METHODS OF BLOCKING EFFLUX PUMP ACTIVITY IN ESCHERICHIA COLI
Thesis
Submitted to
The College of Arts and Sciences of the
UNIVERSITY OF DAYTON
In Partial Fulfillment of the Requirements for
The Degree of
Master of Science in Chemistry
By
Erich Nicholaus Auer
Dayton, Ohio
May 2018
METHODS OF BLOCKING EFFLUX PUMP ACTIVITY IN ESCHERICHIA COLI
Name: Auer, Erich Nicholaus
APPROVED BY:
Matthew E. Lopper, Ph. D. Faculty Advisor Associate Professor
Justin C. Biffinger, Ph. D. Committee Member Assistant Professor
Doug Daniels, Ph. D. Committee Member Executive Director & Research Professor
ii ABSTRACT
METHODS OF BLOCKING EFFLUX PUMP ACTIVITY IN ESCHERICHIA COLI
Name: Auer, Erich Nicholaus University of Dayton
Advisor: Dr. Matthew E. Lopper
Overuse of antibiotics throughout the developed world has contributed significantly to the emergence of antibiotic-resistance in bacteria. Although this helps bacteria it is extremely harmful to humans. As bacteria adapt to the antibiotics, current treatments become ineffective and bacterial infections can become life threatening. One of the mechanisms that bacteria, such as E. coli, use are efflux pumps. Efflux pumps are capable of taking potentially harmful substances, like antibiotics, from inside the bacterial cell to outside of the cell. This allows the bacterium to escape from the damaging effects of the antibiotics. My hypothesis was that natural products, small molecule compounds, or DNA aptamers would be able to bind to some part of the tripartite structure of the AcrA-AcrB –TolC efflux pump and block its activity. The use of natural products and small molecule compounds as potential inhibitors allowed me to determine what substances and structural targets are not viable options. The use of DNA aptamers did show significant potential in blocking efflux pump activity.
iii ACKNOWLEDGMENTS
I would like to start by thanking Dr. Matthew Lopper, my advisor, for allowing me to be a part of his research lab for the past three years. He has been there for every step of this journey and I would not have finished this thesis without his guidance.
I would also like to thank the Office for Graduate Academic Affairs for the financial support. In addition, I would like to thank the University of Dayton Chemistry
Department for support throughout the past five years. Finally, I would like to thank all of the people I have worked with under the direction of Dr. Lopper including: Donald
Mckenna, Robert Leszcynski, Sarah Baxter, CJ Moellering, and Venicia Hawach.
iv TABLE OF CONTENTS
ABSTRACT……………………………………………………………...………………iii
ACKNOWLEDGMENTS………………………………………………………………..iv
LIST OF FIGURES………………………………………………………………………vi
LIST OF TABLES…………………………………………………………………….....vii
LIST OF ABBREVIATIONS…………………………………………………………..viii
CHAPTER 1: INTRODUCTION………….……………………………………………...1
CHAPTER 2: IN SILICO DOCKING OF SMALL MOLECULE COMPOUNDS………7
2.1 Introduction…………………………………………………………………..7 2.2 Experimental Methods………………………………………….…………….8 2.3 Results and Discussion…………………………………………….………..10 2.4 Conclusions……………………………………………………….…………18
CHAPTER 3: NATURAL PRODUCTS…………………………….…………………..19
3.1 Introduction……………………………………………………………….…19 3.2 Experimental Methods………………………………………………………20 3.3 Results and Discussion……………………………………………………...21 3.4 Conclusions………………………………………………………………….23
CHAPTER 4: APTAMERS………………..……………………………..……………...24
4.1 Introduction………………………………………………………………….24 4.2 Experimental Methods……………………………………………………....25 4.3 Results and Discussion……………………………………………………...29 4.4 Conclusions………………………………………………………………….31
CHAPTER 5: GENERAL CONCLUSION…………………………………………...... 33
REFERENCES…………………………………………………………………………..36
v LIST OF FIGURES
Figure 1: Efflux pump overexpression leads to antibiotic resistance…..………………...3
Figure 2: Model of Gram-positive and Gram-negative bacteria efflux pumps…...………4
Figure 3: Binding Sites chosen for in silico efflux pump screening….………………….12
Figure 4: Efflux assay showing inhibition using ZINC06716957…………………….…18
Figure 5: Results of efflux assays with plant extracts……...…………………………….23
Figure 6: Efflux assay showing inhibition using ssDNA aptamers.…………..…………31
vi LIST OF TABLES
Table 1: Small molecule compounds……….…………………………………………..15
Table 2: Mass of plant extracts…………………………………………………………21
vii LIST OF ABBREVIATIONS
DNA Deoxyribonucleic Acid
E. coli Escherichia coli
O.D. Optical density
PBS Phosphate-buffered saline
CPZ Chlorpromazine
DMSO Dimethylsulfoxide
EtBr Ethidium bromide
ABI-PP AcrAB/ MexAB-specific inhibitor of
pyridopyrimidine derivative a.u. Arbitrary fluorescence intensity units
SELEX Systematic Evolution of Ligands by Exponential
enrichment ssDNA Single-stranded deoxyribonucleic acid
viii CHAPTER 1
INTRODUCTION
Antibiotic resistance is a developing problem throughout the world. As society progresses and people use more antibiotics, pathogens are able to mutate in order to avoid detection and destruction. Part of the persistence and survival of bacteria, including
Escherichia coli, is the ability to confer resistance over time. Without this ability, they would struggle to survive for long periods of time.1 As host organisms have evolved and developed natural antimicrobial mechanisms, bacteria have responded with their own protective processes.3 The speed of adaptation within bacterial populations has been so rapid that within two years of the development of penicillin several bacterial strains had already developed resistance.4 This rate of adaptation has not slowed down since it first observed 70 years ago. Currently there are strains of Staphylococcus aureus,
Pseudomonas aeruginosa, and other types of bacteria that are resistant to many antibiotics that are on the market.2 The development of resistance to current antibiotics is a major concern for the health care industry due to the higher costs and the higher risk of permanent damage caused by these drug-resistant pathogens.4
Bacteria can develop resistance to antibiotics in three different ways: acquired, adaptive, and intrinsic resistance.4,5 Acquired resistance refers to any advantage that an organism has gained over a parent, whether it is through the incorporation of DNA or from mutations that occur.6 Adaptive resistance occurs when the microbe changes the
1 way it expresses genes in response to stimuli. The last way in which microbes can develop resistance is through intrinsic resistance. Intrinsic resistance includes the mechanisms that exist with in an organism from the start. Efflux pumps are part of the intrinsic resistance since they are present in bacterial cells as a functional tool used for many purposes. They also become part of the acquired resistance through mutations that effect the levels to which the efflux pumps are expressed. This mutation is when they truly become problematic in our public healthcare system.4,5,7
Efflux pumps are thought to be used in the normal metabolism of bacteria, through functions such as the transport of amino acids, glucose, and cellular signaling.2
These efflux pumps also have the ability to pump out potentially harmful substances such as antibiotics, allowing them to be readily repurposed in resistance mechanisms. In drug resistant bacteria, the expression of efflux pumps increases in order to pump out as much of the antibiotic as possible (Figure 1).2,3 E. coli, along with many types of bacteria, has the ability to remove an extremely wide range of compounds from within the cell. This allows bacteria to be highly resistant to antibiotic agents due to the ability to easily pump out these compounds using the efflux pump apparatus.7 Development of new potential therapies that use chemical efflux pump inhibitors, in combination with available antibiotics, as a viable option to antimicrobial resistant bacteria.2,3
2 Figure 1. Efflux pump overexpression leads to antibiotic resistance: As a potentially harmful substance (red) builds up inside of a bacterial cell, the cell is able to use efflux pumps (blue/ purple) to prevent accumulation of the substance in the cell and protect itself from damage. The end result is a cell that has pumped out the majority of harmful compounds as shown on the right.
Efflux pumps differ in structure depending on the type of bacteria in which they occur. Gram positive bacteria usually have efflux pumps that are a single polypeptide within the cytoplasmic membrane (Figure 2A).2,3,6 Meanwhile gram negative bacterial efflux pumps consist of three parts: an inner membrane protein, an outer membrane protein and a connector that bridges the two (Figure 2B).2 This focused on a gram- negative RND-type pump which consists of AcrA, AcrB, and TolC. AcrA acts as the adaptor protein connecting AcrB, the inner membrane transporter, and TolC, the outer membrane transporter.9 TolC is an outer membrane protein that is connected directly to
AcrA and AcrB. AcrA bridges the intermembrane space, this is known as a membrane- fusion protein and acts as a barrier for where TolC and AcrB fit together within the membrane.
3 A B
TolC
AcrA
AcrB
Figure 2: Model of Gram-positive and Gram-negative bacteria efflux pumps: (A) An efflux pump in a gram-positive bacteria (B) The E. coli efflux pump in a Gram-negative bacteria that bridges the inner and outer membrane of the cell, consisting of AcrA (dark blue), AcrB (purple) and TolC (light blue).
These three protein structures, working in conjunction with one another, form a channel that uses the energy of the proton-motive force to pump substrates out using conformational changes that push them through the protein channel.11 The complex that forms is capable of expelling a large range of compounds from the bacterial cell. This is due to AcrB. AcrB has three separate conformations that allow it to bind to substrates within the cell and then expel it upwards out of the cell by funneling the substrate through
TolC.10 AcrB also has flexibility within its binding pocket that allows molecules to easily enter into it. Once bound into this cavity, proton movement provides the energy to perform the conformational changes and therefore allows AcrB to pump the bound compounds through to TolC.12 TolC is an outer membrane channel that is approximately
100-Å wide when open and it is believed to keep its open conformation throughout the entire pumping process. AcrA is thought to move along with AcrB in a push-pull mechanism that allows the overall assembly to behave as a pump.10
Although it is known how these three proteins work together to expel foreign compounds from E. coli cells, there is still some uncertainty in exactly how the individual
4 components interact with one another. There have been several problems attaining a complete crystal structure of all three proteins working together that has led to this uncertainty. One such problem is that in order to pump molecules out of the cell there must presumably be conformational changes in all three proteins. This means that the determination of a static crystal structure would not be able to tell the whole story. The first proposed structural model includes each of the three components, AcrA, AcrB, and
TolC, with 1:1:1 stoichiometry.13 This was determined using crystal structures of the
AcrA and AcrB, performing cross-linking assays and looking at the spots where the two proteins interacted. It was determined that each subunit of AcrA matched with one subunit of AcrB. TolC and AcrB were also found to be subsequently stabilized by a single AcrA on each subunit showing that there would be no need for a second. Their study has some substantial limitations, including the lack of the complete structure of
AcrA (there were 130 amino acid residues not present in the crystal structure).13 Another study also concluded that a 1:1:1 stoichiometry is sufficient for efflux activity. However, to determine this in vivo they had to heavily modify the proteins, by creating AcrA-AcrB
1:1 fusion proteins, therefore not providing a definitive answer concerning the wildtype stoichiometry. The second proposed structural model has a stoichiometry of 1:2:1, with
AcrA in excess. Experiments such as electron microscopy of the pump and claims that
AcrA exists as a dimer lead to the conclusion of a 1:2:1 stoichiometry, contradicting the other studies.14 While there is evidence supporting both conclusions, based on the structures and spacing of the proteins within the pump, I believe that the 1:1:1 stoichiometry is more likely. This could prove to be important when looking at targets for inhibition.
5 The goal of this research was to identify compounds that are capable of blocking the activity of the AcrA-AcrB-TolC efflux pump. I addressed this using three independent methodologies. The first method used docking studies performed on specific sites of the AcrB and TolC proteins using digital libraries of small molecule compounds in order to find organic compounds with the potential to block activity of the pump. The second strategy was to use edible plant extracts in efflux assays in order to determine if there were any molecules within the plant capable of acting as an efflux pump inhibitor.
The last approach was the use of synthetic nucleic acid aptamers that were selectively chosen to bind to the TolC subunit and inhibit the function of the efflux pump. If we can find a way to restore the usefulness of common antibiotics in clinical settings through efflux pump inhibition, we might be able to help save lives.
6 CHAPTER 2
IN SILICO DOCKING OF SMALL MOLECULE COMPOUNDS
2.1 Introduction
One way of blocking efflux pumps is through the use of small molecule inhibitors. This works by introducing the small molecule compound to the bacterial cells, where the compounds will then bind to specific sites on the efflux pump apparatus and block some function of the efflux pump’s mechanics. This can be through something as simple as blocking the channel formed by the proteins. It can also be as complex as finding a small binding pocket on the protein structures, binding to this pocket and stopping any conformational changes that would otherwise take place as a part of normal efflux pump function.7
In order to make an informed decision on what small molecules to test with the efflux pumps, virtual screening was implemented. Virtual screening takes an area on a three-dimensional rendering of a macromolecule and attempts to place small molecule compounds into this space. The docking space is predetermined by the user based on their specific needs. These small molecule compounds can be obtained from the ZINC database (http://zinc15.docking.org), which contains commercially available libraries of small molecule compounds. The computational software then attempts to place the desired molecules within the designated binding pocket on the protein. The program generates a predicted free energy of binding for each docked molecule that can be used to
7 rank and sort the hits. The more negative the score is, the more likely that molecule is to bind to the target. The binding energies are determined through many factors: electrostatic attraction, electrostatic repulsion, hydrophobic interactions, hydrogen bonds, and rotation of ligand bonds. These binding energies then provide a good starting point for in vivo studies where the validity of in silico studies can be determined.8
Small molecule compounds have shown potential in past studies as a way to block the activity of the efflux pump. Although there have been no compounds studied that have been able to block all activity there are some that are able to decrease the ability of the efflux pumps to expel foreign compounds.7 These studies have provided a foundation for what to test and how we can measure the efflux ability. From this point, this study is looking at more diverse potential binding sites and many more compounds that could be potential inhibitors.
2.2 Experimental Methods
In silico screening: Small molecule docking was performed using Autodock Vina and small molecule libraries from the ZINC database (http://zinc15.docking.org) including: IBScreen natural products (82,852 molecules), Drugbank-approved (1,738 molecules), and Maybridge hit finder (18,359 molecules). High resolution structures of the efflux pump proteins were obtained from the Protein Databank; 3W9H and 2GIF for
AcrB, and 1EK9 for TolC. The searched sites on AcrB were the hydrophobic trap, A-B
Hotspot, PC2 Upper and PC2 Lower (Figure 3). The coordinates for the hydrophobic trap
(3W9H) were: X=+53.8 Å, Y=+31.6 Å, Z=+49.0 Å, with dimensions: X=25 Å, Y=25 Å,
Z=25 Å. The coordinates for the A-B Hotspot (2GIF) were: X=-59.5 Å, Y=+9.0 Å, Z=-
8 88.0 Å, with dimensions: X=35 Å, Y=22 Å, Z=22 Å. The coordinates for the PC2 Upper
(2GIF) were: X=-30.0 Å, Y=+28.0 Å, Z=-29.0 Å, with dimensions: X=25 Å, Y=25 Å,
Z=25 Å. The coordinates for the PC2 Lower (2GIF) were: X=-75.6 Å, Y=-10.0 Å, Z=-
48.5 Å with dimensions: X=25 Å, Y=25 Å, Z=25 Å. The searched site on TolC was
1EK9 Site A which had a search center of X=+36.8 Å, Y=+87.7 Å, Z=+64.8 Å, with dimensions of X=25 Å, Y=25 Å, Z=25 Å.
Efflux Assays: Overnight 3 mL cultures of AG100 E. coli were grown in LB medium at 37C with shaking. These cells were then used to inoculate 20 mL of LB medium to an optical density at 600 nm (O.D.600) of 0.4. The cells grew at 37C until they reached an O.D.600 of 0.6. The cells were transferred to a 1.5 mL microcentrifuge tube and were centrifuged at 21130 g for 1 minute at room temperature. The pellet was washed with 1 mL of phosphate-buffered saline (PBS) and centrifuged for 1 minute at
21130 g at room temperature. The supernatant was discarded and 1 mL of PBS was used to suspend the cells. The cells were then diluted to an O.D.600 of 0.1 using PBS.
The efflux assay was performed using chlorpromazine (CPZ) and dimethylsulfoxide (DMSO) controls. A 3.75 mM stock of CPZ was made along with a
0.045 mg/L stock of ethidium bromide (EtBr). A volume of 140.3 L of the cells in PBS were combined with 3 L of the CPZ stock for a final concentration of 75 M CPZ.
Lastly, 6.67 L of the stock EtBr was added to the mixture, this was then placed in a quartz cuvette and placed in a fluorimeter. The fluorimeter ran for 25 minutes, taking fluorescence measurements every 5 seconds, with the excitation wavelength at 515 nm and the emission wavelength at 600 nm. The cell holder temperature was maintained at
9 37°C. This same procedure was run using 3 µL of DMSO in the place the CPZ as a negative control.
The efflux assay was then performed using small molecule compounds identified as hits in the virtual screens. Each small molecule was dissolved in DMSO to a concentration of 5 nM. A volume of 3 µL was added to each assay so that the final concentration of small molecule compound in the assay was 100 µM. Fluorescence intensity data were collected every five seconds and averaged at one minute intervals.
Each small molecule compound was tested in triplicate. ZINC06716957 was also tested at 200 µM.
2.3 Results and Discussion
Determination of Potential blocking sites: The structures of all three proteins that make up the E. coli efflux pump were considered and five sites were chosen for screening. The first site is located in the channel formed by the three subunits of TolC called 1EK9 Site A (Figure 3A). This site was chosen based on the potential to block the movement of organic materials out of the cell by essentially closing the channel formed by the three units of TolC. The second site chosen is located on AcrB and this binding pocket is located inside the channel formed by the three subunits. Unlike 1EK9 Site A, this binding pocket, known as the hydrophobic trap, is located on the interior of the protein (Figure 3B). This site was chosen based on past studies on the homologous structure MexB that found an efflux pump inhibitor, ABI-PP, that functions by binding in this pocket and preventing rotation of the pump mechanism.16 The third site chosen is also located on AcrB. This binding pocket, called PC2 Upper, is located on the outside
10 surface of the protein in a deep pocket that forms between the subunits (Figure 3C). The rationale behind choosing this binding pocket was that major conformational changes are hypothesized to occur at this location. If the conformational changes were blocked by an inhibitor the pump motion would be blocked. The next binding pocket is called PC2
Lower and it is also located on the outside of AcrB in a deep pocket. This binding pocket is located below the interaction surface between AcrA and AcrB (Figure 3D). Once again, this site was chosen based on the hypothesis that conformational changes that are important for pump function occur at this binding pocket. The fifth binding pocket, called the A-B Hotspot, is a location that is thought to have strong interactions between AcrA and AcrB and any compounds that bind to that site might disrupt the way that the two proteins work together. The interactions between AcrA and AcrB are represented by the colored regions of Figure 3E.
11 Figure 3. Binding Sites chosen for in silico efflux pump screening: (A) The 1EK9 Site A binding pocket, located in the channel formed by the tripartite AcrB structure so that it is in contact with all three subunits. Each subunit of TolC is represented as a different color (B) The hydrophobic trap is located inside each AcrB subunit and was chosen because of past studies performed on Mex-B. This location would be accessible through the substrate translocation channel that the subunits form. Each subunit of AcrB is represented as a different color. (C) The PC2 Upper binding pocket is located on the outside of AcrB. (D) The PC2 Lower binding pocket located on the outside of AcrB, below the interactions between AcrA and AcrB. (E) The A-B Hotspot was chosen in an attempt to block AcrA from interacting with AcrB. In C-E the colored regions on the AcrB represent the interactions that occur between AcrA and AcrB, the darker the color the stronger the interactions are between the two proteins All structures were rendered using PyMol.
In silico screening: The target sites were specified in AutoDock Vina and screened using 3 libraries: IBScreen natural products, Drugbank-approved, and
Maybridge hit finder, using sucrose as the negative control and (ABI-PP) as the positive control for the hydrophobic trap site. This program then took each small molecule compound in the libraries and attempted to fit them into the specified locations. The
AutoDock Vina results were populated into a spreadsheet and arranged from the lowest
12 binding energies to the highest for each binding pocket. The lowest binding energy structures were observed and those that looked like they would fit well in the binding pockets and were soluble enough, based on the LogP values, were ordered for use in efflux assays (Table 1). Small molecules that were highly similar were removed from the potential candidates, usually favoring the one with the more negative binding energies.
This was done by grouping similar structures into families of compounds and then choosing the best candidate of the interesting groups. The majority of compounds that were screened resulted in binding energies of -6.0 kcal/mol to -8.0 kcal/mol which matches the negative control’s binding energy of -6.1 kcal/mol. The major exception to this was in the compounds tested for the hydrophobic trap where the average binding energy was -8.9 kcal/mol. Some of the compounds that were chosen from the screening were unavailable for purchase, which affected the choice of compounds in addition to the previous criteria.
Efflux Assays: In order to monitor the behavior of the efflux pumps within E. coli a marker of some type has to be used. A good marker for activity is something that does not negatively impact the cells functions. In addition, for monitoring efflux activity, markers that have a higher fluorescence inside the cell compared to outside the cell are beneficial to monitor activity. Ethidium bromide is able to do this in bacterial cells because it increases fluorescence when bound to DNA. In order to accurately monitor the movement of ethidium bromide across the cell wall, the E. coli cells are determined to be in log phase growth, in order to ensure that they are actively expressing the efflux pumps.
Once this is done the growth medium of the cells needs to be removed in order to stop cell growth, replication, and prevent active efflux. This allows for the cell to be
13 continuously monitored without any major growth occurring. It also allows the efflux activity to be monitored in comparison to a relatively inactive efflux pump. When the cells are in this state there is not much activity occurring, this allows for any changes in activity to be more readily seen therefore making the assay more sensitive to changes.15
Using ethidium bromide, the efflux assay procedure proved successful as shown by the positive and negative controls (Figure 4). The positive CPZ control showed a significant rise throughout the duration of the assay and produce a hyperbolic-shaped curve, while the DMSO control shows relatively no increase in fluorescence. All of the compounds tested showed a negative result, with the exception of ZINC06716957 (Table
1). ZINC06716957 which showed an increase of approximately 4 arbitrary fluorescence intensity units (a.u.) in comparison to the positive control CPZ, which showed an increase of around 25 a.u. (Figure 4). This positive result at 100 M concentration led to the idea that inhibition of the efflux pump could increase with an increase in concentration. The first attempt to test this hypothesis was with a 500 M concentration of ZINC06716957, this lead to a precipitate forming when combined with the cells in PBS. Due to the precipitate, this concentration could not be tested for efflux activity. Concentrations higher than 200 M all showed the formation of a precipitate at varying degrees based on the concentration of ZINC06716957.
There are several potential reasons that the other small molecule compounds tested did not work. One of these reasons is that they did not bind to the target and were pumped out of the cell by the efflux pump. This could have occurred if the molecule was not physically able to get to the binding pocket, if it was not in the right conformation to bind to the site, or the binding energy was not actually as high as predicted.
14 Table 1: Small Molecule Compounds chosen from in silico screening for ethidium bromide efflux assays in E. coli cells. The LogP values were obtained from the ZINC database. 1EK9 Site A (Figure 3A), Hydrophobic Trap (Figure 3B), PC2 Upper (Figure 3C), PC2 Lower (Figure 3D), A-B Hotspot (Figure 3E). Molecular Binding ZINC ID Structure Target Site LogP Activity Weight Energy
70705362 561.97 1EK9 Site A -10.6 2.94 No
70705428 569.01 1EK9 Site A -10.4 4.05 No
85893430 792.87 1EK9 Site A -10.4 1.28 No
85888725 772.98 1EK9 Site A -10.7 4.47 No
02092998 532.59 1EK9 Site A -10.9 4.59 No
15 70665805 630.74 1EK9 Site A -10.5 4.45 No
Hydrophobic 11665555 442.47 -14.9 5.02 No Trap
Hydrophobic 02202893 533.57 -14.1 7.16 No Trap
04015296 493.478 PC2 Upper -12.4 5.35 No
01612996 587.697 PC2 Upper -10.7 4.1 No
95862733 764.95 PC2 Upper -10.6 2.03 No
70705428 569.017 PC2 Lower -12.6 4.05 No
16 70705478 594.711 PC2 Lower -12.3 5.81 No
03978083 610.751 PC2 Lower -11.7 1.99 No
85878218 620.713 A-B Hotspot -11.1 6.18 No
15968013 525.008 A-B Hotspot -10.9 4.25 No
Hydrophobic 01900625 575.705 -16.2 7.88 No Trap
Hydrophobic 06716957 529.526 -13.6 4.99 Yes Trap
Hydrophobic 36701290 533.578 -13.4 4.24 No Trap
17 Accumulation of EtBr in the Prescence of ZINC06716957
ZINC06716957 CPZ Control 75uM DMSO Control
30
25
20
15
Intensity Intensity (a.u.) 10
5
0 0 5 10 15 20 25 Time (min)
Figure 4: Efflux assay showing inhibition using ZINC06716957: ZINC06716957 was the only compound tested that was able to block efflux pump activity, showing a rise of approximately 4 a.u. compared to the baseline of the DMSO control. Experiments were conducted in triplicate and error bars represent one standard deviation of the mean.
2.4 Conclusions
Although no compounds were found that blocked activity to a significant degree, this study showed that small molecule compounds do have the potential to block efflux pumps. In addition to our results, homologous structures in other species of bacteria have known efflux pump inhibitors showing that this is a worthwhile topic to continue research on. This study was mainly focused on AcrB, moving forward potential sites on
AcrA and more sites on TolC should be considered, in addition to looking further into compounds that could fit within the hydrophobic trap. Based on this research the hydrophobic trap is the only location on AcrB, that was tested, that should continue to be studied moving forward.
18 CHAPTER 3
NATURAL PRODUCTS
3.1 Introduction
Most of the world relies mainly on the use of plant byproducts as a form of medicine.2 These natural sources have been shown to work and to influence modern medicine. There are studies that look into natural compounds that work as antibacterial agents, and are focused on the synthesis of similar structures that have more activity than the natural products. This was observed in experiments such as the use of diospyrin derivatives as antimycobacterial agents.17 A large number of the antibiotic agents that are used today are derivatives of compounds found in plants. The natural ability that plants have to defend themselves against bacteria is not a coincidence; it is a product of evolution. Just like human beings, plants are exposed to bacteria and must find ways to combat them in order to survive. This is done through the development and modification of substances that are able to prevent the growth and spreading of bacteria found in the soil. Since humans are also exposed to bacteria, it makes sense to take advantage of the antibacterial agents that these plants may have naturally created. It is unknown how many antibiotic compounds might exist but there is a possibility that one of these compounds could save many people.2
There are many similarities, and a few big differences, to using small molecule compounds and plant extracts as inhibitors. The use of small molecule compounds as
19 inhibitors is a very focused approach to blocking the efflux pump. In this approach, a predetermined binding pocket is chosen and only a single compound is introduced to bind to that pocket. In contrast, the use of plant extracts is extremely broad. Instead of one compound being present, all of the naturally occurring compounds that are extracted are introduced to the efflux pump, greatly increasing the number of compounds tested. The introduction of these natural products also allows for every potential binding site and every type of efflux pump to be tested, rather than one specific target.5 The major downside to this method is that if a plant extract shows inhibitory activity, multiple more experiments are required in order to determine what natural product was causing the efflux pump inhibition since the extract is likely to be a complex mixture of compounds.
This method has been shown to work in other types of bacteria and some within E. coli.3
The goal of this research was to find additional and potentially more potent natural products efflux pump inhibitors from plant extracts.
3.2 Experimental Methods
Preparation of plant extracts: Purple Sweet potato (Dioscorea alata) and kale
(Brassica oleracea) were obtained from an organic foods market (Whole Foods Market,
Dayton, OH), chopped, dried in an incubator at room temperature for a week, crushed with a mortar and pestle, and stored at -80°C. Samples were then placed in an extraction thimble (masses shown in Table 2). The thimble was placed in the chamber of a Soxhlet extractor and combined with the amount methanol specified in Table 2 in a round bottom flask. The Soxhlet extractor was allowed to run for 24 hours at 65°C to 75°C. The
20 methanol was evaporated at room temperature under compressed air. The resulting extracts (solids and oils) were stored at -80C until use in the efflux assay.
Table 2: The mass of dried plants, the quantity of methanol, and the final mass of extract present from extraction, using the Soxhlet extractor. Dry mass Volume of Sample of plant Mass of Methanol (mL) material (g) extract (g) Purple Sweet Potato 14.9435 60 2.1157 Kale 3.5655 100 1.8807
Apple cider vinegar was centrifuged and the solid was dried at room temperature overnight. The solid was then dissolved in DMSO, at a concentration of 250 mg/mL, in order to test activity through an efflux assay. Similarly, spinach (Spinacia oleracea) was chopped, dried, crushed using a mortar and pestle, and dissolved in DMSO at a concentration of 286 mg/mL. Matcha powder was dissolved in DMSO at a concentration of 100 mg/mL in preparation for the efflux assay.
Efflux Assays: Following the same procedure outlined for the small molecule inhibitors, the plant extracts were tested for their potential efflux blocking activity. The same controls were once again used and 3 µL of each sample was tested for the assays.
The extract of kale had an oil portion that was used for the assay and a solid portion that was dissolved in DMSO, at 250 mg/mL, to be used in a separate efflux assay. The same controls were used for the natural product efflux assays as those used in the small molecule compounds.
3.3 Results and Discussion
The process of determining the best extraction method for the plant materials involved some trial and error. The heating mantles had to be set to the right temperature
21 to prevent violent boiling of the solvent over the 24-hour period. In addition to the temperature, the amount of methanol used as the solvent had to be determined in order to have some solvent always present in the round bottom flask throughout the extraction.
Both the purple sweet potato and kale resulted in enough extract in order to be used in efflux assays since only 3 L of each were needed. For every 1 g of dried purple sweet potato 0.1416 g of extract was produced and for every 1 g of dried kale 0.5275 g of extract was produced.
Some of the plant materials used were not extracted using the Soxhlet extractors because they could be dissolved directly in DMSO. This allowed them to be used directly in efflux assays without the loss of any potentially useful compounds during the extraction process. Due to the natural state of apple cider vinegar, the liquid had to be removed in order to test if there was a useful compound. The liquid was removed in order to avoid introducing an acidic compound into the cells during the efflux assay. The solid portion of the apple cider vinegar, commonly referred to as the “mother” did not have any effect on efflux activity (Figure 5). The dried spinach was also tested by dissolving in
DMSO because it became such a fine powder that it was able to fully dissolve in DMSO without any potential loss of compounds that could occur by extraction. Spinach also showed no change in efflux activity within the E. coli cells (Figure 5). Matcha is found mainly in a powder form that is highly soluble so it was tested dissolved in DMSO. Once again this had no effect on the activity of the efflux pumps (Figure 5).
22 EFFLUX ACCUMULATION ASSAYS FOR NATURAL PRODUCT EXTRACTS
DMSO Control CPZ Control 75uM Apple Cider Vinegar Kale Solid Kale Oil Matcha (DMSO) Purple Sweet Potato Spinach (DMSO) 30
25
20
15
10 INTENSITY (A.U.) INTENSITY 5
0 0 5 10 15 20 25
-5 TIME (MIN)
Figure 5: Results of efflux assays with plant extracts: None of the natural products showed any inhibitory activity in the efflux assays, all having lower intensities than the DMSO control.
3.4 Conclusions
Although none of the plant extracts showed any effect on efflux pump activity, there is still evidence that plants could potentially offer the answer to the rise in antibiotic resistance. Past studies have shown that there are natural products that can block efflux activity such as diospyrin derivatives.17 Other researchers within this lab have found natural products that do have inhibitory activity and are currently in the process of determining what compound is responsible for this activity. Moving forward there are many other plants that could contain compounds that are capable of blocking activity that should be tested to help move toward discovering new antibacterial treatments.
23 CHAPTER 4
APTAMERS
4.1 Introduction
The final method used to find an efflux pump inhibitor for E. coli was the use of aptamers. Aptamers are strands of DNA or RNA that can be bound to specific biological structures, such as proteins. They are chosen to be extremely specific and have a high affinity for a given target. The development of aptamers occurs through a process called
SELEX (Systematic Evolution of Ligands by Exponential enrichment). This method starts with a random pool of single-stranded DNAs that is then systematically selected for their ability to bind to a target. Selected aptamers are then amplified and subjected to further rounds of selection and amplification. This allows for the randomness of the oligonucleotide library to slowly decrease. At the end of SELEX what is left is different strands of DNA that are highly specific for the chosen target.18,19
Aptamers have several advantages that make them a viable therapeutic agent.
They are relatively quick to develop; each round of SELEX can be performed in a matter of days. One of the biggest advantages is that they can be developed to target almost any structure.19 In this study, aptamers were used to target the TolC portion of the efflux pump. This structure was chosen because it lies on the outside of E. coli cells and permits easy access to the aptamers. SELEX was performed with purified TolC protein.
24 4.2 Experimental Methods
Purification of TolC protein: BL21 E. coli cells containing pET29b:tolC and pLvsS plasmids were grown in 6 L of LB containing 50 µg/mL kanamycin and 50 µg/ mL chloramphenicol in a shaking incubator at 37°C and 180 rpm to an O.D.600 of 0.6.
The cells were then induced with 0.5 mM IPTG and grown for an additional 3 hours at
37°C. The cells were then centrifuged at 5,000 × g for 20 minutes at 4°C. The pellets were suspended in 250 mL of Lysis Buffer containing 20 mM Tris-HCl at pH 8.0, 100 mM NaCl, and 1 mM phenylmethylsulfonyl fluoride (PMSF). The cells were sonicated at
70% power attenuation using four 30 second periods of 1 second on and 1 second off.
The lysate was centrifuged twice at 8,000 × g for 30 minutes at 4°C, saving the supernatant after each centrifugation step. The supernatant was centrifuged at 100,605 × g for 90 minutes at 4°C. The pellets were suspended in 15 mL of 1 M Protein
Solubilization Buffer containing 20 mM Tris-HCl at pH 8.0, 100 mM NaCl, 2% (v/v)
Triton X-100, and 30 mM MgCl2. The solution was incubated for 20 minutes at 4°C on a rotating platform. This solution was centrifuged at 3,716 × g for 10 minutes at 4°C. The solution was applied to a Ni2+-NTA agarose column (Qiagen) and equilibrated with Ni2+
Column Equilibration Buffer containing 20 mM Tris-HCl at pH 8.0, 300 mM NaCl, 10 mM imidazole, and 0.2% Triton X-100. The bound proteins were eluted with a 10 mM to
250 mM imidazole gradient at 0.5-1.0 mL/min using Ni2+ Column Elution Buffer containing 20 mM Tris-HCl at pH 8.0, 300 mM NaCl, 250 mM imidazole, and 0.2%
(v/v) Triton X-100. The TolC fractions were concentrated using a Centriprep YM-3 concentrator centrifuged at 2,643 × g at 4°C. The TolC was resolved through a HiPrep
16/60 Sephacryl S-300 column at 0.30 to 0.50 mL/min at 4°C collecting 3 mL fractions,
25 using S-300 Buffer containing 20 mM Tris-HCl at pH 8.0, 300 mM NaCl, and 0.2% (v/v)
Triton X-100. The TolC containing fractions were concentrated using a Centriprep YM-3 concentrator centrifuged at 2,643 g at 4C until a minimum volume was obtained.
Preparation of ssDNA library: 2.5 nmol of the 107-base long random DNA library (5'-CAT ACG ATT TAG GTG ACA CTA TAG (N)60 ATT TCT CCT ACT GGG
ATA GGT GG) was diluted to 1.0 mL using the selection buffer. The selection buffer consisted of 137 mM NaCl, 2.7 mM KCl, 10mM Na2HPO4, 2 mM KH2PO4, and 1.4
MgCl2 adjusted to a pH of 7.4 using HCl. This solution was denatured for 5 min at 95C and allowed to slowly return to room temperature.
Protein SELEX: Imidazole was added to the ssDNA for a final concentration of
20 mM. 10 L of a 50% slurry of Ni2+-NTA agarose beads (Qiagen) were added to the mixture of DNA and imidazole and incubated for 45 minutes at room temperature on a rotating platform. The mixture was pelleted by centrifugation for 6 minutes at 8,000 g and the supernatant was removed. A volume of 10 L (310 pmol) of TolC protein was added to the supernatant and incubated for an additional 45 minutes at room temperature on a rotating platform. A volume of 10 L of Ni2+-NTA agarose beads were added to this and it was allowed to incubate for 30 more minutes at room temperature. The mixture was pelleted for 6 minutes at 8,000 g at room temperature. The pellet was washed with
1 mL of selection buffer and centrifuged again for 6 minutes at 8,000 g at room temperature. The supernatant was discarded and the pellet was suspended in 46 L of sterile water. The solution was heated at 95C for 5 minutes and centrifuged for 6 minutes 8,000 g at room temperature to elute the bound aptamers.
26 Amplification of Aptamer Library: The supernatant from the end of the SELEX procedure was combined with 50 L of GoTaq Green Master Mix, the forward primer
(5'-CAT ACG ATT TAG GTG ACA CTA TAG) at 0.06 M, and the reverse primer (5'-
TCC ACC TAT CCC AGT AGG AGA AAT) at 2.0 M. This mixture was then PCR amplified for 40 cycles using the following steps: it was denatured for 1 minute at 95C for the first round. After the initial denaturing step the cycle began with a 1 minute denaturation at C, a 30 second annealing step at 65C, and a 40 second extension step at 72C. Once it had been cycled 40 times the amplification process ended with a 2 minute extension at 72C and then held at 4C.
The amplified DNA was electrophoresed through an 8% TBE-polyacrylamide gel at 30 mA for approximately 20 minutes. The fluorescent ssDNA bands were cut from the gels and placed in 1.5 mL centrifuge tubes. A volume of 500 L of Qiagen Buffer EB was added and the gel was crushed in solution and allowed to incubate overnight at 4C.
The solution was centrifuged for 2 minutes at 21130 g, and the supernatant was transferred to a fresh tube. The following was added to the supernatant: NaCl to 3 M, 20
g of glycogen, and 1 mL of ethanol. This solution was incubated at -20C for 10 minutes followed by being centrifuged for 10 minutes at 4C at 21130 g. The supernatant was discarded and the remaining pellet was dried overnight at room temperature. In order to prepare for the next round of SELEX, the pellet was suspended in 100 L of milliQ water, 5 L of which was removed and stored. The remaining sample was then diluted to 1.0 mL with selection buffer. The procedure was repeated for a total of 5 rounds of SELEX.
27 Efflux Assay using Aptamers: After the fifth round of SELEX, the pellet was suspended in 100 L of milliQ water. A volume of 5 L of the sample was set aside, and the remaining 95 L was divided into 5 microcentrifuge tubes. A volume of 50 L of
GoTaq green was then added to each tube along with 0.06 M of the forward primer and
2.0 M of the reverse primer. These tubes were then run through the PCR amplification script as stated above. The resulting DNA was electrophoresed through an 8% TBE- polyacrylamide gel. The DNA was extracted from the gel and precipitated as described above until the dry pellet was obtained. The pellet was suspended in 60 L Qiagen Buffer
EB.
The DNA was used in an efflux assay as described earlier, although a volume of
55 L of DNA was used in each assay. The concentration of DNA was determined to be
24.4 ng/L for a total of 1.34 g of DNA used in the efflux assay. This concentration was determined using 3 L and a microplate reader at 260 nm with Qiagen Buffer EB as the blank.
Efflux Assay using a random DNA pool: 2.5 nmol of the 107-base long random
DNA library (5'-CAT ACG ATT TAG GTG ACA CTA TAG (N)60 ATT TCT CCT ACT
GGG ATA GGT GG) was added to two microcentrifuge tubes and each received 50 L of GoTaq Green Master Mix, the forward primer (5'-CAT ACG ATT TAG GTG ACA
CTA TAG) at 0.06 M, and the reverse primer (5'-TCC ACC TAT CCC AGT AGG
AGA AAT) at 2.0 M. These were then PCR amplified using the same scheme as used for the aptamers. NaCl to 3 M, 20 g of glycogen, and 1 mL of ethanol was added to each amplified tube of DNA and incubated at -20C for 10 minutes. The samples were centrifuged for 10 minutes at 4C at 21130 g. The supernatant was discarded and the
28 remaining pellet washed with 1 mL ice-cold 70% ethanol and was dried overnight at room temperature. Each pellet was suspended using 10 µL Qiagen Buffer EB and combined into one microcentrifuge tube. The DNA was diluted using Qiagen Buffer EB to a concentration of 116 ng/µL as determined with the same microplate reader used for the aptamers. 3.53 µg of DNA was used in the efflux assay.
4.3 Results and Discussion
Purification of TolC: The purification of TolC resulted in a protein with a purity of 98%. A purity gel showed no other substances with equal or greater intensity than the
1/100 dilution of TolC on the gel. The final concentration of TolC was determined to be
4.038 mg/mL using a Bovine IgG concentration curve.
Amplification of Aptamer Library: The SELEX procedure was followed for five rounds. During the third round of amplification no pellet was visible after the drying process. The pellet is believed to have been lost during the drying process so I had to go back to the beginning of the previous round of SELEX and re-amplify the DNA using 2
µL of the saved aliquot from the end of round two. Round three was then repeated and the procedure moved forward through the end of round five.
Efflux Assay using Aptamers: The DNA concentration was determined using a plate reader and was adequate for the assay. If this large quantity was not able to block efflux activity then the likelihood of there being any inhibition from the aptamers would be low.
The intensity of the fluorescent signal started high and had a very sudden drop
(Figure 6). This could have been the result of a sudden build-up of ethidium bromide
29 within the cells followed by abrupt efflux. It has not been determined exactly why this happened in this assay but not in others. One potential reason could be that the aptamers are slow to bind to TolC. If this is the case it would explain accumulation and subsequent efflux of the EtBr. Once the fluorescent signal reached a minimum, it began to follow a hyperbolic curve like those seen in past studies. After the first 20 minutes, it was clear that the amount of EtBr within the cell was still rising so the time was extended in order to find the point of saturating in the presence of the aptamer. This point was reached around 130 minutes when the curve began to plateau. At the end of the 130 minutes period the intensity had risen approximately 22 a.u. which is extremely close to the CPZ control used in the small molecule inhibitor study. This is shown in Figure 6 below, along with the random DNA strand control, which showed no rise in intensity. The data showed a significant differentiation from the control and suggest that the efflux pump is being inhibited.
30 Efflux Assay using a ssDNA Aptamer Aptamer CPZ Control DNA Control 30
25
20
15
10 Intensity Intensity (a.u.) 5
0 0 20 40 60 80 100 120 -5 Time (min)
Figure 6: Efflux assay showing inhibition using ssDNA aptamers: Efflux assay using the round 5 resultant aptamer, showed inhibition of the E. coli efflux pump. The controls were tested for a 25-minute period while the aptamer was tested over 130 minutes. The CPZ control shows the error bars associated with the triplicate data available.
4.4 Conclusions
The results of the aptamer study show that the DNA aptamer that was selected for binding to TolC is successful in blocking the efflux pumps, leading to the accumulation of ethidium bromide in the cells. The results of this study are the most promising of the three experiments. This is a good starting point for a potential blocker. From here there are a few things that could be done in order to expand on this work and help to move it toward being therapeutically beneficial. The first is to consider moving forward with more rounds of SELEX. As more rounds of SELEX are performed the aptamers become more concentrated for a specific strand of DNA. The purpose of SELEX is to select for the strand of DNA that binds the best to the protein, if one strand is substantially better binder then the procedure will amplify this strand over another potential candidate. This
31 could prove to be either beneficial and result in a better inhibitor, or could select against the inhibiting strand of DNA. Due to the SELEX procedure being designed to select for the best binder of the entire purified TolC protein, further rounds of SELEX could select for an aptamer that binds better to TolC that is buried within the membrane. Without access to the binding site this TolC binder would no longer cause inhibition of the efflux pump. The other choice would be to take the round five aptamer DNA, clone and sequence it. The downside to this course of action is that there is no way of knowing how many different DNA strands are present.
The best option would be to continue with more rounds of SELEX and start testing for efflux activity after each round. This would allow for the determination of
DNA that has the highest inhibitory ability, even if there is a strand of DNA present that does bind better than the inhibitory strand. Once this maximum inhibitory aptamer is found in efflux assays, the DNA could then be cloned and sequenced resulting in a fewer number of different colonies. This would also result in less efflux assays to be performed with these specific individual strands of DNA.
32 CHAPTER 5
GENERAL CONCLUSION
Antibiotics are no longer as effective as they once were due to the rise of multidrug resistant bacteria. In addition to no longer being as effective as medically necessary, there are few new antibiotics in development. This situation could prove to be a major problem for the future of healthcare. Bacteria have this resistance, in part, because of their ability to overexpress and use efflux pumps that are able to remove a wide range of antibacterial agents from within the cell. To combat this, new therapeutic treatments must be discovered, such as co-agents that are able to block efflux pump activity and restore the effectiveness of the current antibiotics. There are many potential ways to fight efflux pumps, this study focused on three: small molecule compounds, natural products and DNA aptamers.
Small molecule compounds with previously determined chemical structures that are available in large libraries. These libraries can be used with in silico screening to determine the best theoretical binders to pockets within a protein structure. This study focused on areas of AcrB because it is the main subunit of the efflux pump that is responsible for the pump like action used to remove foreign particles. This protein was also chosen because of the inhibitors that have been found for the homologous structure
MexB in Pseudomonas aeruginosa. My results show that the majority of the chosen compounds for in vivo studies do not inhibit efflux activity. However, there is one
33 compound, ZINC06716957, that shows some inhibition of efflux pump activity. This compound is located in the hydrophobic trap and helped lead to the conclusion that this is a worthwhile location on the protein to study further. I think that more locations similar to the hydrophobic trap could potentially show inhibition, and should be considered.
Natural products come from organisms found in nature, such as plants, that offer some major advantages to small molecules. They are less likely to be toxic to humans, are not selectively chosen for only one binding site and are easily obtained. In addition, natural product inhibitors have been found to work in the past on different structures so there is precedence for their use. This study chose commonly found plants, which were dried and extracted from their natural state for use in efflux assays with E. coli. The results showed no inhibition with any of the natural products chosen. Due to past success in this area of study it would be an area that could have future promising results.
DNA aptamers provided the most interesting result of this study. Aptamers are short portions of oligonucleotide that are used to bind to structures such as proteins with high specificity and affinity. This is done through the use of SELEX, a repeating process that slowly removes the strands of ssDNA that do not bind well to the target, in this case
TolC. What is left at the end of SELEX is a small group of distinct ssDNA that can then be used in efflux assays to test for inhibition. The results showed that a 107 base-long ssDNA aptamer was able to provide a significant level of inhibition. This work should be followed up with more rounds of SELEX and begin looking into cloning and sequencing of the DNA that was present and led to inhibition.
All of the methods used to block the E. coli efflux pump had their positives and negatives. Based on the results the small molecule compound approach and the DNA
34 aptamer approach should be further researched. Small molecules have shown success in other studies and have the potential to be very helpful in the fight against multidrug resistant bacteria. However, my results only show that there is potential for this line of study, not a definitive answer for how to inhibit efflux pump activity. The aptamer approach is the most promising line of research and should be the focus moving forward, because it is the only approach that provided a positive result in blocking efflux activity.
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