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.