The role of HOCl-derived N-chloramines in the bacterial cell envelope in host-pathogen interactions

• Applicant: Dr. Lisa Roxanne Knoke
• Subject Area: Medical Microbiology and Mycology, Hygiene, Molecular Infection Biology ; Biochemistry
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 565924663

Neutrophils are the first line of defense against pathogens in humans. Highly specialized in phagocytosis, they kill engulfed pathogens with a cocktail of toxins and oxidants, including the highly toxic hypochlorous acid (HOCl). Besides oxidizing thiols, HOCl forms N-chloramines with free amines. When bacteria encounter HOCl, or HOCl-derived reactive chlorine species such as taurine chloramine, the first target of the oxidants will naturally be the bacterial cell envelope. However, surprisingly little is known about the occurrence of HOCl-derived envelope N‑chloramines during phagocytosis and their effect in the immune response. In in vitro and in vivo studies we have now shown that, like in proteins, HOCl results in chlorination of the membrane lipids’ amino residues found in the head groups of some lipids. These lipid N‑chloramines exhibit oxidative activity against protein thiols and can be dechlorinated back to a free amine by the physiological antioxidant glutathione. While relatively stable in vitro, they are significantly less stable in living cells, suggesting on the one hand active detoxification and on the other hand high reactivity with other cellular components. Besides membrane lipids, the cell envelope of Gram-negative bacteria contains lipopolysaccharides (LPS) in the outer leaflet of the outer membrane, covering the bacterial surface. These LPS molecules activate proinflammatory responses, and their lipid A moiety acts as endotoxin. LPS in E. coli carries ethanolamine modifications and sugar amines, both containing free amines potentially prone to N-chlorination. In preliminary experiments, we have shown that HOCl indeed forms N-chloramines with LPS in living E. coli cells. In the proposed project we want to study the formation and physiological roles of cell envelope derived N-chloramines and want to investigate how bacteria can counteract and repair these modifications. For that, we propose to (A) identify the targets of HOCl in the cell envelopes of pathogenic and non-pathogenic E. coli and host cell membranes. In order to (B) understand the physiological role of cell envelope N-chloramines, we propose to elucidate their immune modulatory activity, as well as their role in eliminating bacteria. Finally, we propose to elucidate (C) how bacteria protect themselves from cell envelope N-chlorination, and how they detoxify this modification by addressing the roles of cellular antioxidant systems for the removal of N-chloramines in vitro and in vivo. Overall, the proposed work will help unraveling the role of HOCl-derived modifications of bacterial cell envelop components in the modulation of the immune response as well as understanding their role in bacterial survival of the immune attack.

• DFG Programme: Research Grants