Data set related to the article: "Klebsiella pneumoniae-OMVs activate death-signaling pathways in Human Bronchial Epithelial Host Cells (BEAS-2B)"

The programmed cell death pathways of apoptosis are important in mammalian cellular protectionfrom infections. The activation of these pathways depends on the presence of membranereceptors that bind bacterial components to activate the transduction mechanism. In addition tobacteria, these mechanisms can be activated by outer membrane vesicles (OMVs). OMVs arespherical vesicles of 20–250 nm diameter, constitutively released by Gram-negative bacteria.They contain several bacterial determinants including proteins, DNA/RNA and proteins, thatactivate different cellular processes in host cells. This study focused on Klebsiella pneumoniae-OMVs in activating death mechanisms in human bronchial epithelial cells (BEAS-2B). Characterizationof purified OMVs was achieved by scanning electron microscopy, nanoparticle trackinganalysis and protein profiling. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay while apoptotic induction was measured by flow cytometryand confirmed by western blotting. The OMVs produced showed a spherical morphology, with adiameter of 137.2 ± 41 nm and a vesicular density of 7.8 × 109 particles/mL Exposure of cellmonolayers to 50 μg of K. pneumoniae-OMV for 14 h resulted in approximately 25 % cytotoxicityand 41.15–41.14 % of cells undergoing early and late apoptosis. Fluorescence microscopyrevealed reduced cellular density, the presence of apoptotic bodies, chromatin condensation, andnuclear membrane blebbing in residual cells. Activation of caspases 􀀀 3 and 􀀀 9 and dysregulationof BAX, BIM and Bcl-xL indicated the activation of mitochondria-dependent apoptosis. Furthermore,a decrease in the antioxidant enzymes superoxide dismutase, catalase and glutathioneperoxidase involved endoplasmic reticulum stress with the potential formation of reactive oxygen species. These findings provide evidence for the role of OMVs in apoptosis and involvement in thepathogenesis of K. pneumoniae infections.细胞凋亡（apoptosis）这一程序性细胞死亡通路，在哺乳动物抵御感染的细胞保护过程中发挥关键作用。此类通路的激活依赖于能够结合细菌组分以启动信号转导机制的膜受体。除细菌外，外膜囊泡（outer membrane vesicles, OMVs）同样可激活上述通路。OMVs为直径20–250 nm的球形囊泡，由革兰氏阴性菌（Gram-negative bacteria）结构性释放，内含蛋白质、DNA/RNA等多种细菌决定簇，可激活宿主细胞内的不同细胞进程。 本研究聚焦于肺炎克雷伯菌（Klebsiella pneumoniae）来源的OMVs对人支气管上皮细胞（BEAS-2B）死亡机制的激活作用。通过扫描电子显微镜、纳米颗粒追踪分析（nanoparticle tracking analysis）及蛋白质谱分析完成了纯化OMVs的表征。采用3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）分析法评估细胞活力，通过流式细胞术（flow cytometry）检测细胞凋亡诱导情况，并以蛋白质印迹法（western blotting）进行验证。 所获OMVs呈球形形态，直径为137.2 ± 41 nm，囊泡浓度为7.8 × 10^9 粒子/mL。将50 μg肺炎克雷伯菌OMVs与细胞单层共培养14 h后，可引发约25%的细胞毒性，且41.15%–41.14%的细胞发生早期及晚期凋亡。荧光显微镜观察显示，残留细胞的细胞密度降低，出现凋亡小体、染色质固缩及核膜出泡现象。半胱天冬酶（caspase）3与9的激活，以及BAX、BIM和Bcl-xL的表达失调，表明线粒体依赖性凋亡通路被激活。此外，抗氧化酶超氧化物歧化酶、过氧化氢酶及谷胱甘肽过氧化物酶的活性下调，提示内质网应激（endoplasmic reticulum stress）伴随活性氧（reactive oxygen species, ROS）的潜在生成。 上述研究结果证实了OMVs在细胞凋亡中的作用，及其在肺炎克雷伯菌感染致病过程中的参与机制。