Year of Publication

2013

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Arts and Sciences

Department

Chemistry

First Advisor

Dr. Yinan Wei

Abstract

Drug resistant bacteria pathogen poses a severe threat to human health. Bacterial drug efflux pumps are transporter proteins involved in the export of antibiotics out of cells. Efflux by transporters is one of the major drug resistant mechanisms. Multidrug efflux pumps can transport multiple classes of antibiotics and are associated with bacteria multiple drug resistance (MDR). Overproduction of these pumps reduces susceptibility of bacteria to a variety of antibiotics. MDR regulators are cytoplasmic proteins that control the expression level of MDR transporters in response to the cellular concentration of antibiotics. This thesis research focuses on three main directions in the area of bacteria drug resistance: the structural and functional study of a MDR transporter, the characterization of a novel MDR regulator protein, and the development of a sensing method for the detection of glycopeptide antibiotics.

Acriflavine resistance protein B (AcrB) in Escherichia coli belongs to resistance nodulation division (RND) superfamily of efflux transporters. It plays an important role in confering multidrug resistance in Gram-negative bacteria. The functional unit of AcrB is a trimer in vivo. However, the relationship between AcrB trimer stability and functionality remains elusive. In chapter 2, a residue that is critical for AcrB trimerization, Pro 223, was identified. The replacement of Pro 223 by other residues destabilized AcrB trimer, and thus decreased its activity. The loss of transport activity could be partially recovered when the AcrB trimer was stabilized by the introduction of a pair of inter-subunit disulfide bond. In chapter 3, a systematically alanine-scanning study of the producing loop (amino acid residues 211-240) was conducted. Five residues in the loop were found to be important for AcrB activity. These residues form a collar or belt in the loop close to the tip. These mutation studies revealed new insight into the conformation of the loop during AcrB trimerization. In chapter 4, residue Arg 780 was identified to be crucial for the pump function of AcrB. The study results indicated that Pro 223 serves as a “wedge” and Arg 780 as a “lock” via hydrogen bonding between the backbone carbonyl oxygen of Pro 223 and side chain of Arg780. Similar as Pro 223, replacement of Arg 780 by other residues drastically decreased the activity of AcrB. Dissociation of the AcrB trimer also contributed to the decrease of activity. However, the introduction of inter-subunit disulfide bond could not restore the function of the mutant, indicating that Arg 780 plays multiples roles in the operation of AcrB.

In chapter 5, a MDR regulator ST1710 from the archaeon Sulfolobus tokodaii, homologous to the multiple-antibiotic resistance repressor (MarR) family bacterial regulators, was characterized in vitro. The binding affinities of ligands and double strand (ds) DNA for ST1710 were measured. The presence of substrates suppressed the interaction between ST1710 and dsDNA, which indicated that ST1710 functioned as a repressor in vivo.

Finally, in chapter 6, a direct fluorescence polarization based method for the detection of glycopeptide antibiotics is developed. Briefly, the acetylated tripeptide L-Lys-D-Ala-D-Ala was labeled with a fluorophore (fluorescein isothiocyanate or AlexaFluor 680) to create a peptide probe. The fluorescence polarization signal of the peptide probe increased upon binding with glycopeptide antibiotics in a concentration dependent manner. The detection is highly selective toward glycopeptide antibiotics. The designed method is expected it to have broad applications in both research and clinical settings.

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