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.


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.
Melstrom
Kurt A
KA
Department of Surgery, Loyola University Medical Center, Maywood, Illinois 60153, USA.
Kozlowski
Ryan
R
Hassett
Daniel J
DJ
Suzuki
Hideki
H
Bates
Donna M
DM
Gamelli
Richard L
RL
Shankar
Ravi
R
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects; D003713:Dendritic Cells / Q000166:cytology / Q000187:drug effects; D005098:Exotoxins / Q000378:metabolism* / Q000494:pharmacology; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics; D008264:Macrophages / Q000166:cytology / Q000187:drug effects; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D011550:Pseudomonas aeruginosa / Q000378:metabolism* / Q000472:pathogenicity*; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D005098:Exotoxins; C524857:OxyR protein, Pseudomonas aeruginosa; D015534:Trans-Activators