Salmonella is an intracellular pathogen that infects a wide range of hosts and can survive in macrophages. An essential mechanism uses by the macrophages to eradicate Salmonella is production of reactive oxygen species. Here, we used proteogenomics to determine the candidate genes and proteins that have a role in resistance of S. Typhimurium to H2O2. For Tn-seq, a highly saturated Tn5 insertion library was grown in vitro under either 2.5 (H2O2L) or 3.5 mM H2O2 (H2O2H). We identified two sets of overlapping genes that are required for resistance of S. Typhimurium to H2O2L and H2O2H, and the results were validated via phenotypic evaluation of 50 selected mutants. The enriched pathways for resistance to H2O2 included DNA repair, aromatic amino acid biosynthesis (aroBK), Fe-S cluster biosynthesis, iron homeostasis and a putative iron transporter system (ybbKLM), flagellar genes (fliBC), H2O2 scavenging enzymes, and DNA adenine methylase. Proteomics revealed that the majority of essential proteins, including ribosomal proteins, were downregulated upon exposure to H2O2. A subset of proteins identified by Tn-seq were analyzed by targeted proteomics, and 70 % of them were upregulated upon exposure to H2O2. The identified candidate genes will deepen our understanding about mechanisms of S. Typhimurium survival in macrophages, and can be exploited to develop new antimicrobial drugs.

* Grew Salmonella Typhimurium under H2O2 till mid-exponential phase * Constructed Tn5 mutant library * Measured growth responses of S. Typhimurium to H2O2 * Selected the mutant library for Tn-seq analysis * Prepared Tn-seq amplicon libraries * Evaluated phenotypic individual single deletion mutants * Protein extracts were analyzed by shotgun proteomics using Bruker Amazon SL ion trap with calptive spray source (LC-ESI-MS/MS) and targeted selected number of proteins using Shimadzu 8050 QQQ based on Tn-seq data shotgun proteomics data.