PT - JOURNAL ARTICLE AU - Sardar Karash AU - Rohana Liyanage AU - Abdullah Qassab AU - Jackson O. Lay, Jr. AU - Young Min Kwon TI - A Comprehensive Assessment of the Genetic Determinants in <em>Salmonella</em> Typhimurium for Resistance to Hydrogen Peroxide AID - 10.1101/115360 DP - 2017 Jan 01 TA - bioRxiv PG - 115360 4099 - http://biorxiv.org/content/early/2017/03/09/115360.short 4100 - http://biorxiv.org/content/early/2017/03/09/115360.full AB - Salmonella is a Gram-negative bacterium that infects a wide range of hosts. Salmonella Typhimurium causes gastroenteritis in human, and can survive and replicate in macrophages. An essential mechanism used by the macrophages to eradicate Salmonella is production of reactive oxygen species. Here, we used proteogenomic approaches 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 in Luria-Bertani broth (LB) medium as well as LB containing 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 result was validated via phenotypic evaluation of 50 selected mutants. The enriched pathways for resistance to H2O2 included DNA repair, aromatic amino acid biosynthesis, Fe-S cluster biosynthesis, iron homeostasis, flagellar genes, H2O2 scavenging enzymes, and DNA adenine methylase. Particularly, we identified aromatic amino acid biosynthesis (aroB, and aroK) and putative iron transporter system (ybbK, ybbL, and ybbM) as novel mechanisms for resistance to H2O2. The proteomics revealed that the majority of essential proteins, including ribosomal proteins, were downregulated upon exposure to H2O2. A subset of the 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 on the mechanisms of S. Typhimurium survival in macrophages, and can be exploited to develop new antimicrobial drugs.IMPORTANCE Salmonella infection is frequently caused by consumption of contaminated food or water. The infection may lead to gastroenteritis or typhoid fever, depending on the Salmonella serovars. Even though the bacterium encounters the immune defense arsenals of the infected host, including reactive oxygen species in phagocytes, the bacterium can survive and replicate. In this study, proteogenomic approaches were used in order to identify the genes and proteins that have a role in resistance to H2O2. In addition to the H2O2 scavenging and degrading enzymes, aromatic amino acid biosynthesis and iron homeostasis were identified among the most important pathways for H2O2 resistance. These findings will deepen our knowledge on the mechanisms of Salmonella survival in phagocytes and other niches with oxidative stress, and also provides novel targets to develop new antimicrobial therapeutics.