title: FipB, an essential virulence factor of Francisella tularensis subsp. tularensis, has dual roles in disulfide bond formation.
abstract: FipB, an essential virulence factor of Francisella tularensis, is a lipoprotein with two conserved domains that have similarity to disulfide bond formation A (DsbA) proteins and the amino-terminal dimerization domain of macrophage infectivity potentiator (Mip) proteins, which are proteins with peptidyl-prolyl cis/trans isomerase activity. This combination of conserved domains is unusual, so we further characterized the enzymatic activity and the importance of the Mip domain and lipid modification in virulence. Unlike typical DsbA proteins, which are oxidases, FipB exhibited both oxidase and isomerase activities. FipA, which also shares similarity with Mip proteins, potentiated the isomerase activity of FipB in an in vitro assay and within the bacteria, as measured by increased copper sensitivity. To determine the importance of the Mip domain and lipid modification of FipB, mutants producing FipB proteins that lacked either the Mip domain or the critical cysteine necessary for lipid modification were constructed. Both strains replicated within host cells and retained virulence in mice, though there was some attenuation. FipB formed surface-exposed dimers that were sensitive to dithiothreitol (DTT), dependent on the Mip domain and on at least one cysteine in the active site of the DsbA-like domain. However, these dimers were not essential for virulence, because the Mip deletion mutant, which failed to form dimers, was still able to replicate intracellularly and retained virulence in mice. Thus, the Mip domains of FipB and FipA impart additional isomerase functionality to FipB, but only the DsbA-like domain and oxidase activity are essential for its critical virulence functions.
authors.affiliation: Department of Medicine, Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, Virginia, USA.
authors.forename: Aiping
authors.initials: A
authors.lastname: Qin
authors.affiliation: Department of Medicine, Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, Virginia, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Medicine, Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, Virginia, USA.
authors.forename: Melinda E
authors.initials: ME
authors.lastname: Clark
authors.affiliation: Department of Medicine, Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, Virginia, USA.
authors.forename: Meaghan M
authors.initials: MM
authors.lastname: Rabideau
authors.affiliation: Department of Medicine, Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, Virginia, USA.
authors.forename: Luis R
authors.initials: LR
authors.lastname: Millan Barea
authors.affiliation: Department of Medicine, Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, Virginia, USA Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, Virginia, USA bjm2r@virginia.edu.
authors.forename: Barbara J
authors.initials: BJ
authors.lastname: Mann
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003300:Copper; D005604:Francisella tularensis / Q000187:drug effects / Q000378:metabolism* / Q000472:pathogenicity; D015971:Gene Expression Regulation, Enzymologic; D007535:Isomerases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D014406:Tularemia / Q000382:microbiology*; D037521:Virulence Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D037521:Virulence Factors; D003300:Copper; D010088:Oxidoreductases; D007535:Isomerases
references.citation: EMBO J. 1995 Jun 1;14(11):2461-70
references.pmid: 7781600
references.citation: Antioxid Redox Signal. 2011 Jun 15;14(12):2399-412
references.pmid: 21250836
references.citation: J Biol Chem. 2005 Mar 25;280(12):11387-94
references.pmid: 15642731
references.citation: J Bacteriol. 2008 Jul;190(13):4584-95
references.pmid: 18469101
references.citation: J Bacteriol. 2013 Apr;195(8):1825-33
references.pmid: 23435972
references.citation: J Proteome Res. 2010 Apr 5;9(4):1995-2005
references.pmid: 20175567
references.citation: Mol Microbiol. 2005 Nov;58(3):669-81
references.pmid: 16238618
references.citation: Antioxid Redox Signal. 2010 Oct;13(8):1231-46
references.pmid: 20367276
references.citation: Proc Natl Acad Sci U S A. 1995 May 23;92(11):4927-31
references.pmid: 7761426
references.citation: Science. 2004 Jan 23;303(5657):534-7
references.pmid: 14739460
references.citation: Microbiology (Reading). 2009 Apr;155(Pt 4):1152-1163
references.pmid: 19332817
references.citation: J Biol Chem. 2009 Apr 10;284(15):10056-66
references.pmid: 19144642
references.citation: Biochemistry. 1995 Apr 18;34(15):5075-89
references.pmid: 7536035
references.citation: J Bacteriol. 2011 Oct;193(19):5498-509
references.pmid: 21803994
references.citation: Cell. 1991 Nov 1;67(3):581-9
references.pmid: 1934062
references.citation: EMBO J. 2001 Apr 2;20(7):1555-62
references.pmid: 11285220
references.citation: J Bacteriol. 2007 Jul;189(13):4739-48
references.pmid: 17449608
references.citation: Biotechniques. 2010 Nov;49(5):831-3
references.pmid: 21091448
references.citation: Nat Rev Microbiol. 2009 Mar;7(3):215-25
references.pmid: 19198617
references.citation: J Mol Biol. 1993 Apr 5;230(3):1097-100
references.pmid: 8478925
references.citation: Microbiology (Reading). 2013 Nov;159(Pt 11):2364-2374
references.pmid: 24014665
references.citation: Infect Immun. 2003 Aug;71(8):4389-97
references.pmid: 12874317
references.citation: PLoS One. 2011;6(9):e24611
references.pmid: 21931773
references.citation: J Biol Chem. 2008 Nov 14;283(46):31469-76
references.pmid: 18782764
references.citation: J Biol Chem. 2012 Jul 20;287(30):25191-202
references.pmid: 22661710
references.citation: Microb Pathog. 2008 Feb;44(2):151-8
references.pmid: 17933489
references.citation: J Biol Chem. 2005 Oct 7;280(40):33785-91
references.pmid: 16087673
references.citation: Infect Immun. 2009 Jan;77(1):152-61
references.pmid: 18981253
references.citation: Protein Expr Purif. 1999 Feb;15(1):34-9
references.pmid: 10024467
references.citation: Trends Microbiol. 2009 Jan;17(1):6-12
references.pmid: 19059781
references.citation: Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7084-8
references.pmid: 7688471
references.citation: J Bacteriol. 2001 Feb;183(3):980-8
references.pmid: 11208797
references.citation: Int J Syst Evol Microbiol. 2007 Nov;57(Pt 11):2665-2669
references.pmid: 17978237
references.citation: Nat Rev Microbiol. 2004 Dec;2(12):967-78
references.pmid: 15550942
references.citation: J Proteome Res. 2009 Nov;8(11):5336-46
references.pmid: 19799467
references.citation: Infect Immun. 2011 Sep;79(9):3683-96
references.pmid: 21690239
references.citation: mBio. 2012 Apr 03;3(2):
references.pmid: 22493033