An evolutionary conserved detoxification system for membrane lipid peroxidation stress in Gram-negative bacteria

How double membraned Gram-negative bacteria overcome lipid peroxidation is virtually unknown.Burkholderia cenocepacia, is a Gram-negative opportunistic pathogen that infects cystic fibrosis and other immunocompromised patients. These bacteria are also highly resistant to multiple antibiotics, and therefore difficult to eradicate. Due to the inflammatory response during infection, these bacteria are exposed to increased oxidative stress. However, the mechanism to resist this stress is not completely clear. B. cenocepacia possess a highly conserved operon that encodes three evolutionary conserved proteins. Two periplasmatic proteins, BcnA and BcnB, both members of the lipocalin family, and an inner-transmembrane protein that resembles the eukaryotic cytochrome b561 (Lipocalin-associated cytochrome oxidase, LcoA). Mutants in lcoA, bcnA but, not in bcnBgenes show increased lipid peroxidation and susceptibility against bactericidal antibiotics. Similar results were shown with a mutant in a gene that produces a conserved cytoplasmatic protein of the aldehyde reductase family, PrsA. Lipid peroxidation in vivo was measured using fluorescent probes that directly interact with lipid peroxidation radicals, showing increased fluorescent at the bacterial poles under peroxidative stress-inducing conditions. Using mass spectrometry and fluorescent microscopy, we also observed that mutants exposed to antibiotic stress display a rearrangement of anionic phospholipids in the membrane. By assessing peroxidation in vivo, we found that BcnA, LcoA and PsrA play a key role in detoxification of peroxidation byproducts arising from membrane lipid peroxidation. We conclude that these proteins constitute an evolutionary conserved, hitherto unrecognized peroxidation detoxification system that protects the bacterial cell envelope from lipid peroxyl radicals.