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Title
Characterization of the structure and interactions of the AcrAB-TolC multi-drug efflux pump in Escherichia coli
Description
The spread of antibiotic resistant bacteria is currently a pressing global health concern, especially considering the prevalence of multi-drug resistance. Efflux pumps, bacterial machinery involved in various active transport functions, are capable of removing a broad range of antibiotics from the periplasmic space and the outer leaflet of the inner membrane, frequently conferring multi-drug resistance. Many aspects of efflux machinery’s structure, functions, and inter-protein interactions are still not fully understood; further characterization of these components of efflux will provide a strong foundation for combating this resistance mechanism. In this project, I further characterize the channel protein TolC as a part of the AcrAB-TolC efflux pump complex in Escherichia coli by first determining the specificity of compensatory mutations in TolC against defective AcrA and AcrB, and then identifying TolC residues that might influence TolC aperture dynamics or stability when altered. Specificity of compensatory mutations was determined using an array of TolC mutants, previously generated from defective AcrA or AcrB, against a different mutant AcrB protein; these new mutant combinations were then analyzed by real-time efflux and antibiotic susceptibility assays. A vancomycin susceptible TolC mutant—a phenotype that has been associated with constitutively open TolC channels—was then used to generate vancomycin-resistant revertants which were evaluated with DNA sequencing, protein quantification by Western blots, and real-time efflux assays to identify residues important for TolC aperture dynamics and protein stability and complex activity. Mutations identified in revertant strains corresponded to residues located in the lower half of the periplasmic domain of TolC; generally, these revertants had poorer efflux than wild-type TolC in the mutant AcrB background, and all revertants had poorer efflux activity than the parental mutant strain.
Date Created
2016-05
Contributors
- McFeely, Megan Elizabeth (Author)
- Misra, Rajeev (Thesis director)
- Haydel, Shelley (Committee member)
- Stout, Valerie (Committee member)
- School of Mathematical and Statistical Sciences (Contributor)
- School of Life Sciences (Contributor)
- Barrett, The Honors College (Contributor)
Topical Subject
Extent
32 pages
Language
eng
Copyright Statement
In Copyright
Primary Member of
Series
Academic Year 2015-2016
Handle
https://hdl.handle.net/2286/R.I.37923
Level of coding
minimal
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System Created
- 2017-10-30 02:50:58
System Modified
- 2021-08-11 04:09:57
- 3 years 2 months ago
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