Date of Award

Spring 2003

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry & Biochemistry

Program/Concentration

Chemistry

Committee Director

Xiao-Hong Nancy Xu

Committee Member

Kenneth Brown

Committee Member

Patricia A. Pleban

Committee Member

Mark Elliot

Call Number for Print

Special Collections LD4331.C45 K97 2003

Abstract

This thesis centers on the study of the xenobiotic efflux system in Pseudomonas aeruginosa, which is a ubiquitous bacterium. It resists many structurally and functionally diverse substrates due to expression of Mex-extrusion pumps, including MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM systems. Despite extensive research, the structure and mechanism of multidrug resistance is unclear (1-9). For example, (i) how do MexA, MexB and OprM proteins assemble to extrude antibiotics? (ii) What is the antibiotic susceptibility of MexA, MexB, and OprM proteins? (iii) How do substrates cross the outer membrane of P. aeruginosa? (iv) Where are antibiotics accumulated inside the cell? This thesis reports a new platform for real-time measurement and observation of MexAB-OprM pump in three strains of P. aeruginosa, PAO4290 (WT, wild-type expression of MexA, MexB, OprM), TNP030#1 (nalB-1, over-expression of MexA, MexB, OprM) and TNP076 (ΔABM, deletion of MexA, MexB, OprM) using nanoparticle optics and single live cell microscopy and spectroscopy. Single nanoparticles were used as molecular probes to study real-time transformation of sizes and dynamics of the pump and for direct observation of real-time transformation of membrane permeability and efflux kinetics of intact cells in the presence of aztreonam and chloramphenicol. This work provides the first direct observation of interplay of membrane permeability and efflux kinetics in real-time at single-cell resolution and suggests that multi-antibiotic resistance may include the induction of transformation of membrane permeability and intrinsic efflux pump machinery. In addition, the results offer the first direct evidence that substrates were not accumulated in the periplasmic space, but in the cytoplasm of live cells. Furthermore, the results demonstrate that nanoparticles inhibit the growth of the bacterium with MIC at 20 pM.

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DOI

10.25777/3kv1-8884

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