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dc.contributor.advisorBrian J. Akerley, Ph.D.
dc.contributor.authorHarrington, Jane Colleen
dc.date2022-08-11T08:08:42.000
dc.date.accessioned2022-08-23T16:04:38Z
dc.date.available2022-08-23T16:04:38Z
dc.date.issued2008-11-14
dc.date.submitted2009-01-30
dc.identifier.doi10.13028/q1cv-fp30
dc.identifier.urihttp://hdl.handle.net/20.500.14038/31726
dc.description.abstractHaemophilus influenzae encounters niches within the human host that are predicted to differ in availability of oxygen and reactive nitrogen species (RNS: nitrite and nitric oxide), which influence the environmental redox state. Previously reported data has indicated that an altered redox condition could serve as a signal recognized by H. influenzae to optimize its survival within host microenvironments. To elucidate the role of redox signaling in virulence, we examined regulation by the FNR homolog of H. influenzae, whose counterpart in E. coli has been reported to be a direct oxygen sensor and a regulator of genes responsible for RNS metabolism and resistance. Many members of the FNR regulon are subject to coordinated transcriptional control by NarP, a regulator in E. coli that is activated by cognate sensor NarQ in response to environmental nitrite. To study the regulatory activities of FNR and NarQ-NarP in H. influenzae, I targeted a gene predicted to be FNR-regulated, nrfA, which encodes nitrite reductase, a periplasmic cytochrome-c involved in anaerobic respiration. The fnr, narP and nrfA mutants were assayed for nitrite reduction, which implicated the roles of FNR, NarP and NrfA in RNS metabolism. Using Western blot detection of an epitope-tagged reporter protein fused to the endogenous nrf promoter (Pnrf-HA), I demonstrate that FNR and NarP, but not NarQ, are required for full activation of the nrf promoter. Additionally, Pnrf-HA expression increases as oxygen becomes depleted and decreases when exposed to high concentrations of nitrite, implying that the nrfpromoter is modulated by environmental redox signals. FNR of E. coli has been implicated in regulation of resistance mechanisms to a reactive nitrogen species, nitric oxide (NO), which is produced by innate immune cells during infection as a host defense mechanism. A mutant lacking FNR is more sensitive to NO exposure and killing by activated macrophages than wild type H. influenzae after anaerobic pre-growth. Mutants of nrfA and narP have been tested and initial experiments have shown both mutants have a lesser NO sensitivity phenotype as compared to the fnr mutant, suggesting that other factors could be involved in FNR-mediated NO resistance in H. influenzae. Upon examination of potential factors that might be involved to this phenotype, we discovered FNR-regulated gene, ytfE, which contributes to defense against nitrosative stress. The fnr and ytfE mutants are more susceptible to killing by activated macrophages indicating that FNR regulation of ytfE might be important for in vivo infection.
dc.language.isoen_US
dc.rightsCopyright is held by the author, with all rights reserved.
dc.subjectHaemophilus influenzae
dc.subjectReactive Nitrogen Species
dc.subjectOxidation-Reduction
dc.subjectDNA-Binding Proteins
dc.subjectMembrane Proteins
dc.subjectEscherichia coli Proteins
dc.subjectAmino Acids, Peptides, and Proteins
dc.subjectBacteria
dc.subjectGenetic Phenomena
dc.titleRegulation of Reactive Nitrogen Species (RNS) Metabolism and Resistance Mechanisms in <em>Haemophilus influenzae</em>: A Dissertation
dc.typeDoctoral Dissertation
dc.identifier.legacyfulltexthttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=1401&amp;context=gsbs_diss&amp;unstamped=1
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/gsbs_diss/401
dc.legacy.embargo2009-12-29T00:00:00-08:00
dc.identifier.contextkey703620
refterms.dateFOA2022-08-30T16:19:09Z
html.description.abstract<p><em>Haemophilus influenzae</em> encounters niches within the human host that are predicted to differ in availability of oxygen and reactive nitrogen species (RNS: nitrite and nitric oxide), which influence the environmental redox state. Previously reported data has indicated that an altered redox condition could serve as a signal recognized by <em>H. influenzae</em> to optimize its survival within host microenvironments. To elucidate the role of redox signaling in virulence, we examined regulation by the FNR homolog of <em>H. influenzae</em>, whose counterpart in <em>E. coli</em> has been reported to be a direct oxygen sensor and a regulator of genes responsible for RNS metabolism and resistance. Many members of the FNR regulon are subject to coordinated transcriptional control by NarP, a regulator in <em>E. coli</em> that is activated by cognate sensor NarQ in response to environmental nitrite. To study the regulatory activities of FNR and NarQ-NarP in <em>H. influenzae</em>, I targeted a gene predicted to be FNR-regulated, <em>nrfA</em>, which encodes nitrite reductase, a periplasmic cytochrome-c involved in anaerobic respiration. The <em>fnr, narP</em> and <em>nrfA</em> mutants were assayed for nitrite reduction, which implicated the roles of FNR, NarP and NrfA in RNS metabolism. Using Western blot detection of an epitope-tagged reporter protein fused to the endogenous <em>nrf</em> promoter (Pnrf-HA), I demonstrate that FNR and NarP, but not NarQ, are required for full activation of the <em>nrf</em> promoter. Additionally, Pnrf-HA expression increases as oxygen becomes depleted and decreases when exposed to high concentrations of nitrite, implying that the <em>nrf</em>promoter is modulated by environmental redox signals.</p> <p>FNR of <em>E. coli</em> has been implicated in regulation of resistance mechanisms to a reactive nitrogen species, nitric oxide (NO), which is produced by innate immune cells during infection as a host defense mechanism. A mutant lacking FNR is more sensitive to NO exposure and killing by activated macrophages than wild type <em>H. influenzae</em> after anaerobic pre-growth. Mutants of <em>nrfA</em> and <em>narP</em> have been tested and initial experiments have shown both mutants have a lesser NO sensitivity phenotype as compared to the <em>fnr</em> mutant, suggesting that other factors could be involved in FNR-mediated NO resistance in <em>H. influenzae</em>. Upon examination of potential factors that might be involved to this phenotype, we discovered FNR-regulated gene, <em>ytfE</em>, which contributes to defense against nitrosative stress. The <em>fnr</em> and <em>ytfE</em> mutants are more susceptible to killing by activated macrophages indicating that FNR regulation of <em>ytfE</em> might be important for <em>in vivo</em> infection.</p>
dc.identifier.submissionpathgsbs_diss/401
dc.contributor.departmentDepartment of Molecular Genetics and Microbiology
dc.description.thesisprogramMolecular Genetics and Microbiology


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