Mallet de Lima Carolina Diettrich, da Conceição Costa Jessica, de Oliveira Lima Santos Sabrina Alves, Carvalho Simone, de Carvalho Laís, Albano Rodolpho Mattos, Teixeira Mauro Martins, Plotkowski Maria Cristina Maciel, Saliba Alessandra Mattos
Departamento de Microbiologia, Imunologia e Parasitologia, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
Cell Microbiol. 2014 Aug;16(8):1244-54. doi: 10.1111/cmi.12280. Epub 2014 Mar 21.
ExoU is an important virulence factor in acute Pseudomonas aeruginosa infections. Here, we unveiled the mechanisms of ExoU-driven NF-κB activation by using human airway cells and mice infected with P. aeruginosa strains. Several approaches showed that PAFR was crucially implicated in the activation of the canonical NF-κB pathway. Confocal microscopy of lungs from infected mice revealed that PAFR-dependent NF-κB activation occurred mainly in respiratory epithelial cells, and reduced p65 nuclear translocation was detected in mice PAFR-/- or treated with the PAFR antagonist WEB 2086. Several evidences showed that ExoU-induced NF-κB activation regulated PAFR expression. First, ExoU increased p65 occupation of PAFR promoter, as assessed by ChIP. Second, luciferase assays in cultures transfected with different plasmid constructs revealed that ExoU promoted p65 binding to the three κB sites in PAFR promoter. Third, treatment of cell cultures with the NF-κB inhibitor Bay 11-7082, or transfection with IκBα negative-dominant, significantly decreased PAFR mRNA. Finally, reduction in PAFR expression was observed in mice treated with Bay 11-7082 or WEB 2086 prior to infection. Together, our data demonstrate that ExoU activates NF-κB by PAFR signalling, which in turns enhances PAFR expression, highlighting an important mechanism of amplification of response to this P. aeruginosa toxin.
外切酶U是铜绿假单胞菌急性感染中的一种重要毒力因子。在此，我们通过使用人气道细胞和感染铜绿假单胞菌菌株的小鼠，揭示了外切酶U驱动核因子κB（NF-κB）激活的机制。多种方法表明，血小板活化因子受体（PAFR）在经典NF-κB信号通路的激活中起关键作用。对感染小鼠肺部的共聚焦显微镜检查显示，PAFR依赖的NF-κB激活主要发生在呼吸道上皮细胞中，在PAFR基因敲除小鼠或用PAFR拮抗剂WEB 2086处理的小鼠中，检测到p65核转位减少。多项证据表明，外切酶U诱导的NF-κB激活调节PAFR表达。首先，通过染色质免疫沉淀法评估，外切酶U增加了PAFR启动子上p65的占有率。其次，在转染不同质粒构建体的培养物中进行的荧光素酶测定表明，外切酶U促进p65与PAFR启动子中的三个κB位点结合。第三，用NF-κB抑制剂Bay 11-7082处理细胞培养物，或转染IκBα负显性突变体，可显著降低PAFR mRNA水平。最后，在感染前用Bay 11-7082或WEB 2086处理的小鼠中观察到PAFR表达降低。总之，我们的数据表明，外切酶U通过PAFR信号激活NF-κB，进而增强PAFR表达，突出了对这种铜绿假单胞菌毒素反应放大的重要机制。
