To obtain insight in the molecular mechanisms of excretion systems for drugs, antibiotics and toxins in pro- and eukaryotic cells and to manipulate these systems in order to control the effects of toxic compounds on these cells.
Studies on the E. coli haemolysin secretion system have revealed a dual function of the secretion signal of HlyA: the N-terminal portion is involved in recognition of the HlyB, D translocator while the C-terminal portion is involved in the final folding of the secreted molecules of haemolysin. Fusion proteins of the HlyA C-terminal were constructed to define different stages of the translocation process. HlyD has been successfully overproduced and antibodies have been raised against this protein which have been used to demonstrate that HlyD interacts with HlyB in the inner membrane, spans the periplasm and forms a tight seal with the outer membrane, allowing the translocation of HlyA directly to the medium. HlyB has also been overproduced and can be purified.
The antibiotic chloramphenicol is pumped out of the hyphae of Streptomyces lividans by a specific membrane protein. The cmir gene encoding efflux of chloramphenicol was cloned in a Streptomyces vector and transformed in a mutant lacking the cmir gene. This cmir gene is located next to a novel KscA which can also be induced by chloramphenicol. This gene encodes a protein with two membrane spanning helices which functions as a potassium channel. To assess the interaction of KscA and Cml patch clam studies are conducted, Histidine tagged proteins have been constructed and cloned in E. coli. The proteins have been isolated, purified and used to raise antibodies. The proteins will be reconstituted in liposomes individual and together and these proteoliposomes will be used for binding and transport studies.
The interaction of the oleandomycin transporter Ole B of Streptomyces antibioticus with the substrate and ATP was analysed. These studies revealed that conformational changes occur upon interaction of Ole B with ATP, Mg and oleandomycin. Ole B showed ATPase activity only in the presence of Mg. The ATPase activity was not stimulated by the presence of substrate. Nitrobenzodiaxol but not vanadate was an effective inhibitor. The genes encoding a second ABC transporter excreting the antitumour drug mithramycin have been sequenced. Two protein components of the system have been identified: an ATP-binding protein and an hydrophobic membrane protein.
Metabolic energy-dependent efflux of erytrhomycin in Staphylococcus aureus is mediated by an ATP-binding protein Msr A. MsrA functions in the absence of products from other upstream or downstream located genes. Deletion of the control region of MsrA leads to constitutive expression of erythromycin resistance and also of streptogramin resistance. This latter resistance is also present in the absence of MsrA. The two ABC regions of MsrA do not function independently. MsrA has been overexpressed in E coli and polyclonal antibodies have been raised against MsrA. Furthermore ATP binding to MsrA was directly demonstrated.
Studies of the multidrug resistance P-glycoproteins have shown that P-gp plays an important role in the regulation of cell wolume. Phosphorylation of the linker region of P-gp is not essential for regulation of drug transport but plays a role in the regulation of cell volume. Topology studies of P-gp expressed in E. coli showed that the protein is misfolded in this organism. P-gp has been purified close to homogeneity and functionally reconstituted. 2D-crystals of P-gp have been obtained and are currently analysed.
Two multidrug resistance systems from Lactococcus lactis have been characterised: a proton motive force driven drug/proton antiport system LmrP and ABC-transporter LmrA. This latter is the prokaryotic, structural and functional homolog of MDR1. It extrudes a similar spectrum of drugs as MDR1 and the activity is inhibited by similar inhibitors. LmrP and LmrA have been functionally expressed in E. coli. Functional analysis of both systems showed that the substrates bind with the transport protein in the cytoplasmic leaflet of the membrane and is subsequently extruded in an energy-dependent process across the membrane to the external medium.
Funding SchemeCSC - Cost-sharing contracts
OX3 9DU Oxford
91405 Gometz La Ville
LS9 7TF Leeds