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 (Delta 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 microg/100 microL) 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 microg/100 microL PAO1 supernatant protein, cellular proliferation was significantly reduced to 4.2 +/- 2.8% compared to untreated controls, while the DeltaoxyR supernatant protein remained at 103.3 +/- 4.0% of untreated controls (P < 0.05). Similar significant results were seen in the U937-, P388D1-, and ER-MP20(+)-derived macrophage cells. CONCLUSIONS: Taken together, our data indicate that OxyR regulates the secretion of potent cytotoxic factors by P. aeruginosa.
BACKGROUND:Nosocomial infections often lead to sepsis and multisystem organ failure in critically injured patients, including burn and traumapatients. 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 (Delta 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 microg/100 microL) 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 microg/100 microL PAO1 supernatant protein, cellular proliferation was significantly reduced to 4.2 +/- 2.8% compared to untreated controls, while the DeltaoxyR supernatant protein remained at 103.3 +/- 4.0% of untreated controls (P < 0.05). Similar significant results were seen in the U937-, P388D1-, and ER-MP20(+)-derived macrophage cells. CONCLUSIONS: Taken together, our data indicate that OxyR regulates the secretion of potent cytotoxic factors by P. aeruginosa.
Authors: Lynne R Usher; Roderick A Lawson; Ian Geary; Christopher J Taylor; Colin D Bingle; Graham W Taylor; Moira K B Whyte Journal: J Immunol Date: 2002-02-15 Impact factor: 5.422
Authors: Kuzhali Muthu; Jiangping Deng; Fred Romano; L-K He; Richard Gamelli; Ravi Shankar; Stephen B Jones Journal: J Neuroimmunol Date: 2005-08 Impact factor: 3.478
Authors: Geoffrey L Winsor; Raymond Lo; Shannan J Ho Sui; Korine S E Ung; Shaoshan Huang; Dean Cheng; Wai-Kay Ho Ching; Robert E W Hancock; Fiona S L Brinkman Journal: Nucleic Acids Res Date: 2005-01-01 Impact factor: 16.971