Show simple item record

dc.contributor.authorHittle, Lauren Elizabeth
dc.date.accessioned2014-08-25T12:38:52Z
dc.date.available2014-08-25T12:38:52Z
dc.date.issued2014
dc.identifier.urihttp://hdl.handle.net/10713/4176
dc.descriptionUniversity of Maryland, Baltimore. Molecular Microbiology and Immunology. Ph.D. 2014en_US
dc.description.abstractPseudomonas aeruginosa (PA) is a free-living Gram-negative opportunistic pathogen associated with long-term airway infections of patients with cystic fibrosis (CF). One early adaptation to the CF lung is production of a pro-inflammatory lipid A containing palmitate. Increasing inflammation leads to an increase in tissue destruction and lung function, ultimately resulting in premature death of the patient. PagP, the biosynthetic enzyme responsible for this CF-specific modification of PA lipid A, had not yet been identified in PA. We identified PA1343 as a candidate gene with palmitoyltransferase activity. A deletion mutant was constructed and analyzed for changes in lipid A structure. The ΔPA1343 mutant strain did not synthesize any palmitoylated lipid A species. Activity studies confirmed pro-inflammatory properties of palmitoylated lipid A as well as showed protection against specific cationic antimicrobial peptides. Subsequent experiments focused on elucidating the mechanism by which PA PagP expression and activity through the two-component system, PhoP/Q were regulated. PA is known to replicate in the oxygen limited mucus plugs in patients' lungs. Growth of PA under oxygen limitation resulted in the synthesis of palmitoylated lipid A species. Further, deletion of phoP, phoQ, and pagP all resulted in a loss of palmitoylation under anaerobic growth conditions. Anaerobic respiration in PA requires the involvement of the enzymes of the denitrification pathway. To determine if components of these pathways were stressors of PhoP/Q, palmitoyltransferase activity was determined after growth in media containing nitrate and nitrite. PA specifically lacking the nitrite reductase, NirS, was unable to synthesize palmitoylated lipid A structures. The importance of the NirS component was believed to be due to enzyme activity and not merely structural interaction. Targeting of the active site of Nir, the heme d1 component confirmed the active enzyme was necessary. Growth of the nirS mutant, a NO deficient strain, was grown with the NO over-producing norC mutant. This restored the lipid A phenotype confirming the production of NO leads to anaerobic lipid A palmitoylation. Collectively, this data indicates production of subinhibitory concentrations of NO within the CF lung contributes to disease progression by increasing signals leading to modification but not bacterial clearance.en_US
dc.language.isoen_USen_US
dc.subjectnirSen_US
dc.subject.meshLipid Aen_US
dc.subject.meshNitric Oxideen_US
dc.subject.meshPseudomonas aeruginosaen_US
dc.titleAnaerobic Lipid A Palmitoylation in Pseudomonas aeruginosa is Regulated by Nitric Oxide Productionen_US
dc.typedissertationen_US
dc.contributor.advisorErnst, Robert K.
refterms.dateFOA2019-02-19T18:15:22Z


Files in this item

Thumbnail
Name:
Hittle_umaryland_0373D_10564.pdf
Size:
21.68Mb
Format:
PDF

This item appears in the following Collection(s)

Show simple item record