@article{XIONG2024, 
author = {Junzhi XIONG and Hua YU and Xingmin WANG and Xiaomei HE and Qian DAI and Jing QIU},
title = {Role of T3SS in promoting Pseudomonas aeruginosa internalization in pulmonary epithelial cells via ERK/ROS signaling pathway},
year = {2024},
journal = {Journal of Army Medical University},
volume = {46},
number = {22},
pages = {2493-2504},
keywords = {Pseudomonas aeruginosa, type three secretion system, ERK/ROS signaling pathway, internalization of PA in pulmonary epithelial cells},
url = {https://www.sciopen.com/article/10.16016/j.2097-0927.202407056},
doi = {10.16016/j.2097-0927.202407056},
abstract = {ObjectiveTo explore the role and underlying mechanism of type Ⅲ secretion system (T3SS) in regulating the internalization of Pseudomonas aeruginosa (PA) into pulmonary epithelial cells.MethodsThe human non-small cell lung cancer A549 cells were infected with or without PA strains, including wild-type PAO1 (a standard experimental PA strain), △exsA (knockout of the critical activator for T3SS genes), △pscJ (T3SS secretion-defective strain) and PAO1-E (EGTA-induced high expression of T3SS genes). The A549 cells pretreated with ERK inhibitor U0126 or reactive oxygen species (ROS) inhibitor apocynin (APO)/N-acetyl-L-cysteine (NAC) were infected with PAO1 or PAO1-E strain. Thus, the experiment was grouped as follows: the mock-treated group, PAO1- or PAO1-E-infected group, inhibitor-treated group, and PAO1/PAO1-E plus inhibitor-treated group. Extracellular bacteria were killed by gentamicin, and the cell lysates were diluted and then plated on PA screening plates. Bacterial amounts were detected by counting colony-forming units (CFUs). The production of ROS was analyzed using fluorescent probe labeling and flow cytometry. The activation of the ERK pathway was detected by Western blotting.ResultsCompared with the PAO1-infected group, the intracellular bacteria and ROS level in △exsA- or △pscJ-infected cells were lower (P < 0.05, P < 0.01), so was the generation of ROS(P < 0.01); In contrast, those of the PAO1-E strain-infected cells displayed an opposite trend (P < 0.01). Compared with the PAO1- or PAO1-E-infected group, the cells pretreated with APO/NAC followed by PAO1 or PAO1-E infection showed reduced intracellular bacterial amounts (P < 0.01). Compared to the PAO1-infected A549 cells, the phosphorylation level of ERK was increased in the △exsA- or △pscJ-infected cells (P < 0.01), while that level was suppressed in the PAO1-E-treated cells (P < 0.01). Compared with the PAO1-infected group, the PAO1-infected cells pretreated with U0126 displayed reduced ERK activation, elevated ROS production, and increased intracellular counts of PAO1 (P < 0.01).ConclusionT3SS-mediated inhibition of the ERK pathway promotes the production of ROS and the internalization of PA in lung epithelial cells.}
}