The MexCD-OprJ multidrug efflux pump of Pseudomonas aeruginosa confers resistance to a range of antimicrobials. Although not expressed under normal laboratory conditions, exposure to the membrane-active biocides, chlorhexidine or benzalkonium chloride, results in mexCD-oprJ expression. This suggests that membrane disruption provides the inducing signal. Consistent with this, increased mexCD-oprJ expression was demonstrated in the presence of additional membrane-damaging agents including polymyxin B, ethanol, SDS, EDTA, the organic solvents n-hexane and p-xylene, and the antimicrobial peptides melittin, V8 and V681. MexCD-OprJ expression was initially verified through increased resistance to known MexCD-OprJ antimicrobial substrates and subsequently using a mexC-lacZ transcriptional fusion and RT-PCR. Since the P. aeruginosa sigma factor AlgU is responsive to envelope stress, it was of interest to ascertain whether AlgU is capable of mediating this increased mexCD-oprJ expression. Thus, the impact of AlgU loss on mexCD-oprJ expression in response to membrane-damaging agents was assessed in a algU strain. In contrast with above, little or no mexCD-oprJ expression (assessed using resistance to MexCD-OprJ antimicrobial substrates, the mexC-lacZ transcriptional fusion and RT-PCR) occurred in response to membrane-damaging agents in the algU strain, consistent with AlgU playing a role in the envelope stress inducibility of mexCD-oprJ. Overall, envelope stress, and the ability to react to this stress through AlgU, appears to play an important role in mexCD-oprJ induction. This suggests an important role for MexCD-OprJ in alleviating envelope stress, independent of its ability to export and provide resistance to antimicrobials. A gene, PA4596, whose product shows substantial homology to the NfxB repressor of mexCD-oprJ expression, occurs downstream of mexCD-oprJ and shows AlgU-dependence and chlorhexidine inducibility, suggesting a role in the chlorhexidine-induced, AlgU-mediated expression of mexCD-oprJ. Thus, the impact of PA4596 loss on mexCD-oprJ expression was assessed. Paradoxically, the loss of PA4596 increases mexCD-oprJ expression in wild-type cells in response to chlorhexidine treatment (as assessed through RT-PCR), while its loss compromises mexCD-oprJ expression in an nfxB mutant. Nonetheless, this suggests that PA4596 is involved in the induction of mexCD-oprJ and that its ability to induce mexCD-oprJ differs depending on the state of nfxB. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2007-07-31 12:03:52.535
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OKQ.1974/494 |
| Date | 02 August 2007 |
| Creators | Campigotto, Aaron James |
| Contributors | Queen's University (Kingston, Ont.). Theses (Queen's University (Kingston, Ont.)) |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | 2748513 bytes, application/pdf |
| Rights | This 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. |
| Relation | Canadian theses |
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