Genetical and biochemical study of neutrophil chemotactic inhibitor of bacteria as a juvenile periodontal pathogen

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Actinobacillus actinomycetemcomitans is a major pathogen associated with juvenile periodontitis and produces a number of virulence factors including neutrophil chemotactic inhibitory factor which was reported in 1991 but has not so far been clarified. In this project we to study the fine properties of the factor and clone the gene encoding the factor. This factor is proteinous, heat-stable (90 ℃ 5 min), and resistant for acidic, basic, and organic solvent-containing conditions. We purified the factor (Mw ; about 10K) using TMS-column chromatography with HPLC from the heat-treated cytosolic fraction as a starting material. The sequence of N-terminal 20 amino acid residues of the factor is identical and similar to those of DNA-binding protein HU of Pasteurella multocida and Haemophilus influenzae, respectively. We could not obtain the complete sequence of DNA encoding HU of A. actinomycetemcomitans from DNA database because the DNA sequencing project for this bacterium dose not finish. However, we obtained partial sequence of the DNA, and we try to carry out the cloning the HU gene using PCR method and colony hybridization technique, and also we try the expression of recombinant HU in Escherichia coli.We performed another cloning procedure for inhibitory factor-encoding DNA, but did not succeed. We screened the activities of cytosol of recombinant E. coli containing the gene library on the vector plasmids.We found the another unique inhibitor for the neutrophil chemotaxis. This is sanguiin H-11 , a tannin isolated from Chinese medicinal plant. The mechanism of the inhibition of has been studying.