New way to fight against antibiotic resistance: reconstruction of a three-component efflux pump – ASSEMBLY

After several decades of continued success of antibiotic therapy against bacterial infections, we are now facing an increasing threat: the accelerated evolution of antibiotic resistance in important human pathogens and the scarcity of new anti-infective drug families under development. For this reason development of original solutions is mandatory.
There are four main mechanisms by which bacteria exhibit resistance to antibiotic: (i) drug inactivation or modification; (ii) alteration of target site; (iii) Alteration of metabolic pathway and (iv) reduction of drug accumulation by increasing active efflux. Efflux pumps recognize a large panel of substrates including all antibiotic families. In Gram negative bacteria, this mechanism involves the functional assembly of three different membrane proteins.
The classical approach largely already explored with E. coli efflux pump TolC/AcrA/AcrB, consist in targeting the function of the sole RND protein, but without any exploitable success so far. As an alternative, preventing their assembly represents an original approach never been yet investigated, mainly because of the misunderstanding of the mechanism of their assembly at the molecular level. The improvement of our knowledge on the mechanism of pump assembly will permit the development and design of new drugs in the future.
P. aeruginosa is an opportunistic pathogen and a leading cause of hospital-acquired infections associated with high morbidity and mortality. This bacteria is the third cause of nosocomial infection after E. coli and S. aureus. Twelve genetically distinct systems have been described in P. aeruginosa genome among which four have been biochemically characterized. Each one is composed of three proteins, two (the RND and the OMF) being membrane proteins localized in the two opposing membranes, and the third one (MFP), periplasmic, being anchored in the inner membrane.
Increasing evidences show that the complete pump formation is necessary for the efflux to be efficient. Therefore, we want to characterize the conditions of formation and the mechanisms of these pumps and to describe the different binding interfaces. Those information will be of main importance for future development of "complex blockers" avoiding the protein/protein recognition and/or rigidifying the different protein partners preventing their required structural adaptability.

This project is organized in three main axes at the physics/biology interface that could be summarized as follows:
- In vitro and in vivo analysis of the different protein complex. The stoichiometry of the different protein partners will be first determined two by two by different approaches (BN-PAGE, FRAPP, cryo-EM…) in different membrane substitute, exploring different pH. Then the relative importance of the three proteins in this assembly and their sequence of assembly will be characterised using the same techniques, but adding alternatively the third partner. The consequences of the eventual mutations suggested to stabilize the assembly will be analyse in vivo by complementation experiments.
- In silico analysis of the different partners by molecular simulations (normal mode analysis and molecular dynamics simulations) using different resolution levels to analyse their intrinsic dynamics. The rationale is to underline the possible important functional movements performed by the pump components. Construction of a tripartite model and description of the interface between each complex will allow us to suggest mutations stabilizing formation of the complex.
- Structure determination of each protein by X-ray crystallography to be used for the complete edifice structure determination by cryo-electron microscopy and tomography.
The final purpose of this proposal is to obtain crucial information about the assembly and opening of efflux pumps in order to pave new ways to fight against antibiotic resistance.