DESCRIPTION: Periodontitis, an inflammatory disease of tissues in the subgingival crevice, is associated with a dramatic shift in the subgingival microflora towards Gram negative organisms. We have focused on studying the virulence properties of one of these periodontal pathogens, the bacterium Actinobacillus actinomycetemcomitans (Aa). This bacterium has been strongly implicated in localized aggressive periodontitis and in several adult periodontal disorders. Aa is a facultative anaerobe that is capable of colonizing both an anaerobic microenvironment, like the diseased periodontal pocket, and an aerobic environment, like the blood stream. Thus, Aa has evolved virulence mechanisms that allow it to shift between these two environments. Using two-dimensional protein gels, we have shown that anaerobic growth induces the synthesis of dozens of Aa proteins, including leukotoxin (a presumed virulence protein that kills neutrophils). Numerous other proteins are repressed under anaerobic conditions but induced in the presence of oxygen. By generating defined mutations in An, we have shown that the aerobic/anaerobic regulation of most of these proteins is controlled by the Fnr or ArcAB pathways, as expected. Nevertheless, interestingly, the regulation of at least eight other proteins, including leukotoxin, was not controlled by Fnr or ArcAB. Since Aa does not encode significant homologues to any other bacterial oxygen regulatory proteins, we posit that a subset of Aa genes will be controlled by one (or more) transcriptional regulatory pathways which have not previously been described in Aa or in any other bacterial system. The full complement of Aa genes that are differentially synthesized in aerobic versus anaerobic growth will be identified using DNA microarrays (Specific Aim I). Importantly, we will also identify the set of oxygen-responsive genes, like leukotoxin, that are regulated by the previously unidentified, non-Fnr/non-ArcA pathway(s). In Specific Aim II, a systematic and comprehensive molecular genetic approach will be employed to identify this potentially novel regulatory pathway and the transcription factors involved. Finally, the interactions of our newly identified oxygen regulatory proteins with redox-regulated Aa promoters will be characterized (Specific Aim III). This will allow us to begin to develop the first molecular models for the mechanisms of non-Fnr/non-ArcAB aerobic/anaerobic regulation in a periodontal pathogen. The research proposed is highly significant because it will reveal the players in a new regulatory pathway regulating the adaptation of An, and possibly other periodontal pathogens, to the anaerobic subgingival microenvironment. Characterizing undefined, potentially new transcriptional regulatory pathways is important because the proteins involved may serve as targets for future drug development.