Probing Pal-TolB Interactions Using Site-directed Mutagenesis.

Extensive research has suggested that Peptidoglycan-associated Lipoprotein (Pal) from Escherichia coli (E. coli) plays a role in E. coli sepsis. When exposed to blood sera, for example, Pal is released from the E.coli outer membrane (OM). Released Pal has been found to be toxic and inflammatory, capable of inducing the release of pro-inflammatory cytokines. Pal is tethered to the OM via an N-terminal lipid moiety and non-covalently binds to peptidoglycan (PG) through a site that overlaps with its binding site for TolB, a periplasmic protein and a key member of the Tol-Pal complex. Due to the many interactions with these cellular components, the mechanism of Pal's release from E. coli is not well understood. Here, we discuss our production and analysis of a site-directed mutant of Pal (K150E), which we propose to disrupt the Pal-TolB interaction. The effects of the mutation are assessed using immunoblotting and enzyme-linked immunosorbent assays, the results of which we hope will provide insight into the Pal-TolB interaction and the mechanism of Pal release from E. coli.