Biogenesis of bacterial autotransporter proteins

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We have been using an autotransporter produced by E. coli O157:H7 called EspP as a model protein to study autotransporter biogenesis. We showed several years ago that after the EspP passenger domain is translocated across the OM it is released into the extracellular milieu through an autocatalytic cleavage reaction that involves the activation of an asparagine residue. In one major line of investigation we have been examining the mechanism by which the EspP passenger domain is translocated across the OM. It was originally proposed that the passenger domain is secreted through a channel formed by the covalently linked beta domain (whence the name autotransporter), but results that we obtained from both biochemical and structural studies appear to be inconsistent with this hypothesis. We found that the insertion of a small linker into the EspP passenger domain effectively creates a translocation intermediate by transiently stalling translocation near the site of the insertion. By 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 that are trapped in the periplasm (the space between the two cell membranes) interact with the chaperones SurA and Skp. The EspP-BamA interaction is short-lived and can only be detected when passenger domain translocation is stalled. These results support a model in which molecular chaperones prevent misfolding of the passenger domain prior to its secretion and the Bam complex plays a major role in facilitating both the integration of the beta domain into the OM and the translocation of the passenger domain across the OM. We also found that periplasmic chaperones and specific components of the Bam complex interact with the EspP beta domain in a temporally and spatially regulated fashion. While the chaperone Skp initially interacts with the entire beta domain, BamA, BamB and BamD subsequently interact with discrete beta domain regions. BamB and BamD remain bound to the beta domain longer than BamA and therefore appear to function at a later stage of assembly. Our results suggest that the hitherto enigmatic BamB and BamD proteins play a direct role in the membrane integration of autotransporter beta domains and possibly other beta barrel proteins. Interestingly, we also obtained evidence that the completion of beta domain assembly is regulated by an intrinsic checkpoint mechanism that requires the completion of passenger domain secretion. Recently, we have been examining the energetics of passenger domain secretion. Because the periplasm lacks ATP, the source of energy that drives passenger domain secretion is unknown. The prevailing model postulates that vectorial folding of the passenger domain in the extracellular space facilitates unidirectional secretion. We used a chimeric protein composed of the receptor-binding domain (RD) of the Bordetella pertussis adenylate cyclase toxin CyaA fused to the C-terminus of EspP to test this hypothesis. The RD is a highly acidic, repetitive polypeptide that is intrinsically disordered in the absence of calcium. Surprisingly, we found that the RD moiety is efficiently secreted when it remained in an unfolded conformation. Furthermore, we found that neutralizing or reversing the charge of acidic amino acid clusters stalls translocation in the vicinity of the altered residues. These results challenge the vectorial folding model and, taken together with an analysis of naturally occurring passenger domain sequences, provide evidence that a net negative charge plays a significant role in driving the translocation reaction. In separate studies we also obtained evidence that the folding of the native EspP passenger domain helps to drive translocation, but that a significant fraction of the energy required for secretion must be derived from another source.