Functional and biochemical characterization of Pseudomonas aeruginosa MsbA

Lipopolysaccharide (LPS) of Pseudomonas aeruginosa is a major constituent of the outer leaflet of the outer membrane and an important virulence factor of this microorganism. LPS consists of three parts: the lipid A, core oligosaccharides and O antigens. The biosynthesis and assembly of this macromolecule is a highly complex process and takes place at the cytoplasm and the periplasmic space. The lipid A and the core oligosaccharides are synthesized and assembled at the cytoplasmic side of the inner membrane and then translocated to the periplasmic side of this membrane where it serves as the acceptor of the O antigens. This thesis focuses on characterizing open reading frame PA4997 from P. aeruginosa genome and testing the hypothesis that this gene encodes an ATP-binding cassette transporter, termed MsbApa , which is essential for viability of P. aeruginosa. Attempts to construct a knockout mutant of msbApa were unsuccessful since such a mutation is lethal to the bacterial organism. In cross complementation experiments, my results showed that msbA from Escherichia coli (msbAEc) could not substitute for msbApa. MsbA pa was over-expressed as a histidine-fusion protein, solubilized with dodecyl-beta-D-maltoside, purified by cobalt-chelation affinity chromatography and used for biochemical characterization. MsbApa exhibited tight regulation of its intrinsic ATP hydrolysis activity when in the liposome-reconstituted form as compared to the detergent-solubilized form. The activity of MsbApa could be strongly stimulated by wild-type core-lipid A prepared from P. aeruginosa and to a lesser extent by truncated lipid A-core. In addition, my results suggested an important role for the phosphate moieties of the core-lipid A molecule for its translocation by MsbApa. In conclusion, results from this thesis demonstrated that MsbApa possesses properties that are consistent with proteins of the superfamily of ATP-binding cassette transporters. The fact that MsbApa showed differences when compared to MsbA Ec, served to shed light on our understanding of these ABC-transporters; in particular, the communication between the membrane spanning domain and the nucleotide binding domain which could account for the differences observed between these two homologues.