Evolutionary Divergence of Aggregatibacter actinomycetemcomitans

Abstract

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.

Keywords: aggressive periodontitis; genetic variation; genomic islands; genomic structural variation; horizontal gene transfer; phylogeny.

Figure 1.

Phylogenetic tree based on 397 concatenated core genes (335,400 base pairs) of 31 strains of Aggregatibacter actinomycetemcomitans and 2 strains of Aggregatibacter aphrophilus according to the maximum likelihood method. A. actinomycetemcomitans and A. aphrophilus were clearly segregated. RHAA-1 is distinguished from the human strains of A. actinomycetemcomitans. Strains from the same individuals (i.e., SCC1398/SCC4092, I23C/S23A, SCC2302/AAS4a, and SCC393/A160) were closest to one another than to other strains. Five clades, designated as e′, b, c, e/f, and a/d, were distinguished among A. actinomycetemcomitans strains from human. The tree further indicates a greater similarity between clades b and c and between clades a/d and e/f.

Figure 2.

Percentage of gene content differences among 5 Aggregatibacter actinomycetemcomitans clades and between A. actinomycetemcomitans and Aggregatibacter aphrophilus. A heat map indicator of percentage difference is provided to the right of the matrices. The number in each box represents the percentage range of gene content between strains of one clade (left) and the other (upper). See Appendix Figure 1 for details of the analysis by strains.

Figure 3.

Classification of genes based on their distributions in Aggregatibacter actinomycetemcomitans (Aa) and Aggregatibacter aphrophilus (A. aphr) and within each clade of Aa. These groups are as follows: core 1 (found in all Aa and A. aphr strains), variable 1 (found in some but not all Aa and A. aphr strains), variable 1.1 (found only in A. aphr but not in Aa), variable 1.2 (found in some strains of both species), variable 1.3 (found only in Aa but not in A. aphr), core 2 (found in all Aa), variable 2 (found in some but not all Aa), and variables 2.1 to 2.4 (genes specific to each of clades a/d/e/f, b/c, and e′).

Figure 4.

Clusters of Orthologous Groups gene classification of the core 1 and variable 1 gene groups identified in this study. The core 1 and variable 1 groups are defined in Figure 3. A total of 3,220 genes were identified by whole genome sequencing of Aggregatibacter aphrophilus and Aggregatibacter actinomycetemcomitans. The histograms show the percentage of genes assigned to the major groups (A; upper panel) and/or functional categories (B; lower panel) of Clusters of Orthologous Groups. A higher percentage of genes in variable 1 were assigned to poorly characterized (73.41%) or function unknown (67.66%) than in core 1 (24.26% and 17.08%, respectively).

Figure 5.

Nucleotide sequence homology of serotype-specific gene clusters of serotype e and e′ strains. The 18 open reading frames (ORFs) of the homologous serotype e–specific gene clusters SA2876 (serotype e) and SC1083 (serotype e′) are shown. The percentage identity between the ORFs of the strains is provided in the middle. The ORFs with sequence identify ≤95% are represented by light gray arrows.