The increasing drug resistance among pathogenic bacteria is a major problem. This project is an integrated attempt to study the two general and synergistic mechanisms that bacteria use in order to prevent the access of antibiotics and chemotherapeutic agents to their targets within the bacterial cell. Such drugs must enter the bacterium and bind to the targets in order to inhibit and kill the cell. This process is made difficult by the bacteria by first building a low permeability surface membrane. An outstanding example is the outer membrane of Pseudomonas aeruginosa or Acinetobacter spp, with their exceptionally low-permeability channels or porins. The molecular mechanisms that make the channel protein to show such a low permeability appear to be in the peculiar folding process of these porins, and the project will examine the details of this process. Another example is the mycobacterial outer membrane or cell wall with its unique lipid composition, which makes the entry of lipophilic drugs apparently quite difficult. The project will examine the lipid composition of this membrane in a quantitative manner, and the effect of the lipid composition on permeability. The second mechanism for preventing drug access is the active efflux. Thus, the bacteria can overproduce multidrug efflux pumps of surprisingly wide specificity to actively pump out drugs before they reach the target. The project will examine the AcrB pump of Escherichia coli, which pumps out most of the commonly used antibiotics, with the sole exception of aminoglycosides. It will try to elucidate the path the drug molecule follows inside the large AcrB transporter, and the details of interaction between AcrB and its accessory proteins AcrA and TolC in the adjacent compartments of periplasm and outer membrane. The effect of the presence of drugs on the assembly of AcrB with AcrA and TolC will also be examined. Many of the studies will utilize a covalently linked trimer version of AcrB, so that each component monomer can be fixed in the precise conformation representing a finite step in the transport process. It is hoped that these studies will result in a rather complete and quantitative understanding of the drug entry (and expulsion) process in gram-negative bacteria and mycobacteria, and will help in the design of better antimicrobial agents and also better inhibitors of the efflux pumps.

Public Health Relevance

There is a rapid and continuing increase in drug resistance among bacterial pathogens, which have often become resistant to a large number of agents, sometimes even to most of the available agents. One major mechanism underlying this phenomenon is the active efflux (pumping out) of drugs coupled with the outer membrane permeability barrier, which limits the influx of drugs. This project focusing on molecular mechanisms of both of these components is hoped to contribute significantly in preventing drug resistance in bacteria.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Study Section
Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
Program Officer
Korpela, Jukka K
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University of California Berkeley
Schools of Arts and Sciences
United States
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Nikaido, Hiroshi (2018) RND transporters in the living world. Res Microbiol 169:363-371
Kinana, Alfred D; Vargiu, Attilio V; May, Thithiwat et al. (2016) Aminoacyl ?-naphthylamides as substrates and modulators of AcrB multidrug efflux pump. Proc Natl Acad Sci U S A 113:1405-10
Kinana, Alfred D; Vargiu, Attilio V; Nikaido, Hiroshi (2016) Effect of site-directed mutations in multidrug efflux pump AcrB examined by quantitative efflux assays. Biochem Biophys Res Commun 480:552-557
Sugawara, Etsuko; Kojima, Seiji; Nikaido, Hiroshi (2016) Klebsiella pneumoniae Major Porins OmpK35 and OmpK36 Allow More Efficient Diffusion of ?-Lactams than Their Escherichia coli Homologs OmpF and OmpC. J Bacteriol 198:3200-3208
Soparkar, Ketaki; Kinana, Alfred D; Weeks, Jon W et al. (2015) Reversal of the Drug Binding Pocket Defects of the AcrB Multidrug Efflux Pump Protein of Escherichia coli. J Bacteriol 197:3255-64
Nobre, Thatyane M; Martynowycz, Michael W; Andreev, Konstantin et al. (2015) Modification of Salmonella Lipopolysaccharides Prevents the Outer Membrane Penetration of Novobiocin. Biophys J 109:2537-2545
Li, Xian-Zhi; Pl├ęsiat, Patrick; Nikaido, Hiroshi (2015) The challenge of efflux-mediated antibiotic resistance in Gram-negative bacteria. Clin Microbiol Rev 28:337-418
Vargiu, Attilio V; Ruggerone, Paolo; Opperman, Timothy J et al. (2014) Molecular mechanism of MBX2319 inhibition of Escherichia coli AcrB multidrug efflux pump and comparison with other inhibitors. Antimicrob Agents Chemother 58:6224-34
Bansal-Mutalik, Ritu; Nikaido, Hiroshi (2014) Mycobacterial outer membrane is a lipid bilayer and the inner membrane is unusually rich in diacyl phosphatidylinositol dimannosides. Proc Natl Acad Sci U S A 111:4958-63
Opperman, Timothy J; Kwasny, Steven M; Kim, Hong-Suk et al. (2014) Characterization of a novel pyranopyridine inhibitor of the AcrAB efflux pump of Escherichia coli. Antimicrob Agents Chemother 58:722-33

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