Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

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Abstract

Type three secretion systems enable bacterial pathogens to inject effectors into the cytosol of eukaryotic hosts to reprogram cellular functions. It is technically challenging to label effectors and the secretion machinery without disrupting their structure/function. Herein, we present a new approach for labeling and visualization of previously intractable targets. Using genetic code expansion, we site-specifically labeled SsaP, the substrate specificity switch, and SifA, a here-to-fore unlabeled secreted effector. SsaP was secreted at later infection times; SsaP labeling demonstrated the stochasticity of injectisome and effector expression. SifA was labeled after secretion into host cells via fluorescent unnatural amino acids or non-fluorescent labels and a subsequent click reaction. We demonstrate the superiority of imaging after genetic code expansion compared to small molecule tags. It provides an alternative for labeling proteins that do not tolerate N- or C-terminal tags or fluorophores and thus is widely applicable to other secreted effectors and small proteins.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data of Figure 4A-B, Figure 2-figure supplement 5, Figure 3-figure supplement 1, Figure 3-figure supplement 4, Figure 3-figure supplement 5, Figure 4-figure supplement 1 are included.

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Moirangthem Kiran Singh

Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, United States

Competing interests
The authors declare that no competing interests exist.
Parisa Zangoui

Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, United States

Competing interests
The authors declare that no competing interests exist.
Yuki Yamanaka

School of Life Dentistry, Nippon Dental University School of Life Dentistry at Tokyo, Tokyo, Japan

Competing interests
The authors declare that no competing interests exist.
Linda J Kenney

Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, United States

For correspondence
likenney@utmb.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-8658-0717

Funding

Ministry of Education, Singapore (MOE2018-T2-1-038)

Linda J Kenney

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.