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.

Aggregatibacter actinomycetemcomitans Leukotoxin

“…In a recent study (Haubek et al, 2008) in Morocco, individuals who harbored the JP2 strains had significantly increased risk of periodontal attachment loss than those with the 652 strains. However, minimally leukotoxic strains are also found in persons with LAP (Kaplan et al, 2002;Fine et al, 2007). Fine et al (2007) studied adolescents in Newark, NJ, and found that the majority of individuals who developed bone loss did not harbor the JP2 strain in their mouths during the study period.…”

Regulation of Aggregatibacter ( Actinobacillus ) actinomycetemcomitans Leukotoxin Secretion by Iron

“…To confirm that ltxA synthesis is not affected by iron, we determined the levels of ltxA mRNA produced under normal and excess-iron growth conditions by quantitative real-time PCR (qRT-PCR) using a quantitative RT-PCR ReadyMix kit according to the manufacturer's protocol (Sigma, St. Louis, MO) ( Table 1). Highly leukotoxic strains JP2 (36,41) and NJ4500, a fresh clinical isolate (9,21), were grown in AAGM broth with and without 300 M FeCl 3 . Relative to the expression of housekeeping genes glyA (29) and gapdh (34) ( Table 1), we found that the ratio of ltxA mRNA levels under excess-iron conditions to those under normal growth conditions was approximately 1 ( Table 2).…”

Virulence Mechanisms of Leukotoxin from Aggregatibacter actinomycetemcomitans

“…Molecular genetics has demonstrated a degree of biodiversity in the oral microflora and A. actinomycetemcomitans is genetically heterogeneous and comprises distinct clonal lineages that may have different virulence potentials (Kittichotirat et al, 2011). Seven serotypes have been identified among A. actinomycetemcomitans isolates; each serotype represents a distinct clonal lineage (Kaplan et al, 2002;Kilian et al, 2006;Takada et. al., 2010).…”