title: Identification of disulfide bond isomerase substrates reveals bacterial virulence factors.
abstract: Bacterial pathogens are exposed to toxic molecules inside the host and require efficient systems to form and maintain correct disulfide bonds for protein stability and function. The intracellular pathogen Francisella tularensis encodes a disulfide bond formation protein ortholog, DsbA, which previously was reported to be required for infection of macrophages and mice. However, the molecular mechanisms by which F. tularensis DsbA contributes to virulence are unknown. Here, we demonstrate that F. tularensis DsbA is a bifunctional protein that oxidizes and, more importantly, isomerizes complex disulfide connectivity in substrates. A single amino acid in the conserved cis-proline loop of the DsbA thioredoxin domain was shown to modulate both isomerase activity and F. tularensis virulence. Trapping experiments in F. tularensis identified over 50 F. tularensis DsbA substrates, including outer membrane proteins, virulence factors, and many hypothetical proteins. Six of these hypothetical proteins were randomly selected and deleted, revealing two novel proteins, FTL_1548 and FTL_1709, which are required for F. tularensis virulence. We propose that the extreme virulence of F. tularensis is partially due to the bifunctional nature of DsbA, that many of the newly identified substrates are required for virulence, and that the development of future DsbA inhibitors could have broad anti-bacterial implications.
authors.affiliation: Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, Toledo, OH, 43614, USA.
authors.forename: Guoping
authors.initials: G
authors.lastname: Ren
authors.forename: Matthew M
authors.initials: MM
authors.lastname: Champion
authors.forename: Jason F
authors.initials: JF
authors.lastname: Huntley
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004195:Disease Models, Animal; D004220:Disulfides / Q000378:metabolism*; D005604:Francisella tularensis / Q000201:enzymology* / Q000378:metabolism*; D017353:Gene Deletion; D007535:Isomerases; D008809:Mice, Inbred C3H; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism*; D016019:Survival Analysis; D014406:Tularemia / Q000382:microbiology / Q000473:pathology; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D004220:Disulfides; D050505:Mutant Proteins; D037521:Virulence Factors; D007535:Isomerases; D019704:Protein Disulfide-Isomerases