. Oxidative stress has been implicated in a wide range of human pathologies, and it is also determinative in the ecology of microbial communities and the microbicidal actions of the cell-based immune system. However, its molecular nature is incompletely understood. In discussions of oxidative stress, the presence of reactive oxygen species (ROS) is often conflated with the accumulation of protein disulfide bonds. The model bacterium Escherichia coli provides circumstantial evidence to support this view. E. coli responds to H2O2 exposure by inducing several redoxins devoted to the reduction of disulfide bonds, and its growth during superoxide stress is possible only if a rapidly imported cysteine precursor is provided. Yet we and others have shown that the primary ROS?superoxide and hydrogen peroxide?do not oxidize typical cysteine residues at an impactful rate. In this proposal we seek to identify the connection between the presence of these oxidants and the disruption of cellular thiol status. We have begun by delineating the processes that control the intracellular level of cysteine. We have already identified the routes and extremely high rate of cystine entry into E. coli, and in Aim 1 we propose to do the same for cysteine. We will then examine the devices that help to curb cysteine over-accumulation: its efflux via AlaE, and its degradation by YhaM. This approach provides necessary context for interpreting the effects of cysteine supplementation upon the phenotypes of oxidative stress.
In Aim 2 we seek to identify the major mechanisms by which oxidative stress can generate disulfide bonds. We will test three widely suspected sources of general disulfide-bond formation: iron-catalyzed thiol oxidation, copper-catalyzed thiol oxidation, and the inadvertent dissemination of disulfide bonds by the primary peroxidase, AhpCF. We will then examine site-specific thiol oxidation that we have observed when H2O2 attacks iron-based enzymes. In particular, we will test whether the induction of redoxins is an important step in the repair of these enzymes.
In Aim 3, we seek to explain why superoxide-stressed cells can only grow if they contain high levels of cysteine. Superoxide disrupts enzymic iron-sulfur clusters and triggers the mismetallation of mononuclear iron enzymes. We have shown that the slow step in cluster repair is the delivery of iron, and our recent work suggests that intracellular cysteine mobilizes iron to the enzymes that need it. Further, cysteine is uniquely effective at extracting zinc from mismetallated iron enzymes, which is the rate-limiting step in their repair. Thus our analysis of superoxide stress has revealed opportunities for cysteine to restore the functions of pathways that superoxide otherwise blocks. In aggregate, the successful completion of these aims will reveal how thiol status is connected to oxidative stress. This would fill a large, perplexing hole in our view of how oxidants damage cells and how cells defend themselves against them. It will also finally provide a rational framework for considering the use of thiol antioxidants to diagnose or suppress oxidative stress.

Public Health Relevance

. Oxidativestressisasourceofhumanpathology,butitsmolecularnatureis notfullyunderstood.UsingthemodelsystemEscherichiacoli,wewilltestthe widelyheldtheorythattheimpactofoxidantsiscountervailedbytheactionsof thiolsasantioxidants.Thesuccessfulresolutionofthisquestionwillguideworkers whowishtousesuchcompoundsinthediagnosisandtreatmentofoxidativestress.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Prokaryotic Cell and Molecular Biology Study Section (PCMB)
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Melillo, Amanda A
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University of Illinois Urbana-Champaign
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Korshunov, Sergey; Imlay, James A (2018) Quantification of Hydrogen Sulfide and Cysteine Excreted by Bacterial Cells. Bio Protoc 8:
Li, Xin; Imlay, James A (2018) Improved measurements of scant hydrogen peroxide enable experiments that define its threshold of toxicity for Escherichia coli. Free Radic Biol Med 120:217-227
Korshunov, Sergey; Imlay, Karin R C; Imlay, James A (2016) The cytochrome bd oxidase of Escherichia coli prevents respiratory inhibition by endogenous and exogenous hydrogen sulfide. Mol Microbiol 101:62-77
Chonoles Imlay, Karin R; Korshunov, Sergey; Imlay, James A (2015) Physiological Roles and Adverse Effects of the Two Cystine Importers of Escherichia coli. J Bacteriol 197:3629-44
Martin, Julia E; Waters, Lauren S; Storz, Gisela et al. (2015) The Escherichia coli small protein MntS and exporter MntP optimize the intracellular concentration of manganese. PLoS Genet 11:e1004977
Imlay, James A (2015) Transcription Factors That Defend Bacteria Against Reactive Oxygen Species. Annu Rev Microbiol 69:93-108
Imlay, James A (2014) The mismetallation of enzymes during oxidative stress. J Biol Chem 289:28121-8
Sobota, Jason M; Gu, Mianzhi; Imlay, James A (2014) Intracellular hydrogen peroxide and superoxide poison 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase, the first committed enzyme in the aromatic biosynthetic pathway of Escherichia coli. J Bacteriol 196:1980-91
Gu, Mianzhi; Imlay, James A (2013) Superoxide poisons mononuclear iron enzymes by causing mismetallation. Mol Microbiol 89:123-34
Imlay, James A (2013) The molecular mechanisms and physiological consequences of oxidative stress: lessons from a model bacterium. Nat Rev Microbiol 11:443-54

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