A Comprehensive Assessment of the Genetic Determinants in Salmonella Typhimurium for Resistance to Hydrogen Peroxide Using Proteogenomics

Salmonella is an intracellular pathogen infecting a wide range of hosts and can survive in macrophages. An essential mechanism used by 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 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 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 H₂O₂ resistance included DNA repair, aromatic amino acid biosynthesis (aroBK), Fe-S cluster biosynthesis, iron homeostasis and a putative iron transporter system (ybbKLM), and H₂O₂ scavenging enzymes. Proteomics revealed that the majority of essential proteins, including ribosomal proteins, were downregulated upon exposure to H₂O₂. On the contrary, a subset of conditionally essential proteins identified by Tn-seq were analyzed by targeted proteomics, and 70% of them were upregulated by H₂O₂. The identified genes will deepen our understanding on S. Typhimurium survival mechanisms in macrophages, and can be exploited to develop new antimicrobial drugs.