The goal of this project is to identify new virulence factors of oral bacteria. This search concentrates on proteins having catalytic activities harmful to host cells and tissues. These proteins will be classified as protein toxins if they have the ability to damage cell membranes or to penetrate to the cytosol and disrupt metabolic processes. In the current year, most of the activity of this project evolved into more detailed examinations of individual toxins, with some of the results being reported in other project descriptions. Effort under this project provides support for molecular biology procedures and the logistical and technical support for the detailed studies of individual toxins, while continuing to develop systems capable of detecting and analyzing new virulence factors. A fusion protein system derived from components of anthrax toxin was used to internalize the osteotoxin derived from Bordetella avium, beta-cystathionase, into cultured cells. Fusions with the anthrax toxin retained enzymatic activity and were toxic to cells. These fusion proteins can be used to determine if the osteotoxin is more potent on the plasma membrane or in the cytosol. Mouse macrophage cell line systems were developed for study of phagocytosis of toxin-producing bacteria. Methods for measuring bacterial uptake, growth, and for killing of the macrophages were examined. This system will be used to search for other virulence factors involved in bacterial invasion and persistance in tissues.
