Hypochlorous acid (HOCl)-derived lipid N-chloramines in host-microbe interactions

When bacteria encounter neutrophils, the first line of defence against pathogens in humans, they are phagocytised and killed by a cocktail of oxidants, including the highly toxic hypochlorous acid (HOCl), produced by the neutrophils in the phagolysosome. In vitro studies have shown that, like in proteins, HOCl results in the chlorination of the membrane lipids" amino residues found in the head groups of some lipids. However, reliable methods for the in vivo detection of lipid N-chloramines are lacking and hence their physiological importance is still under investigation.

Here, we used the dansyl derivative dansyl sulfinic acid (DANSO₂H) to investigate N-chlorination of membrane lipids in living bacteria and mammalian cells exposed to HOCl and in vitro studies with model membranes to identify target lipids and activity of N-chloramines towards biomolecules.

First, we showed that exposure of living mammalian (THP1) or bacterial cells (E. coli) to HOCl results in the formation of lipid N-chloramines in cell membranes using DANSO₂H-derivatization followed by thin layer chromatography (TLC). Furthermore, HOCl treatment of living cells also results in protein N-chlorination. In vitro studies of model membranes in large unilamellar vesicles (LUV) single out phosphatidylethanolamine (PE), a major phospholipid in E. coli, also present in the mammalian cytoplasmic membrane, as a main target of HOCl. These N-chloramines were reversed by treatment of chlorinated LUV with the cellular antioxidant glutathione, indicating a role of this cellular reduction system in counteracting N-chloramines. To investigate the activity of chlorinated LUVs towards biomolecules, we used the redox active protein roGFP2. Exclusively PE-containing, HOCl-treated LUVs oxidize the probe, suggesting that lipid N-chloramines exhibit oxidative capacity. Taken together, our experiments suggest that lipid N-chloramines have a biologically relevant oxidative activity, and hence potentially accelerate the host immune response similar to protein N-chloramines. Furthermore, this work lays the foundation for future experiments aiming at their occurrence and biological role(s) in host pathogen interactions.