Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors
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 approac...
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doaj-785ee42ba4b44a6daed999e8c15cafed2021-06-15T15:31:21ZengeLife Sciences Publications LtdeLife2050-084X2021-06-011010.7554/eLife.67789Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectorsMoirangthem Kiran Singh0https://orcid.org/0000-0001-9620-5454Parisa Zangoui1https://orcid.org/0000-0003-2875-6726Yuki Yamanaka2Linda J Kenney3https://orcid.org/0000-0002-8658-0717Mechanobiology Institute, T-Lab, 5A Engineering Drive 1, National University of Singapore, Singapore, Singapore; Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, United StatesMechanobiology Institute, T-Lab, 5A Engineering Drive 1, National University of Singapore, Singapore, Singapore; Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, United StatesMechanobiology Institute, T-Lab, 5A Engineering Drive 1, National University of Singapore, Singapore, SingaporeMechanobiology Institute, T-Lab, 5A Engineering Drive 1, National University of Singapore, Singapore, Singapore; Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, United StatesType 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.https://elifesciences.org/articles/67789genetic code expansionSalmonella-induced filamentsnon-canonical amino acidsSalmonellaclick chemistrysubstrate specificity switch |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Moirangthem Kiran Singh Parisa Zangoui Yuki Yamanaka Linda J Kenney |
spellingShingle |
Moirangthem Kiran Singh Parisa Zangoui Yuki Yamanaka Linda J Kenney Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors eLife genetic code expansion Salmonella-induced filaments non-canonical amino acids Salmonella click chemistry substrate specificity switch |
author_facet |
Moirangthem Kiran Singh Parisa Zangoui Yuki Yamanaka Linda J Kenney |
author_sort |
Moirangthem Kiran Singh |
title |
Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors |
title_short |
Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors |
title_full |
Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors |
title_fullStr |
Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors |
title_full_unstemmed |
Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors |
title_sort |
genetic code expansion enables visualization of salmonella type three secretion system components and secreted effectors |
publisher |
eLife Sciences Publications Ltd |
series |
eLife |
issn |
2050-084X |
publishDate |
2021-06-01 |
description |
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. |
topic |
genetic code expansion Salmonella-induced filaments non-canonical amino acids Salmonella click chemistry substrate specificity switch |
url |
https://elifesciences.org/articles/67789 |
work_keys_str_mv |
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