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Aggregatibacter actinomycetemcomitans as an early colonizer of oral tissues: epithelium as a reservoir?

ABSTRACT: This study examined in vivo and in vitro colonization by Aggregatibacter actinomycetemcomitans, an organism highly associated with aggressive periodontitis. Thirteen volunteers (5 were A. actinomycetemcomitans positive for buccal epithelial cells [BECs] and teeth, 5 were A. actinomycetemcomitans positive for teeth only, and 3 were A. actinomycetemcomitans-negative controls) had two mandibular stents fabricated. Each stent contained 3 removable hydroxyapatite (HA) tooth surrogates. One HA square was removed from a stent at 5 time points over 7 h to assess the transfer of A. actinomycetemcomitans from teeth or BECs to HA. Streptococcus, Actinomyces, A. actinomycetemcomitans, and total anaerobic counts were evaluated on each square over time. In vitro experiments evaluated binding, desorption, transfer, and reattachment of A. actinomycetemcomitans wild-type and mutant strains to BECs and saliva-coated HA (SHA). Streptococcus and Actinomyces formed 80% of the cultivable flora on HA in all subjects. Transfer of A. actinomycetemcomitans to HA was not seen in subjects with A. actinomycetemcomitans on teeth only. All 5 subjects with A. actinomycetemcomitans on BECs showed transfer of A. actinomycetemcomitans to HA. In vitro, A. actinomycetemcomitans desorbed from BECs and transferred to SHA. A. actinomycetemcomitans binding to SHA was irreversible and did not transfer to BECs. The adhesin Aae showed specificity for BECs. Fimbrial mutants showed the greatest reduction in binding to SHA. A. actinomycetemcomitans migrated from BECs to HA in vivo and to SHA in vitro; however, A. actinomycetemcomitans movement from teeth and SHA to BECs did not occur. In vivo, A. actinomycetemcomitans colonized HA within 6 h and thus can be considered an early colonizer. BECs are a likely reservoir for A. actinomycetemcomitans tooth colonization.

SUBMITTER: Fine DH

PROVIDER: S-EPMC3008435 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Aggregatibacter actinomycetemcomitans as an early colonizer of oral tissues: epithelium as a reservoir?

Fine Daniel H DH Markowitz Kenneth K Furgang David D Velliyagounder Kabilan K

Journal of clinical microbiology 20100929 12

This study examined in vivo and in vitro colonization by Aggregatibacter actinomycetemcomitans, an organism highly associated with aggressive periodontitis. Thirteen volunteers (5 were A. actinomycetemcomitans positive for buccal epithelial cells [BECs] and teeth, 5 were A. actinomycetemcomitans positive for teeth only, and 3 were A. actinomycetemcomitans-negative controls) had two mandibular stents fabricated. Each stent contained 3 removable hydroxyapatite (HA) tooth surrogates. One HA square ...[more]

PMID: 20881174

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Project description:Aggregatibacter actinomycetemcomitans is a Gram-negative bacterial pathogen associated with severe periodontitis and nonoral diseases. Clinical isolates of A. actinomycetemcomitans display a rough (R) colony phenotype with strong adherent properties. Upon prolonged culturing, nonadherent strains with a smooth (S) colony phenotype emerge. To date, most virulence studies on A. actinomycetemcomitans have been performed with S strains of A. actinomycetemcomitans, whereas the virulence of clinical R isolates has received relatively little attention. Since the extracellular proteome is the main bacterial reservoir of virulence factors, the present study was aimed at a comparative analysis of this subproteome fraction for a collection of R isolates and derivative S strains, in order to link particular proteins to the virulence of A. actinomycetemcomitans with serotype b. To assess the bacterial virulence, we applied different infection models based on larvae of the greater wax moth Galleria mellonella, a human salivary gland-derived epithelial cell line, and freshly isolated neutrophils from healthy human volunteers. A total number of 351 extracellular A. actinomycetemcomitans proteins was identified by mass spectrometry, with the S strains consistently showing more extracellular proteins than their parental R isolates. A total of 50 known extracellular virulence factors was identified, of which 15 were expressed by all investigated bacteria. Importantly, the comparison of differences in exoproteome composition and virulence highlights critical roles of 10 extracellular proteins in the different infection models. Together, our findings provide novel clues for understanding the virulence of A. actinomycetemcomitans and for development of potential preventive or therapeutic avenues to neutralize this important oral pathogen. IMPORTANCE Periodontitis is one of the most common inflammatory diseases worldwide, causing high morbidity and decreasing the quality of life of millions of people. The bacterial pathogen Aggregatibacter actinomycetemcomitans is strongly associated with aggressive forms of periodontitis. Moreover, it has been implicated in serious nonoral infections, including endocarditis and brain abscesses. Therefore, it is important to investigate how A. actinomycetemcomitans can cause disease. In the present study, we applied a mass spectrometry approach to make an inventory of the virulence factors secreted by different clinical A. actinomycetemcomitans isolates and derivative strains that emerged upon culturing. We subsequently correlated the secreted virulence factors to the pathogenicity of the investigated bacteria in different infection models. The results show that a limited number of extracellular virulence factors of A. actinomycetemcomitans have central roles in pathogenesis, indicating that they could be druggable targets to prevent or treat oral disease.

Project description:Gram-negative facultative Aggregatibacter actinomycetemcomitans is an oral pathogen associated with periodontitis. The genetic heterogeneity among A. actinomycetemcomitans strains has been long recognized. This study provides a comprehensive genomic analysis of A. actinomycetemcomitans and the closely related nonpathogenic Aggregatibacter aphrophilus. Whole genome sequencing by Illumina MiSeq platform was performed for 31 A. actinomycetemcomitans and 2 A. aphrophilus strains. Sequence similarity analysis shows a total of 3,220 unique genes across the 2 species, where 1,550 are core genes present in all genomes and 1,670 are variable genes (accessory genes) missing in at least 1 genome. Phylogenetic analysis based on 397 concatenated core genes distinguished A. aphrophilus and A. actinomycetemcomitans. The latter was in turn divided into 5 clades: clade b (serotype b), clade c (serotype c), clade e/f (serotypes e and f), clade a/d (serotypes a and d), and clade e' (serotype e strains). Accessory genes accounted for 14.1% to 23.2% of the A. actinomycetemcomitans genomes, with a majority belonging to the category of poorly characterized by Cluster of Orthologous Groups classification. These accessory genes were often organized into genomic islands (n = 387) with base composition biases, suggesting their acquisitions via horizontal gene transfer. There was a greater degree of similarity in gene content and genomic islands among strains within clades than between clades. Strains of clade e' isolated from human were found to be missing the genomic island that carries genes encoding cytolethal distending toxins. Taken together, the results suggest a pattern of sequential divergence, starting from the separation of A. aphrophilus and A. actinomycetemcomitans through gain and loss of genes and ending with the divergence of the latter species into distinct clades and serotypes. With differing constellations of genes, the A. actinomycetemcomitans clades may have evolved distinct adaptation strategies to the human oral cavity.

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Aggregatibacter actinomycetemcomitans as an early colonizer of oral tissues: epithelium as a reservoir?

Publications

Aggregatibacter actinomycetemcomitans as an early colonizer of oral tissues: epithelium as a reservoir?

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