The Inverse Autotransporter Intimin Exports Its Passenger Domain via a Hairpin Intermediate

Philipp Oberhettinger,Jack C Leo,Dirk Linke,Ingo B Autenrieth,Monika S Schütz

doi:10.1074/jbc.m114.604769

Abstract

Autotransporter proteins comprise a large family of virulence factors that consist of a β-barrel translocation unit and an extracellular effector or passenger domain. The β-barrel anchors the protein to the outer membrane of Gram-negative bacteria and facilitates the transport of the passenger domain onto the cell surface. By inserting an epitope tag into the N terminus of the passenger domain of the inverse autotransporter intimin, we generated a mutant defective in autotransport. Using this stalled mutant, we could show that (i) at the time point of stalling, the β-barrel appears folded; (ii) the stalled autotransporter is associated with BamA and SurA; (iii) the stalled intimin is decorated with large amounts of SurA; (iv) the stalled autotransporter is not degraded by periplasmic proteases; and (v) inverse autotransporter passenger domains are translocated by a hairpin mechanism. Our results suggest a function for the BAM complex not only in insertion and folding of the β-barrel but also for passenger translocation.

Highlights

Intimin exports its C-terminal passenger domain through an N-terminal ␤-barrel onto the bacterial surface
We could show that (i) at the time point of stalling, the ␤-barrel appears folded; (ii) the stalled autotransporter is associated with BamA and SurA; (iii) the stalled intimin is decorated with large amounts of SurA; (iv) the stalled autotransporter is not degraded by periplasmic proteases; and (v) inverse autotransporter passenger domains are translocated by a hairpin mechanism
By introducing an HA tag after position 453 in intimin, we have produced a mutant of an inverse autotransporter that is stalled in autotransport

Summary

Background

Intimin exports its C-terminal passenger domain through an N-terminal ␤-barrel onto the bacterial surface. The necessity of additional energy sources has been discussed [20, 21] Another model suggests the involvement of the BAM complex in the insertion of the ␤-barrel domain of autotransporters and in the export of the passenger domain to the cell surface. We created a stalled type Ve autotransporter intermediate of the adhesin intimin of enteropathogenic E. coli (EPEC) O127:H6 by inserting a double HA tag after amino acid position 453 This position is located in the D00 domain, which comprises the N-terminal part of the extracellular passenger domain (Fig. 1A) [25]. We could identify transient interaction partners, and by different approaches, we were able to confirm that the transport of the passenger domain occurs by adopting a hairpin conformation, suggesting that hairpin formation might be a general feature of all type V secretion systems possibly with the exception of two-partner secretion

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION