T3SS chaperone of the CesT family is required for secretion of the anti-sigma factor BtrA in <i>Bordetella pertussis</i>

<i>Bordetella pertussis</i> is a Gram-negative, strictly human re-emerging respiratory pathogen and the causative agent of whooping cough. Similar to other Gram-negative pathogens, <i>B. pertussis</i> produces the type III secretion system, but its role in the pathogenesis of <i>B. pertussis</i> is enigmatic and yet to be elucidated. Here, we combined RNA-seq, LC-MS/MS, and co-immunoprecipitation techniques to identify and characterize the novel CesT family T3SS chaperone BP2265. We show that this chaperone specifically interacts with the secreted T3SS regulator BtrA and represents the first non-flagellar chaperone required for the secretion of an anti-sigma factor. In its absence, secretion but not production of BtrA and most T3SS substrates is severely impaired. It appears that the role of BtrA in regulating T3SS extends beyond its activity as an antagonist of the sigma factor BtrS. Predictions made by artificial intelligence system AlphaFold support the chaperone function of BP2265 towards BtrA and outline the structural basis for the interaction of BtrA with its target BtrS. We propose to rename BP2265 to BtcB for the <i>Bordetella</i> type III chaperone of BtrA. In addition, the absence of the BtcB chaperone results in increased expression of numerous flagellar genes and several virulence genes. While increased production of flagellar proteins and intimin BipA translated into increased biofilm formation by the mutant, enhanced production of virulence factors resulted in increased cytotoxicity towards human macrophages. We hypothesize that these phenotypic traits result indirectly from impaired secretion of BtrA and altered activity of the BtrA/BtrS regulatory node.百日咳博德特菌（Bordetella pertussis）是一类革兰氏阴性、严格人类嗜性的再发呼吸道病原体，亦是百日咳的致病菌。与其他革兰氏阴性病原体类似，百日咳博德特菌（B. pertussis）拥有III型分泌系统（type III secretion system, T3SS），但其在百日咳博德特菌致病过程中的作用仍不明朗，尚未得到阐明。本研究结合RNA测序（RNA-seq）、液相色谱-串联质谱（LC-MS/MS）以及免疫共沉淀（co-immunoprecipitation）技术，鉴定并表征了新型CesT家族III型分泌系统分子伴侣BP2265。研究证实，该分子伴侣可与分泌型III型分泌系统调控蛋白BtrA特异性结合，亦是首个被发现的、参与抗σ因子分泌的非鞭毛分子伴侣。在缺失该分子伴侣的情况下，BtrA以及多数III型分泌系统底物的分泌过程会受到严重损害，但其蛋白合成并未受到影响。研究表明，BtrA对III型分泌系统的调控作用远超其作为σ因子BtrS拮抗剂的活性范畴。人工智能系统阿尔法折叠（AlphaFold）的预测结果佐证了BP2265对BtrA的分子伴侣功能，并阐明了BtrA与其靶标BtrS相互作用的结构基础。我们提议将BP2265重命名为BtcB，即百日咳博德特菌BtrA的III型分泌系统分子伴侣。此外，BtcB分子伴侣的缺失会导致大量鞭毛基因与多种毒力基因的表达水平显著上调。鞭毛蛋白与紧密素BipA的表达量提升，使得突变株的生物膜形成能力增强；而毒力因子的过量产生则会提升其对人巨噬细胞的细胞毒性。我们推测，上述表型特征间接源于BtrA的分泌受损以及BtrA/BtrS调控节点的活性改变。