Title: Multidrug Efflux Pumps and Type I Secretion Systems of Gram-negative Bacteria

Project leader: Christian Andersen (since 11/01)

Staff: Georg Polleichtner (since 01/02), Johannes Stegmeier (since 03/02), Bettina Mühling (since 04/02), and Christian Hotz (since 02/04)

Objectives: To understand the function of multidrug efflux pumps and type I secretion system of Gram-negative bacteria, which are responsible for drug resistance and the export of pathogenic factors

Approach: Multidrug efflux pumps and the type I protein secretion system are tripartite export machineries. A complex formed by an inner membrane (IM) transporter and a periplasmic adaptor protein contacts an outer membrane (OM) channel-tunnel (see figure 1). Interaction with the adaptor protein leads to an opening of the periplasmic entrance of channel-tunnel prerequisite for a successful export. We analyze the adaptor proteins with bioinformatical methods, categorize the diffuse adaptor protein family and predict their structure. Models of adaptor proteins are used to simulate the adaptor protein/channel-tunnel interaction. By mutational analysis we verify our models. By reconstitution of the pore forming channel-tunnels in black lipid bilayers we characterize the electrophysiological properties of diverse channel-tunnel proteins. In vivo and in vitro we thus investigate the influence of mutations or diverse substances on the interactions between adaptor proteins and channel-tunnels.

Fig. 1: Model of a channel-tunnel dependent export apparatus. Interaction with the adaptor protein opens the entrance of the channel-tunnel allowing export of proteins or drugs. In contrast to the channel-tunnel the structure of the adaptor protein is unknown.

Progress: We have categorized the diffuse family of adaptor proteins and used this information and structural predictions to build models of several adaptor proteins of Escherichia coli, which all interact with the channel-tunnel TolC. We established a knock out strain collection to investigate the HlyBD/TolC hemolysin secretion system and the AcrAB/TolC efflux pump of E. coli. We could show that a putative coiled coil structure of the adaptor proteins form the interaction site with the channel-tunnel TolC. We have cloned channel-tunnel proteins of E. coli, Haemophilus influenzae, Ralstonia eutropha, and Borrelia burgdorferi for expression of the proteins in E. coli.

Significance: Multidrug efflux pumps are widespread across Gram-negative bacteria and are often responsible for their multidrug resistance. Type I secretion systems export pathogenic factors like the hemolysin of E. coli or the adenylate cyclase toxin of Bordetella pertussis. The knowledge of the molecular basis of the mechanism can help to design substances, which can inhibit the function of these export systems.

Future projects: In future we want to characterize interaction sites of other adaptor proteins. The aim is to confirm our computer models of the adaptor proteins by solving the structure of a member of the adaptor protein family. Further characterization of the interaction sites between adaptor proteins and channel-tunnels should help to find substances which bind to one of the partners and inhibit interaction. This should lead to an increased drug sensitivity and a defect in the export of proteins secreted by the type I secretion system. Beside the E. coli export systems we want to investigate the multidrug efflux pump of Haemophilus influenzae.

Collaborations: Dr. J. Reidl, University Würzburg; Prof. Dr. R. Benz, University Würzburg; Prof. Dr. D. Nies, University Halle; Prof. Dr. I. Henderson, University Leeds; Prof. Dr. M. Vinas, University Barcelona

Publications:

Current external funding: Emmy Noether fellowship by the DFG