10.1021/ja209008w.s001
Shixian Lin
Shixian
Lin
Zhenrun Zhang
Zhenrun
Zhang
Hao Xu
Hao
Xu
Lin Li
Lin
Li
She Chen
She
Chen
Jie Li
Jie
Li
Ziyang Hao
Ziyang
Hao
Peng R. Chen
Peng R.
Chen
Site-Specific Incorporation of Photo-Cross-Linker and Bioorthogonal Amino Acids into Enteric Bacterial Pathogens
American Chemical Society
2011
bioorthogonal functionalities
enteric pathogens
secretion efficiency
vivo client proteins
Shigella type 3 secretion effector
code expansion strategy
acid stress
Bioorthogonal Amino Acids
pathogenesis mechanisms
shHdeA
cause infections
OspF
Shigella acid chaperone HdeA
extracellular space
enteropathogenic Escherichia coli
2011-12-21 00:00:00
Journal contribution
https://acs.figshare.com/articles/journal_contribution/Site_Specific_Incorporation_of_Photo_Cross_Linker_and_Bioorthogonal_Amino_Acids_into_Enteric_Bacterial_Pathogens/2569636
Enteric bacterial pathogens are known to effectively pass through the extremely acidic mammalian stomachs and cause infections in the small and/or large intestine of human hosts. However, their acid-survival strategy and pathogenesis mechanisms remain elusive, largely due to the lack of tools to directly monitor and manipulate essential components (e.g., defense proteins or invasive toxins) participating in these processes. Herein, we have extended the pyrrolysine-based genetic code expansion strategy for encoding unnatural amino acids in enteric bacterial species, including enteropathogenic Escherichia coli, Shigella, and Salmonella. Using this system, a photo-cross-linking amino acid was incorporated into a Shigella acid chaperone HdeA (shHdeA), which allowed the identification of a comprehensive list of in vivo client proteins that are protected by shHdeA upon acid stress. To further demonstrate the application of our strategy, an azide-bearing amino acid was introduced into a Shigella type 3 secretion effector, OspF, without interruption of its secretion efficiency. This site-specifically installed azide handle allowed the facile detection of OspF’s secretion in bacterial extracellular space. Taken together, these bioorthogonal functionalities we incorporated into enteric pathogens were shown to facilitate the investigation of unique and important proteins involved in the pathogenesis and stress-defense mechanisms of pathogenic bacteria that remain exceedingly difficult to study using conventional methodologies.