The purified Shigella IpaB and Salmonella SipB translocators share biochemical properties and membrane topology

Summary An essential early event in Shigella and Salmonella pathogenesis is invasion of non‐phagocytic intestinal epithelial cells. Pathogen entry is triggered by the delivery of multiple bacterial effector proteins into target mammalian cells. The Shigella invasion plasmid antigen B (IpaB), which inserts into the host plasma membrane, is required for effector delivery and invasion. To investigate the biochemical properties and membrane topology of IpaB, we purified the native full‐length protein following expression in laboratory Escherichia coli. Purified IpaB assembled into trimers via an N‐terminal domain predicted to form a trimeric coiled‐coil, and is predominantly α‐helical. Upon lipid interaction, two transmembrane domains (residues 313–333 and 399–419) penetrate the bilayer, allowing the intervening hydrophilic region (334–398) to cross the membrane. Purified IpaB integrated into model, erythrocyte and mammalian cell membranes without disrupting bilayer integrity, and induced liposome fusion in vitro. An IpaB‐derived 162 residue α‐helical polypeptide (IpaB418−580) is a potent inhibitor of IpaB‐directed liposome fusion in vitro and blocked Shigella entry into cultured mammalian cells at 10−8 M. It is also a heterologous inhibitor of Salmonella invasion protein B (SipB) activity and Salmonella entry. In contrast, IpaB418−580 failed to prevent the contact‐dependent haemolytic activity of Shigella. These findings question the proposed direct link between contact‐dependent haemolysis and Shigella entry, and demonstrate that IpaB and SipB share biochemical properties and membrane topology, consistent with a conserved mode of action during cell entry.