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

eLife

Yuki Yamanaka¹  Moirangthem Kiran Singh²  Parisa Zangoui²  Linda J Kenney² 

[1] Mechanobiology Institute, T-Lab, 5A Engineering Drive 1, National University of Singapore, Singapore, Singapore;Mechanobiology Institute, T-Lab, 5A Engineering Drive 1, National University of Singapore, Singapore, Singapore;Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, United States;

关键词: genetic code expansion;    Salmonella;    non-canonical amino acids;    Salmonella;    click chemistry;    substrate specificity switch;    Other;   

DOI  :  10.7554/eLife.67789

来源: eLife Sciences Publications, Ltd

【 摘 要 】

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.

【 授权许可】

CC BY