STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF MULITDRUG RESISTANCE TRANSPORTER AND REGULATOR

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

Functional Relevance of AcrB Trimerization in Pump Assembly and Substrate Binding

AcrB is a multidrug transporter in the inner membrane of Escherichia coli. It is an obligate homotrimer and forms a tripartite efflux complex with AcrA and TolC. AcrB is the engine of the efflux machinery and determines substrate specificity. Active efflux depends on several functional features including proton translocation across the inner membrane through a proton relay pathway in the transmembrane domain of AcrB; substrate binding and migration through the substrate translocation pathway; the interaction of AcrB with AcrA and TolC; and the formation of AcrB homotrimer. Here we investigated two aspects of the inter-correlation between these functional features, the dependence of AcrA-AcrB interaction on AcrB trimerization, and the reliance of substrate binding and penetration on protein-protein interaction. Interaction between AcrA and AcrB was investigated through chemical crosslinking, and a previously established in vivo fluorescent labeling method was used to probe substrate binding. Our data suggested that dissociation of the AcrB trimer drastically decreased its interaction with AcrA. In addition, while substrate binding with AcrB seemed to be irrelevant to the presence or absence of AcrA and TolC, the capability of trimerization and conduction of proton influx did affect substrate binding at selected sites along the substrate translocation pathway in AcrB

AcrB Trimer Stability and Efflux Activity, Insight from Mutagenesis Studies

The multidrug transporter AcrB in Escherichia coli exists and functions as a homo-trimer. The assembly process of obligate membrane protein oligomers, including AcrB, remains poorly understood. In a previous study, we have shown that individual AcrB subunit is capable of folding independently, suggesting that trimerization of AcrB follows a three-stage pathway in which monomers first fold, and then assemble. Here we destabilized the AcrB trimer through mutating a single Pro (P223) in the protruding loop of AcrB, which drastically reduced the protein activity. We replaced P223 separately with five residues, including Ala, Val, Tyr, Asn, and Gly, and found that AcrBP223G was the least active. Detailed characterization of AcrBP223G revealed that the protein existed as a well-folded monomer after purification, but formed a trimer in vivo. The function of the mutant could be partly restored through strengthening the stability of the trimer using an inter-subunit disulfide bond. Our results also suggested that the protruding loop is well structured during AcrB assembly with P223 served as a “wedge” close to the tip to stabilize the AcrB trimer structure. When this wedge is disrupted, the stability of the trimer is reduced, accompanied by a decrease of drug efflux activity

Comparison of the expression levels of WT AcrB and AcrB_P223G.

<p><b>A.</b> Western blot analysis of membrane vesicles extracted from BW25113Δ<i>acrB</i> expressing WT AcrB (WT) or AcrB_P223G (P223G). Each sample was diluted 1, 5, and 25 folds. <b>B.</b> SDS-PAGE analysis of purified WT AcrB and AcrB_P223G. The expression levels of the WT and AcrB_P223G are similar.</p

Limited trypsin digestion of purified WT AcrB (left) and AcrB_P223G (right).

<p>Lanes 2 to 5 were samples taken at 10, 20, 40, and 60 min into the digestion, respectively. Lane 1 was a control sample in which no trypsin was added. WT AcrB was digested slower than AcrB_P223G.</p

CD spectra of purified WT AcrB and AcrB_P223G.

<p><b>A.</b> Wavelength scans at the far UV region of WT AcrB (black) and AcrB_P223G (grey) superimposed well onto each other, indicating that the two proteins had similar secondary structure contents. <b>B.</b> Temperature denaturation curves of WT AcrB (black) and AcrB_P223G (grey). The ellipticity values monitored at 222 nm were normalized to the reading at 4°C. The thermal stabilities of the two proteins were similar.</p

Sequence alignment of the loop.

<p>The numbers indicate positions of the starting and ending residues in the sequence of <i>E. coli</i> AcrB. Asterisks, colons and periods indicate identical, conserved and semi-conserved residues, respectively. The sequences are: EC, AcrB from <i>E. coli</i>; PA, MexB from <i>Pseudomonas aeruginosa PAO1</i>; NM, MtrD from <i>Neisseria meningitidis 8013</i>; HC, hypothetical protein HcanM9_00968 from <i>Helicobacter canadensis MIT 98-5491</i>; SE, acridine efflux pump from <i>Salmonella enterica subsp. enterica serovar Typhimurium str. LT2</i>; LL, acriflavine resistance protein B from <i>Legionella longbeachae D-4968</i>; SM, hydrophobe/amphiphile efflux-1 (HAE1) from <i>Tenotrophomonas maltophilia R551-3</i>; MC, RND system efflux pump AcrB from <i>Moraxella catarrhalis RH4</i>.</p

Disulfide trapping analysis of AcrB tertiary structure.

<p><b>A.</b> Schematic illustration of the blocking-reducing-labeling procedure. <b>B.</b> The locations of reporter Cys pairs in the structure of AcrB were highlighted using black circles and blue ball-and-stick models. Residue numbers of the Cys mutations were marked. <b>C.</b> AcrB tertiary structure as revealed by the disulfide trapping method. The extents of disulfide bond formation for each reporter Cys pair were very similar in AcrB_P223G as compared to WT AcrB. Therefore, the overall conformation, or tertiary structure, of AcrB_P223G was very similar to that of WT AcrB.</p