Helen Schreiner

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.

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

ABSTRACT 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.

Colonization and persistence of rough and smooth colony variants of Actinobacillus actinomycetemcomitans in the mouths of rats

Fresh isolates of Actinobacillus actinomycetemcomitans (Aa) bind avidly to surfaces in vitro, but existing in vivo studies of the adherence of Aa are limited. This study had two goals: (1) to compare the oral colonization of two isogenic strains of Aa-CU1010, a clinical isolate that expresses the adherent phenotype, and CU1012, a minimally adherent laboratory variant-and (2) to check for phenotypic reversion of these strains in a clinical setting. Rifampicin-resistant strains, developed for tracking in Sprague-Dawley rats, were tested in vitro to determine their stability and binding. In study 1, after antibiotic suppression, six rats (group I) received CU1010 in their feed. The eight rats in group II received CU1012 in their feed and four were supplemented by oral swabbing and four by gastric gavage. Group III consisted of three sham-inoculated controls. All rats were inoculated for 4 days. Microbiological data were collected at 1, 4 and 8 weeks after inoculation. Supporting data were supplied by antibody titres and clinical measures of alveolar bone loss. Study 2 consisted of six rats in each of three groups as above, but tagged strains of Aa were delivered by food alone. At all time-points in both studies, Aa was absent before inoculation and controls had no Aa or antibody to Aa. In study 1, all six rats in group I yielded positive cultures for Aa at 8 weeks. In group II, five of eight had positive cultures for Aa at 1 week, two of eight at 4 weeks and none had Aa at 8 weeks (P < or =0.001). All six rats in group I had serum anti-Aa titres compared to group II, where titres were seen in four of eight rats (P < or =0.015). In vitro data paralleled those found in vivo. No phenotypic reversion of either strain was seen in vivo. In study 2, four of six rats in group I showed Aa and had titres to Aa, while no other animals showed Aa at any time. The model provides convincing evidence that, unlike laboratory variants, clinical isolates colonize, persist and integrate into an already established, albeit reduced, econiche.

Aggregatibacter actinomycetemcomitans Infection Enhances Apoptosis In Vivo through a Caspase-3-Dependent Mechanism in Experimental Periodontitis

ABSTRACT The purpose of this study was to test the hypothesis that diabetes aggravates periodontal destruction induced by Aggregatibacter actinomycetemcomitans infection. Thirty-eight diabetic and 33 normal rats were inoculated with A. actinomycetemcomitans and euthanized at baseline and at 4, 5, and 6 weeks after inoculation. Bone loss and the infiltration of polymorphonuclear leukocytes (PMNs) in gingival epithelium were measured in hematoxylin-eosin-stained sections. The induction of tumor necrosis factor alpha (TNF-α) was evaluated by immunohistochemistry and of apoptotic cells by a TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay. After A. actinomycetemcomitans infection, the bone loss in diabetic rats was 1.7-fold and the PMN infiltration 1.6-fold higher than in normoglycemic rats (P < 0.05). The induction of TNF-α was 1.5-fold higher and of apoptotic cells was up to 3-fold higher in diabetic versus normoglycemic rats (P < 0.05). Treatment with a caspase-3 inhibitor significantly blocked noninflammatory cell apoptosis induced by A. actinomycetemcomitans infection in gingival epithelium and connective tissue (P < 0.05). These results provide new insight into how diabetes aggravates A. actinomycetemcomitans-induced periodontal destruction in rats by significantly increasing the inflammatory response, leading to increased bone loss and enhancing apoptosis of gingival epithelial and connective tissue cells through a caspase-3-dependent mechanism. Antibiotics had a more pronounced effect on many of these parameters in diabetic than in normoglycemic rats, suggesting a deficiency in the capacity of diabetic animals to resist infection.

Adaptive immune response in osteoclastic bone resorption induced by orally administered Aggregatibacter actinomycetemcomitans in a rat model of periodontal disease

There is mounting evidence that innate and adaptive immunity are critical for periodontal disease-mediated bone resorption. These studies examined the role of B and CD4 T cells in adaptive immunity of rats infected with Aggregatibacter actinomycetemcomitans (Aa). Sprague-Dawley male rats were fed Aa-containing mash or control-mash for 2 weeks. B and CD4 T cells were obtained from draining lymph nodes at 2, 4 and 12 weeks, postinoculation. Quantitative polymerase chain reaction-based messenger RNA expression was conducted for 89 cytokine family genes. Disease-relevance of the differentially expressed genes was assessed using a biological interaction pathway analysis software. B and CD4 T cells of Aa-infected rats increased and were activated, resulting in enhanced isotype-switched serum immunoglobulin G by 2 weeks postinoculation. Bone resorption was evident 12 weeks after Aa-feeding. In B cells, interleukin-2 (IL-2), macrophage-inhibiting factor, IL-19, IL-21, tumor necrosis factor (TNF), CD40 ligand (CD40L), CD70, bone morphogenetic protein 2 (BMP2), BMP3, and BMP10 were upregulated early; while IL-7, Fas ligand (FasL), small inducible cytokine subfamily E1, and growth differentiation factor 11 (GDF11; BMP11) were upregulated late (12 weeks). BMP10 was sustained throughout. In CD4 T cells, IL-10, IL-16, TNF, lymphotoxin-beta (LTbeta), APRIL, CD40L, FasL, RANKL and osteoprotegerin were upregulated early, whereas IL-1beta, IL-1RN, IL-1F8, IL-24, interferon-alpha1, GDF11 (BMP11), and GDF15 were upregulated late (12 weeks). Adaptive immunity appears crucial for bone resorption. Several of the deregulated genes are, for the first time, shown to be associated with bone resorption, and the results indicate that activated B cells produce BMP10. The study provides a rationale for a link between periodontal disease and other systemic diseases.

Bacterial Infection Increases Periodontal Bone Loss in Diabetic Rats Through Enhanced Apoptosis

Periodontal disease is the most common osteolytic disease in humans and is significantly increased by diabetes mellitus. We tested the hypothesis that bacterial infection induces bone loss in diabetic animals through a mechanism that involves enhanced apoptosis. Type II diabetic rats were inoculated with Aggregatibacter actinomycetemcomitans and treated with a caspase-3 inhibitor, ZDEVD-FMK, or vehicle alone. Apoptotic cells were measured with TUNEL; osteoblasts and bone area were measured in H&E sections. New bone formation was assessed by labeling with fluorescent dyes and by osteocalcin mRNA levels. Osteoclast number, eroded bone surface, and new bone formation were measured by tartrate-resistant acid phosphatase staining. Immunohistochemistry was performed with an antibody against tumor necrosis factor-α. Bacterial infection doubled the number of tumor necrosis factor-α-expressing cells and increased apoptotic cells adjacent to bone 10-fold (P < 0.05). Treatment with caspase inhibitor blocked apoptosis, increased the number of osteoclasts, and eroded bone surface (P < 0.05); yet, inhibition of apoptosis resulted in significantly greater net bone area because of an increase in new bone formation, osteoblast numbers, and an increase in bone coupling. Thus, bacterial infection in diabetic rats stimulates periodontitis, in part through enhanced apoptosis of osteoblastic cells that reduces osseous coupling through a caspase-3-dependent mechanism.

Aggregatibacter actinomycetemcomitans-induced bone loss and antibody response in three rat strains.

BACKGROUND The aim of this study is to compare the colonization, immunoglobulin (Ig) G response, and alveolar bone loss in Aggregatibacter actinomycetemcomitans (Aa)-inoculated Fawn Hooded Hypertensive (FHH), Dahl Salt-Sensitive (DSS), and Brown Norway (BN) rats. METHODS Each rat strain was divided into wild-type Aa-inoculated and non-inoculated control groups. Blood taken at 12 weeks after inoculation was assessed for Aa-specific IgG antibodies by an enzyme-linked immunosorbent assay. Colonization was assessed 12 weeks postinoculation. Bone loss was estimated by measuring the distance from the cemento-enamel junction (CEJ) to the alveolar bone crest (ABC) at 20 molar sites. Colonization and antibody levels were compared by using the Student t test. Diseased rats were defined as having two sites per quadrant with CEJ-ABC distances that were significantly greater than the control CEJ-ABC distances. RESULTS The Aa colonization of FHH rats was significantly higher than in other strains (P <0.05). The Aa-specific IgG levels in the DSS Aa-inoculated group were significantly higher than in its control group (P <0.05). Only FHH rats showed Aa disease-associated bone loss (P = 0.0021). CONCLUSIONS Aa colonized and caused more disease in FHH rats than in the other rat strains. The rat strains each responded differently to the same Aa strain.

A Comparison of Aggregatibacter actinomycetemcomitans (Aa) Virulence Traits in a Rat Model for Periodontal Disease

Our aim was to explore the effects of Cytolethal Distending toxin (Cdt) in a well established rat model of periodontal disease where leukotoxin (LtxA) was thought to have no known effect. In vitro studies, were used to assess CdtB activity using Aa Leukotoxin as a negative control. These studies showed that both CdtB and LtxA (unexpectedly) exerted significant effects on CD4+ T cells. As a result we decided to compare the effects of these two prominent Aa virulence factors on bone loss using our rat model of Aa-induced periodontitis. In this model, Aa strains, mutant in cdtB and ltxA, were compared to their parent non-mutant strains and evaluated for colonization, antibody response to Aa, bone loss and disease. We found that bone loss/disease caused by the ltxA mutant strain, in which cdtB was expressed, was significantly less (p<0.05) than that due to the wild type strain. On the other hand, the disease caused by cdtB mutant strain, in which ltxA was expressed, was not significantly different from the wild type strain. This data indicates that Aa LtxA exerts a greater effect on bone loss than Cdt in this rat model of periodontal disease and supports the utility of this model to dissect specific virulence factors as they relate to immunopathology in studies of Aa-induced disease.