Secretion of a bacterial virulence factor is driven by the folding of a C-terminal segment Janine H. Peterson, Pu Tian, Raffaele Ieva, Nathalie Dautin 1 , and Harris D. Bernstein 2 Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 Edited by Thomas J. Silhavy, Princeton University, Princeton, NJ, and approved September 2, 2010 (received for review June 30, 2010) (14, 15). Furthermore, a stalled passenger domain translocation Autotransporters are bacterial virulence factors consisting of an intermediate can be cross-linked to BamA, a subunit of a protein N-terminal “ passenger domain ” that is secreted in a C- to-N-terminal complex (Bam complex) that facilitates the integration of β barrel direction and a C-terminal “ β domain ” that resides in the outer proteins into the OM (12, 16). These observations have led to the membrane (OM). Although passenger domain secretion does not proposal that the secretion of the passenger domain and mem- appear to use ATP, the energy source for this reaction is unknown. brane integration of the β domain are facilitated by the Bam Here, we show that ef fi cient secretion of the passenger domain of complex in a concerted reaction (6, 12, 14). In this model, the the Escherichia coli O157:H7 autotransporter EspP requires the sta- β domain is required for secretion because it targets the passenger ble folding of a C-terminal ≈ 17-kDa passenger domain segment. domain to the Bam complex. We found that mutations that perturb the folding of this segment The source of energy used for passenger domain secretion do not affect its translocation across the OM but impair the secre- is also unknown. Although the periplasm is devoid of ATP, it is tion of the remainder of the passenger domain. Interestingly, an conceivable that passenger domain translocation is driven by an examination of kinetic folding mutants strongly suggested that unidenti fi ed inner membrane (IM) protein that utilizes ATP hy- the ≈ 17-kDa segment folds in the extracellular space. By mutage- drolysis or the IM membrane potential. Such a protein might act nizing the ≈ 17-kDa segment, we also fortuitously isolated a unique directly on the passenger domain or interact with periplasmic or translocation intermediate. Analysis of this intermediate suggests OM factors. As an alternative, the folding of the passenger do- that a heterooligomer that facilitates the membrane integration of main in the extracellular space might promote translocation, possibly by acting as a Brownian ratchet (17). Consistent with this OM proteins (the Bam complex) also promotes the surface expo- hypothesis, it has been shown that two different puri fi ed pas- sure of the ≈ 17-kDa segment. Our results provide direct evidence senger domains fold slowly in vitro and contain a protease- that protein folding can drive translocation and help to clarify the resistant ≈ 20- to 25-kDa C-terminal segment whose sequence mechanism of autotransporter secretion. is conserved (5, 18, 19). The data raise the possibility that a C- terminal stable core might nucleate vectorial folding of the β helix. autotransporters | Bam complex | outer membrane | protein folding | The exact function of this C-terminal region, however, is unclear. protein translocation Although in one case the introduction of speci fi c mutations into this region appears to inhibit passenger domain secretion (20), in A utotransporters are a large superfamily of virulence factors several other cases deletion of this segment perturbs passenger produced by Gram-negative bacteria that consist of two domain folding and stability but does not clearly affect secretion domains, an N-terminal passenger domain that frequently exceeds (19, 21, 22). Furthermore, recent studies suggest that the β helix 100 kDa and a C-terminal ≈ 30-kDa β domain (reviewed in ref. 1). folds in a concerted process involving the whole protein rather The passenger domain is secreted into the extracellular space, than a stepwise process that requires the formation of a stable C- where it mediates the virulence function of the protein, and is terminal core (23). often released from the cell surface by a subsequent proteolytic In this study, we reexamined the role of the C-terminal seg- cleavage. Structural and bioinformatic studies strongly suggest that ment of the passenger domain in secretion by using the Escher- almost all passenger domains form an elongated β helix (2 – 5). The ichia coli O157:H7 autotransporter EspP as a model protein. The β domain forms a 12-stranded β barrel that is localized to the outer EspP passenger domain is released from the cell surface by an membrane (OM) (6 – 8). The pore of the β barrel is traversed by an intrabarrel cleavage after the completion of translocation (14, α -helical segment that typically protrudes into the extracellular 24). By analyzing mutants of EspP we obtained evidence that space and connects the β domain to the passenger domain. In a ≈ 17-kDa C-terminal fragment is initially exposed on the cell some cases, however, the α -helical segment is cleaved in an surface and that the stable folding of this segment in the extra- intrabarrel reaction that releases the passenger domain and leaves cellular space is the rate-limiting step in the translocation of the only a small α -helical fragment inside the β barrel (7, 9). The rest of the passenger domain. The results demonstrate a clear α -helical segment appears to be incorporated into the pore of the coupling of protein folding and secretion and strongly suggest β barrel (which presumably acquires considerable tertiary struc- that at least part of the passenger domain is secreted as an un- ture in the periplasm) before its integration into the OM (10). folded polypeptide. During the course of our experiments we Recent work has shown that passenger domains are secreted isolated an early translocation intermediate in which only the in a C-to-N-terminal direction (11, 12), but the mechanism of ≈ 17-kDa C-terminal fragment was exposed. Examination of this MICROBIOLOGY secretion is unclear. Based on the observation that the deletion intermediate strongly suggested that the initial phase of trans- of the β domain abolishes secretion, it was originally proposed location is facilitated by the Bam complex. that the passenger domain is secreted through a channel formed by the covalently linked β domain (13). In this model, the C terminus of the passenger domain fi rst inserts into the β domain pore as a hairpin, and N-terminal segments progressively slide Author contributions: J.H.P., P.T., R.I., N.D., and H.D.B. performed research; P.T., R.I., and past a static strand. Because the β domain pore is only ≈ 10 Å in H.D.B. designed research; P.T., R.I., and H.D.B. analyzed data; and H.D.B. wrote the paper. diameter (6 – 8), the hairpin would most likely be in a fully ex- The authors declare no con fl ict of interest. tended conformation until translocation is complete, at which This article is a PNAS Direct Submission. point the C terminus of the passenger domain would form an 1 Present address: Department of Biology, Catholic University, 620 Michigan Avenue, NE, α helix. More recent data, however, have challenged the self- Washington, DC 20064. transport or “ autotransporter ” hypothesis. Most notably, folded 2 To whom correspondence should be addressed. E-mail: harris_bernstein@nih.gov. polypeptides that cannot fi t into the β barrel pore have been This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. shown to be ef fi ciently secreted via the autotransporter pathway 1073/pnas.1009491107/-/DCSupplemental. | | | | www.pnas.org/cgi/doi/10.1073/pnas.1009491107 PNAS October 12, 2010 vol. 107 no. 41 17739 – 17744
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