Cytotoxicity of Pseudomonas Secreted Exotoxins Requires OxyR Expression

Background Nosocomial infections often lead to sepsis and multisystem organ failure in critically injured patients, including burn and trauma patients. A better understanding of the bacterial response to the host immune system is essential to develop better antimicrobials against pathogens. Pseudomonas aeruginosa combats host-initiated oxidant stress through expression of the transactivating factor, OxyR. Here we have tested the premise that OxyR regulates Pseudomonal cytotoxicity through secreted exotoxin production. Materials and methods Wild-type P. aeruginosa (PAO1) and a deletion mutant lacking the oxyR gene (Δ oxyR ) were grown for 18 h in Luria broth and the supernatant containing the secreted products was removed using centrifugation. Secreted proteins were isolated using ammonium sulfate precipitation. ER-MP20 + myeloid progenitor cells were harvested from the bone marrow of C57Blk/6J mice. These cells were differentiated into dendritic cells and macrophages. Various concentrations (0–20 μg/100 μL) of the bacterial proteins were added to the medium and cells were allowed to differentiate for 7 days. Cellular viability was then assayed using a proliferation assay. These studies were repeated on two other macrophage cell lines, human U937 and murine P388D1. Results At a protein concentration of 5 μg/100 μL PAO1 supernatant protein, cellular proliferation was significantly reduced to 4.2 ± 2.8% compared to untreated controls, while the Δ oxyR supernatant protein remained at 103.3 ± 4.0% of untreated controls ( P + -derived macrophage cells. Conclusions Taken together, our data indicate that OxyR regulates the secretion of potent cytotoxic factors by P. aeruginosa .