Formation of the β-sheet at the extracellular opening of the intimin β-barrel domain is necessary for stability and efficient passenger secretion

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Sarma Kandanur, SP, Breuer, L, Renschler, FA, Schuetz, MS and Leo, JC ORCID: https://orcid.org/0000-0002-7066-7527, 2025. Formation of the β-sheet at the extracellular opening of the intimin β-barrel domain is necessary for stability and efficient passenger secretion. bioRxiv.

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Official URL: https://doi.org/10.1101/2025.05.11.653311

Abstract

Attaching and effacing pathogens, such as enteropathogenic and enterohaemorrhagic E. coli, rely on an adhesin, intimin, for attachment to enterocytes and downstream effects leading to actin pedestal formation. Intimin belongs to the inverse autotransporter family (type 5e secretion systems), which secrete the extracellular adhesive domain or passenger via a hairpin intermediate. While many of the next steps in secretion are now understood, how the hairpin initially forms is not known. We sought to investigate this by making point mutations at several positions in the β-barrel domain of intimin, as this domain forms at least part of the secretion pore. We made the mutations in a wild-type background and in a stalled intermediate caught in the hairpin conformation, which allowed us to uncouple passenger secretion from hairpin formation. Surprisingly, most of the point mutations did not have an appreciable effect on hairpin formation or passenger secretion, and larger changes such as replacing the entire linker region with a flexible glycine-serine stretch showed only a modest reduction in passenger secretion. By contrast, mutations affecting a small β-sheet at the extracellular face of the intimin β-barrel between two extracellular loops and the C-terminus of the linker had a more pronounced effect on secretion, and abolishing this β-sheet prevented hairpin formation and led to complete loss of the wild-type protein. Our results show that the intimin β-barrel is remarkably tolerant to changes and that the β-sheet on the extracellular side of this domain plays a central role in passenger secretion and protein stability.

Item Type:	Working paper
Description:	Platform: bioRxiv
Creators:	Sarma Kandanur, S.P., Breuer, L., Renschler, F.A., Schuetz, M.S. and Leo, J.C.
Publisher:	bioRxiv
Date:	11 May 2025
Identifiers:	Number Type 10.1101/2025.05.11.653311 DOI 2446665 Other
Rights:	The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
Divisions:	Schools > School of Science and Technology
Record created by:	Jeremy Silvester
Date Added:	05 Jun 2025 08:26
Last Modified:	05 Jun 2025 08:26
URI:	https://irep.ntu.ac.uk/id/eprint/53695