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dc.contributor.authorHay, Thomas
dc.contributor.otherQueen's University (Kingston, Ont.). Theses (Queen's University (Kingston, Ont.))en
dc.date2013-02-26 13:32:39.307en
dc.date.accessioned2013-02-26T21:41:04Z
dc.date.available2013-02-26T21:41:04Z
dc.date.issued2013-02-26
dc.identifier.urihttp://hdl.handle.net/1974/7828
dc.descriptionThesis (Master, Microbiology & Immunology) -- Queen's University, 2013-02-26 13:32:39.307en
dc.description.abstractChemotherapeutic treatment of Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, is substantially challenged by several membrane-spanning, multidrug-efflux pumps of the three-component RND family. Of these pumps, MexXY-OprM contributes to the intrinsic resistance of this organism by exporting clinically relevant antibiotics, most notably the ribosome-targeting aminoglycosides. Overproduction of MexXY-OprM is the most common mechanism providing pan-aminoglycoside resistance to P. aeruginosa cystic fibrosis clinical isolates. The mexXY genes are located in an operon, the expression of which is induced by ribosome-targeting antimicrobials. The mexXY operon is negatively regulated by MexZ, a repressor protein encoded by the divergently-transcribed gene mexZ. A second gene, PA5471, is also induced by ribosome-targeting antibiotics and is required for antibiotic induction of mexXY expression. One possibility is that PA5471 interacts with MexZ to alleviate repression of mexXY, thereby providing a mechanism for PA5471-dependent drug inducibility of mexXY. PA5471 interaction with MexZ was confirmed using a bacterial two-hybrid assay. To identify residues/regions of PA5471 important for interaction with MexZ, random chemical mutagenesis of the mexZ and PA5471 genes was carried out and the effects of these mutations on interaction of their protein products was assessed using the bacterial two-hybrid assay. Mutations of PA5471 that compromised interaction with MexZ included P68S, G76C, R216C, R221W, R221Q, G231D, and G252S, which occur within or in close proximity to a predicted surface-exposed α-helix of a PA5471 structural model that may contribute to the MexZ-interaction domain. Representative mutations P68S, G76C, R216C and R221W were placed into the chromosome of P. aeruginosa to assess their impact on drug-inducible mexXY expression. All of these mutations significantly reduced mexX upregulation in the presence of spectinomycin, where mutations R216C and R221W resulted in the near complete ablation of this antibiotic induction. These data suggest that PA5471 acts as a direct antirepressor of MexZ and that this interaction is key to mexXY upregulation in response to ribosome-targeting induction signals.en_US
dc.languageenen
dc.language.isoenen_US
dc.relation.ispartofseriesCanadian thesesen
dc.rightsThis publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.en
dc.subjectEffluxen_US
dc.subjectMultidrug resistanceen_US
dc.subjectPseudomonas aerguinosaen_US
dc.subjectPA5471en_US
dc.subjectMexZen_US
dc.subjectMexXY-OprMen_US
dc.titlePA5471 modulation of the Pseudomonas aeruginosa mexXY multidrug efflux pump operon repressor MexZ: Identification of important interaction residues and domainsen_US
dc.typethesisen_US
dc.description.degreeMasteren
dc.contributor.supervisorPoole, R. Keithen
dc.contributor.departmentMicrobiology and Immunologyen


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