The main cause of antibiotic resistance of Gram-negative bacteria is active efflux of drugs from cells by multidrug efflux (MDR) transporters. MDR transporters from Resistance-Nodulation-cell Division (RND) superfamily possess an astonishing breadth of substrate specificity. The key mechanistic advantage of RND pumps is that they capture antibiotics in the periplasm and extrude them across the outer membrane of Gram-negative bacteria. This activity is possible due to the concerted action of the RND pumps and proteins belonging to the Membrane Fusion Protein (MFP) family. MFPs are absolutely required for multidrug resistance of Gram-negative pathogens. However, how MFPs enable drug efflux remains unclear. The long term goal is to understand the mechanism of drug efflux in Gram-negative bacteria. The objective of this application is to characterize the biochemical mechanism of MFPs. Our central hypothesis is that MFPs in Gram-negative bacteria play a dual role. On one hand, MFPs are functional subunits of transporters and are required to initiate transport cycles. On the other hand, these proteins are needed to create a physical link and coordinate actions between components of MDR complexes located in two different membranes. The approach used to test this hypothesis is to investigate the mechanistic properties of AcrA and compare them to MFPs functioning with multidrug efflux transporters belonging to different families of proteins. We will pursue three specific aims: (i) Investigate the mechanism of MFP-dependent transport reaction;(ii) Investigate the stability and specificity of interactions between MFPs and their cognate transporters;(iii) Investigate functional interactions of structurally diverse MFPs with the outer membrane. Under the first aim, we will characterize the kinetics and energetics of native and mutant efflux pumps using already proven transport in intact cells and in vitro reconstitution approaches. Under the second and third aims, surface plasmon resonance and in vivo cysteine accessibility approaches will be used to characterize functional interactions between MFPs and two other components of drug efflux complexes: the inner membrane transporters and the outer membrane channels. The expected outcome of the proposed studies is the mechanistic understanding how MFPs function in transport of substrates across two membrane envelope of Gram-negative bacteria. This contribution is significant because MFPs are absolutely required for antibiotic resistance and their function could be targeted in development of effective inhibitors of multidrug efflux transporters.
This application is focused on the most troubling form of antibiotic resistance in bacteria - multidrug resistance, which is caused by activities of efflux transporters. Multidrug efflux transporters are important targets in drug discovery and development programs. Understanding the biochemical mechanism of these transporters will greatly facilitate the development of new strategies to combat multidrug resistant bacteria.
|Hwang, Hyea; Paracini, Nicolò; Parks, Jerry M et al. (2018) Distribution of mechanical stress in the Escherichia coli cell envelope. Biochim Biophys Acta Biomembr 1860:2566-2575|
|Picard, Martin; Tikhonova, Elena B; Broutin, Isabelle et al. (2018) Biochemical Reconstitution and Characterization of Multicomponent Drug Efflux Transporters. Methods Mol Biol 1700:113-145|
|Zgurskaya, Helen I; Rybenkov, Valentin V; Krishnamoorthy, Ganesh et al. (2018) Trans-envelope multidrug efflux pumps of Gram-negative bacteria and their synergism with the outer membrane barrier. Res Microbiol 169:351-356|
|Amaro, Rommie E; Baudry, Jerome; Chodera, John et al. (2018) Ensemble Docking in Drug Discovery. Biophys J 114:2271-2278|
|López, Cesar A; Travers, Timothy; Pos, Klaas M et al. (2017) Dynamics of Intact MexAB-OprM Efflux Pump: Focusing on the MexA-OprM Interface. Sci Rep 7:16521|
|Yue, Zhi; Chen, Wei; Zgurskaya, Helen I et al. (2017) Constant pH Molecular Dynamics Reveals How Proton Release Drives the Conformational Transition of a Transmembrane Efflux Pump. J Chem Theory Comput 13:6405-6414|
|Haynes, Keith M; Abdali, Narges; Jhawar, Varsha et al. (2017) Identification and Structure-Activity Relationships of Novel Compounds that Potentiate the Activities of Antibiotics in Escherichia coli. J Med Chem 60:6205-6219|
|Abdali, Narges; Parks, Jerry M; Haynes, Keith M et al. (2017) Reviving Antibiotics: Efflux Pump Inhibitors That Interact with AcrA, a Membrane Fusion Protein of the AcrAB-TolC Multidrug Efflux Pump. ACS Infect Dis 3:89-98|
|Ntreh, Abigail T; Weeks, Jon W; Nickels, Logan M et al. (2016) Opening the Channel: the Two Functional Interfaces of Pseudomonas aeruginosa OpmH with the Triclosan Efflux Pump TriABC. J Bacteriol 198:3176-3185|
|Weeks, Jon W; Nickels, Logan M; Ntreh, Abigail T et al. (2015) Non-equivalent roles of two periplasmic subunits in the function and assembly of triclosan pump TriABC from Pseudomonas aeruginosa. Mol Microbiol 98:343-56|
Showing the most recent 10 out of 34 publications