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PubMed1995

Lactoferrin interaction with Actinobacillus actinomycetemcomitans.

The interaction of lactoferrin with Actinobacillus actinomycetemcomitans was examined in a 125I-labeled protein binding assay. The binding of human and bovine lactoferrins reached maximum within 1 h. Lactoferrin binding to the bacterium was pH-dependent and reversible. Scatchard analysis indicated the existence of two different types of binding sites on the bacterium, one with a high affinity constant k alpha approximately 8.8 x 10(-7) M) and the other with a low one (k alpha approximately 1.8 x 10(-6) M). Bacteria in the exponential phase of growth showed higher binding than cells in the stationary phase. Bacteria grown in medium containing serum and/or lysed erythrocytes bound lactoferrin to a lesser extent. Heat-inactivated serum, lysed erythrocytes and other proteins such as mucin and laminin inhibited lactoferrin binding to A. actinomycetemcomitans in a competitive binding assay. Sodium dodecyl sulfate polyacrylamide-gel electrophoresis and Western blot analysis of the cell envelope as well as the outer membrane of A. actinomycetemcomitans revealed lactoferrin-reactive protein bands at 29 kDa and 16.5 kDa. The 29-kDa band displayed a heat-modifiable lactoferrin-reactive form with a molecular weight of 34 kDa. Neither proteinase K-treated cell envelope nor lipopolysaccharide of this bacterium showed reactivity with lactoferrin. These data suggests a specific interaction of lactoferrin with outer membrane proteins of A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans↗
PubMed1995

Actinobacillus actinomycetemcomitans genotypes in relation to serotypes and periodontal status.

Actinobacillus actinomycetemcomitans is prevalent in periodontitis but is found in some periodontally healthy individuals as well. The arbitrarily primed polymerase chain reaction (AP-PCR) was used to fingerprint clinical A. actinomycetemcomitans isolates of different serotypes to determine the association between individual clonal types and periodontal conditions. Fifteen different AP-PCR genotypes were distinguished among 93 A. actinomycetemcomitans isolates from 86 uncohabiting individuals with adult periodontitis, localized juvenile periodontitis or no periodontal destruction. The 3 most common AP-PCR genotypes accounted for 68% of the isolates. Seven of the remaining AP-PCR genotypes were found only in periodontitis. The isolates of a given AP-PCR genotype usually belonged to the same serotype. The distribution of the AP-PCR genotypes among serotype b isolates seemed to differ among the subject groups. The results revealed a major genetic dissimilarity between A. actinomycetemcomitans serotypes and suggested a relationship between some A. actinomycetemcomitans clones and periodontal disease.

Aggregatibacter actinomycetemcomitans↗
PubMed1998

Putative heat shock protein 70 gene from Actinobacillus actinomycetemcomitans: molecular cloning and sequence analysis of its gene.

We have cloned and sequenced two overlapping fragments of chromosomal DNA from Actinobacillus actinomycetemcomitans. The nucleotide sequence contained two open reading frames. The deduced amino acid sequences of the two open reading frames showed significant homology with the heat shock proteins hsp70 and hsp40 of other organisms respectively. The upstream open reading frame consisted of 1902 bp, corresponding to 634-amino-acid residues. The CAA codon for glutamines was frequently seen in hsp70, i.e., in 30 of 32 glutamines (93.8%). The spacing region between the two open reading frames was unusually long compared with other prokaryotic organisms. A number of unique and distinguishing features of the sequences in the hsp70 family were verified, and it was found that a particular spacing sequence between the hsp70 and hsp40 gene loci can be used to identify A. actinomycetemcomitans from the periodontal pocket.

Aggregatibacter actinomycetemcomitans↗
PubMed1998

Differential clinical treatment outcome after systemic metronidazole and amoxicillin in patients harboring Actinobacillus actinomycetemcomitans and/or Porphyromonas gingivalis.

48 adult patients with untreated periodontitis harboring subgingival Actinobacillus actinomycetemcomitans and/or Porphyromonas gingivalis as assessed by PCR were randomly assigned to receive full-mouth scaling alone (control) or scaling with systemic metronidazole plus amoxicillin and supragingival irrigation with chlorhexidine digluconate (test). In patients harboring A. actinomycetemcomitans intraorally at baseline, the adjunctive antimicrobial therapy resulted in a significantly higher incidence of probing attachment level (PAL) gain of 2 mm or more compared to scaling alone over 12 months (p<0.05). In addition, suppression of A. actinomycetemcomitans in subgingival plaque below detectable levels was associated with an increased incidence of PAL gain. In contrast, patients initially harboring P. gingivalis but not A. actinomycetemcomitans in the oral cavity showed a significantly higher incidence of PAL loss following adjunctive antimicrobial therapy compared to scaling alone (p<0.05). When the presence of pathogens at baseline was disregarded in the analysis, adjunctive antimicrobial therapy did not significantly enhance clinical treatment outcome. The results indicated that adults with untreated periodontitis harboring A. actinomycetemcomitans may benefit from the adjunctive antimicrobial therapy for a minimum of 12 months, whereas, the regimen may adversely affect the clinical treatment outcome of patients harboring P. gingivalis but not A. actinomycetemcomitans.

Adult↗
PubMed1995

Immunoglobulin G2 antibodies promote neutrophil killing of Actinobacillus actinomycetemcomitans.

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.

Agammaglobulinemia↗
PubMed1995

Directed genomic integration in Actinobacillus actinomycetemcomitans: generation of defined leukotoxin-negative mutants.

To develop targeted gene integration in the periodontal pathogen Actinobacillus actinomycetemcomitans, a ColE1-based, spectinomycin-resistant plasmid containing a segment of the leukotoxin gene was electroporated into strain JP2. In all of the stable spectinomycin-resistant transformants that arose, the plasmid had recombined into the genomic leukotoxin locus since ColE1-based vectors cannot replicate extrachromosomally in A. actinomycetemcomitans. Directed genomic integration was then used to construct a leukotoxin-negative strain by transforming the leukotoxin-producing strain JP2 with a ColE1-based plasmid containing an internal fragment of the leukotoxin gene. Cytotoxicity assays proved that these transformants had < 0.1% of the leukotoxin activity of the parental strain. These results demonstrate that integration-based approaches can be used for generating isogenic mutants in specific virulence genes in A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans↗
PubMed2002

Natural transformation and DNA uptake signal sequences in Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans is a member of the family Pasteurellaceae and a major causative agent of periodontitis. While several genera from this family are known to be competent for transformation, A. actinomycetemcomitans has yet to be fully characterized. Here we show that the competence of A. actinomycetemcomitans is remarkably similar to that of Haemophilus influenzae. In addition to having a similar frequency of transformation as H. influenzae, A. actinomycetemcomitans competence could also be induced at least 100-fold by cyclic AMP, suggesting that, as in H. influenzae, at least some competence genes are regulated by catabolite repression. Even more intriguing was the discovery of a putative A. actinomycetemcomitans DNA uptake signal sequence (USS) virtually identical to the USS of H. influenzae. Moreover, we provide evidence that this sequence functions in the same capacity as that from H. influenzae; the sequence appears to be required and sufficient for DNA uptake in a variety of assays. Finally, we have taken advantage of this system to develop a simple, highly efficient competence-based method for generating site-directed mutations in the wild-type fimbriated A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans↗
PubMed1994

Actinobacillus actinomycetemcomitans in adult periodontitis. II. Characterization of isolated strains and effect of mechanical periodontal treatment.

Ten systemically healthy subjects (ages 28 to 60 years) with untreated moderate to severe periodontal disease and evidence of presence of A. actinomycetemcomitans underwent standard mechanical periodontal treatment consisting of oral hygiene instruction and systematic deep scaling and root planing. Before, and 4 to 5 weeks after treatment, clinical measurements and separate subgingival microbiological samples were taken from the mesial and distal aspect of every tooth, with the exception of the third molars. A. actinomycetemcomitans could still be detected in all patients after treatment. In 9 of the 10 patients, all tested isolates from both examinations were of a single type. Two patients carried serotype a; 2 serotype b; 2 serotype c; and 1 serotype e. Two individuals showed only non-typeable isolates lacking serotype a, b, c, d, or e specific antigens. Another subject was colonized by serotype c and, in addition, yielded a non-typeable isolate. Persistence of A. actinomycetemcomitans after treatment was significantly correlated with the frequency of A. actinomycetemcomitans before treatment (P < 0.001) and the mean probing depth before treatment (P < 0.05). No serotype-specific patterns of treatment outcome could be recognized. The analysis of the site specific effect of treatment showed a significant relationship between post treatment levels of A. actinomycetemcomitans and both probing depth reduction as well as attachment gain. Individuals showing evidence of A. actinomycetemcomitans in a multitude of sites appeared to be more difficult to treat than patients with few positive sites only. Within such individuals, the deeper pockets showed the greater resistance to eradication of A. actinomycetemcomitans.

Adult↗
PubMed2005

Identification of a genomic island of Actinobacillus actinomycetemcomitans.

BACKGROUND: Horizontal gene transfer (HGT) is a process by which bacteria acquire genes from organisms of distant taxa. HGT is now recognized as a major driving force in the evolution of bacterial pathogens. Through this process, bacteria may accumulate blocks of DNA such as genomic islands (GEIs) that encode fitness or virulence factors. The periodontal pathogen A. actinomycetemcomitans has been known to exhibit variable virulence potential. It is postulated that GEIs may play a role in modifying the virulence potential of A. actinomycetemcomitans. This study was initiated to identify and determine the distribution of GEIs in A. actinomycetemcomitans. METHODS: Forty-seven A. actinomycetemcomitans strains of serotypes a through f were examined. Strain-specific variant DNA in the genomes of A. actinomycetemcomitans was identified by polymerase chain reaction (PCR) genomic mapping and sequenced to identify GEIs. The distribution of the GEIs among test strains of A. actinomycetemcomitans was determined by PCR analysis and Southern hybridization assays. RESULTS: An approximately 22 kb GEI of A. actinomycetemcomitans, designated AAI-1, was identified in five serotype b strains. The AAI-1 exhibits low %G+C and encodes proteins of phage, restriction modification systems, mobile elements, and other hypothetical proteins of unknown functions. The insertion of AAI-1 was found to cause truncation of A. actinomycetemcomitans genes at the insertion site. CONCLUSIONS: Some A. actinomycetemcomitans strains may harbor GEIs, which were acquired via HGT by the bacteria. The GEIs may increase the gene repertoire of A. actinomycetemcomitans. However, the insertion of the GEIs in A. actinomycetemcomitans may also cause truncation and inactivation of resident genes at the insertion sites. The virulence significance of such gain and loss of genes in A. actinomycetemcomitans remains to be determined.

Aggregatibacter actinomycetemcomitans↗
PubMed2001

Actinobacillus actinomycetemcomitans harbours type IV secretion system genes on a plasmid and in the chromosome.

Nine contiguous genes encoding a potential type IV secretion system have been identified in the chromosome of Actinobacillus actinomycetemcomitans strain VT747 and on a plasmid (pVT745) in strain VT745. Seven of these genes encode predicted proteins that share significant homology with type IV secretion proteins in Bordetella pertussis (ptl operon), Brucella melitensis biovar suis and Agrobacterium tumefaciens (virB operons), where they are involved in protein secretion, pathogen intracellular survival and multiplication, and DNA transport, respectively. Results of previous studies have demonstrated that pVT745 is a conjugative plasmid and that a secondary plasmid, pMMB67, can be mobilized from strain VT745. Given these results, it was hypothesized that (1) the type IV secretion genes on pVT745 are responsible for these two functions and (2) the type IV VT747 chromosomal genes also play a role in the transport of DNA. Wild-type and mutant strains of VT745 were evaluated for their conjugative abilities. Wild-type mating efficiency was 10(-6) transconjugants per donor, while the mutant strain yielded no transconjugants. Wild-type VT745 harbouring a co-resident plasmid, pMMB67, mobilized pMMB67 at a frequency of 10(-6), while VT747 was unable to mobilize this plasmid. These results support the hypothesis that the plasmid-encoded type IV secretion system on pVT745 is involved in DNA transport. However, the chromosomally encoded secretion system may not play a role in DNA transport in strain VT747. While the precise function of these chromosomal genes in strain VT747 has not been determined, Northern blot analyses demonstrated that these genes are expressed in both ACT: actinomycetemcomitans strains VT745 and VT747.

Aggregatibacter actinomycetemcomitans↗
PubMed2001

Energy metabolism of Actinobacillus actinomycetemcomitans during anaerobic and microaerobic growth in low- and high-potassium continuous culture.

Actinobacillus actinomycetemcomitans, a member of the gamma subclass of the Proteobacteria, has been implicated as the agent responsible for human periodontitis. In this study, A. actinomycetemcomitans 301-b was grown in fructose-limited chemostat cultures under anaerobic [redox potential (E(h))<-400 mV] and microaerobic (E(h)= -200 mV) conditions to characterize its energy metabolism. Effects of K(+) and Na(+) on growth and metabolism were also examined. In a control medium containing 5.2 mM K(+) and 24 mM Na(+), the molar growth yield on fructose (Y(fructose)) of microaerobic cultures was 1.3 times higher than the yield of anaerobic cultures at D < or =0.10 h(-1), but the difference in the Y(fructose) between microaerobic and anaerobic cultures decreased at D< or =0.10 h(-1). When the ATP yield from fermentation was estimated from the amounts of fructose consumed and acetate formed, the value of the microaerobic culture (2.49 mol ATP produced per mol fructose consumed) was lower than the anaerobic value [3.13 mol ATP (mol fructose)(-1)]. Therefore, ATP production from fermentation could not account for the increase in the Y(fructose) at D > 0.10 h(-1) and thus additional ATP was expected to be generated via respiration. Assuming that the Y(ATP) (g cells formed per mol ATP synthesized) was similar between anaerobic and microaerobic cultures, the estimated ATP yield from respiration was between 1.2 and 2.0 mol ATP (mol fructose)(-1) below D=0.10 h(-1) and decreased to 0.3 mol ATP (mol fructose)(-1) when D was increased to 0.19 h(-1). Such growth-rate-dependent decreases in the Y(fructose) and the estimated ATP production from respiration were also observed in a high-Na(+) (5.2 mM K(+) and 106 mM Na(+)) culture but not in a high-K(+) (81 mM K(+) and 24 mM Na(+)) culture. In the high-K(+) culture, the microaerobic Y(fructose) was 1.4-2.0 times higher than the anaerobic value and the respiration-derived ATP yield was estimated to be between 1.2 and 1.9 mol ATP (mol fructose)(-1) over a wide range of dilution rate. These results suggest that higher concentrations of extracellular K(+) are required for the respiration to occur in rapidly growing cells of A. actinomycetemcomitans.

Actinobacillus Infections↗
PubMed1993

Identification of Actinobacillus actinomycetemcomitans: polymerase chain reaction amplification of lktA-specific sequences.

Actinobacillus actinomycetemcomitans has been strongly implicated in the etiology of localized juvenile periodontitis. Techniques used in the identification of this periodontal pathogen include cultural, biochemical, immunological and DNA hybridization analysis. In this study, we report the use of polymerase chain reaction (PCR) to amplify unique sequences of A. actinomycetemcomitans. Specific oligonucleotide primers LKT2 and LKT3 were designed to hybridize to the A. actinomycetemcomitans lktA gene, which encodes leukotoxin, a putative A. actinomycetemcomitans virulence factor. The LKT2 and LKT3 primers amplified lktA-specific sequences from all 12 A. actinomycetemcomitans strains tested. In another set of experiments, 13 other bacterial species, most of which are normal residents of the oral cavity, were tested with these primers. These PCR amplifications also contained 2 additional primers, RRN4 and RRN5, which served as positive controls; RRN4 and RRN5 were designed to amplify specific sequences of eubacterial 16S ribosomal DNA (rDNA). PCR amplifications of all bacterial species tested, including A. actinomycetemcomitans, yielded 16S rDNA-specific DNA fragments. Furthermore, each bacterial species tested, with the exception of A. actinomycetemcomitans, failed to amplify lktA sequences. The LKT and RRN primers were used in further PCR experiments to detect A. actinomycetemcomitans directly from gingival fluid samples. The results clearly demonstrate the simplicity, rapidity, specificity and accuracy of the LKT primers in the identification of A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans↗
PubMed1993

Actinobacillus actinomycetemcomitans recovery from extracrevicular locations of the mouth.

Associations between recovery of Actinobacillus actinomycetemcomitans from samples of subgingival plaque, and samples of buccal mucosa, tongue and unstimulated saliva were studied in 107 subjects. Ten subjects had gingivitis, 18 localized juvenile periodontitis, 45 rapidly progressive periodontitis and 32 adult periodontitis. Two children suffered from prepubertal periodontitis. Heterogeneity tests for associations in different study populations yielded nonsignificant results. Mantel-Haenszel's common odds ratios were 52.9, 37.2 and 19.8 for respective associations between pooled subgingival samples, and cheek, saliva and tongue samples. Significant McNemar's chi-square of 5.88, 11.25 and 16.96 for respective associations pointed to secondary occurrence of A. actinomycetemcomitans in extracrevicular samples. Multiple linear regression yielded a significant influence of the number of deep periodontal pockets of 7 mm or more and a negative influence of the diagnosis "adult periodontitis" on the log-transformed number of colony-forming units of A. actinomycetemcomitans in samples from cheek mucosa in patients infected with the organism. Extracrevicular occurrence of A. actinomycetemcomitans seems to reflect total subgingival numbers of the organism. Especially sampling cheek mucosa appears to be a promising tool in the diagnosis of a periodontal infection with A. actinomycetemcomitans.

Adolescent↗
PubMed1998

Expression of iron binding proteins and hemin binding activity in the dental pathogen Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans was found to express a polypeptide immunologically related to the Neisseria gonorrhoeae FbpA iron binding protein. In addition, the expression of hitB and hitC homologs was detected by Northern blot analysis. This periodontal pathogen also expresses a polypeptide homologous to the 31-kDa Haemophilus influenzae protein, which shows amino acid sequence homology with the FimA and YfeA proteins from Streptococcus parasanguis and Yersinia pestis, respectively. Both A. actinomycetemcomitans protein homologs were located within the periplasmic space, and their synthesis was regulated by the iron and hemin concentration of the culture medium. Southern and Western blot analysis together with molecular cloning revealed the presence of a Fur-like repressor, which may control the iron regulation of gene expression in this bacterium. Cultivation in the presence of hemin or Congo red revealed the ability of this organism to bind hemin. This binding activity was further confirmed by isolating Escherichia coli DH5 alpha clones that produced red and brown colonies on agar plates containing Congo red and hemin, respectively, after transformation with an A. actinomycetemcomitans gene library.

Aggregatibacter actinomycetemcomitans↗
PubMed1996

Occurrence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia in juvenile periodontitis.

The occurrence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia in subgingival plaque in 24 juvenile periodontitis patients was determined using DNA probe. 36 samples of subgingival plaque from 36 pockets having > or = 6 mm depth, > or = 3 mm of loss of attachment, and bleeding on probing and/or suppuration were taken from 18 patients with localized juvenile periodontitis (LJP, age range 12-24 years); and 12 samples from 6 patients with generalized juvenile periodontitis (GJP, age range 23-26 years). As control, an equal numbers of samples from healthy sites in the same patients were studied. P. gingivalis was found in 17 of 18 LJP patients, and in 31 of 36 diseased sites in those patients. P. intermedia was found in 15 out of the 18 LJP patients and in 28 of the 36 diseased sites. A. actinomycetemcomitans was present in 7 of the 18 LJP patients, and in 9 of the 36 diseased sites, and was not found in any GJP patients. All GJP patients had P. gingivalis (11 out of 12 diseased sites) and P. intermedia (all of the diseased sites). None of the three bacterial species was detected in healthy sites of GJP patients, and were found in healthy sites in only 2 of 18 LJP patients. The high prevalence and high levels of P. gingivalis and P. intermedia found in the LJP and GJP patients studied, suggest that there are populations affected by juvenile periodontitis in which this type of periodontitis is more associated with these species than with A. actinomycetemcomitans.

Adolescent↗