Role of T3SS in promoting Pseudomonas aeruginosa internalization in pulmonary epithelial cells via ERK/ROS signaling pathway

To explore the role and underlying mechanism of type Ⅲ secretion system (T3SS) in regulating the internalization of Pseudomonas aeruginosa (PA) into pulmonary epithelial cells.

The 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.

Compared 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).

T3SS-mediated inhibition of the ERK pathway promotes the production of ROS and the internalization of PA in lung epithelial cells.