Probing the Function of Bordetella bronchiseptica Adenylate Cyclase Toxin by Manipulating Host Immunity

We have examined the role of adenylate cyclase-hemolysin (CyaA) by constructing an in-frame deletion in the Bordetella bronchiseptica cyaA structural gene and comparing wild-type and cyaA deletion strains in natural host infection models. Both the wild-type strain RB50 and its adenylate cyclase toxin deletion (ΔcyaA) derivative efficiently establish persistent infections in rabbits, rats, and mice following low-dose inoculation. In contrast, an inoculation protocol that seeds the lower respiratory tract revealed significant differences in bacterial numbers and in polymorphonuclear neutrophil recruitment in the lungs from days 5 to 12 postinoculation. We next explored the effects of disarming specific aspects of the immune system on the relative phenotypes of wild-type and ΔcyaA bacteria. SCID, SCID-beige, or RAG-1(−/−) mice succumbed to lethal systemic infection following high- or low-dose intranasal inoculation with the wild-type strain but not the ΔcyaA mutant. Mice rendered neutropenic by treatment with cyclophosphamide or by knockout mutation in the granulocyte colony-stimulating factor locus were highly susceptible to lethal infection by either wild-type or ΔcyaA strains. These results reveal the significant role played by neutrophils early in B. bronchiseptica infection and by acquired immunity at later time points and suggest that phagocytic cells are a primary in vivo target of the Bordetella adenylate cyclase toxin.

本研究通过对支气管败血波氏杆菌（Bordetella bronchiseptica）的cyaA结构基因构建框内缺失突变体，并在天然宿主感染模型中对比野生型菌株与cyaA缺失菌株的表型，以探究腺苷酸环化酶溶血素（adenylate cyclase-hemolysin, CyaA）的功能。野生型菌株RB50及其腺苷酸环化酶毒素缺失（ΔcyaA）突变株，在低剂量接种后均可在兔、大鼠与小鼠体内成功建立持续性感染。与之相反，采用接种于下呼吸道的实验方案时，在接种后第5至12天，两组菌株在肺部的细菌载量与多形核中性粒细胞募集水平均存在显著差异。随后，本研究进一步探讨了抑制免疫系统特定功能组分对野生型与ΔcyaA菌株相对表型的影响。采用高剂量或低剂量鼻内接种野生型菌株后，重症联合免疫缺陷（SCID）、SCID-beige或RAG-1(-/-)小鼠均会发生致死性全身性感染，而接种ΔcyaA突变株的小鼠则无此现象。经环磷酰胺处理或粒细胞集落刺激因子基因座敲除而导致中性粒细胞减少的小鼠，无论接种野生型还是ΔcyaA菌株，均极易发生致死性感染。上述结果揭示了中性粒细胞在支气管败血波氏杆菌感染早期的关键作用，适应性免疫在感染后期起到重要调控功能，同时提示吞噬细胞是波氏杆菌属腺苷酸环化酶毒素的体内主要作用靶点。