The development and spread of antibiotic resistance in bacteria is a universal threat to both humans and animals. New therapies against multidrug resistant infections are urgently needed. The majority of currently available antibiotics have low efficacy against Gram-negative pathogens because of active efflux of drugs from cells by multidrug efflux transporters. These transporters are promising targets in development of small molecule efflux inhibitors (EPIs) that could be used in combinations with antibiotics to improve their efficacy against Gram-negative pathogens. Our long-term goal is to understand the molecular mechanism of drug efflux in Gram-negative bacteria and to develop approaches to inhibit multidrug efflux transporters. During the previous funding period, using a combination of biochemical, genetic and biophysical approaches we have reconstructed a sequence of events leading to the assembly of active drug efflux complexes and characterized the roles of each component in this process. Our findings exposed a previously unknown vulnerability of multidrug pumps that could be targeted in development of new inhibitors. The objective of this application is to characterize this vulnerability in molecular details and to discover new effectiv inhibitors of drug efflux in Gram-negative bacteria. The central hypothesis is that periplasmic membrane fusion proteins control the transition in efflux pumps from the dormant to the active state, and that inhibition of this transition is an effective way to block multidrug efflux in Gram negative pathogens. The experimental approach is based on molecular analyses of biochemical properties of efflux complexes in the presence of EPIs and the rational design of new inhibitors. We will pursue three specific aims: (i) to investigate the activation of multidrug efflux pumps;(i) to investigate the mechanisms of drug efflux inhibition;(iii) to identify new allosteric inhibitor of drug efflux transporters. The expected outcome of the proposed studies is detailed understanding of how multidrug efflux pumps are activated and new allosteric EPIs acting on this critical step in drug efflux. This contribution is significant because EPIs are expected to restore activities of already existing antibiotics and expand therapeutic options against multidrug resistant infections.

Public Health Relevance

Infections caused by Gram-negative pathogens are notoriously difficult to treat with antibiotics. The major cause of multiple antibiotic resistance n clinical isolates is active efflux of antibiotics from cells. The proposal is focused on understanding the molecular mechanism of multidrug efflux and finding effective means to inhibit this mechanism.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI052293-11A1
Application #
8786960
Study Section
Special Emphasis Panel (ZRG1-IDM-N (02))
Program Officer
Korpela, Jukka K
Project Start
2002-07-01
Project End
2019-07-31
Budget Start
2014-08-01
Budget End
2015-07-31
Support Year
11
Fiscal Year
2014
Total Cost
$441,603
Indirect Cost
$125,291
Name
University of Oklahoma Norman
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
848348348
City
Norman
State
OK
Country
United States
Zip Code
73019
Zgurskaya, Helen I; Weeks, Jon W; Ntreh, Abigail T et al. (2015) Mechanism of coupling drug transport reactions located in two different membranes. Front Microbiol 6:100
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
Zgurskaya, Helen I; Löpez, Cesar A; Gnanakaran, S (2015) Permeability Barrier of Gram-Negative Cell Envelopes and Approaches To Bypass It. ACS Infect Dis 1:512-522
Yang, Liang; Lu, Shuo; Belardinelli, Juan et al. (2014) RND transporters protect Corynebacterium glutamicum from antibiotics by assembling the outer membrane. Microbiologyopen 3:484-96
Lu, Shuo; Zgurskaya, Helen I (2013) MacA, a periplasmic membrane fusion protein of the macrolide transporter MacAB-TolC, binds lipopolysaccharide core specifically and with high affinity. J Bacteriol 195:4865-72
Krishnamoorthy, Ganesh; Tikhonova, Elena B; Dhamdhere, Girija et al. (2013) On the role of TolC in multidrug efflux: the function and assembly of AcrAB-TolC tolerate significant depletion of intracellular TolC protein. Mol Microbiol 87:982-97
Tikhonova, Elena B; Zgurskaya, Helen I (2013) Assessment of multidrug efflux assemblies by surface plasmon resonance. Methods Mol Biol 966:133-55
Lu, Shuo; Zgurskaya, Helen I (2012) Role of ATP binding and hydrolysis in assembly of MacAB-TolC macrolide transporter. Mol Microbiol 86:1132-43
Yamada, Yoichi; Tikhonova, Elena B; Zgurskaya, Helen I (2012) YknWXYZ is an unusual four-component transporter with a role in protection against sporulation-delaying-protein-induced killing of Bacillus subtilis. J Bacteriol 194:4386-94
Tikhonova, Elena B; Yamada, Yoichi; Zgurskaya, Helen I (2011) Sequential mechanism of assembly of multidrug efflux pump AcrAB-TolC. Chem Biol 18:454-63

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