Repeats-in-Toxin (RTX) proteins of Gram-negative bacteria are excreted through the type I secretion system (T1SS) that recognizes non-cleavable C-terminal secretion signals. These are preceded by arrays of glycine and aspartate-rich nonapeptide repeats grouped by four to eight β strands into blocks that fold into calcium-binding parallel β-roll structures. The β-rolls are interspersed by linkers of variable length and sequence and the organization of multiple RTX repeat blocks within large RTX domains remains unknown. Here we examined the structure and function of the RTX domain of Bordetella pertussis adenylate cyclase toxin (CyaA) that is composed of five β-roll RTX blocks. We show that the non-folded RTX repeats maintain the stability of the CyaA polypeptide in the Ca²⁺-depleted bacterial cytosol and thereby enable its efficient translocation through the T1SS apparatus. The efficacy of secretion of truncated CyaA constructs was dictated by the number of retained RTX repeat blocks and depended on the presence of extracellular Ca²⁺ ions. We further describe the crystal structure of the RTX blocks IV–V of CyaA (CyaA_1372–1681) that consists of a contiguous assembly of two β-rolls that differs substantially from the arrangement of the RTX blocks observed in RTX lipases or other RTX proteins. These results provide a novel structural insight into the architecture of the RTX domains of large RTX proteins and support the “push-ratchet” mechanism of the T1SS-mediated secretion of very large RTX proteins.

革兰氏阴性细菌的毒素重复（RTX）蛋白通过识别不可裂解的C端分泌信号的I型分泌系统（T1SS）排出。在这些序列之前，排列有甘氨酸和富含天冬氨酸的九肽重复序列，这些重复序列由4至8条β链分成折叠成钙结合的平行β卷结构的嵌段。β-rolls散布着长度和序列可变的接头，在大型RTX域内多个RTX重复模块的组织仍然未知。在这里，我们检查了百日咳博德特氏菌腺苷酸环化酶毒素（CyaA）的RTX结构域和结构，该毒素由5个β卷RTX嵌段组成。我们显示非折叠的RTX重复序列可维持Ca ^2+中CyaA多肽的稳定性耗竭的细菌胞质溶胶，从而使其能够通过T1SS装置高效转运。截短的CyaA构建体分泌的功效由保留的RTX重复序列的数目决定，并取决于细胞外Ca ²⁺离子的存在。我们进一步描述了CyaA（CyaA _{1372 – 1681}）的RTX嵌段IV–V的晶体结构，该结构由两个β-roll的连续组装组成，该组装与在RTX脂肪酶或其他RTX蛋白质中观察到的RTX嵌段的排列方式有很大不同。这些结果为大型RTX蛋白的RTX域的结构提供了新颖的结构见解，并支持T1SS介导的大型RTX蛋白分泌的“推棘”机制。