title: Identification of secreted bacterial proteins by noncanonical amino acid tagging.
abstract: Pathogenic microbes have evolved complex secretion systems to deliver virulence factors into host cells. Identification of these factors is critical for understanding the infection process. We report a powerful and versatile approach to the selective labeling and identification of secreted pathogen proteins. Selective labeling of microbial proteins is accomplished via translational incorporation of azidonorleucine (Anl), a methionine surrogate that requires a mutant form of the methionyl-tRNA synthetase for activation. Secreted pathogen proteins containing Anl can be tagged by azide-alkyne cycloaddition and enriched by affinity purification. Application of the method to analysis of the type III secretion system of the human pathogen Yersinia enterocolitica enabled efficient identification of secreted proteins, identification of distinct secretion profiles for intracellular and extracellular bacteria, and determination of the order of substrate injection into host cells. This approach should be widely useful for the identification of virulence factors in microbial pathogens and the development of potential new targets for antimicrobial therapy.
authors.affiliation: Division of Chemistry and Chemical Engineering, Department of Bioengineering, and Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, Pasadena, CA 91125.
authors.forename: Alborz
authors.initials: A
authors.lastname: Mahdavi
authors.forename: Janek
authors.initials: J
authors.lastname: Szychowski
authors.forename: John T
authors.initials: JT
authors.lastname: Ngo
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Sweredoski
authors.forename: Robert L J
authors.initials: RL
authors.lastname: Graham
authors.forename: Sonja
authors.initials: S
authors.lastname: Hess
authors.forename: Olaf
authors.initials: O
authors.lastname: Schneewind
authors.forename: Sarkis K
authors.initials: SK
authors.lastname: Mazmanian
authors.forename: David A
authors.initials: DA
authors.lastname: Tirrell
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D000900:Anti-Bacterial Agents / Q000737:chemistry; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D008718:Methionine-tRNA Ligase / Q000737:chemistry; D008828:Microbiological Techniques; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009154:Mutation; D009646:Norleucine / Q000737:chemistry; D020543:Proteome; D040901:Proteomics / Q000379:methods; D013997:Time Factors; D037521:Virulence Factors; D015008:Yersinia enterocolitica / Q000378:metabolism
chemical_list: D000596:Amino Acids; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D020543:Proteome; D037521:Virulence Factors; D009646:Norleucine; D008718:Methionine-tRNA Ligase
keywords: BONCAT; Yop; click chemistry; proteomics