Date of Graduation

8-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Biological Sciences

Advisor

Young Min Kwon

Committee Member

Daniel Lessner

Second Committee Member

Jeffrey Lewis

Third Committee Member

Ravi Barabote

Abstract

Salmonella is an intracellular pathogen that infects a wide range of hosts. The infected host utilizes reactive oxygen species (ROS) and iron-restriction to eliminate the pathogen. 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 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. Further, we assessed genomic of S. Typhimurium under gradient iron-restricted conditions using Tn-seq. In addition to conditionally essential genes that mediate the pathogen survival under iron-restricted conditions, we found ROS-dependent essential genes. Based on this, we expand ROS-antibiotic mediated killing model, which asserts that bactericidal antibiotics induce ROS formation and ultimately contributes to cell death. We show that impairment of many essential genes with transposons, without antibiotic interference, induce ROS formation and the death of these mutants can be ceased through an iron chelator. Tn-seq reveals that one-third of S. Typhimurium essential genome are ROS-dependent, far beyond antibiotic targets, as they can grow very slowly in iron-restricted conditions. Interestingly, majority of antibiotic target genes are ROS-dependent. We propose that ROS-independent essential genes may be better targets for antibiotic development because the cells die immediately following the disruption of the essential gene. This work expands our knowledge about mechanisms of S. Typhimurium survival in macrophages, the role of ROS in cell death following essential gene disruption, and provides novel targets for development of new antibiotics.

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