains the protease active site, whereas the beta component appears to have a role in adherence and hemagglutination processes. DNA sequences homologous to the coding region for the RI beta component are present at multiple loci on the P. gingivalis chromosome and may represent a family of related genes. In this report, we describe the cloning, sequence analysis, and characterization of one of these homologous loci isolated in plasmid pJM7. The 6,041-bp P. gingivalis DNA fragment in pJM7 contains a major open reading frame of 3,291 bp with coding potential for a protein with an Mr 118,700. An internal region of the deduced sequence (V304 to N768) shows 98% identity to the beta domain of PrpRI, and the recombinant product of pJM7 is immunoreactive with an antibody specific to the RI beta component. The N terminus of the deduced sequence has regional similarity to TonB-linked receptors which are frequently involved in periplasmic translocation of hemin, iron, colicins, or vitamin B12 in other bacteria. We have therefore designated this gene tla (TonB-linked adhesin). In contrast to the parent strain, an isogenic mutant of P. gingivalis W50 in which the tla was insertionally inactivated was unable to grow in medium containing low concentrations of hemin (<2.5 mg liter(-1)), and hemin-depleted cells of this mutant failed to respond to hemin in an agar diffusion plate assay. These data suggest a role for this gene product in hemin acquisition and utilization. Furthermore, the mutant produced significantly less arginine- and lysine-specific protease activities than the parent strain, indicating that there may be a regulatory relationship between tla and other members of this gene family.

The relationship between colonization and haemagglutination inhibiting and B cell epitopes of Porphyromonas gingivalis.

Passive immunization with the monoclonal antibody 61BG1.3 selectively prevents colonization by Porphyromonas gingivalis in humans (Booth V, Ashley FP, Lehner T. Infect Immun 1996; 64:422-7). The protective MoAb recognizes the beta component of the RI protease of P. gingivalis which is formed by proteolytic processing of a polyprotein precursor termed PrpR1. This subunit is both a haemagglutinin and an antigen which is recognized by sera from patients with periodontitis. In this study the relationship was investigated between a colonization epitope which is recognized by the MoAb 61BG1.3, a haemagglutinating and B cell epitope which are recognized by sera from patients with periodontitis. B cell epitopes were mapped by Western blotting with a series of truncated recombinant polypeptides spanning the adhesion domain within residues 784-1130 of PrpR1 and by ELISA using a panel of synthetic peptides spanning the same sequence. The epitope which is recognized by the protective MoAb was mapped within residues 907-931 of PrpR1, while serum responses of patients were directed predominantly to the adjacent carboxy-terminal sequence within residues 934-1042. The haemagglutinating epitope was mapped to residues 1073-1112. In view of our previous findings that the MoAb 61BG1.3 prevents colonization of P. gingivalis in vivo and inhibits haemagglutination, these two epitopes may be in proximity in the native protein. Active or passive immunization strategies which target the protective or haemagglutinating epitopes of the adhesion domain of PrpR1 may provide a means of preventing infection with P. gingivalis.

Characterization of an adherence and antigenic determinant of the ArgI protease of Porphyromonas gingivalis which is present on multiple gene products.

This study was performed to characterize the antigen(s) recognized by a panel of monoclonal antibodies (MAbs) produced to be specific for Porphyromonas gingivalis whole cells which we had previously shown to bind to epitopes recognized by sera from periodontitis patients. Preliminary data had suggested that the arginine-specific proteases of P. gingivalis (ArgI, ArgIA, and ArgIB) contained the antigenic determinants of four of these antibodies (MAbs 1A1, 2B/H9, 7D5, and 3B1). The location of the binding sites was examined with purified P. gingivalis enzymes and recombinant regions of the ArgI polyprotein expressed by subclones of the prpR1 gene in Escherichia coli XL-1 Blue cells. All four antibodies were reactive with protein determinants within the beta subunit, a hemagglutinin and/or adhesin component, of the ArgI dimer. MAb 1A1 strongly inhibited the agglutination of human erythrocytes by P. gingivalis W50 culture supernatant, suggesting that the binding site for this antibody contains residues which are critical for the interaction with the erythrocyte surface. The determinant for MAb 1A1 was examined further by construction of a set of truncated forms of the beta component expressed as fusion proteins with glutathione S-transferase at the N terminus. Analysis of these constructs mapped the binding site for MAb 1A1 to PrpRI residues G-907 to T-931, GVSPKVCKDV TVEGSNEFAP VQNLT. Western blot (immunoblot) analysis of P. gingivalis whole-cell proteins demonstrated that MAb 1A1 reacts with several proteins in the Mr range of 20,000 to 120,000. Furthermore, an oligonucleotide probe corresponding to the coding sequence for the region of the ArgI beta component containing the MAb 1A1 binding site hybridized to multiple bands on genomic digests of P. gingivalis DNA. These data indicate that the MAb 1A1 epitope may be a component of a binding domain common to multiple gene products of this organism and may thus represent a functionally important target of the host's specific immune response to P. gingivalis in periodontal disease.

Oro-dental bacteria in various atherosclerotic arteries.

Chlamydia pneumoniae DNA has been detected in at least 40% of all major arteries affected by atherosclerosis, but several other microorganisms have also been detected. In this study, diseased vessels were evaluated for the presence of the DNA of seven oro-dental bacteria and two nonoral bacteria. A polymerase chain reaction technique was employed using primer pairs based on 16S rRNA genes. Of 32 specimens tested, 10 (31.2%) were DNA positive: seven for Actinobacillus actinomycetemcomitans and three for Prevotella intermedia. The DNA was found in specimens from the aorta and the iliac, internal mammary and coronary arteries. Eleven (35.4%) of 31 specimens had been shown previously to be positive for Chlamydia pneumoniae DNA. A mixture of chlamydiae and oro-dental bacteria was found in three cases. These findings may have implications for antibiotic prophylaxis of coronary heart disease if directed solely at Chlamydia pneumoniae.

Variable carbohydrate modifications to the catalytic chains of the RgpA and RgpB proteases of Porphyromonas gingivalis W50.

Proteases of Porphyromonas gingivalis are considered to be important virulence determinants of this periodontal bacterium. Several biochemical isoforms of arginine-specific proteases are derived from rgpA and rgpB. HRgpA is a heterodimer composed of the catalytic alpha chain noncovalently associated with a beta adhesin chain derived from the C terminus of the initial full-length translation product. The catalytic alpha chain is also present as a monomer (RgpA) either free in solution or associated with membranes. rgpB lacks the coding region for the adhesin domain present in rgpA and yields only monomeric forms (RgpB) which again may be soluble or membrane associated. In this study, the catalytic chains of this unusual group of enzymes are shown to be differentially modified by the posttranslational addition of carbohydrate. A monoclonal antibody (MAb 1B5) raised to the monomeric RgpA did not react with the corresponding recombinant RgpA alpha chain expressed in Escherichia coli but was immunoreactive with P. gingivalis lipopolysaccharide. MAb 1B5 also reacted with the membrane-associated forms of RgpA and RgpB but not with the heterodimeric HRgpA and the soluble form of RgpB. RgpA treated with denaturants was capable of binding to MAb 1B5 whereas treatment with periodate abolished this binding, suggesting the presence of carbohydrate residues within the epitope. Chemical deglycosylation abolished immunoreactivity with MAb 1B5 and caused a approximately 30% reduction in the size of the membrane-associated enzymes. Monosaccharide analysis of HRgpA and RgpA demonstrated 2.1 and 14.4%, respectively, carbohydrate by weight of protein. Furthermore, distinct differences were detected in their monosaccharide compositions, indicating that these protease isoforms are modified not only to different extents but also with different sugars. The variable nature of these additions may have a significant effect on the structure, stability, and immune recognition of these protease glycoproteins.