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

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 domai...

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Bibliographic Details
Published in:The Journal of biological chemistry 2015-01, Vol.290 (3), p.1837-1849
Main Authors: Oberhettinger, Philipp, Leo, Jack C., Linke, Dirk, Autenrieth, Ingo B., Schütz, Monika S.
Format: Article
Language:English
Subjects:
Adhesin
Adhesins, Bacterial - chemistry
Adhesins, Bacterial - physiology
Bacterial Pathogenesis
Biological Transport
Cell Membrane - metabolism
Cloning, Molecular
Cross-Linking Reagents - chemistry
Epitopes - chemistry
Escherichia coli - metabolism
Escherichia coli Proteins - chemistry
Escherichia coli Proteins - physiology
HeLa Cells
Humans
Intimin, Type Ve Secretion, Autotransporter, beta Barrel Protein, Hairpin
Membrane Protein
Microbial Pathogenesis
Microbiology
Microscopy, Fluorescence
Molecular Chaperones - chemistry
Mutagenesis, Site-Directed
Mutation
Outer Membrane
Peptide Hydrolases - chemistry
Protein Binding
Protein Folding
Protein Structure, Secondary
Protein Structure, Tertiary
Surface Properties
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Summary: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.Intimin exports its C-terminal passenger domain through an N-terminal β-barrel onto the bacterial surface. Insertion of an epitope tag at the very N terminus of the passenger domain stalls the export of the passenger. The intimin passenger adopts a hairpin conformation during translocation. Our results confirm the hairpin model of inverse autotransport where the passenger is translocated from the N to the C terminus.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M114.604769