Studies of the Flp operon in A. actinomycetemcomitans

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DESCRIPTION: (provided by applicant) Actinobacillus actinomycetemcomitans (Aa) is a gram-negative bacterium whose primary habitat in humans is the subgingival sulcus. The association between Aa and aggressive periodontitis in adolescents (e.g. localized juvenile penodontitis, LJP) provides the most compelling evidence for bacterial specificity in periodontitis. Fresh isolates with a "rough," adherent colony phenotype spontaneously and irreversibly switch to a non-adherent, smooth colony phenotype when grown in broth. Recent preliminary studies in rats found only the rough phenotype cells capable of colonizing the oral cavity. The rough phenotype is primarily associated with numerous bundle-forming fimbriae on the cell surface. These fimbriae are encoded by a fimbrial operon (the flp operon) that has recently been identified in Aa. Transposon analysis has demonstrated that this operon is important in fimbrial expression. It likely influences phase variation, biofilm formation, and possibly leukotoxin association with the bacterial cell. Although the Flp fimbrillin subunit encoded by the first gene of this operon shares homology with type V-like fimbrillin, it is unique in several respects, especially its small size (6.5 kb). The focus of our study on regulation will be the 5' end of the operon, the flp to tadA sequence, based on previous complementation studies in transposon mutants. One of the SPECIFIC AIMS of this proposal is 1.) to determine the molecular basis of fimbrial phase variation through the study of transcription, translation, and cell localization of Flp fimbrillin subunit. By comparing the DNA sequence, transcription, and cell localization of the flp subunit in rough and spontaneous isogenic smooth variants, we will identify at what molecular level fimbrial expression is interrupted in the smooth variant, and identify potential cis regulatory sequences. Our preliminary transcriptional analysis of these genes suggests there may be more than one polycistronic message and more than one promoter within the operon. Our goal is 2.) to perform functional analysis of potential promoter regions and to determine the transcriptional organization of the flp operon through the study of selected deletion mutants. The experiments proposed in this application, when successfully completed, will provide the essential preliminary results for subsequent experiments to define the basis of fimbrial regulation in Aa. The information gained by these studies will ultimately aid in the development of novel strategies for preventing colonization of Aa and subsequent periodontal disease.