Author ORCID Identifier
Year of Publication
Doctor of Philosophy (PhD)
Arts and Sciences
Dr. Yinan Wei
The Resistant Nodulation Division (RND) super family member, tripartite AcrA-AcrB-TolC efflux pump, is a major contributor in conferring multidrug-resistance in Escherichia coli. The structure of the pump complex, and drug translocation by functional rotation mechanism have been widely studied. Despite of all these data, the dynamics of the assembly process of the pump and AcrB during functional rotation in the process of drug efflux remains poorly understood. My thesis focuses on understanding the pump assembly process, dynamics of AcrB in functional rotation mechanism, and also investigate the mechanism of degradation of AcrB facilitated by a C-terminal ssrA tag.
In the first project, I studied the impact of relative flexibility at the inter-subunit interface utilizing disulfide bond crosslinking, minimum inhibitory concentration (MIC), and EtBr efflux assay. Six inter-subunit disulfide links were inserted into the periplasmic domain of AcrB using site-directed mutagenesis. Based on results from MIC measurement, the double Cys mutants tested led to equal or higher susceptibility to AcrB substrates compared to their corresponding single mutants. EtBr accumulation assays was conducted utilizing Dithiothreitol (DTT) as the reducing agent. In two cases, the activities of the double Cys-mutants were partially restored by DTT reduction, indicating the importance of relative inter-subunit movement in the respective location for function. In addition, crosslinking at the other 4 sites did not have such an effect.
In the second project, I tested the effect of over-expressing functionally defective pump components in wild-type E. coli cells to probe the pump assembly process. The incorporation of defective components is expected to reduce the complex's efflux efficiency and lead to the so-called “dominant negative” effect. The study examined two groups of mutants defective in different aspects and found that none of them demonstrated the expected dominant-negative effect, even at concentrations many folds higher than their genomic counterpart. Based on the data, the assembly of the AcrAB-TolC complex appears to have a proof-read mechanism that effectively eliminated the formation of the futile pump complex.
Moreover, I utilized a novel tool- transposons library creation in studying the possible other proteases that contribute to the degradation of the AcrB-ssrA. Using the next-generation sequencing, I identified the already known clpX gene, and MIC and western blot analysis confirmed the results. While this result demonstrated the effectiveness of the strategy, the current library size is too small and does not yield novel genes related to AcrB-ssrA degradation.
Digital Object Identifier (DOI)
Rajapaksha, Prasangi Irosha, "Investigation of Multidrug Efflux Transporter AcrB in Escherichia coli: Assembly, Degradation and Dynamics" (2021). Theses and Dissertations--Chemistry. 142.