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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI009644-44
Application #
8432500
Study Section
Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
Program Officer
Korpela, Jukka K
Project Start
1976-03-01
Project End
2016-02-29
Budget Start
2013-03-01
Budget End
2014-02-28
Support Year
44
Fiscal Year
2013
Total Cost
$627,358
Indirect Cost
$218,656
Name
University of California Berkeley
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Sugawara, Etsuko; Nikaido, Hiroshi (2014) Properties of AdeABC and AdeIJK efflux systems of Acinetobacter baumannii compared with those of the AcrAB-TolC system of Escherichia coli. Antimicrob Agents Chemother 58:7250-7
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
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
Kojima, Seiji; Nikaido, Hiroshi (2014) High salt concentrations increase permeability through OmpC channels of Escherichia coli. J Biol Chem 289:26464-73
Kinana, Alfred D; Vargiu, Attilio V; Nikaido, Hiroshi (2013) Some ligands enhance the efflux of other ligands by the Escherichia coli multidrug pump AcrB. Biochemistry 52:8342-51
Kojima, Seiji; Nikaido, Hiroshi (2013) Permeation rates of penicillins indicate that Escherichia coli porins function principally as nonspecific channels. Proc Natl Acad Sci U S A 110:E2629-34
Nikaido, Hiroshi; Pages, Jean-Marie (2012) Broad-specificity efflux pumps and their role in multidrug resistance of Gram-negative bacteria. FEMS Microbiol Rev 36:340-63
Nikaido, Hiroshi (2011) Structure and mechanism of RND-type multidrug efflux pumps. Adv Enzymol Relat Areas Mol Biol 77:1-60
Bansal-Mutalik, Ritu; Nikaido, Hiroshi (2011) Quantitative lipid composition of cell envelopes of Corynebacterium glutamicum elucidated through reverse micelle extraction. Proc Natl Acad Sci U S A 108:15360-5
Husain, Fasahath; Bikhchandani, Mihir; Nikaido, Hiroshi (2011) Vestibules are part of the substrate path in the multidrug efflux transporter AcrB of Escherichia coli. J Bacteriol 193:5847-9

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