calque

From Structure to Function: Influx and Efflux Systems

May 6, 2009 to May 8, 2009
Location : Cagliari c/o SLACS-CNR, Italy

Organisers

  • Matteo Ceccarelli (University of Cagliari, Italy)
  • Ulrich Kleinekathoefer (Jacobs University Bremen, Germany)
  • Paolo Ruggerone (University of Cagliari, Italy)

Supports

   CECAM



EU RTN FP06

Regione Sardegna

Description

Gram-Negative bacteria can resist the action of antibiotics by simply decreasing the internal concentration of antibiotics. This is achieved by controlling both the influx and efflux through the outer membrane. Two classes of structures are involved, i.e., general porins (influx) and efflux pumps (efflux). The former are membrane channels that work by simple diffusion driven by the gradient concentration, while the latter span both the inner and outer membrane and have a more complex mechanism that requires energy to work against the gradient of the concentration (nikaido). Both porins and efflux pumps play an important role in a number of resistance mechanisms that bacteria have developed against a large spectrum of antibiotics, often belonging to different families. This phenomenon is known as bacterial multidrug resistance (MDR).

The serious concern due to the spread of MDR bacteria outside hospitals as well as due to some re-emerging pathogens such as the tuberculosis appears in a moment where almost no new drugs are in preparation and most pharmaceutical companies have abandoned this research field: nowadays we need a new generation of antibiotics able to overcome the different mechanisms of resistance (Payne, Vicente). Any of such new solutions to bacterial resistance has to take care of both influx and efflux systems: in order to kill bacteria it is necessary but no sufficient to design new antibiotics which efficiently diffuse through the influx systems. It is necessary that the concentration of antibiotics inside the bacteria has not to be reduced as a consequence of the efflux system. Thus, this latter should be inhibited, but this alone is not sufficient to kill bacteria, one always needs an antibiotic.

As already shown, all the processes involved in efflux and influx subtly depend on electrostatics and other microscopic interactions at the level of single amino acids (Nestorovich). Therefore, the path leading to the next generation of antibiotics passes through the knowledge of the molecular processes and structures governing resistance.

Nowdays, all-atom simulations have achieved the maturity of supporting experimental studies in many areas of science. In particular, through this successful approach, detailed information can be obtained about structure, dynamics and energetics of the system of interest. These properties are fundamental for the rational drug-design (Barker).

Influx and efflux systems are responsible of a large part of the resistance. Therefore, the design of new drugs requires the knowledge of the structure and functioning of both, porins and efflux pumps. The X-ray structures of several bacterial porins, e.g. OmpF and OmpC, have been solved at high resolution and represent an excellent starting point for theoretical and experimental investigation (Cowan et al, Baslé et al.). However, for many other porins, extracted from resistant strains, only primary sequences are known. This point leads to the first issue to address in the proposed workshop: is the combined scheme bioinformatics+MD able to provide reasonable structures for unknown porins? Are these predictions good enough to be used in drug-design?

For efflux pumps the problem is even more challenging: besides the little number of known structures, it is not clear how and through which paths the drugs are extruded from the bacteria. In addition, in the case of the RND pumps (the major superfamily of gram-negative bacteria), the way that the subunits are spatially organized (with respect to the two spanning membranes) is poorly understood (Higgins, Lomoskaya et al). This leads to the second topic of our workshop: how can simulations help to decipher the spatial organization of efflux pumps and their structure-function relationship in general?

References

Nikaido, H.: Prevention of drug access to bacterial targets: permeability barriers and active efflux. Science 264, 382-388 (1994)

Payne, D. J., M. N. Gwynn, D. J. Holmes & D. L. Pompeiano: Drugs for bad bugs: confronting the challenges of antibacterial discovery. Nature Rev. Drug Discovery 6, 29-40 (2007)

Vicente M., J. Hodgson, O. Massidda, T. Tonjum, B. Henriques-Normark & E. Z. Ron: The fallacies of hope: will we discover new antibiotic to combat pathogenic bacteria in time? FEMS Microbiol. Rev. 30, 841-852 (2006)

Nestorovich, E. M., C. Danelon, M. Winterhalter & S. M. Bezrukov: Designed to penetrate: time-resolved interaction of single antibiotic molecules with bacterial pores. Proc. Natl. Acad. Sci. USA. 99, 9789-9794 (2002)

Barker, J. J.: Antibacterial drug discovery and structure-based design. Drug. Discov. Today 11, 391-404 (2006)

Cowan S. W., T. Schirmer, G. Rummel, M. Steiert, R.A. Pauptit, J. N. Jansonius & J. P. Rosenbusch: Crystal structures explain functional properties of two E. coli porins. Nature 358, 727-733 (1992)

Baslé A., G. Rummel, P. Storici, J. P. Rosenbusch & Tilman Schirmer: Crystal structure of osmoporin OmpC from E. coli at 2.0 Å. J. Mol. Biol. 362, 933-942 (2006)

Higgins, C. F: Multiple molecular mechanisms for multidrug resistance transporters. Nature 446, 749-757 (2007)

Lomovskaya, O., Totrov M.: Vacuuming the Periplasm. J. Bacteriol. 187, 1879-1883 (2005)

C. Chimerel, L. Movileanu, S. Pezeshki, M. Winterhalter, U. Kleinekathöfer, Transport at the nanoscale: temperature dependence of ion conductance, Eur. Biohys. J. (in press, available online)

Ceccarelli, M., C. Danelon, A. Laio & M. Parrinello: Microscopic mechanism of antibiotics translocation through a porin. Biophys. J. 87, 58–64 (2004)

Mach, T., P. Neves, E. Spiga, H. Weingart, M. Winterhalter, P. Ruggerone, M. Ceccarelli and P. Gameiro: Facilitated permeation of antibiotic across membrane channels – Interaction of the quinolone moxifloxacin with the OmpF channel. J. Am. Chem. Soc. 130, 13301-13309 (2008)