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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI052293-10
Application #
8386931
Study Section
Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
Program Officer
Korpela, Jukka K
Project Start
2002-07-01
Project End
2014-11-30
Budget Start
2012-12-01
Budget End
2014-11-30
Support Year
10
Fiscal Year
2013
Total Cost
$341,091
Indirect Cost
$110,767
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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