The role of N-chlorination, a novel oxidative post-translational modification, in host-pathogen interactions and chronic inflammation

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Hypochlorous acid (HOCl), produced by immune cells during the so-called oxidative burst, is a highly toxic oxidant. To survive their encounter with HOCl-producing immune cells, bacteria have evolved numerous defense mechanisms against this antibacterial compound. In 2014, we found that RidA is a bacterial redox-regulated chaperone that helps Escherichia coli to survive HOCl exposure. This chaperone is activated by N-chlorination, a novel HOCl-induced post-translational modification. We also found that this N-chlorination is fully reversible through NADPH-dependent reduction by the thioredoxin system. Since then, we and others have discovered more proteins that undergo N-chlorination upon exposure to HOCl, some of bacterial (like CnoX) and others of human origin (like serum albumin and other plasma proteins). These N-chlorinated proteins have been shown to have both immunomodulatory as well as protective properties. However, until now, most of these findings are based on in vitro observations. A regulatory role in host-pathogen interactions and potentially chronic inflammation of this modification is, thus, only implied. Here we propose to develop the chemical tools that will allow us, for the first time, to study the (patho-)physiological role of N-chlorination in vivo, too. At first, we plan to biochemically characterize protein N-chloramine reduction by thioredoxin in order to identify the molecular mechanism that links N-chlorination to the thiol-disulfide homeostasis. This will reveal the underpinnings of the integration of N-chlorination, as a regulatory modification, in host-pathogen interactions and chronic inflammation. In parallel, we intend to synthesize a set of probes for the detection and quantification of protein N-chloramines. We plan to characterize these probes and develop a chemoproteomic methodology to quantify the extent of protein N-chlorination in vivo, based on our prior experience in quantitative redox proteomics. With these tools we will then be able to determine the role of the thioredoxin and glutathione system in cellular N-chloramine homeostasis in E. coli exposed to HOCl and immune cells. Additionally, we will, for the first time, attempt to detect protein N-chlorination in samples from patients suffering from juvenile idiopathic arthritis, determining the clinical relevance of this novel post translational modification under severe chronic inflammation.