Interaction of an autotransporter passenger domain with BamA during its translocation across the bacterial outer membrane

Autotransporters are a superfamily of virulence factors produced by Gram-negative bacteria consisting of a large N-terminal extracellular domain (“passenger domain”) and a C-terminal β barrel domain (“β domain”). The mechanism by which the passenger domain is translocated across the outer membrane (OM) is unknown. Here we show that the insertion of a small linker into the passenger domain of the Escherichia coli O157:H7 autotransporter EspP effectively creates a translocation intermediate by transiently stalling translocation near the site of the insertion. Using a site-specific photocrosslinking approach, we found that residues adjacent to the stall point interact with BamA, a component of a heterooligomeric complex (Bam complex) that catalyzes OM protein assembly, and that residues closer to the EspP N terminus interact with the periplasmic chaperones SurA and Skp. The EspP–BamA interaction was short-lived and could be detected only when passenger domain translocation was stalled. These results support a model in which molecular chaperones prevent misfolding of the passenger domain before its secretion and the Bam complex catalyzes both the integration of the β domain into the OM and the translocation of the passenger domain across the OM in a C- to N-terminal direction.