Sequential and spatially restricted interactions of assembly factors with an autotransporter β domain

Abstract

Autotransporters are bacterial virulence factors that consist of an N-terminal extracellular ("passenger") domain and a C-terminal β barrel domain ("β domain") that resides in the outer membrane. Here we used an in vivo site-specific photocrosslinking approach to gain insight into the mechanism by which the β domain is integrated into the outer membrane and the relationship between β domain assembly and passenger domain secretion. We found that periplasmic chaperones and specific components of the β barrel assembly machinery (Bam) complex interact with the β domain of the Escherichia coli O157:H7 autotransporter extracellular serine protease P (EspP) in a temporally and spatially regulated fashion. Although the chaperone Skp initially interacted with the entire β domain, BamA, BamB, and BamD subsequently interacted with discrete β domain regions. BamB and BamD remained bound to the β domain longer than BamA and therefore appeared to function at a later stage of assembly. Interestingly, we obtained evidence that the completion of β domain assembly is regulated by an intrinsic checkpoint mechanism that requires the completion of passenger domain secretion. In addition to leading to a detailed model of autotransporter biogenesis, our results suggest that the lipoprotein components of the Bam complex play a direct role in the membrane integration of β barrel proteins.