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PubMed2002

The role of macrophages in the induction of murine immune response to Actinobacillus actinomycetemcomitans.

The aim of this study was to determine the role of macrophages in the Actinobacillus actinomycetemcomitans-induced murine immune response. BALB/c mice were given carrageenan solution by intraperitoneal injection before immunisation with heat-killed A. actinomycetemcomitans. Mice immunised with antigens and phosphate-buffered saline served as positive and negative controls, respectively. One week after the last immunisation, the delayed-type hypersensitivity (DTH) response was assessed by measurement of footpad swelling. Serum IgG and IgM anti-A. actinomycetemcomitans antibody levels and culture supernate levels of interferon (IFN)-gamma were determined by ELISA. The diameter of abscess formation was determined every 5 days. Sham-immunised spleen cells were transferred to carrageenan-untreated recipients (groups A and B) and to carrageenan-treated recipients (group D). Antigen-immunised spleen cells were transferred to carrageenan-untreated (group C) and carrageenan-treated (group E) recipients. The carrageenan-treated recipients in groups F and G received macrophages from antigen- and sham-immunised mice respectively. All mice except those in group A were immunised with antigen 24 h after cell transfer. After 1 week, a partial suppression of DTH response, reduced levels of IFN-gamma, serum IgG and IgM anti-A. actinomycetemcomitans antibodies and delayed healing were seen in carrageenan-treated mice when compared with the positive control. The immune response to A. actinomycetemcomitans in groups A, B and D was lower than that in groups C and E. Healing of the lesion in the former groups was also delayed when compared with the latter groups. The immune response and the healing of the lesion could be partially restored in carrageenan-treated mice that received antigen-pulsed macrophages (group F) but not in those that received naive macrophages (group G). These results suggest that macrophages play a partial role in the induction of the murine immune response to A. actinomycetemcomitans.

Abscess↗
PubMed1996

PCR primers for the amplification of the 16S rRNA gene of oral bacteria and for the specific identification of Actinobacillus actinomycetemcomitans.

The aim of this study was to develop a PCR reaction specific to Actinobacillus actinomycetemcomitans, which targets a widely conserved gene of this bacterium. Two sets of primers were designed based on published sequences of the 16S rRNA of several microorganisms. The first set amplifies a major part of the 16S small subunit rRNA gene of several strains of bacteria commonly found in the periodontal pocket. This reaction produced a 1306 bp-long product and served as a positive control. The second set was specific to A. actinomycetemcomitans and produced a 449 bp-long product. H. aphrophilus and E. coli yielded positive results with the control primers and negative results with the A. actinomycetemcomitans-specific primers. DNA-DNA hybridization was used to validate the identify of the amplified sequences. B. cereus, which is a common contaminator in the laboratory, and human DNA did not generate PCR products in either reaction. The developed primers seen useful for the identification of A. actinomycetemcomitans strains.

Aggregatibacter actinomycetemcomitans↗
PubMed2000

Secretion of RTX leukotoxin by Actinobacillus actinomycetemcomitans.

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.

Aggregatibacter actinomycetemcomitans↗
PubMed2003

Expression cloning of a periodontitis-associated apoptotic effector, cagE homologue, in Actinobacillus actinomycetemcomitans.

To study anti-bacterial immunity and to identify critical bacterial antigens associated with specific periodontal infection, we screened the genomic library of Actinobacillus actinomycetemcomitans, a major Gram(-) anaerobe causing human periodontitis, by expression cloning using disease-associated periodontal CD4(+)T cells derived from HuPBL-engrafted NOD/SCID mice. Here, we report one of the novel genes identified and designated, cagE homologue (in short: cagE) of A. actinomycetemcomitans, which encodes a putative bacterial type IV secretion system with significant homology to Helicobacter pylori CagE and Agrobacterium tumefaciens VirB4. All serum samples from A. actinomycetemcomitans-infected periodontitis patients, but not from the healthy controls, readily recognized CagE by ELISA and Western blot, suggesting its biological and clinical significance. The CagE protein, upon secretion, elicited significant apoptosis on primary human epithelia, endothelia, osteoblasts, and T cells by 4-12h in vitro. Importantly, both cagE(-) mutant strain and N-terminus truncated CagE protein drastically reduced (p<0.001) the induction of apoptosis on human epithelia in vitro. These data strongly suggest that a novel effector protein, CagE in A. actinomycetemcomitans, induces apoptosis of human cells and destructive immunity, thereby it may play an important role in the pathogenesis of A. actinomycetemcomitans-mediated infections.

Adult↗
PubMed1993

Genetic relationship of strains of Haemophilus aphrophilus, H. paraphrophilus, and Actinobacillus actinomycetemcomitans studied by ribotyping.

Strains of H. aphrophilus, H. paraphrophilus, and A. actinomycetemcomitans are phenotypically very similar. Ribotyping of 24 strains of H. aphrophilus, 22 strains of H. paraphrophilus, 8 strains of A. actinomycetemcomitans, and one strain each of the species Pasteurella aerogenes, H. parahaemolyticus, and the genus Capnocytophaga was studied using a non-radioactive digoxigenin labelled probe based on E. coli 16S- and 23S-ribosomal RNA. Restriction fragments were generated using restriction enzyme EcoRI. The ribotypes were analysed by a numerical approach using UPGMA clustering. Two major clusters were seen: One contained all A. actinomycetemcomitans strains, the other all H. aprophilus and all except one H. paraphrophilus strain intermingled between each other. The H. paraphrophilus strain not found in the H. aphrophilus/H. paraphrophilus cluster, the H. parahaemolyticus, P. aerogenes, and the Capnocytophaga strains clustered separately from each other and the two major clusters. The H. paraphrophilus strain with the deviating ribotype was atypical in other respects: it neither did ferment lactose nor mannose and it was isolated from a deer in contradiction to the remaining H. paraphrophilus strains, which were human isolates. This study supports the view that H. aphrophilus and H. paraphrophilus should be regarded as one species separated from A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans↗
PubMed2001

Protective humoral immunity induced by surface-associated material from Actinobacillus actinomycetemcomitans in mice.

The aim of the present study was to determine the role of antibodies specific to anti-surface-associated material from Actinobacillus actinomycetemcomitans (anti-SAM-Aa) in an infection induced by this periodontopathogen in mice. When SAM-Aa obtained by saline extraction of A. actinomycetemcomitans Y4 was separated on one-dimensional gel electrophoresis, this constituent contained antigen fragments with molecular weights ranging from 14000 to 79000. Immunoblot analysis revealed that increased antigen dose/immunization resulted in increased numbers of antigen epitopes recognized by serum antibodies of the immunized mice. Rapid healing of the primary lesions and high levels of specific IgG antibodies after challenge with live A. actinomycetemcomitans were seen in the immunized mice, especially at the highest-dose level of 100 microg/immunization. Transfer of SAM-Aa-immunized, but not the SAM-Aa-immunized and adsorbed, serum prior to challenge with live bacteria led to rapid healing of the lesions in the recipient mice. Increased phagocytosis of A. actinomycetemcomitans by murine macrophages (RAW264.7 cells) was observed when this periodontopathogen was opsonized by the SAM-Aa-immunized, but not SAM-Aa-immunized and adsorbed, serum. These results suggest that in mice, SAM-Aa antigens may induce protective antibodies by acting, at least, as an opsonin against challenge with live A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans↗
PubMed2002

In vitro antimicrobial susceptibility of oral strains of Actinobacillus actinomycetemcomitans to seven antibiotics.

BACKGROUND/AIMS: Periodontal infections with Actinobacillus actinomycetemcomitans seem to be refractory to conventional therapy. The aim of the present study was to test the in vitro susceptibilities of A. actinomycetemcomitans strains to a panel of seven orally administrable antibiotics. METHODS: A total of 60 isolates of A. actinomycetemcomitans recovered from 43 individuals with gingivitis or periodontitis were tested. In addition, laboratory strains UP-6 and JP2 were analysed. The E-test was employed in order to determine minimal inhibitory concentrations (MIC) of antibiotics ampicillin/sulbactam, roxithromycin, azithromycin, doxycycline, metronidazole, ciprofloxacin, and moxifloxacin. RESULTS: A. actinomycetemcomitans was highly susceptible to both fluoro-quinolones (MIC90 of 0.006 microgram/mL of ciprofloxacin and 0.032 microgram/mL of moxifloxacin). Good susceptibilities were found for ampicillin/sulbactam and doxycycline (MIC90 of 0.75 microgram/mL and 1 microgram/mL, respectively), and moderate susceptibilities for azithromycin (MIC90 of 3 microgram/mL). Most strains were resistant to metronidazole and roxithromycin. Cluster analysis revealed two larger clusters of A. actinomycetemcomitans strains with the smaller cluster assembling isolates with significantly higher MICs of most antibiotics. CONCLUSIONS: Due to reported favourable pharmacokinetics, the fluoro-quinolone moxifloxacin appeared to be a promising candidate for adjunctive systemic antibiotic therapy in periodontal infections with A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans↗
PubMed2005

Intragenomic recombination in the highly leukotoxic JP2 clone of Actinobacillus actinomycetemcomitans.

The highly leukotoxic JP2 clone of Actinobacillus actinomycetemcomitans is strongly associated with aggressive periodontitis in adolescents of African descent. DNA fingerprinting using the frequently cutting restriction enzyme MspI and multilocus sequence typing (MLST) showed that five strains of this clone were genetically virtually identical, although ribotyping of the six rrn genes and EcoRI RFLP analysis of the seven IS150-like elements revealed differences. PCR analyses demonstrated that these multi-copy sequences are subject to intragenomic homologous recombination, resulting in translocations or large inversions. The genome rearrangements were reflected in differences among 25 strains representing the JP2 clone in DNA fingerprinting using the rare-cutting restriction enzyme XhoI and resolved by PFGE. XhoI DNA fingerprinting provides a tool for studying local epidemiology, including transmission of this particularly pathogenic clone of A. actinomycetemcomitans.

Actinobacillus Infections↗
PubMed1996

Characterization of Actinobacillus actinomycetemcomitans hemolysin.

Twenty out of 33 Actinobacillus actinomycetemcomitans strains formed hemolytic colonies on horse blood agar plates under anaerobic conditions. The hemolytic activity found in A. actinomycetemcomitans strain 137HE was examined. This activity was detected in the late exponential to early stationary phases of growth. Human erythrocytes were the most susceptible, followed by rabbit, sheep, horse and swine red blood cells. The majority of activity was detected in the cell-associated vesicle fraction. Zwitterionic detergent 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS) extract from whole cells was semipurified by ammonium sulfate precipitation, preparative isoelectric focusing (IEF) and gel-filtration chromatography to yield a major band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular mass of 12 kDa. Heating at 80 C for 30 min and treatment with proteinase K or trypsin resulted in complete disappearance of the hemolytic activity. Sulphydryl reagents enhanced activity and small amounts of cholesterol inhibited it. In summary, we demonstrated the presence of hemolysin in A. actinomycetemcomitans, and examined and characterized it.

Aggregatibacter actinomycetemcomitans↗
PubMed1993

Evaluating two methods for fingerprinting genomes of Actinobacillus actinomycetemcomitans.

The arbitrary primer polymerase chain reaction (AP-PCR) and Southern blot restriction fragment length polymorphism (RFLP) were used to genotype the periodontal pathogen A. actinomycetemcomitans. Total genomic DNA from 73 strains was extracted by conventional methods. Three random-sequence 10-base oligonucleotide primers were chosen for AP-PCR. The amplified DNA products were separated electrophoretically in a 1% agarose gel containing ethidium bromide and the banding patterns were compared among different strains. For RFLP analysis, DNA was digested with EcoRI, separated on a 0.8% agarose gel and transferred to a nylon membrane. The membrane was probed with a previously characterized 5.2 kilobases (kb) DNA fragment cloned from A. actinomycetemcomitans strain Y4. The probe was labeled with digoxigenin, and hybridized fragments were detected with anti-digoxigenin antibody. AP-PCR produced 4-10 DNA bands in the 0.5-5 kb regions and distinguished 9, 13 or 17 genotypes, depending on the specific primer used. Southern blot RFLP analysis revealed 12 hybridization patterns consisting of 1 or 2 DNA fragments (2-23 kb). The addition of the Southern blot analysis to the AP-PCR analysis gave rise to a total of 30 DNA profiles among the 73 A. actinomycetemcomitans study strains. The results indicate that both AP-PCR and Southern blot analysis are useful in clonal analysis of A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans↗
PubMed1993

Killing of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus by human polymorphonuclear leukocytes in serum and saliva.

The ability of polymorphonuclear leukocytes from human peripheral blood to kill Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus was examined with fresh isolates and laboratory strains from each species (5 strains within each group) under different conditions. Bacterial cells were mixed with a polymorphonuclear leukocyte suspension in the presence of either active serum or heat-inactivated serum or active serum together with sterile-filtered saliva. Surviving bacteria were determined by counting the number of bacterial colony-forming units in the mixtures after a 60-min incubation at 37 degrees C. Mixtures without polymorphonuclear leukocytes served as controls for the evaluation of the degree of killing of the bacteria. In general, A. actinomycetemcomitans resisted phagocytic killing to a greater extent than H. aphrophilus, and the killing of the former species mainly depended on the presence of heat-labile serum components, probably complement factors. Laboratory strains of A. actinomycetemcomitans were more easily killed than fresh isolates. The presence of saliva in the reaction mixtures decreased the degree of killing. However, strain-dependent variations in the killing were found under either condition. The leukotoxic activity of A. actinomycetemcomitans strains, determined by a [51Cr]-release assay, was not correlated with the resistance of these strains to the phagocytic killing. The results point out a strain-dependent difference in the ability of A. actinomycetemcomitans to evade the inflammatory response associated with polymorphonuclear leukocytes. This difference may constitute a potential virulence factor for this periodontopathogen. Furthermore, the leukotoxicity of the strains is not the main determinant that modifies the interaction of A. actinomycetemcomitans with human neutrophils.

Aggregatibacter actinomycetemcomitans↗