Adenylate Cyclase Toxin Promotes Internalisation of Integrins and Raft Components and Decreases Macrophage Adhesion Capacity

Bordetella pertussis, the bacterium that causes whooping cough, secretes an adenylate cyclase toxin (ACT) that must be post-translationally palmitoylated in the bacterium cytosol to be active. The toxin targets phagocytes expressing the CD11b/CD18 integrin receptor. It delivers a catalytic adenylate cyclase domain into the target cell cytosol producing a rapid increase of intracellular cAMP concentration that suppresses bactericidal functions of the phagocyte. ACT also induces calcium fluxes into target cells. Biochemical, biophysical and cell biology approaches have been applied here to show evidence that ACT and integrin molecules, along with other raft components, are rapidly internalized by the macrophages in a toxin-induced calcium rise-dependent process. The toxin-triggered internalisation events occur through two different routes of entry, chlorpromazine-sensitive receptor-mediated endocytosis and clathrin-independent internalisation, maybe acting in parallel. ACT locates into raft-like domains, and is internalised, also in cells devoid of receptor. Altogether our results suggest that adenylate cyclase toxin, and maybe other homologous pathogenic toxins from the RTX (Repeats in Toxin) family to which ACT belongs, may be endowed with an intrinsic capacity to, directly and efficiently, insert into raft-like domains, promoting there its multiple activities. One direct consequence of the integrin removal from the cell surface of the macrophages is the hampering of their adhesion ability, a fundamental property in the immune response of the leukocytes that could be instrumental in the pathogenesis of Bordetella pertussis.

百日咳博德特菌（Bordetella pertussis）是引发百日咳的病原菌，其分泌的腺苷酸环化酶毒素（adenylate cyclase toxin, ACT）需在细菌胞质中经历翻译后棕榈酰化修饰方可激活。该毒素靶向表达CD11b/CD18整合素受体的吞噬细胞，可将自身催化性腺苷酸环化酶结构域递送至靶细胞胞质，快速升高细胞内环腺苷酸（cAMP）浓度，进而抑制吞噬细胞的杀菌功能。此外，ACT还可诱导靶细胞产生钙流反应。
本研究通过生物化学、生物物理及细胞生物学实验手段证实，ACT与整合素分子及其他脂筏组分，可通过依赖于毒素诱导的钙升高的过程被巨噬细胞快速内吞。毒素触发的内吞事件存在两条不同的入胞途径：氯丙嗪敏感的受体介导内吞作用，以及不依赖网格蛋白的内吞作用，二者或可并行发挥功能。即便在缺失该受体的细胞中，ACT仍可定位于类似脂筏的结构域并被内吞。
综合上述实验结果，本研究提示腺苷酸环化酶毒素——乃至其所属的RTX（毒素重复序列，Repeats in Toxin）家族的其他同源致病性毒素——或具备内在能力，可直接且高效地插入类似脂筏的结构域，从而在该处完成其多种功能活动。
从巨噬细胞表面移除整合素，会直接削弱其黏附能力；而黏附能力是白细胞免疫应答的核心属性，这一效应或在百日咳博德特菌的致病机制中发挥关键作用。