Abdalla Maher Y, Hoke Traci, Seravalli Javier, Switzer Barbara L, Bavitz Melissa, Fliege Jill D, Murphy Peter J, Britigan Bradley E
Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska, USA
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA.
Infect Immun. 2017 Aug 18;85(9). doi: 10.1128/IAI.00176-17. Print 2017 Sep.
causes lung infections in patients with cystic fibrosis (CF). The quinolone signal (PQS) compound is a secreted virulence factor that contributes to the pathogenicity of We were able to detect PQS in sputum samples from CF patients infected with but not in samples from uninfected patients. We then tested the hypothesis that PQS induces oxidative stress in host cells by determining the ability of PQS to induce the production of reactive oxygen species (ROS) in lung epithelial cells (A549 and primary normal human bronchial epithelial [NHBE]) cells and macrophages (J774A.1 and THP-1). ROS production induced by PQS was detected with fluorescent probes (dichlorodihydrofluorescein diacetate, dihydroethidium, and MitoSOX Red) in conjunction with confocal microscopy and flow cytometry. PQS induced ROS production in lung epithelial (A549 and NHBE) cells and macrophages (J774A.1 and THP-1 cells). NHBE cells were sensitive to PQS concentrations as low as 500 ng/ml. PQS significantly induced early apoptosis ( < 0.05, = 6) in lung epithelial cells, as measured by annexin/propidium iodide detection by flow cytometry. However, no change in apoptosis upon PQS treatment was seen in J774A.1 cells. Heme oxygenase-1 (HO-1) protein is an antioxidant enzyme usually induced by oxidative stress. Interestingly, incubation with PQS significantly reduced HO-1 and NrF2 expression in A549 and NHBE cells but increased HO-1 expression in J774A.1 cells ( < 0.05, = 3), as determined by immunoblotting and densitometry. These PQS effects on host cells could play an important role in the pathogenicity of infections.
导致囊性纤维化（CF）患者发生肺部感染。喹诺酮信号（PQS）化合物是一种分泌型毒力因子，它有助于[某种病原体]的致病性。我们能够在感染[该病原体]的CF患者痰液样本中检测到PQS，但在未感染患者的样本中未检测到。然后，我们通过测定PQS诱导肺上皮细胞（A549和原代正常人支气管上皮[NHBE]细胞）和巨噬细胞（J774A.1和THP-1）中产生活性氧（ROS）的能力，来检验PQS是否在宿主细胞中诱导氧化应激这一假设。使用荧光探针（二氯二氢荧光素二乙酸酯、二氢乙锭和MitoSOX Red）结合共聚焦显微镜和流式细胞术检测PQS诱导的ROS产生。PQS在肺上皮（A549和NHBE）细胞以及巨噬细胞（J774A.1和THP-1细胞）中诱导ROS产生。NHBE细胞对低至500 ng/ml的PQS浓度敏感。通过流式细胞术用膜联蛋白/碘化丙啶检测发现，PQS在肺上皮细胞中显著诱导早期凋亡（P<0.05，n = 6）。然而，在J774A.1细胞中，PQS处理后凋亡未见变化。血红素加氧酶-1（HO-1）蛋白是一种通常由氧化应激诱导的抗氧化酶。有趣的是，通过免疫印迹和光密度测定法确定，与PQS孵育显著降低了A549和NHBE细胞中HO-1和NrF2的表达，但增加了J774A.1细胞中HO-1的表达（P<0.05，n = 3）。PQS对宿主细胞的这些作用可能在[该病原体]感染的致病性中起重要作用。
