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 nucleotide sequence of the cloned 1ktCA genes was determined. While the sequence of these clones differs at several positions from 1ktCA of Aa JP2, the activity of LktA is unaffected. 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 Ma1E-LktA fusion protein, from which the LktA domain will be purified and used to prepare an anti-LktA antibody. We propose to identify the sites of transcription initiation and termination for the Aa 1kt operon, and to use Northern hybridizations to investigate the environmental factors that might affect LktA synthesis (temperature, Ca2+, 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 form diseased individuals will be included in this survey.
