Importance of novel myeloperoxidases-derived oxidants during neutrophil-Salmonella interactions – NeutrOX

Neutrophils are the most abundant leukocytes and represent the first line of defence against pathogenic bacteria. Their antimicrobial activity relies on their ability to trigger a myeloperoxidase (MPO)-dependent oxidative burst. Concomitant to the well-documented production of hypochlorous acid (HOCl), other highly oxidizing MPO products have been proposed to form upon bacterial infection. Among the potential candidates, evidence for the implication of N-chlorotaurine (N-ChT) and urate hydroperoxide (UA-OOH) has been only obtained in vitro. N-ChT was shown to be generated by the reaction between taurine (an abundant amino acid derivative) and HOCl, while UA-OOH was proposed to be produced by the MPO-catalysed oxidation of urate (derived from purines catabolism) in an oxidative stress situation.
NeutrOX is a multidisciplinary project gathering analytical and organic chemists, microbiologists and immunologists. In this proposal, we propose to decipher for the first time the in vivo antimicrobial activity of these candidates MPO-controlled pro-oxidants using Salmonella as a model.
By following the Salmonella-neutrophil interactions, NeutrOX aims at investigating two fundamental questions related to the neutrophil-mediated oxidative burst: 1) Are N-ChT and UA-OOH effective MPO-derived oxidants forming during Salmonella infection? 2) How does Salmonella respond to these identified oxidants?
First, following chemical synthesis and purification of N-ChT and UA-OOH, robust analytical and spectrometric methods will be set up to detect these species in varying biological media. This broad array of optimized techniques will be applied afterwards to monitor N-ChT and UA-OOH formation in human neutrophils infected by Salmonella.
Second, we will characterize the in vitro antimicrobial activities of N-ChT and UA-OOH against Salmonella and we will identify bacterial MPO-mediated-stress response, an important step to better understand the pathogen adaptation and survival pathways. We will benefit from this molecular study to develop biological detection probes called bacterial “biosensors” (promoters fused to a gene encoded for fluorescent protein) whose expressions are modulated by the presence of each MPO-derived oxidant. These biosensors will represent new and efficient tools for the in vivo detection of such oxidants at a single-bacterial level and will be compared to the analytical and spectrometric methods for N-ChT and UA-OOH detection.
The ultimate aim of NeutrOX will be to investigate the in vivo characterization and relevance of newly identified MPO-products in a mouse model of salmonellosis and to determine their respective contributions to Salmonella infection host response.
Information and concepts obtained during this project will provide novel insights into the importance of the oxidative burst during the host innate immune response. Moreover, understanding how Salmonella cope with the MPO-derived oxidants could provide information for potential drug targets identification and open the way to new antibacterial compounds design.