Control of the human cell cycle by a bacterial protein, gapstatin

The oral gram-negative bacterium Actinobacillus actinomycetemcomitans is a major pathogen in human periodontal disease. Saline extraction releases a range of surface-associated components from this bacterium, including one which exhibits potent anti-proliferative activity as assessed by its capacity to inhibit DNA synthesis by human and other mammalian cells. Cultures incubated with this bacterial fraction for a prolonged period comprise a high proportion of cells containing a 4n level of DNA. Studies using hydroxyurea-synchronized cultures showed that cells treated with the surface-associated fraction were arrested in the G2 phase of the cell cycle and did not enter mitosis. This G2/M blockade was observed only when the bacterial fraction was added to the cells during early S phase. Our data also suggest that the active bacterial component binds to surface receptors expressed by the human cells and may act by a novel mechanism which involves down-regulation of cyclin B1 expression. The anti-proliferative activity of the bacterial fraction, purified by a combination of ammonium sulphate precipitation, HPLC anion exchange and gel filtration, has been shown to be an 8 kDa protein, which we have called gapstatin. Purified gapstatin was shown to be responsible for the the inhibitory effects of the surface-associated fraction on mammalian cells.