This proposal addresses the molecular basis for fimbrial expression by Actinobacillus actinomycetemcomitans (Aa). It is stated that the periodontal pathogen Aa expresses fimbriae when freshly isolated from the oral cavity. Fimbriate Aa are adherent in broth culture, while non-fimbriated Aa are much less adherent. This behavior led many investigators to reason that fimbriae are an important virulence factor for Aa. Fimbrial expression by Aa may be important to that organism during colonization of the periodontal tissues. The long term objectives of this proposal are to determine the genetic organization of the fimbrial genes and to ascertain the molecular basis for regulation of this potential virulence factor.
Specific Aim 1 is to isolate and clone the Aa fimbrial gene(s). Aa fimbrial clones will be selected from genomic libraries of DNA from fimbriated strain UTP001 using PCR and immunologic probes. A genomic library, constructed in a broad range mobilization cosmid vector, will be used in complementation studies with a non-fimbriated, rifampicin resistant, Aa strain generated from UTP001.
Specific Aim 2 is designed to determine the DNA sequence and transcriptional organization of the Aa fimbrial gene(s). Fimbrial gene mapping and sequencing will be accomplished by DNA restriction analysis, Sanger sequencing, and by analysis of fimbrial RNA transcripts. Fimbrial RNA will be studied by Northern analysis, S1 nuclease, and primer extension mapping to determine the transcriptional organization, locate the transcriptional start site, and the promoter region of the gene.
Specific Aim 3 will define the environmental growth conditions that direct regulation of fimbrial expression in Aa strain UTP001, and to determine the molecular basis for the regulation. The regulatory effects of environmental growth conditions on fimbrial expression will be studied first at the phenotypic level, then at the molecular level. Molecular characterization studies will begin as soon as initial DNA sequence and transcriptional organization are ascertained. It is felt that understanding the molecular basis for fimbrial expression by Aa will enable researchers to develop better therapies to combat this pathogen in the most susceptible patient populations.
