Reconstitution of surface lipoprotein translocation through the slam translocon

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Abstract

Surface lipoproteins (SLPs) are peripherally attached to the outer leaflet of the outer membrane in many Gram-negative bacteria, playing significant roles in nutrient acquisition and immune evasion in the host. While the factors that are involved in the synthesis and delivery of SLPs in the inner membrane are well characterized, the molecular machinery required for the movement of SLPs to the surface are still not fully elucidated. In this study, we investigated the translocation of a surface lipoprotein TbpB through a Slam1-dependent pathway. Using purified components, we developed an in vitro translocation assay where unfolded TbpB is transported through Slam1 containing proteoliposomes, confirming Slam1 as an outer membrane translocon. While looking to identify factors to increase translocation efficiency, we discovered the periplasmic chaperone Skp interacted with TbpB in the periplasm of Escherichia coli. The presence of Skp was found to increase the translocation efficiency of TbpB in the reconstituted translocation assays. A knockout of Skp in Neisseria meningitidis revealed that Skp is essential for functional translocation of TbpB to the bacterial surface. Taken together, we propose a pathway for surface destined lipoproteins, where Skp acts as a holdase for Slam-mediated TbpB translocation across the outer membrane.

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All data generated or analysed during this study are included in the manuscript and supporting files

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Minh Sang Huynh

Department of Biochemistry, University of Toronto, Toronto, Canada

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0002-9541-6441
Yogesh Hooda

MRC Laboratory of Molecular Biology, University of Cambridge, Cambridge, United Kingdom

Competing interests
Yogesh Hooda, is a co-author on a patent, 'Slam polynucleotides and polypeptides and uses thereof' - patent number WO2017136947A1..
Yuzi Raina Li

Department of Biochemistry, University of Toronto, Toronto, Canada

Competing interests
No competing interests declared.
Maciej Jagielnicki

Department of Biochemistry, University of Toronto, Toronto, Canada

Competing interests
No competing interests declared.
Christine Chieh-Lin Lai

Department of Biochemistry, University of Toronto, Toronto, Canada

Competing interests
Christine Chieh-Lin Lai, is a co-author on a patent, 'Slam polynucleotides and polypeptides and uses thereof' - patent number WO2017136947A1..
Trevor F Moraes

Department of Biochemistry, University of Toronto, Toronto, Canada

For correspondence
trevor.moraes@utoronto.ca

Competing interests
Trevor F Moraes, is a co-author on a patent, 'Slam polynucleotides and polypeptides and uses thereof' - patent number WO2017136947A1..

"This ORCID iD identifies the author of this article:" 0000-0001-9883-6145

Funding

Canadian Institutes of Health Research (PJT-148795)

Trevor F Moraes

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.