Progress 06/01/17 to 05/31/20
Outputs
Target Audience:The target audience is researchers studying the mechanism for transport, drug efflux by bacteria, or virulence factors of human and animal pathogens. Our current target audience of researchers were provided data and results in the form of posters presenting work in progress. One manuscript is available as a preparing on BioRxiv and is under review at Antimicrobial Agents and Chemotherapy. Another is to be submitted to the journal Cell. Results have been presented in invited seminars at Texas Tech. Several planned presentationspostponed due to the pandemic are currently scheduled in virtual or in-person format for 2021. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One graduate student has worked on this project with assistance from 4 part-time undergraduate researchers. These students have received training in expression testing and membrane protein purification;biosafety when working with recombinant DNA; bacterial microplate growth assay and Biolog assay design, controls, execution, and analysis of mid- and high-throughput assays; design, execution, and analysis of dilution plate microbial growth assays on solid media; analysis of bacterial carbon source utilization, and consequences for respiration and the proton motive force; electrophysiology-based transport assays; sample optimization, data acquisition, and analysis of protein NMR spectra. They have all also worked on preparing the figures and writing and revising the manuscript currently under review at Antimicrobial Agents and Chemotherapy, and the nearly complete draft of the second manuscript. Due to Covid-19, planned poster presentations were cancelled but the work will be presented in 2021 as these poster sessions are rescheduled in either virtual or in-person formats. How have the results been disseminated to communities of interest?We will present theresults at several graduate and undergraduate poster session that have been rescheduled for 2021 due to Covid-19. The work was presented at Texas Tech in an invited seminar and will be presented at rescheduled meetings and seminars in 2021, including international venues (International Conference on Magnetic Resonance in Biological Systems virtual seminar in Jan. 2021 and ETH Zurich virtual seminar in March 2021). One paper is under review at Antimicrobial Agents and Chemotherapy and available now as a preprint athttps://www.biorxiv.org/content/10.1101/2021.01.06.425668v1. Another paper has complete figures and nearly complete draft and will be submitted to Cell shortly. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The small multidrug resistance (SMR) transporters perform proton/drug antiport, resulting in proton-driven efflux of toxic substrates. Our previous work suggested EmrE could also perform proton-coupled symport, which would actively pump compounds into bacteria leading to drug susceptibility rather than resistance. Progress on Aim 1 and 2: We have now screened three different SMR homologs from Staphylococcus aureus, Pseudomonas aeruginosaand Francisella tularensis. These were screened in parallel with E. coli EmrE, as well as E. coli Gdx. Gdx is an SMR family member thas been functionally re-assigned as a metabollite exporter rather than mutidrug resistance transporter and thus serves as a control transporter from the opposite branch of the SMR family. These Biolog assays revealed that ALL of these transporters confer resistance to some substrates and susceptibility to others. This data confirmsour hypothesis that the SMR family transporter functional promiscuity does not just encompass export of a broad range of substrates, but also includes different modes of transport (uniport and or symport) for other substrates that result in this multidrug "resistance" family of transporters conferring susceptibility to some classes of small molecules. These functional assays were performed using Biolog screening of 204 compounds andcompared the metabolic function (readout of NADH production using a colorimetric indicator) of E. coli expressing either the WT version of the SMR transporter or a functionally dead point mutant (E14Q or E13Q). Upon exposure to some compounds, E. coli expressing the WT transporter had higher metabolic activity than E. coli expressing the non-functional transporter. This indicates that the function of the SMR transporter is beneficial and is consistent with conferring resistance to the small molecule. However, there were some compounds for which the opposite outcome was observed -E. coli expressing the WT transporter had lower metabolic activity than E. coli expressing the non-functional transporter. This indicates that active transporter is detrimental to the bacteria, indicating that the SMR function results in susceptibility rather than resistance. All five SMR homologs showed more than a dozen hits out of 200 compounds tested, and both phenotypes were observed for all five transporters with roughly equal numbers of resistance and susceptibility hits. Thus, susceptibility is not a minor phenotype. Not surprisingly given the expected polyspecificity of these multidrug transporters and our prior biophysical data, many substrates were "hits" across multiple transporters, but each transporter also had unique hits. This was expected because there are differences in the primary amino acid sequence of these transporters in regions known to be important for substrate binding, and mutagenesis of EmrE demonstrated that significant differences in substrate specificity can result from single amino acid substitution. Since resistance or susceptibility in our study is driven purely by the identity of the SMR substrate, this raises the question of whether it is possible to design small molecules that selective trigger susceptibility that could be used as antiseptics or antibiotics. We have new grants pending that follow up this question of whether SMR transporters could be a novel antibiotic target. This data confirms our hypothesis that the Small Multidrug "Resistance" transporters don't always conferresistance. Furthermore, this behavior is widespread across the family, including SMR homologs from human and livestockpathogens. These results are part of the manuscript is under review atAntimicrobial Agents andChemotherapy and available on BioRxiv. Interestingly, some of the Biolog hits were multivalent metal ions, opening up a new question of whether the SMR transporters can transport metal ions or siderophore-bound metal ions. A potential role for SMR transporters in metal ion uptake has never been considered previously. After our Biolog screening, we confirmed hits using full growth curves. In addition, we performed liposomal flux assays to assess transport activity in vitrousing solid-supported membrane electrophysiology. These experiments report the net movement of charge into or out of a proteoliposome when transport is initiated. Interestingly, they showed that harmane, one of the susceptibility substrates we identified is probably not actually transported, but rather triggers uncoupled proton uniport. This implies that the susceptibility phenotype arises from uncontrolled proton leak leading to rundown of the PMF.This is an entirely novel result - and potentially a novel way to selectively trigger PMF run down by targeting a protein rather than the membrane directly. This paper is nearly ready for submission to Cell. As part of that manuscript, we also include NMR experiments demonstrating that there is a second substrate binding site in EmrE. Previously, EmrE has been hypothesized to function by a single-site alternating-access model, where glutamate 14 (E14) in transmembrane helix 1 defines the single substrate and proton binding site. Our NMR data now unambiguously confirms prior work from 20 years ago suggesting a second substrate binding site in the C_terminal tail and loop regions of EmrE. Together with our biophysical studies funded through another grant, this leads to the clear hypothesis that this binding site is part of a secondary "gate" in EmrE that regulates access to the primary E14 binding site. This represents a fundamental change in the mmechanistic models for EmrE and an increase in complexity that may be important for understanding how such a small transporter can actively transport such diverse substrates with reasonable efficiency. Progress on Aim 3: We have successfully expressed, purifiedand reconstitutedinto liposomes and bicelles the SMR homologs fromStaphylococcus aureusandPseudomonas aeruginosa with sufficient yield, purity, and homogeneity to perform NMR experiments. This opens up more detailed biophysical investigation of these other SMR transporters. The NMR data obtained to date show that there are fundamental differences in either the strucural symmetry or timescale of motion in these homologs compared to the well-studied EmrE.We have new grant proposals pending to further explore the structural and dynamics similarities and differences across the SMR family using these homologs. This work will provide insight into the molecular basis of substrate promiscuity and mechanistic promiscuity in this unusual transporter family that can recognize a broad range of small molecule substrates but where these substrates can trigger transport activity with opposing biological outcomes of resistance or susceptibility.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2021
Citation:
https://www.biorxiv.org/content/10.1101/2021.01.06.425668v1
Under review at Antimicrobial Agents and Chemotherapy
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:The target audience is researchers studying the mechanism for transport, drug efflux by bacteria, or virulence factors of human and animal pathogens. Our current target audience of researchers were provided data and results in the form of posters presenting work in progress. Two manuscripts are nearly ready for submission. One will be submitted to the journal Cell and one to Antimicrobial Agents and Chemotherapy. Results have been presented in invited seminars at UCSF, University of Massachusetts-Amherst, and University of Wisconsin Raper Symposium in Microbiology. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? One graduate student has worked on this project with assistance from 4 part-time undergraduate researchers. These students have received training in vector design, cloning, and testing of the suitability of the construct for different assays; biosafety when working with recombinant DNA; bacterial microplate growth assay and respiration assay design, controls, execution, and analysis; design, execution, and analysis of dilution plate microbial growth assays on solid media; analysis of bacterial carbon source utilization, and consequences for respiration and the proton motive force. How have the results been disseminated to communities of interest? We have presented preliminary results of this work in progress at several graduate and undergraduate poster session with a broad range of researchers from across campus, at invited talks at UCSF and University of Massachusetts at Amherst, and at the Raper Symposium in Microbiology at UW-Madison. What do you plan to do during the next reporting period to accomplish the goals?We plan to complete the preparation of manuscripts for publication as the project comes to completion. I am also using the data generated by this project to actively seek additional support to assess the feasibility of targeting SMR transporters to combat antibiotic resistance due to efflux.
Impacts
What was accomplished under these goals?
Thesmall multidrug resistance (SMR)transportersperform proton/drug antiport,resulting in proton-driven efflux of toxic substrates. Our previous worksuggested EmrE could also performproton-coupled symport, which would actively pump compoundsintobacteria leading to drug susceptibility rather than resistance. 1. Screening of two different SMR homologs fromStaphylococcus aureusandFrancisella tularensisin parallel withE. coliEmrErevealed thatallof these transporters confer resistan­­ce to some substrates and susceptibility to others, thus confirming our hypothesis. We compared the metabolic function ofE. coliexpressing either the WT version of the SMR transporter or a functionally dead point mutant (E14Q or E13Q). Upon exposure to some compounds,E. coliexpressing the WT transporter did have higher metabolic activity thanE. coliexpressing the non-functional transporter as expected for a drug resistance phenotype. However, there were some compounds for whichE. coliexpressing the WT transporter hadlowermetabolic activity thanE. coliexpressing the non-functional transporter. This indicates that active transporter is detrimental to the bacteria, consistent with a susceptibility phenotype. All three SMR homologs showed more than a dozen hits out of 200 compounds tested, andboth phenotypes were observed for all three transporters. Not surprisingly given the expected polyspecificity of these multidrug transporters and our prior biophysical data, many substrates were "hits" across multiple transporters, but the phenotype was sometimes consistent and sometimes divergent (ie. one transporter conferred susceptibility to a compound while another conferred resistance to the same compound).This data confirms our hypothesis that the Small Multidrug "Resistance" transporters don't always confer resistance. Furthermore, this behavior is widespread across the family, including SMR homologs from human and livestock pathogens. These results are part of the manuscript that is in final preparation for submission toAntimicrobial Agents and Chemotherapy. 2.Proton-coupled antiport activity is required for EmrE to confer drug resistancein vivo, since with an inwardly direct proton motive force, only drug/proton antiport will result in active pumping of toxic compounds out of the bacterial cytoplasm. By analogy we had expected that susceptibility would arise due to proton coupled symport, which would actively pump toxic molecules into bacteria. Some of our other mechanistic studies of EmrE also suggested that the C-terminal tail acts as a secondary gate, preventing proton leak in the absence of drug. After our Biolog screening, we confirmed hits using full growth curves. Expression of functional EmrE results in a 10-fold increase in ethidium IC50as expected for resistance, whileexpression of functional EmrE results in a 3fold decrease in harmance IC50 indicating EmrE-mediated susceptibility. Furthermore, we performed liposomal flux assays to assess transport using solid-supported membrane electrophysiology. These experimentsreport the net movement of charge into or out of a proteoliposome when transport is initiated and revealedthat charge movement always occur down the proton gradient, regardless of whether the drug gradient is in the same direction or the opposite direction. This suggests that harmane is triggering uncontrolled proton leak, rather than coupled symport. We usedNMR to confirm direct biding of harmane to EmrE and discovered that harmane does not bind primarily near E14 like antiport substrates, but rather binds to theloops and C-terminal tail. This is exactly the site of the proposed secondary gate to prevent proton leak. Thus, our combinedin vivoandin vitroexperiments indicate that a wild-type multidrug "resistance" transporter can confer susceptibility rather than resistance to some substrates through a novel mode of action by which the substrate breaks the proton coupling mechanism and results in uncontrolled proton leak, resulting in rundown of the proton motive force and impaired bacterial growth. This provides an entirely new mechanism to potential target antibiotic resistance due to efflux, and is currently being compiled into a manuscript for submission toCell.
Publications
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:The target audience is researchers studying the mechanism of transport, drug efflux by bacteria, or virulence factors of human and animal pathogens. Our current target audience of researchers were provided data and results in the form of posters presenting work in progress. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One graduate student has worked on this project with assistance from 4 part-time undergraduate researchers. These students have received training in vector design, cloning, and testing of the suitability of the construct for different assays; biosafety when working with recombinant DNA; bacterial microplate growth assay and respiration assay design, controls, execution, and analysis; design, execution, and analysis of dilution plate microbial growth assays on solid media; analysis of bacterial carbon source utilization, and consequences for respiration and the proton motive force. How have the results been disseminated to communities of interest?We have presented preliminary results of this work in progress at several graduate and undergraduate poster session with a broad range of researchers from across campus. This has provided initial dissemination of the data and feedback on our results and conclusions. What do you plan to do during the next reporting period to accomplish the goals?We will publish the results already obtained and described above. We will extend our carbon source, pH and salt tolerance experiments to the other SMR transporters proposed in the initial proposal. Now that assay conditions are worked out, it is straightforward to repeat the experiments with a plasmid expressing different transporters. We will perform the functional phenotyping experiments described in Aim 1 using the expression system and glucose, salt and pH conditions optimized from our current data to ensure that the results are due to active pumping of the tested compound and not due to metabolic or environmental effects of the assay conditions.
Impacts
What was accomplished under these goals?
We accomplished three goals this year relevant to Aims 1 and 2 of the proposal. 1. We tested several additional vectors for EmrE expression in order to ensure the optimal construct for all of our in vivo assays in E. coli. Expression of membrane proteins at high levels can be very toxic. Our initial experiments used leaky expression from a high copy number plasmid that was IPTG-inducible (lac promoter) to observe the effect of SMR activity on the growth of E. coli MG1655 EmrE (MG1655 strain of E. coli with the native EmrE gene deleted). Induction with IPTG results in high expression of EmrE or another of the small multidrug resistance (SMR) proteins under study in this project, and this is toxic to E. coli, resulting in cell death. We therefore switched to a low copy number plasmid (pWB1, designed in our lab to combine a pTrc99 backbone with a p15A origin of replication) and did not use IPTG. This results in low-level "leaky" expression of the small multidrug efflux pump, and this is not toxic to the bacteria but does result in a clear phenotype - expression of functional SMR renders the bacteria resistant to toxic levels of ethidium bromide, while expression of non-functional (E14A) SMR does not confer resistance. We tried additional vectors that would provide better, inducible control of expression using arabinose promotors, but could not achieve any expression of EmrE or other SMRs in the presence of arabinose. We will follow up this unusual observation in the future by screening effects of arabinose on SMR activity in the assays of Aim 1. We settled on using the pWB1 vector since western blot experiments testing expression level under several different conditions showed no variation in EmrE expression using this system despite the lack of explicit control over expression level. These experiments, while time consuming, were important to establish the rigor and reproducibility of all of our assays and ensures that the phenotypes observed in the compound resistance/susceptibility assays of Aim 1 and 2 are due to SMR activity. 2. We tested the effect of carbon source on the growth phenotype of E. coli expressing EmrE. This follows up our previous observation that EmrE high-level expression in E. coli is robust when the carbon source is glucose, but results in cell death and no expression at all when the carbon source is pyruvate. We compared the growth of E. coli expressing functional and non-functional (E14A) EmrE and saw no significant difference on any carbon source. Although bacterial growth in general was strongly affected by the type of carbon source (glucose, glycerol, pyruvate) and quantity (1, 2, 4, 8 g/L of M9 minimal media), expression of functional EmrE was not beneficial nor detrimental to growth. We are now replicating these experiments to confirm the results and performing western blots to ensure that EmrE expression was the same throughout these experiments. This will test whether expression of active EmrE transporter is only detrimental at high expression levels (as in our previous studies) or low level expression is inhibited by changing the carbon source. 3. We tested the effect of salt and pH on the growth of E. coli expressing functional EmrE. This follows up previous reports suggesting the native function of EmrE and other SMR pumps is for efflux of compounds important in bacterial osmoregulation (response to water stress), halotolerance (salt tolerance), and pH tolerance. Since E. coli and oher food-borne pathogens may experience very different pH, salt, etc. in different environments in the human body this is a natural hypothesis and explanation for why SMR pumps may contribute to bacterial virulence. Prior published data (Bay & Turner, J. Bacteriology 194:5941, 2012) indicated that EmrE reduced the pH tolerance of BW25113 E. coli at neutral and alkaline pH in minimal media. In direct contrast to those results, we found that expression of functional EmrE in MG1655 resulted in increased growth at neutral and alkaline pH. There are two differences: the bacterial strain and our use of plasmid that expressed EmrE only, while the prior work had a 22 amino acid C-terminal myc-His tag extension. Our lab recently discovered that protonation of the C-terminal histidine residue of WT EmrE is important for proton coupling and ay act as a secondary gate for transport (Thomas, et al, J Biological Chemistry 293:19137, 2018), and this contradictory result has two implications: 1) it reinforces the potential functional role of the C-terminal residues in the SMR transporters and 2) suggests that the results of previous functional assays, almost all of which were performed with the 22 amino acid C-terminal extension must be re-examined to ensure that the tage did not alter the functional profile. We also tested the salt tolerance and confirmed the prior finding that functional EmrE has no effect on salt tolerance of E. coli in minimal media, at least within the small range of hyperosmotic conditions that E. coli can tolerate in this media.
Publications
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Progress 06/01/17 to 09/30/17
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During the three month reporting period, two graduate students have worked on this project in turn with assistance from two part-time undergraduate students. These students have received training in vector design, cloning techniques, and experimental design of bacterialin vivogrowth assays and development of control experiments to test whether the observed phenotype is due to the actual function of the cloned SMR pump. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will complete the initial experiments with the EmrE pump described above and publish the results, assuming that the remaining controls and replicates bear out our initial findings. We will continue to work on Aims 1 and 2 as proposed in the initial project plan. With the equipment installed, vector design and test experiments completed, everything is in place to allow steady progress toward these goals. We expect to complete Aim2 and make significant progress on the more extensive Aim 1 during the next year.
Impacts
What was accomplished under these goals?
During the initial 3 months of this project, we acquired the equipment (microplate reader) necessary to accomplish Aims 1 and 2 of the project and ensured that it was working properly. Weacquired the genes and cloned the vector necessary to express each of the small multidrug resistance (SMR) pumps in E. coli. Our initial vector design proved to be inefficient for cloning multiple SMR pumps and failed our initial control experiments with the EmrE SMR pump, with poor control of expression levels. We therefore redesigned the vector, cloned the EmrE pump into and performed initial control experiments again. The new vector design works well and cloning of the remaining SMR pumps is now in progress. Initial test of EmrE function focused on the effect of EmrE pump expression onE. coligrowth phenotype, and resistance/suseptibility to known EmrE drug substrates as well as guanidinium and urea, since thesenovel substrates of the SugE SMR pump werediscovered in December 2016. Guanidinium and urea were reported to be SugE but not EmrE substrates, however we discovered novel effects of these substrates onE. coligrowth phenotype suggesting that they may indeed be EmrE substrates as well. We are replicating these results and refining our controlexperiment panel to confirm that they are indeed EmrE substrates before publication. We will then proceed with the rest of Aim 1 and 2 as proposed, using our refined vector and panel of control experiments as we screen the functional phenotype of the EmrE pump against the remaining substrates and screen the remaining SMR pump targets.
Publications
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