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
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; Yamada, Yoichi; Zgurskaya, Helen I (2011) Sequential mechanism of assembly of multidrug efflux pump AcrAB-TolC. Chem Biol 18:454-63
Modali, Sita D; Zgurskaya, Helen I (2011) The periplasmic membrane proximal domain of MacA acts as a switch in stimulation of ATP hydrolysis by MacB transporter. Mol Microbiol 81:937-51
Dhamdhere, Girija; Zgurskaya, Helen I (2010) Metabolic shutdown in Escherichia coli cells lacking the outer membrane channel TolC. Mol Microbiol 77:743-54
Dhamdhere, Girija; Krishnamoorthy, Ganesh; Zgurskaya, Helen I (2010) Interplay between drug efflux and antioxidants in Escherichia coli resistance to antibiotics. Antimicrob Agents Chemother 54:5366-8
Zgurskaya, Helen I (2009) Multicomponent drug efflux complexes: architecture and mechanism of assembly. Future Microbiol 4:919-32
Zgurskaya, Helen I; Yamada, Yoichi; Tikhonova, Elena B et al. (2009) Structural and functional diversity of bacterial membrane fusion proteins. Biochim Biophys Acta 1794:794-807
Ge, Qiang; Yamada, Yoichi; Zgurskaya, Helen (2009) The C-terminal domain of AcrA is essential for the assembly and function of the multidrug efflux pump AcrAB-TolC. J Bacteriol 191:4365-71
Dastidar, Vishakha; Mao, Weimin; Lomovskaya, Olga et al. (2007) Drug-induced conformational changes in multidrug efflux transporter AcrB from Haemophilus influenzae. J Bacteriol 189:5550-8
Tikhonova, Elena B; Devroy, Vishakha K; Lau, Sze Yi et al. (2007) Reconstitution of the Escherichia coli macrolide transporter: the periplasmic membrane fusion protein MacA stimulates the ATPase activity of MacB. Mol Microbiol 63:895-910

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