| Autor: |
Karash S; Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR, 72701, USA., Liyanage R; Department of Chemistry, University of Arkansas, Fayetteville, AR, 72701, USA.; Statewide Mass Spectrometry Facility, Fayetteville, AR, 72701, USA., Qassab A; Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR, 72701, USA., Lay JO Jr; Department of Chemistry, University of Arkansas, Fayetteville, AR, 72701, USA.; Statewide Mass Spectrometry Facility, Fayetteville, AR, 72701, USA., Kwon YM; Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR, 72701, USA. ykwon@uark.edu.; Department of Poultry Science, University of Arkansas, Fayetteville, AR, 72701, USA. ykwon@uark.edu. |
| Abstrakt: |
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 H 2 O 2 . For Tn-seq, a saturated Tn5 insertion library was grown in vitro under either 2.5 (H 2 O 2 L) or 3.5 mM H 2 O 2 (H 2 O 2 H). We identified two sets of overlapping genes required for resistance of S. Typhimurium to H 2 O 2 L and H 2 O 2 H, and the results were validated via phenotypic evaluation of 50 selected mutants. The enriched pathways for H 2 O 2 resistance included DNA repair, aromatic amino acid biosynthesis (aroBK), Fe-S cluster biosynthesis, iron homeostasis and a putative iron transporter system (ybbKLM), and H 2 O 2 scavenging enzymes. Proteomics revealed that the majority of essential proteins, including ribosomal proteins, were downregulated upon exposure to H 2 O 2 . 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 H 2 O 2 . The identified genes will deepen our understanding on S. Typhimurium survival mechanisms in macrophages, and can be exploited to develop new antimicrobial drugs. |
