Purification of periodontopatogenic bacterial toxin which induces apoptosis in B cells and identification of its signaling molecules

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Cellular microbiology is a new science comprising microbiology, cell biology and molecular biology. Its primary focus is on the interactions between bacteria and host cells which give rise to pathology but which also may maintain health. Recent studies have shown that bacteria can control many aspects of eukaryotic cell behavior including cell movement and shape, cell proliferation and apoptosis. Actinobacillus actinomycetemcomitans, the etiologic agent of juvenile periodontitis, produces several exotoxins including leukotoxin and cytolethal distending toxin (CDT). We discovered a novel toxin (NTX) which affects the cell cycle of host cells (Infect. Immun. 1998 : 5980-7). In this study, we analyzed the mechanisms of cell cycle arrest induced by this NTX. NTX was purified from the culture supematant of A. actinomycetemcomitans, by four-step procedure : ammonium sulfate precipitation ; POROS HQ/M column chromatography ; polymyxin B matrix column chromatography ; and Mono-Q column chromatography. For the experimental control, recombinant CDT was obtained from the sonicate extracts of E.coli expressing A. actinomycetemcomitans CDT. Both NTX and CDT blocked the cell cycle progression at G2/M phase in mouse hybridoma HS-72 cells with induction of cell cycle regulatory protein p21 WAF1/CIP1. Intracellular responses of host cells against these exotoxins would be important for understanding how this periodontopathogen initiates periodontal pocket formation and plays a role in the development of periodotitis.