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.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Special Emphasis Panel (ZRG1-IDM-N (02))
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Korpela, Jukka K
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University of Oklahoma Norman
Schools of Arts and Sciences
United States
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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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