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

细胞微生物学是一门集微生物学、细胞生物学和分子生物学于一体的新兴科学。它的主要重点是细菌和宿主细胞之间的相互作用，这种相互作用会引起病理，但也可能维持健康。近年来的研究表明，细菌可以控制真核细胞的许多方面的行为，包括细胞的运动和形状，细胞的增殖和凋亡。放线菌是青少年牙周炎的病原，产生多种外毒素，包括白细胞毒素和细胞致死膨胀毒素（CDT）。我们发现了一种新的毒素（NTX），它影响宿主细胞的细胞周期(感染。免疫学杂志。1998:5980-7)。在本研究中，我们分析了这种NTX诱导细胞周期阻滞的机制。从放线菌培养液中纯化NTX，采用硫酸铵沉淀法；POROS HQ/M柱色谱法；多粘菌素B基质柱层析；和Mono-Q柱色谱法。作为实验对照，从表达放线菌comitans CDT的大肠杆菌超声提取物中获得重组CDT。NTX和CDT均通过诱导细胞周期调节蛋白p21 WAF1/CIP1，阻断小鼠杂交瘤HS-72细胞G2/M期的细胞周期进程。宿主细胞对这些外毒素的细胞内反应对于了解这种牙周病原如何引发牙周袋形成并在牙周炎的发展中发挥作用非常重要。

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.