MOLECULAR-GENETICS AND THE ANALYSIS OF LEUKOTOXIN IN A-ACTINOMYCETEMCOMITANS

THE PRIMARY PATHOGEN INVOLVED in certain forms of early-onset periodon titis is A. actinomycetemcomitans. Among its numerous potential virule nce 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 geneti c approaches are being established. Although there has been significan t progress in developing plasmids and bacteriophage as E. coil/A. acti nomycetemcomitans shuttle vectors, more work needs to be done. Tn5-bas ed transposon mutagenesis has been shown to work in this organism. Tar geted mutagenesis is now possible in A. actinomycetemcomitans; exogeno usly introduced DNA recombines efficiently with the homologous chromos omal locus. These techniques have been applied to studies of leukotoxi n. Targeted mutagenesis has been used to construct leukotoxin negative mutants that are otherwise isogenic with their leukotoxin-producing p arent strain. These mutants can be tested in animal models to ascertai n the in vivo role of leukotoxin in A. actinomycetemcomitans pathogene sis. Gene targeting has also been used to make strains in which the le ukotoxin promoter is regulating the synthesis of a beta-galactosidase reporter gene. Such strains have been used to show that leukotoxin syn thesis increases in cells grown anaerobically, but that several other environmental changes had little effect on leukotoxin synthesis; Final ly, plasmid shuttle vectors with leukotoxin promoters from various str ains of A. actinomycetemcomitans fused to reporter genes have been use d in cis/trans tests to show that leukotoxin promoter sequences and st rain-specific trans-acting factors are important in determining why di fferent strains of A. actinomycetemcomitans produce different levels o f leukotoxin.