Stenotrophomonas maltophilia is a Gram-negative opportunistic pathogen that can chronically colonize the lungs of people with cystic fibrosis (CF) and is associated with lethal pulmonary hemorrhage in immunocompromised patients. Its secreted virulence factors include the extracellular serine proteases StmPR1, StmPR2, and StmPR3. To explore the impact of secreted virulence determinants on pulmonary mucosal defenses in CF, we examined the secretome of human CFBE41o- bronchial epithelial cells in response to treatment with S. maltophilia K279a cell culture supernatant (CS) using a liquid-chromatography-tandem mass spectrometry (LC-MS/MS) based label-free quantitative (LFQ) shotgun proteomics approach for global profiling of the cell secretome. Secretome analysis identified upregulated pathways mainly relating to biological adhesion and epithelial cell signaling in infection, whereas no specific pathways relating to the immune response were enriched. Further exploration of the potentially harmful effects of K279a CS on CF bronchial epithelial cells, demonstrated that K279a CS caused CFBE41o- cell condensation and detachment, reversible by the serine protease inhibitor PMSF. K279a CS also decreased trans-epithelial electrical resistance in CFBE41o- cell monolayers suggestive of disruption of tight junction complexes (TJC). This finding was corroborated by an observed increase in fluorescein isothiocyanate (FITC) dextran permeability and by demonstrating PMSF-sensitive degradation of the tight junction proteins ZO-1 and occludin, but not JAM-A or claudin-1. These observations demonstrating destruction of the CFBE41o- TJC provide a novel insight regarding the virulence of S. maltophilia and may explain the possible injurious effects of this bacterium on the CF bronchial epithelium and the pathogenic mechanism leading to lethal pulmonary hemorrhage.

嗜麦芽窄食单胞菌是革兰氏阴性机会病原体，可长期定居于囊性纤维化（CF）人的肺部，并与免疫功能低下的患者致命的肺出血有关。它分泌的毒力因子包括胞外丝氨酸蛋白酶StmPR1，StmPR2和StmPR3。为了探索分泌的毒力决定因素对CF中肺粘膜防御的影响，我们检查了人CFBE41支气管上皮细胞的分泌基因组，以应对CF的治疗。嗜麦芽孢杆菌使用基于液相色谱-串联质谱（LC-MS / MS）的无标记定量（LFQ）gun弹枪蛋白质组学方法对K279a细胞培养上清液（CS）进行细胞分泌组的整体分析。分泌组分析确定了上调的途径，主要与感染中的生物粘附和上皮细胞信号传导有关，而与免疫反应有关的特定途径没有被丰富。进一步探索K279a CS对CF支气管上皮细胞的潜在有害作用，证明K279a CS导致CFBE41o细胞凝结和脱落，可被丝氨酸蛋白酶抑制剂PMSF逆转。K279a CS还降低了CFBE41o细胞单层的跨上皮电阻，提示紧密连接复合物（TJC）破裂。观察到的异硫氰酸荧光素（FITC）右旋糖酐渗透性增加以及证实紧密连接蛋白ZO-1和occludin对PMSF敏感的降解（但对JAM-A或claudin-1却无影响）证实了这一发现。这些观察结果表明CFBE41o-TJC的破坏提供了关于毒力的新见解。嗜麦芽孢杆菌 并可以解释该细菌对CF支气管上皮的可能伤害作用以及导致致命性肺出血的致病机制。