Elucidating the role of the Actinobacillus actinomycetemcomitans leukotoxin. The leukotoxin from the periodontal pathogen A. actinomycetemcomitans is considered a putative virulence factor as it specifically lyses the host defense cells, polymorphonuclear leukocytes and monocytes. The objective of this project is to localize the leukotoxin in A. actinomycetemcomitans and to use genotypic markers (including the leukotoxin gene itself) to correlate pathogenicity among various isolates. In doing so, the potential virulence of the leukotoxin in periodontal diseases will be better understood and the genotypic markers will serve as valuable tools in future epidemiologic studies of A. actinomycetemcomitans in periodontal diseases. To do this, the following specific aims and methodology have been proposed: 1. Determine whether genes are present in leukotoxin operon that encode proteins which may be involved in the transport and secretion of the leukotoxin. A 6kb DNA fragment located downstream of the leukotoxin gene was cloned and 3.6kb sequenced. Two open reading frames were found and the predicted proteins were very similar to """"""""transport"""""""" proteins in other toxin producing pathogens. 2. Assess whether transport proteins are functional by mutational analysis. First, the leukotoxin will be localized by analyzing various cell fractions by Western blot analysis, a chromium release assay (a functional assay) and possibly, immunoelectron microscopy. Next, the two transport genes will be mutated and plasmids containing the mutated constructs will be transformed back into A. actinomycetemcomitans such that one gets integrative replacement. The genetically manipulated constructs will be retested for localization of the leukotoxin. 3. Correlate the genotypic variabilities among A. actinomycetemcomitans isolates with the levels of the leukotoxin and pathogenicity of these strains. A panel of probes was generated consisting of three random genomic probes and two potential virulence related probes. These probes detected genotypic differences among A. actinomycetemcomitans laboratory strains, clinical isolates and isolates cultured from monkeys using a restriction fragment length polymorphism (RFLP) analysis. Secondly, the genotypic presentation was compared with the phenotypic expression of the A. actinomycetemcomitans isolates. To evaluate the phenotypic traits, the strains were biochemically categorized and serotyped. Finally, to correlate pathogenicity, the isolates' ability to produce leukotoxin was measured by Western blots or a Chromium release assay.
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