A systematic and comprehensive search was begun to identify new protein virulence factors and toxins secreted by bacteria considered to be associated with periodontal disease. A collection of strains from patients with HIV and periodontal disease was sampled so as to obtain fresh disease isolates. To date, fourteen of these anaerobic species have been successfully grown and culture supernatants collected. Culture supernatants were assayed for adenylate cyclase activity; no positives were identified. Use of an internal standard of Bacillus anthracis adenylate cyclase showed that several standard media used to grow the bacterial inhibited enzyme activity. This inhibition was shown to be due to the presence of Tween 80 in the media. Media were altered to omit the detergent, thereby removing the inhibitory activity in this and potentially in other assays. Reagents and protocols were prepared for assay of the supernatants for protein tyrosine phosphatase and for ADP-ribosylation. Additional cultured cell lines were obtained that can be used for cytotoxicity tests.
