Role of Bordetella pertussis virulence factors in pathogenesis of whooping cough.

1.为了建立一种检测百日咳的高灵敏度方法，我们尝试生成针对多胺化Rho gtpase的抗体，并从多胺化Rho免疫家兔的血清中提取。然而，该血清对完整Rho的抗体效价高于多胺化血清。然后，我们尝试用兔抗dnt抗体，采用ELISA法直接检测皮肤坏死毒素。该方法可以检测到低至0.2 ng/ml的DNT。用该方法测定了10株百日咳杆菌和支疱杆菌培养上清液中DNT的浓度。培养上清液中DNT的含量随着细菌的增殖而增加，在百日咳杆菌和结核分枝杆菌中DNT的含量分别达到平均5.4 ng/ml和2.0 ng/ml。这些结果表明，DNT在细菌外环境中以完全有效的浓度存在，并可能影响感染博德特拉氏菌的组织。此外，在寻找上呼吸道组织靶细胞的过程中，我们发现猪气管平滑肌细胞对DNT敏感。综上所示，我们推测，有效剂量的DNT从感染气管内的生物体中释放出来，通过上皮到达平滑肌细胞，使它们容易收缩，导致百日咳中的阵发性咳嗽。博德特拉菌体外感染模型的病理生理分析。我们检查了由博德特拉菌感染引起的气管上皮细胞系的改变，发现大鼠肺泡上皮L2细胞对实验性百日咳感染的反应表现出特征性的形态学改变，而对支气管结核杆菌感染则没有反应，这意味着L2细胞可以作为百日咳感染的体外细胞模型。少

1.The function of dermonecrotic toxin(DNT) as a cause of whooping coughTo establish a highly sensitive method detecting DNT, we attempted to generate antibody against polyaminated Rho GTPases, which are yielded by DNT action, and obtained serum from rabbits immunized with polyaminated Rho. The serum, however, showed higher antibody titer against intact Rho than polyaminated one. Then, we attempted to directly detect dermonecrotic toxin by ELISA method, using rabbit anti-DNT antibody. This method made it possible to detect DNT at a low concentration as little as 0.2 ng/ml. Using this method, we determined the concentrations of DNT in culture supernatants of 10 different strains of Bordetella pertussis and B.bronchiseptica. The amounts of DNT in the culture supernatants were found to increase in accordance with proliferation of the organisms and to reach to an average of 5.4 ng/ml and 2.0 ng/ml for B.pertussis and B.bronchiseptica, respectively. These results indicate that DNT is present … More at fully effective concentrations in the extrabacterial milieu and likely affect the tissues which are infected with Bordetella spp. In addition, in the course of searching for target cells in the upper respiratory tissues, we found that tracheal smooth muscle cells from pig are sensitive to DNT. Taken together, we speculate that an effective dose of DNT is liberated from the organisms in the infected trachea, reach to smooth muscle cells through the epithelium, and make them easily constrictive, leading to the paroxysmal cough in whooping cough.2.Pathophysiologic analysis of Bordetella infections in in vitro models.We examined alterations caused by Bordetella infection in various lines of cells derived from the tracheal epithelium and found that rat alveolar epithelial L2 cells showed a characteristic morphological change in response to experimental infection with B.pertussis but not with B.bronchiseptica, implying that L2 cells could be useful as an in vitro cellular model for B.pertussis infection. Less