Pathogenic oral bacteria are often associated with the progression of oral diseases, such as periodontitis. The Gram- negative bacterium Actinobacillus actinomycetemcomitans (Aa)colonizes periodontal sites and produces a leukotoxin that is likely to play an important role in periodontitis. Understanding the role of the Aa leukotoxin and other virulence factors in oral disease requires knowledge of the conditions under which such factors are produced, and how they act. Because of its probable role in periodontal disease, we have initiated studies of the genetics and mechanism of cytotoxicity of the Aa leukotoxin, LktA. LktCA has been isolated from Aa ATCC 29524 by PCR amplification, and we have developed fluorescent and isotopic assays for cytotoxicity. We have shown that cytoplasmic extracts of recombinant E. coli carrying Aa lktCA contain a protein that is cytotoxic to a human pre-monocyte cell line. The protein has been partially purified by S-sepharose chromatography, and is stabilized in the presence of denaturing agents (4M urea, 1% CHAPS). We are using recombinant DNA techniques to generate a histidine-labeled protein, which will be purified by Ni-NTA chromotography and used to prepare an anti-LKtA antibody. The sites of transcription initiation for Aa lktCA have been identified. We have identified Ca2+ as an extracellular factor that modulates activity of the lkt promoter in Aa strain JP2. We propose to use Northern hybridizations to investigate other environmental factors that might affect LktA synthesis (temperature, C02, and others). We plan to use DNA hybridization as a tool to identify other oral bacteria that might produce leukotoxins. Genomic DNA from normal oral flora as well as organisms frequently isolated from diseased individuals will be included in this survey.
