Molecular Genetics and the Analysis of Leukotoxin in A. actinomycetemcomitans

The primary pathogen involved in certain forms of early-onset periodontitis is A. actinomycetemcomitans. Among its numerous potential virulence factors is a leukotoxin that kills certain host defense cells. In order to analyze the regulation and in vivo function of leukotoxin and other virulence factors in A. actinomycetemcomitans, molecular genetic approaches are being established. Although there has been significant progress in developing plasmids and bacteriophage as E. colilA. actinomycetemcomitans shuttle vectors, more work needs to be done. Tn5-based transposon mutagenesis has been shown to work in this organism. Targeted mutagenesis is now possible in A. actinomycetemcomitans; exogenously introduced DNA recombines efficiently with the homologous chromosomal locus. These techniques have been applied to studies of leukotoxin. Targeted mutagenesis has been used to construct leukotoxin negative mutants that are otherwise isogenic with their leukotoxin-producing parent strain. These mutants can be tested in animal models to ascertain the in vivo role of leukotoxin in A. actinomycetemcomitans pathogenesis. Gene targeting has also been used to make strains in which the leukotoxin promoter is regulating the synthesis of a β-galactosidase reporter gene. Such strains have been used to show that leukotoxin synthesis increases in cells grown anaerobically, but that several other environmental changes had little effect on leukotoxin synthesis. Finally, plasmid shuttle vectors with leukotoxin promoters from various strains of A. actinomycetemcomitans fused to reporter genes have been used in cis/trans tests to show that leukotoxin promoter sequences and strain-specific trans-acting factors are important in determining why different strains of A. actinomycetemcomitans produce different levels of leukotoxin. J Periodontol 1996;67:309-316.