NADPH Oxidase Contributes to Resistance against Aggregatibacter actinomycetemcomitans-Induced Periodontitis in Mice.

Aggregatibacter actinomycetemcomitans is a Gram-negative commensal bacterium of the oral cavity which has been associated with the pathogenesis of periodontitis with severe alveolar bone destruction. The role of host factors such as reactive oxygen and nitrogen intermediates in periodontal A. actinomycetemcomitans infection and progression to periodontitis is still ill-defined. Therefore, this study aimed to analyze the role of NADPH oxidase and inducible nitric oxide synthase (iNOS) in a murine model of A. actinomycetemcomitans-induced periodontitis. NADPH oxidase-deficient (gp91phox knockout [KO]), iNOS-deficient (iNOS KO), and C57BL/6 wild-type mice were orally infected with A. actinomycetemcomitans and analyzed for bacterial colonization at various time points. Alveolar bone mineral density and alveolar bone volume were quantified by three-dimensional micro-computed tomography, and the degree of tissue inflammation was calculated by histological analyses. At 5 weeks after infection, A. actinomycetemcomitans persisted at significantly higher levels in the murine oral cavities of infected gp91phox KO mice than in those of iNOS KO and C57BL/6 mice. Concomitantly, alveolar bone mineral density was significantly lower in all three infected groups than in uninfected controls, but with the highest loss of bone density in infected gp91phox KO mice. Only infected gp91phox KO mice revealed significant loss of alveolar bone volume and enhanced inflammatory cell infiltration, as well as an increased number of osteoclasts. Our results indicate that NADPH oxidase is important to control A. actinomycetemcomitans infection in the murine oral cavity and to prevent subsequent alveolar bone destruction and osteoclastogenesis.

Role of exopolysaccharide in Aggregatibacter actinomycetemcomitans-induced bone resorption in a rat model for periodontal disease.

Aggregatibacter actinomycetemcomitans a causative agent of periodontal disease in humans, forms biofilm on biotic and abiotic surfaces. A. actinomycetemcomitans biofilm is heterogeneous in nature and is composed of proteins, extracellular DNA and exopolysaccharide. To explore the role played by the exopolysaccharide in the colonization and disease progression, we employed genetic reduction approach using our rat model of A. actinomycetemcomitans-induced periodontitis. To this end, a genetically modified strain of A. actinomycetemcomitans lacking the pga operon was compared with the wild-type strain in the rat infection model. The parent and mutant strains were primarily evaluated for bone resorption and disease. Our study showed that colonization, bone resorption/disease and antibody response were all elevated in the wild-type fed rats. The bone resorption/disease caused by the pga mutant strain, lacking the exopolysaccharide, was significantly less (P < 0.05) than the bone resorption/disease caused by the wild-type strain. Further analysis of the expression levels of selected virulence genes through RT-PCR showed that the decrease in colonization, bone resorption and antibody titer in the absence of the exopolysaccharide might be due to attenuated levels of colonization genes, flp-1, apiA and aae in the mutant strain. This study demonstrates that the effect exerted by the exopolysaccharide in A. actinomycetemcomitans-induced bone resorption has hitherto not been recognized and underscores the role played by the exopolysaccharide in A. actinomycetemcomitans-induced disease.

Tight-adherence genes of Actinobacillus actinomycetemcomitans are required for virulence in a rat model.

Actinobacillus actinomycetemcomitans is a Gram-negative coccobacillus that has been associated with localized aggressive periodontitis and infections of the heart, brain, and urinary tract. Wild-type clinical isolates have the remarkable ability to adhere tenaciously and nonspecifically to solid surfaces such as glass, plastic, and hydroxyapatite. Adherence by A. actinomycetemcomitans is mediated by the tight-adherence (tad) gene locus, which consists of 14 genes (flp-1-flp-2-tadV-rcpCAB-tadZABCDEFG). All but 2 of the genes have been shown to be required for the secretion and assembly of long, bundled Flp1 fibrils. To test whether the tad locus is required for colonization and disease, we developed a rat model for periodontal disease. To mimic the natural route of infection, Sprague-Dawley rats were inoculated orally by adding bacteria directly to their food for 8 days. After inoculation with wild-type or mutant strains defective in adherence (flp-1 and tadA), the rats were assessed for colonization of the oral cavity and pathogenesis. Wild-type A. actinomycetemcomitans was able to colonize and persist for at least 12 weeks in the oral cavity, elicit a humoral immune response, and cause significant bone loss in rats. In contrast, rats fed flp-1 or tadA mutant strains showed no bone loss and their immune responses were indistinguishable from those of the uninoculated controls. These results demonstrate the critical importance of the tad locus in the colonization and pathogenesis of A. actinomycetemcomitans.

Population structure and genetic diversity of Actinobacillus actinomycetemcomitans strains isolated from localized juvenile periodontitis patients.

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.

Phenotypic variation in Actinobacillus actinomycetemcomitans during laboratory growth: implications for virulence.

This study examined alteration of specific virulence traits associated with phenotypic changes seen when a low-passage disease-associated and well maintained parent strain of Actinobacillus actinomycetemcomitans was compared to a laboratory-grown spontaneous variant/mutant. Clinical isolates of A. actinomycetemcomitans recovered from periodontitis patients typically grow as rough, adherent colonies on primary culture but undergo transformation to smooth, non-adherent colonies following repeated passage in vitro. The relationship of these phenotypic changes to the virulence of the organism or to the processes that underlie this transformation are not understood. A fresh clinical isolate, designated strain CU1000, was obtained from the first molar site of a patient with classical signs of localized juvenile periodontitis and used as the parent strain to study virulence-related phenotypes. Following several passages of CU1000 on selective agar, a spontaneous variant that demonstrated smooth, opaque, non-adherent colonies was isolated and designated strain CU1060. This study compared the properties of these two strains with respect to colony morphology, autoaggregation, surface appendages, adherence to saliva-coated hydroxyapatite (SHA), LPS chemotype and activity, induction of fibroblast proteinase activity and antigenic properties. CU1000 demonstrated rough, raised, star-positive colonies which upon electron microscopic examination revealed the presence of large, flexible, bundled fibrils. In addition, CU1000 showed adherence to SHA, several unique protein antigens and elevated endotoxin and fibroblast proteinase activity. CU1060, on the other hand, showed minimal adherence to SHA and fewer reactive proteins compared to the fresh clinical isolates. This strain formed smooth, opaque colonies on agar, showed minimal fibril formation and limited endotoxin and fibroblast-proteinase-inducing activity. These findings demonstrate that clinical isolates of A. actinomycetemcomitans undergo significant virulence-reducing phenotypic alterations during in vitro passage and support the need to study this organism in its clinical form.