ABSTRACT

Airway epithelial cells are the first line of defense against respiratory pathogens. Porcine bacterial pathogens, such as Bordetella bronchiseptica, Actinobacillus pleuropneumoniae, Glaesserella (Haemophilus) parasuis, and Pasteurella multocida, breach this barrier to lead to local or systematic infections. Here, we demonstrated that respiratory bacterial pathogen infection disrupted the airway epithelial intercellular junction protein, E-cadherin, thus contributing to impaired epithelial cell integrity. E-cadherin knocking-out in newborn pig tracheal cells via CRISPR/Cas9 editing technology confirmed that E-cadherin was sufficient to suppress the paracellular transmigration of these porcine respiratory bacterial pathogens, including G. parasuis, A. pleuropneumoniae, P. multocida, and B. bronchiseptica. The E-cadherin ectodomain cleavage by these pathogens was probably attributed to bacterial HtrA/DegQ protease, but not host HtrA1, MMP7 and ADAM10, and the prominent proteolytic activity was further confirmed by a serine-to-alanine substitution mutation in the active center of HtrA/DegQ protein. Moreover, deletion of the htrA gene in G. parasuis led to severe defects in E-cadherin ectodomain cleavage, cell adherence and paracellular transmigration in vitro, as well as bacterial breaking through the tracheal epithelial cells, systemic invasion and dissemination in vivo. This common pathogenic mechanism shared by other porcine respiratory bacterial pathogens explains how these bacterial pathogens destroy the airway epithelial cell barriers and proliferate in respiratory mucosal surface or other systemic tissues.

摘要

气道上皮细胞是抵抗呼吸道病原体的第一道防线。猪细菌病原体，例如支气管败血博德特氏菌、胸膜肺炎放线杆菌、副猪格氏杆菌（嗜血杆菌）和多杀性巴氏杆菌，突破这个障碍导致局部或系统性感染。在这里，我们证明呼吸道细菌病原体感染破坏了气道上皮细胞间连接蛋白 E-钙粘蛋白，从而导致上皮细胞完整性受损。通过 CRISPR/Cas9 编辑技术敲除新生猪气管细胞中的 E-cadherin，证实 E-cadherin 足以抑制这些猪呼吸道细菌病原体的细胞旁迁移，包括副猪嗜血杆菌、胸膜肺炎放线菌、多杀假单胞菌和B. 支气管败血症. 这些病原体对 E-cadherin 胞外域的切割可能归因于细菌 HtrA/DegQ 蛋白酶，而不是宿主 HtrA1、MMP7 和 ADAM10，并且通过活性中心的丝氨酸到丙氨酸取代突变进一步证实了突出的蛋白水解活性。 HtrA/DegQ 蛋白。此外，G. parasuis 中htrA基因的缺失导致 E-cadherin 胞外域切割、细胞粘附和体外细胞旁迁移的严重缺陷，以及细菌突破气管上皮细胞、全身侵袭和体内传播。. 其他猪呼吸道细菌病原体共有的这种常见致病机制解释了这些细菌病原体如何破坏气道上皮细胞屏障并在呼吸道粘膜表面或其他全身组织中增殖。