Date of Award
Doctor of Philosophy (PhD)
Xiao-Hong Nancy Xu
Lesley H. Greene
Christopher J. Osgood
Multidrug membrane transporters (efflux pumps) in both prokaryotes and eukaryotes are responsible for ineffective treatment of a wide variety of diseases, including infections and cancer, underscoring the importance of better understanding of their structures and functions for design of effective therapies. Despite extensive studies over decades, their underlying molecular mechanisms remain largely unknown. In this dissertation, we focus on the study of structures and functions of multidrug membrane transporters, including ATP-binding cassette transporter (BmrA) and Resistance-Nodulation-Cell Division transporter (MexA,B-OprM) in gram-positive and gram-negative bacteria ( Bacillus subtilis and Pseudomonas aeruginosa), respectively. We fused EGFP with N-terminus and C-terminus of BmrA to construct BmrA-EGFP fusion proteins and characterized their efflux functions in four strains of bacterial cells (Ct-BmrA-EGFP, Nt-BmrA-EGFP, BmrA and ▵BmrA, B. subtilis) using fluorescence microscopy and spectroscopy. The results show that BmrA-EGFP fusion proteins exhibit the similar efflux kinetics of BmrA and retain the efflux functions of BmrA. Modeled structures of the fusion proteins show a highly flexible linker region connecting EGFP with BmrA, suggesting a minimal obstruction of EGFP to BmrA. These two new strains of BmrA-EGFP offer the possibility for one to visualize and study efflux function and mechanisms of BmrA transporters. We further studied the size-dependent efflux functions of BmrA-EGFP and BmrA using single plasmonic nanoparticle imaging probes and spectroscopy and found size-dependent efflux kinetics of BmrA. Using similar approaches, we fused EGFP with MexA,B-OprM transporters and characterized their efflux functions using fluorescence spectroscopy and single NP plasmonic spectroscopy. The results show that EGFP fused MexA,B-OprM only partially retains its original efflux functions. Structural analysis of fusion protein suggests possible steric hindrance of EGFP toward the conformation changes of MexA,B-OprM, which led to the steric effect upon their efflux functions. These important findings offer new insights into the structural and functional studies of multidrug membrane transporters (efflux pumps), and demonstrate the powerful new nanobiotechnology for better understanding of multidrug resistance.
"Structural and Functional Study of Multidrug Membrane Transporters"
(2013). Doctor of Philosophy (PhD), dissertation, , Old Dominion University, DOI: 10.25777/ghdx-gp13