A geographically homogeneous population of 83 subjects, from 21 families with localized juvenile periodontitis (LJP), and 35 healthy control subjects was monitored, over a 5-year period, for the presence of the periodontal pathogen Actinobacillus actinomycetemcomitans. Restriction fragment length polymorphism (RFLP) analysis was used to monitor the distribution of genetic variants of this bacterium in LJP-susceptible subjects that converted from a healthy to a diseased periodontal status. A. actinomycetemcomitans was cultured from 57% of the LJP family members accessioned into the study. Nine of 36 LJP-susceptible subjects, in seven families, developed signs of periodontal destruction. All but one of these conversion subjects harbored A. actinomycetemcomitans. Bacterial variants representative of a single RFLP group (II) showed the strongest correlation with conversion (P < 0.002). Six of nine conversion subjects were infected with A. actinomycetemcomitans from this group. RFLP group II variants also prevailed in 8 of 22 probands but were absent in the 35 healthy control subjects. In contrast to the selective distribution of group II variants is diseased individuals, variants belonging to RFLP groups XIII and XIV were found exclusively in the control subjects. Thus, the use of RFLP to type clinical isolates of A. actinomycetemcomitans has resulted in the identification of genetic variants that predominate in LJP and health. These results indicate that studies concerned with the pathogenicity of this bacterium in LJP should be focused on the group II variants.
Actinobacillus actinomycetemcomitans strains with enhanced levels of production of leukotoxin are characterized by a 530-bp deletion from the promoter region of the leukotoxin gene operon. Previous isolates with this deletion constituted a single clone belonging to serotype b, although they displayed minor differences among each other. We have analyzed the geographic dissemination of this clone by examining 326 A. actinomycetemcomitans isolates from healthy and periodontally diseased individuals as well as from patients with different types of extraoral infections originating from countries worldwide. A total of 38 isolates, all belonging to the same clone, showed the 530-bp deletion. Comparison of a 440-bp sequence from the promoter region of the leukotoxin gene operon from 10 of these strains revealed complete identity, which indicates that the deletion originates from a single mutational event. This particular clone was exclusively associated with localized juvenile periodontitis (LJP). In at least 12 of 28 families from which the clone was isolated, more than one family member had LJP. Notably, all the subjects carrying this clone had a genetic affiliation with the African population. These observations suggest that juvenile periodontitis in some adolescents with an African origin is associated with a disseminating clone of A. actinomycetemcomitans.
The periodontal pathogen Actinobacillus actinomycetemcomitans produces a leukotoxin that is considered a primary virulence factor in localized juvenile Periodontitis (LJP). Select strains of the bacterium contain a 530-bp deletion in the promoter region of the leukotoxin gene operon which results in enhanced transcription of the leukotoxin. DNA hybridization and polymerase chain reaction (PCR) were used to examine genetic variants of A. actinomycetemcomitans in 24 LJP-susceptible children from 21 families having a history of the disease and 34 control children from non-LJP families. A significant association was found between the detection of variants that had a deletion in the leukotoxin promoter region, indicative of a high level expression leukotoxin genotype, and conversion from a healthy periodontal status to disease. Subjects harboring A. actinomycetemcomitans of this genotype were more likely to convert to LJP than those subjects who had variants containing the full length leukotoxin promoter region (odds ratio = 22.50, 95% C.I.). These findings support the concept that highly virulent strains or clonal types of periodontal pathogens play a major role in the initiation of periodontal disease in susceptible hosts.
Periodontitis; juvenile/epidemiology; Actinobacillus actinomycetemcomitans; leukotoxin; epidemiology; polymerase chain reaction; polymorphism; restriction fragment length
The phylogeny of 20 Actinobacillus actinomycetemcomitans strains isolated from patients with localized juvenile periodontitis (LJP) was investigated by using partial sequence analysis of 16S rRNA genes, arbitrarily primed PCR (AP-PCR), and four additional PCR assays that amplified polymorphic regions in the leukotoxin (lkt), cytolethal distending toxin (cdt), major fimbrial subunit (flp-1), and serotype-specific O polysaccharide gene clusters. Our analysis also included four strains isolated from healthy subjects and nine reference strains. We found that A. actinomycetemcomitans strains comprised three major phylogenetic lineages. One lineage consisted of serotype b strains, a second lineage consisted of serotype c strains, and a third lineage consisted of serotype a, d, e, and f strains. 16S rRNA sequences within each lineage were highly conserved (<1% base substitutions), whereas sequences between lineages were exceptionally divergent (1.9 to 5.0% substitutions). Two strains exhibited 16S rRNA sequences that were even more distantly related to those of the three major lineages (2.7 to 6.7% substitutions), indicating that additional minor lineages or variants exist. The distribution of 16S rRNA sequences and lkt, cdt, flp-1, and AP-PCR genotypes was consistent with a clonal population structure, with little evidence of assortative recombination between strains of different serotypes. Strains from all three major lineages were recovered from LJP patients, suggesting that phylogenetically diverse strains of A. actinomycetemcomitans carry pathogenic potential.
Objective: The aim of this study was to investigate subgingival infection frequencies of Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans strains with genetic variation in Chinese chronic periodontitis (CP) patients and to evaluate its correlation with clinical parameters. Methods: Two multiplex polymerase chain reaction (PCR) assays were developed to detect the 16SrDNA, collagenase (prtC) and fimbria (fimA) genes of P. gingivalis and the 16SrDNA, leukotoxin (lktA) and fimbria-associated protein (fap) genes of A. actinomycetemcomitans in 60 sulcus samples from 30 periodontal healthy subjects and in 122 subgingival plaque samples from 61 patients with CP. The PCR products were further T-A cloned and sent for nucleotide sequence analysis. Results: The 16SrDNA, prtC and fimA genes of P. gingivalis were detected in 92.6%, 85.2% and 80.3% of the subgingival plaque samples respectively, while the 16SrDNA, lktA and fap genes of A. actinomycetemcomitans were in 84.4%, 75.4% and 50.0% respectively. Nucleotide sequence analysis showed 98.62%~100% homology of the PCR products in these genes with the reported sequences. P. gingivalis strains with prtC+/fimA+ and A. actinomycetemcomitans with lktA+ were predominant in deep pockets (>6 mm) or in sites with attachment loss ≥5 mm than in shallow pockets (3~4 mm) or in sites with attachment loss ≤2 mm (P<0.05). P. gingivalis strains with prtC+/fimA+ also showed higher frequency in gingival index (GI)=3 than in GI=1 group (P<0.05). Conclusion: Infection of P. gingivalis with prtC+/fimA+ and A. actinomycetemcomitans with lktA+ correlates with periodontal destruction of CP in Chinese. Nonetheless P. gingivalis fimA, prtC genes and A. actinomycetemcomitans lktA gene are closely associated with periodontal destruction, while A. actinomycetemcomitans fap gene is not.
Porphyromonas gingivalis; Actinobacillus actinomycetemcomitans; Strain; Periodontitis; PCR
The purpose of this study was to assess the sensitivity and specificity of the PCR in detecting Actinobacillus actinomycetemcomitans. The PCR's detection capability was compared with those of three other methods: culture-enhanced PCR (CE-PCR), colony hybridization (CH), and conventional culture with presumptive biochemical identification. A 285-bp stretch of the leukotoxin gene lktA of A. actinomycetemcomitans was amplified by PCR with primers TT-15 and TT-16. For CH, the PCR product was labeled with digoxigenin and used as a hybridization probe. Nucleotide sequence analysis of the PCR product of A. actinomycetemcomitans 1D4 and 1664 and three clinical isolates revealed complete homology among the tested strains, with only one base substitution (at position 1344) in comparison with the published sequence. With artificially infected subgingival plaque, the detection limit of PCR for A. actinomycetemcomitans was 10(3) CFU/ml of plaque suspension. Culturing subgingival plaque on tryptic soy-serum-bacitracin-vancomycin agar prior to PCR (CE-PCR) improved the limit of detection to 10(2) CFU/ml. Analysis of subgingival plaque samples from 35 patients with periodontal disease and 10 periodontally healthy subjects revealed that CE-PCR and CH had the highest overall rate of A. actinomycetemcomitans detection (both 58%), followed by PCR and culture (both 42%). With CH as the "gold standard", the sensitivities of CE-PCR, PCR, and culture were 88, 65, and 58%, respectively; the specificities were 84, 89, and 79%, respectively. The CE-PCR provided acceptable positive and negative predictive values (> or = 70%) when the prevalence of A. actinomycetemcomitans varied between 30 and 70%. PCR alone provided comparable predictive values over a narrower range of prevalence rates (30 to 50%), while culture did not afford acceptable predictive values at any prevalence rate. PCR and CE-PCR were found to be superior to culture with presumptive biochemical identification and should be the preferred methods for the detection of A. actinomycetemcomitans in subgingival plaque.
A high-molecular-weight polysaccharide-containing antigen was isolated from a phenol-water extract of Actinobacillus actinomycetemcomitans ATCC 43718 (formerly Y4) by gel permeation chromatography in lipopolysaccharide (LPS)-disaggregating buffer. The polysaccharide antigen formed a precipitin band with rabbit serotype b-specific antiserum but not with rabbit antisera to serotype a or c. Electroblotted serotype b antigen was probed with serum from a patient with localized juvenile periodontitis (LJP), resulting in a diffuse "smear" in the upper region of the lane. By utilizing an enzyme-linked immunosorbent assay, it was demonstrated that the geometric mean immunoglobulin G antibody titer to the serotype b polysaccharide was significantly higher in sera from LJP patients than in sera from periodontally healthy individuals. Moreover, LJP antibody titers to the serotype b polysaccharide exhibited age-dependent variation. Double immunodiffusion analysis revealed that the serotype b antigen formed a line of identity with low-molecular-weight LPS following reaction with serotype b-specific antiserum. Incubation of LJP serum in the presence of a lipid-free polysaccharide moiety obtained by mild acid hydrolysis of LPS from A. actinomycetemcomitans Y4 markedly reduced immunoglobulin G titer to the serotype b antigen. In contrast, solubilized lipid A was only weakly inhibitory. The results of this study indicate that the serotype b-specific determinant of A. actinomycetemcomitans resides in the polysaccharide moiety of LPS and represents a major target for immunoglobulin G antibody in serum of LJP subjects colonized by this organism.
Patients with localized juvenile periodontitis (LJP) often have high titers of antibody reactive with the serotype-specific immunodominant carbohydrate antigen of Actinobacillus actinomycetemcomitans serotype b. The vast majority of this A. actinomycetemcomitans serotype b-specific antibody is immunoglobulin G2 (IgG2). The present study was undertaken to determine whether the overall total levels of IgG2 in the sera of LJP patients are elevated. LJP patients and nonperiodontitis (NP) controls matched for age, race (black and white), and gender were studied. Additional controls included patients with adult periodontitis (AP) and patients similar in age to LJP patients but with the more-severe, generalized form of early-onset periodontitis (SP). Sera from over 700 periodontally characterized subjects were examined by using radial immunodiffusion to quantitate IgG2 as well as IgG1, -3, and -4, which were included for comparison. Serum IgG2 levels increased with age, and this was most dramatic around puberty. Black subjects in all periodontal groups had nearly 1 mg more IgG2 per ml than their white counterparts. Serum IgG2 levels were elevated (about 30 to 40%) in LJP patients of both races compared with their age- and race-matched NP controls (P < 0.01). In contrast, SP patients and AP patients had IgG2 levels comparable to their age- and race-matched NP controls. No other IgG subclass concentration correlated with periodontal diagnosis except for IgG3, which was elevated in white LJP patients. We reason that the high levels of serum IgG2 in LJP may be helpful in localizing periodontal destruction.
The present study was performed to estimate the observed frequencies of the immunoglobulin heavy-chain (Gm) and light-chain (Km) allotypes among patients with early-onset periodontitis (EOP) and their effect on the IgG2 subclass responses against Actinobacillus actinomycetemcomitans Y4 and Porphyromonas gingivalis 381, respectively. Sixty-nine EOP patients, including 11 with localized juvenile periodontitis (LJP), 19 who had LJP, 15 with LJP-rapidly progressing periodontitis (RPP), and 24 with RPP, were examined for the Gm and Km allotypes by a hemagglutination inhibition test. Levels of immunoglobulin G2 (IgG2) antibodies against the two organisms were determined by enzyme-linked immunosorbent assay. Fifty race- and age-matched, periodontally healthy subjects were also included as a control group. The observed frequencies of the Gm haplotype afnb and Km(1) were significantly higher in the RPP and LJP groups, respectively. The G2m(n)+ group of those with RPP and the Km(1)+ group of those with LJP had significantly higher levels of IgG2 antibodies to A. actinomycetemcomitans and P. gingivalis, respectively. The results indicate that linkage disequilibrium of the G2m(n) locus in RPP patients or the Km(1) locus in LJP patients may be associated with high IgG2 antibody responses to the respective bacteria. It was reasoned that the IgG2 antibody responses are associated with the immunoglobulin allotypes. The function of IgG2 antibodies in their reaction to different bacterial antigens may be interpreted as either protective or nonprotective in the two different types of EOP (i.e., LJP and RPP).
The leukotoxin produced by Actinobacillus actinomycetemcomitans has been implicated in the etiology of localized juvenile periodontitis. To initiate a genetic analysis into the role of this protein in disease, we have cloned its gene, lktA. We now present the complete nucleotide sequence of the lktA gene from A. actinomycetemcomitans. When the deduced amino acid sequence of the leukotoxin protein was compared with those of other proteins, it was found to be homologous to the leukotoxin from Pasteurella haemolytica and to the alpha-hemolysins from Escherichia coli and Actinobacillus pleuropneumoniae. Each alignment showed at least 42% identity. As in the other organisms, the lktA gene of A. actinomycetemcomitans was linked to another gene, lktC, which is thought to be involved in the activation of the leukotoxin. The predicted LktC protein was related to the leukotoxin/hemolysin C proteins from the other bacteria, since they shared a minimum of 49% amino acid identity. Surprisingly, although actinobacillus species are more closely related to pasteurellae than to members of the family Enterobacteriaciae, LktA and LktC from A. actinomycetemcomitans shared significantly greater sequence identity with the E. coli alpha-hemolysin proteins than with the P. haemolytica leukotoxin proteins. Despite the overall homology to the other leukotoxin/hemolysin proteins, the LktA protein from A. actinomycetemcomitans has several unique properties. Most strikingly, it is a very basic protein with a calculated pI of 9.7; the other toxins have estimated pIs around 6.2. The unusual features of the A. actinomycetemcomitans protein are discussed in light of the different species and target-cell specificities of the hemolysins and the leukotoxins.
Actinobacillus acrinomycetemcomirans isolates from periodontal pockets were examined for restriction fragment-length polymorphism using a characterized 4.7-kb DNA probe. A total of 6 patterns of RFLP was found in 133 isolates originating from 12 subjects. No relatedness was found between RFLP types and serotypes. Different periodontal sites within the same subject and different individuals within the same family sometimes showed only one type of A. actinomycetemcomitans RFLP. When members among the same family showed 2 RFLP types, children were always infected with the A. acfinomycefemcomitans strains found in at least one of the parents. These findings support the concept of familial spread of A. actinomycetemcomitans. A. actinomycetemcomitans RFLP type B, corresponding to reference strain JP2, seems to be particularly virulent, as indicated from the presence of RFLP type B in 3 subjects who converted from a healthy periodontal state to localized juvenile periodontitis. RFLP type B was not detected in any of the 21 A. acrinomycetemcomitans-infected patients with adult periodontitis. The RFLP method seems to be useful in determining the epidemiology and possibly the potential virulence of periodontal strains of A. actinomycetemcomitans.
juvenile periodontitis; epidemiology; virulence; restriction fragment-length polymorphism; DNA probes; Actinobaccillus actinomycetemcomitans
Localized juvenile periodontitis (LJP) is characterized by severe, early-onset, molar and incisor bone loss; neutrophil chemotaxis disorders; and a high prevalence of Actinobacillus actinomycetemcomitans infection. LJP is further characterized by significant familial aggregation of the disease. Recent work in our laboratory has demonstrated the selective depletion of a surface glycoprotein of 110,000 Mr (GP110) from LJP neutrophils by using surface labeling with [14C]formaldehyde and autofluorography. The function of GP110 is unknown; however, it does not appear to be a chemotactic factor receptor. Rather, it is bound by a monoclonal antibody (NCD-1) that recognizes a neutrophil differentiation antigen and which itself alters neutrophil chemotactic and secreting functions. To quantify GP110 on LJP and normal neutrophils, fluorescein-labeled NCD-1 was bound to neutrophils and the amount of fluorescence was evaluated by using cytofluorography. Our results indicate that there is a quantifiable reduction (40%) of GP110 on the surface of LJP and GJP neutrophils, compared with controls. Other patients with neutrophil defects express normal quantities of GP110, suggesting disease specificity. Our data suggest that GP110 may be a useful disease marker for LJP and may provide a useful probe for the study of neutrophil chemotactic function and dysfunction.
The epithelial cell invasiveness of Actinobacillus actinomycetemcomitans strains of different restriction fragment-length polymorphism (RFLP) groups associated with disease conversion and asymptomatic carrier status in localized juvenile periodontitis was examined. Twenty clinical isolates were studied for their ability to invade KB monolayers, using the quantitative gentamicin killing assay. Five isolates were found to be invasive; five were not invasive; and the other 10 did not invade better than an invasion negative control Haemophilus aphrophilus strain ATCC 19415. Using probe-specific DNA fingerprinting, 11 strains were assigned to RFLP group II (disease–associated); 4 to RFLP type XIII (carrier status-associated); and the others to groups III, IV, V and VII. Eight isolates, all RFLP group II, were leukotoxin producers as determined by PCR amplification of the lkt promoter region. No correlation was found between invasiveness and RFLP group. Leukotoxin production was more associated with noninvasive than invasive strains.
Actinobacillus actinomycetemcomitans; invasion; KB epithelial cell; restriction fragment-length polymorphism; leukotoxin
The cell envelope of Actinobacillus actinomycetemcomitans includes a number of outer membrane proteins (OMPs) which appear to be important targets for immunoglobulin G (IgG) antibodies in sera from localized juvenile periodontitis (LJP) patients. In this study, we examined the subclass distribution of IgG antibodies reactive to the 16.6- and 29-kDa OMPs of A. actinomycetemcomitans in sera from LJP patients and periodontally healthy individuals. Antibody responses were determined in a quantitative enzyme-linked immunosorbent assay that employed human IgG subclass-restricted monoclonal antibodies. High-titer LJP sera (93% black; geometric mean titer, 32,673) were found to contain significantly elevated levels of IgG1, IgG2, and IgG3 antibodies to the 29-kDa OMP of A. actinomycetemcomitans, compared with those of low-titer LJP sera (mean titer, 1,421) and sera from periodontally healthy, race-matched control subjects. The concentration of IgG2 antibody to this protein was greater than or equal to the corresponding IgG1 concentration in 7 of 14 high-titer sera, although mean IgG1 and IgG2 concentrations were not significantly different. The concentrations of IgG1 and IgG2 antibodies to the 16.6-kDa protein were also significantly elevated in LJP sera, although of considerably lesser magnitude than that observed for the 29-kDa protein. The IgG2 response to the 29-kDa protein could not be attributed to the presence of IgG2 antibodies to lipopolysaccharide contaminants or to Fc-binding activity, nor does this molecule appear to be a glycoprotein. Hence, LJP subjects produce IgG2 antibodies, as well as IgG1 and IgG3 antibodies, directed to at least one of the major OMPs of A. actinomycetemcomitans.
Sera from patients with localized juvenile periodontitis (LJP) often contain markedly elevated immunoglobulin G (IgG) antibody titers to serospecific determinants of the lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans. The objective of the present study was to define the subclass distribution of the IgG antibody response of LJP patients to this key cell envelope antigen. IgG subclass antibody responses to A. actinomycetemcomitans LPS were quantified in an enzyme-linked immunosorbent assay with human IgG subclass-restricted monoclonal antibodies. Serum antibody concentrations were calculated by heterologous interpolation of a dose-response curve constructed by using human-mouse chimeric antibodies. Sixteen of 17 LJP serum samples tested contained significantly greater concentrations of IgG2 than IgG1 antibodies reactive toward A. actinomycetemcomitans LPS. Geometric mean antibody concentrations of IgG1 and IgG2 were 7.8 and 136.5 micrograms/ml, respectively, among LJP patients with elevated IgG titers to LPS (94% of whom were black). However, both IgG1 and IgG2 antibody concentrations were significantly greater than the corresponding values obtained from sera from LJP patients with low IgG titers to LPS. Among LJP patients with elevated IgG titers to A. actinomycetemcomitans LPS, serum IgG2 concentration and total IgG concentration were also significantly elevated compared with both low-titered LJP sera and sera from periodontally healthy race-matched controls. The results of this study indicate that the humoral response of a predominantly black population of LJP patients to A. actinomycetemcomitans includes the production of LPS-reactive IgG antibodies which are primarily of the IgG2 subclass.
Actinobacillus actinomycetemcomitans is a gram-negative bacterium that has been implicated in the etiology of several forms of periodontitis, especially localized juvenile periodontitis. A potent leukotoxin (Lkt) is produced by most A. actinomycetemcomitans isolates from patients with periodontal disease, but some isolates are leukotoxin nonproducing (Lkt-). The molecular bases for the differences in leukotoxin expression are being explored to clarify the role of leukotoxin in pathogenesis. We have previously cloned the leukotoxin structural gene, lktA, from the leukotoxin-producing (Lkt+) strain JP2 and have shown that it is linked to three other genes, lktB, lktC, and lktD, whose gene products are thought to be required for activation and localization of the leukotoxin. These genes have now been used in Southern blot analysis to demonstrate that Lkt- strains, like Lkt+ strains, contain all four genes of the lkt gene cluster. While restriction fragment length polymorphisms were detected, they did not correlate with toxin phenotype. RNA blot analysis demonstrated that Lkt+ strains produced two transcripts, one 9.3 kb in length and the other 4.3 kb. They encode lktCABD and lktCA. respectively. Lkt- strains contained significantly lower levels of the 4.3-kb transcript with no discernible 9.3-kb message. The leukotoxic activity of the A. actinomycetemcomitans strains, measured by chromium release assays, correlated with the lkt RNA content. Therefore, a major component of leukotoxin regulation is at the level of RNA transcription or stability. Interestingly, the lkt RNAs in JP2 are regulated during growth phase, being greatly reduced in cells approaching stationary phase. Thus, the regulation of lkt RNA can be affected by both genotype and environment.
Eleven strains of Actinobacillus actinomycetemcomitans isolated from cases of systemic infections, local abscesses, and periodontitis were identified by genetic assays using the leukotoxin gene as the target. We have developed a polymerase chain reaction (PCR) assay, based on the leukotoxin structural gene of this pathogen, which clearly identified all tested strains of A. actinomycetemcomitans and separated them from the closely related Haemophilus aphrophilus as well as other bacterial species. Furthermore, DNA-DNA hybridization was performed with the cloned partial leukotoxin structural gene (lktA) as a probe, which again clearly distinguished A. actinomycetemcomitans from H. aphrophilus, parts of the normal oral flora, and species harboring RTX (repeats in toxin) family-related cytotoxins. The PCR fragment amplified from the leukotoxin structural gene gave results similar to those given by the cloned leukotoxin gene when used as a probe in hybridization experiments. The hybridization and PCR assays described here are fundamental improvements for the identification of A. actinomycetemcomitans.
Sera of localized juvenile periodontitis (LJP) patients colonized by Actinobacillus actinomycetemcomitans serotype b often contain markedly elevated levels of immunoglobulin G (IgG) antibodies to serospecific determinants in the O polysaccharide of lipopolysaccharide (LPS), as well as to outer membrane proteins of this species. IgG antibodies in LJP sera are known to opsonize A. actinomycetemcomitans for subsequent phagocytosis and killing by human neutrophils. The objective of this study was to determine whether outer membrane proteins or serospecific determinants in LPS are the primary target for opsonic IgG antibodies in LJP sera. An A. actinomycetemcomitans serotype b O-polysaccharide affinity column was constructed and subsequently used to purify LPS-specific IgG antibodies from LJP serum. The affinity-purified anti-LPS IgG antibodies were enriched in content of IgG2 (66.2%, compared with 37.0% in the total IgG fraction) and were immunospecific for A. actinomycetemcomitans serotype b LPS. In an opsonophagocytic assay using neutrophils from donors who were homozygous for the H131 allotype of Fcy receptor IIa (CD32), it was found that LPS-specific IgG antibodies exhibited substantially greater opsonic activity toward A. actinomycetemcomitans serotype b than an LJP IgG fraction that was depleted of LPS-reactive antibodies but contained antibodies against outer membrane proteins of this species. The results of this study indicate that serospecific determinants in the O polysaccharide of A. actinomycetemcomitans serotype b are a principal target for opsonic antibodies in sera of LJP subjects.
Sera from patients with localized juvenile periodontitis (LJP) often contain markedly elevated levels of immunoglobulin G2 (IgG2) antibodies reactive to cell envelope constituents of Actinobacillus actinomycetemcomitans. The objective of this study was to determine if these IgG2 antibodies are capable of supporting phagocytosis and killing of A. actinomycetemcomitans by human neutrophils. Polyclonal IgG2 antibodies were prepared from high-titer LJP serum by affinity chromatography, yielding a preparation which was > 99% subclass restricted and retained immunoreactivity to A. actinomycetemcomitans antigens. Affinity-purified IgG2 antibodies were evaluated by an in vitro opsonophagocytic assay that employed neutrophils obtained from donors who were homozygous for the H131 allotype of Fc gamma receptor type IIa (CD32), which efficiently binds human IgG2 antibodies. Affinity-purified IgG2 antibodies from LJP serum but not from sera of periodontally healthy individuals promoted phagocytosis and killing of A. actinomycetemcomitans. The expression of IgG2-dependent opsonic activity required the presence of complement. Incubation of A. actinomycetemcomitans with neutrophils in the presence of an optimal concentration of LJP IgG2 (50 micrograms/ml) and 5% hypogammaglobulinemic serum (as a complement source) resulted in a > 1 log10 reduction in bacterial viability within 30 min. The opsonic activity of IgG2 antibodies was found to be comparable to that observed with affinity-purified IgG1 antibodies. Moreover, IgG1 antibodies interacted synergistically with IgG2 antibodies in promoting opsonophagocytosis of A. actinomycetemcomitans. The results of this study indicate that LJP serum contains IgG2 antibodies which, when employed in conjunction with neutrophils that express Fc gamma receptors capable of recognizing this subclass, are opsonic for A. actinomycetemcomitans.
The leukotoxin produced by Actinobacillus actinomycetemcomitans has been implicated in the etiology of juvenile periodontitis. To initiate a genetic analysis of the role of this protein in disease, we have cloned the leukotoxin gene in Escherichia coli. Recombinant colonies carrying toxin gene sequences were isolated by screening a genomic A. actinomycetemcomitans library with a DNA probe for the leukotoxin gene from a related bacterium, Pasteurella haemolytica. To demonstrate that the cloned A. actinomycetemcomitans DNA contained a functional leukotoxin gene, protein extracts of E. coli containing the A. actinomycetemcomitans clone were tested directly for leukotoxic activity against human cell lines in chromium release assays. A construct containing the entire cloned region produced a functional toxin. No cytotoxicity was seen when extracts from cells containing plasmids with deletions in the putative coding region were used. Furthermore, the toxin produced by the cloned gene has the same target cell specificity as the leukotoxin extracted directly from A. actinomycetemcomitans. These results indicate that sequences encoding a functional leukotoxin have been cloned and are expressed in E. coli. Southern blot analysis of DNA from leukotoxin-producing (Lkt+) and non-leukotoxin-producing (Lkt-) strains indicated that the Lkt- strain also contained a copy of the gene.
The oral bacterium Actinobacillus actinomycetemcomitans is implicated as a causative agent of localized juvenile periodontitis (LJP). A. actinomycetemcomitans is classified into five serotypes (a to e) corresponding to five structurally and antigenically distinct O polysaccharide (O-PS) components of their respective lipopolysaccharide molecules. Serotype b has been reported to be the dominant serotype isolated from LJP patients. We determined the lipopolysaccharide O-PS structure from A. actinomycetemcomitans CU1000, a strain isolated from a 13-year-old African-American female with LJP which had previously been classified as serotype b. The O-PS of strain CU1000 consisted of a trisaccharide repeating unit composed of l-rhamnose and 2-acetamido-2-deoxy-d-galactose (molar ratio, 2:1) with the structure →2)-α-l-Rhap-(1–3)-2-O-(β-d-GalpNAc)-α-l-Rhap-(1→. O-PS from strain CU1000 was structurally and antigenically distinct from the O-PS molecules of the five known A. actinomycetemcomitans serotypes. Strain CU1000 was mutagenized with transposon IS903φkan, and three mutants that were deficient in O-PS synthesis were isolated. All three transposon insertions mapped to a single 1-kb region on the chromosome. The DNA sequence of a 13.1-kb region surrounding these transposon insertions contained a cluster of 14 open reading frames that was homologous to gene clusters responsible for the synthesis of A. actinomycetemcomitans serotype b, c, and e O-PS antigens. The CU1000 gene cluster contained two genes that were not present in serotype-specific O-PS antigen clusters of the other five known A. actinomycetemcomitans serotypes. These data indicate that strain CU1000 should be assigned to a new A. actinomycetemcomitans serotype, designated serotype f. A PCR assay using serotype-specific PCR primers showed that 3 out of 20 LJP patients surveyed (15%) harbored A. actinomycetemcomitans strains carrying the serotype f gene cluster. The finding of an A. actinomycetemcomitans serotype showing serological cross-reactivity with anti-serotype b-specific antiserum suggests that a reevaluation of strains previously classified as serotype b may be warranted.
Localized juvenile periodontitis (LJP) runs in families, and a predisposition to develop disease appears to be inherited in an autosomal dominant fashion. Patients with LJP have elevated levels of serum immunoglobulin G2 (IgG2), and this is most striking in black LJP patients. We hypothesized that the markedly elevated serum IgG2 levels related to LJP status and race may be attributable to a fundamental difference in the response of black LJP leukocytes. To test this possibility, leukocytes from black LJP patients, black non-periodontitis (NP) controls, and white NP controls were cultured with a nonspecific mitogen (pokeweed mitogen) which stimulates immunoglobulin production. The levels of IgG2 produced were measured using an enzyme-linked immunosorbent assay. The results revealed that the serum IgG2 level differences among black LJP patients and white and black NP subjects were reproducible in peripheral blood leukocytes in vitro. Analysis revealed that B cells from the LJP patients appeared to be predisposed to produce high levels of IgG2. Further analysis supported the concept that the high IgG2 responses of B cells from black LJP patients were regulated by monocytes. Replacing the monocytes in cultures from white NP subjects with LJP monocytes from black patients resulted in production of IgG2 at levels that were comparable with those produced by the LJP B cells from black patients. In short, B cells from black LJP patients produce elevated levels of IgG2 in vitro, and at least part of this elevation appears to be attributable to regulation via the LJP monocytes.
Actinobacillus actinomycetemcomitans, the etiologic agent for localized juvenile periodontitis and certain other human infections, such as endocarditis, expresses a leukotoxin that acts on polymorphonuclear leukocytes and macrophages. Leukotoxin is a member of the highly conserved repeat toxin (RTX) family of bacterial toxins expressed by a variety of pathogenic bacteria. While the RTX toxins of other bacterial species are secreted, the leukotoxin of A. actinomycetemcomitans is thought to remain associated with the bacterial cell. We have examined leukotoxin production and localization in rough (adherent) and smooth (nonadherent) strains of A. actinomycetemcomitans. We found that leukotoxin expressed by the rough, adherent, clinical isolate CU1000N is indeed cell associated, as expected. However, we were surprised to find that smooth, nonadherent strains of A. actinomycetemcomitans, including Y4, JP2 (a strain expressing a high level of toxin), and CU1060N (an isogenic smooth variant of CU1000N), secrete an abundance of leukotoxin into the culture supernatants during early stages of growth. After longer times of incubation, leukotoxin disappears from the supernatants, and its loss is accompanied by the appearance of a number of low-molecular-weight polypeptides. The secreted leukotoxin is active, as evidenced by its ability to kill HL-60 cells in vitro. We found that the growth phase and initial pH of the growth medium significantly affect the abundance of secreted leukotoxin, and we have developed a rapid (<2 h) method to partially purify large amounts of leukotoxin. Remarkably, mutations in the tad genes, which are required for tight nonspecific adherence of A. actinomycetemcomitans to surfaces, cause leukotoxin to be released from the bacterial cell. These studies show that A. actinomycetemcomitans has the potential to secrete abundant leukotoxin. It is therefore appropriate to consider a possible role for leukotoxin secretion in the pathogenesis of A. actinomycetemcomitans.
The purpose of this study was to examine the leukotoxin promoter types of
Aggregatibacter actinomycetemcomitans clones in subjects with
generalized aggressive periodontitis (GAgP) and in their family members (FM).
Material and Methods
Thirty-five patients with GAgP (33.9±7.1 years), 33 of their FM (22.8±11.4 years),
and 41 patients with chronic periodontitis (CP) (44.1±9.4 years) were clinically
analyzed using the plaque index, gingival index, probing depth (PD), and clinical
attachment level (CAL). Subgingival biofilm samples were collected from four
interproximal periodontal sites (>PD and >CAL) of each patient. The presence
of A. actinomycetemcomitans and its leukotoxic clone was
confirmed by polymerase chain reaction (PCR).
A. actinomycetemcomitans was observed in 23 (51.1%) GAgP patients and 16 (30.1%)
CP patients. Thirty-seven (94.8%) patients showed minimally leukotoxic strains and
2 (5.1%) showed highly leukotoxic strains. In the FM group, 10 (30.3%) had
aggressive periodontitis (AgP), 12 (36.3%) had CP, 11 (33.3%) were periodontally
healthy or had gingivitis, and 12.2% were A. actinomycetemcomitans positive.
Greater full mouth PD and CAL were observed in GAgP patients positive for the
bacteria than those negative for it (p<;0.05), and the presence of A.
actinomycetemcomitans positively correlated with GAgP (Odds ratio, 3.1; confidence
interval, 1.4-7.0; p=0.009).
The presence of A. actinomycetemcomitans was associated with the
clinical condition of GAgP, with most patients exhibiting a generalized form of
the disease and minimally leukotoxic clones. Most of the relatives of GAgP
patients presented either CP or AgP.
Aggregatibacter actinomycetemcomitans; Leukotoxin; Periodontitis
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.