title: Comparative proteome analysis of spontaneous outer membrane vesicles and purified outer membranes of Neisseria meningitidis.
abstract: Outer membrane vesicles (OMVs) of Gram-negative bacteria receive increasing attention because of various biological functions and their use as vaccines. However, the mechanisms of OMV release and selective sorting of proteins into OMVs remain unclear. Comprehensive quantitative proteome comparisons between spontaneous OMVs (SOMVs) and the outer membrane (OM) have not been conducted so far. Here, we established a protocol for metabolic labeling of neisserial proteins with (15)N. SOMV and OM proteins labeled with (15)N were used as an internal standard for proteomic comparison of the SOMVs and OMs of two different strains. This labeling approach, coupled with high-sensitivity mass spectrometry, allowed us to comprehensively unravel the proteome of the SOMVs and OMs. We quantified the relative distribution of 155 proteins between SOMVs and the OM. Complement regulatory proteins, autotransporters, proteins involved in iron and zinc acquisition, and a two-partner secretion system were enriched in SOMVs. The highly abundant porins PorA and PorB and proteins connecting the OM with peptidoglycan or the inner membrane, such as RmpM, MtrE, and PilQ, were depleted in SOMVs. Furthermore, the three lytic transglycosylases MltA, MltB, and Slt were less abundant in SOMVs. In conclusion, SOMVs are likely to be released from surface areas with a low local abundance of membrane-anchoring proteins and lytic transglycosylases. The enrichment of complement regulatory proteins, autotransporters, and trace metal binding and transport proteins needs to be explored in the context of the pathogenesis of meningococcal disease.
authors.affiliation: Institute for Hygiene and Microbiology, University of Würzburg, Würzburg, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Lappann
authors.forename: Andreas
authors.initials: A
authors.lastname: Otto
authors.forename: Dörte
authors.initials: D
authors.lastname: Becher
authors.forename: Ulrich
authors.initials: U
authors.lastname: Vogel
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D009345:Neisseria meningitidis / Q000235:genetics / Q000378:metabolism*; D009587:Nitrogen Isotopes; D018272:Porins; D021381:Protein Transport; D020543:Proteome
chemical_list: D001425:Bacterial Outer Membrane Proteins; D009587:Nitrogen Isotopes; D018272:Porins; D020543:Proteome
references.citation: J Exp Med. 1976 Apr 1;143(4):741-58
references.pmid: 56416
references.citation: PLoS Pathog. 2010 Jul 29;6(7):e1001027
references.pmid: 20686663
references.citation: PLoS Genet. 2007 Feb 16;3(2):e23
references.pmid: 17305430
references.citation: Nat Methods. 2008 Apr;5(4):319-22
references.pmid: 18345006
references.citation: Proteomics. 2008 May;8(10):2062-76
references.pmid: 18491319
references.citation: J Bacteriol. 2007 Nov;189(22):7968-76
references.pmid: 17873034
references.citation: J Proteome Res. 2010 Oct 1;9(10):5197-208
references.pmid: 20687614
references.citation: Mol Microbiol. 2013 Aug;89(3):433-49
references.pmid: 23750848
references.citation: Microbiology (Reading). 2003 Jul;149(Pt 7):1849-1858
references.pmid: 12855736
references.citation: Mol Microbiol. 2007 Jan;63(2):545-58
references.pmid: 17163978
references.citation: Microbes Infect. 2006 Jul;8(8):2088-97
references.pmid: 16824779
references.citation: J Bacteriol. 1980 Jan;141(1):169-76
references.pmid: 6766438
references.citation: Clin Microbiol Rev. 2000 Jan;13(1):144-66, table of contents
references.pmid: 10627495
references.citation: NIPH Ann. 1991 Dec;14(2):195-207; discussion 208-10
references.pmid: 1812432
references.citation: Bioinformatics. 2010 Jul 1;26(13):1608-15
references.pmid: 20472543
references.citation: J Proteome Res. 2006 Sep;5(9):2339-47
references.pmid: 16944946
references.citation: J Gen Microbiol. 1977 Apr;99(2):333-41
references.pmid: 406355
references.citation: Anal Chem. 2003 Dec 15;75(24):6912-21
references.pmid: 14670053
references.citation: Vaccine. 2010 Oct 8;28(43):6970-6
references.pmid: 20732470
references.citation: Annu Rev Microbiol. 2010;64:163-84
references.pmid: 20825345
references.citation: Mol Microbiol. 2004 Feb;51(4):1027-37
references.pmid: 14763978
references.citation: Mol Microbiol. 2010 Mar;75(6):1355-71
references.pmid: 20180907
references.citation: Mass Spectrom Rev. 2008 Nov-Dec;27(6):535-55
references.pmid: 18421767
references.citation: Proteomics. 2006 Mar;6(6):1856-66
references.pmid: 16456881
references.citation: Infect Immun. 2007 Mar;75(3):1364-72
references.pmid: 17158897
references.citation: Environ Microbiol. 2013 Feb;15(2):347-54
references.pmid: 23227894
references.citation: J Immunol. 2006 Jul 1;177(1):501-10
references.pmid: 16785547
references.citation: Microbiol Mol Biol Rev. 2010 Mar;74(1):81-94
references.pmid: 20197500
references.citation: Appl Environ Microbiol. 2013 Mar;79(6):1874-81
references.pmid: 23315742
references.citation: Int J Med Microbiol. 2006 Aug;296(4-5):187-95
references.pmid: 16600681
references.citation: Proteomics. 2007 Sep;7(17):3143-53
references.pmid: 17787032
references.citation: Mol Microbiol. 2006 Dec;62(5):1292-309
references.pmid: 17121595
references.citation: Microb Pathog. 1995 Feb;18(2):81-96
references.pmid: 7643745
references.citation: Proc Natl Acad Sci U S A. 2010 Feb 23;107(8):3770-5
references.pmid: 20133713
references.citation: Genome Biol. 2009;10(10):R110
references.pmid: 19818133
references.citation: J Proteome Res. 2005 Jan-Feb;4(1):91-5
references.pmid: 15707362
references.citation: J Clin Invest. 1992 Mar;89(3):816-23
references.pmid: 1541674
references.citation: Science. 2000 Mar 10;287(5459):1809-15
references.pmid: 10710307
references.citation: J Bacteriol. 1989 May;171(5):2499-505
references.pmid: 2496108
references.citation: J Proteome Res. 2013 Apr 5;12(4):1898-908
references.pmid: 23410224
references.citation: J Biol Chem. 2011 Jan 14;286(2):1269-76
references.pmid: 21056982
references.citation: PLoS One. 2013;8(1):e54314
references.pmid: 23372704
references.citation: Mol Microbiol. 2013 Jan;87(2):254-68
references.pmid: 23163582
references.citation: Genome Biol. 2007;8(7):R136
references.pmid: 17617894
references.citation: J Clin Microbiol. 1998 Sep;36(9):2465-70
references.pmid: 9705375
references.citation: Nat Commun. 2010;1:137
references.pmid: 21266987
references.citation: Infect Immun. 2004 Apr;72(4):1914-9
references.pmid: 15039310
references.citation: PLoS Genet. 2012;8(4):e1002626
references.pmid: 22511878
references.citation: Vaccine. 1996 Jul;14(10):1001-8
references.pmid: 8873395
references.citation: J Exp Med. 1974 Jul 1;140(1):87-104
references.pmid: 4134571
references.citation: J Biol Chem. 2005 Nov 18;280(46):38383-94
references.pmid: 16103114
references.citation: Proteomics. 2011 Aug;11(16):3424-9
references.pmid: 21751344
references.citation: Vaccine. 2010 Jul 5;28(30):4810-6
references.pmid: 20483197
references.citation: Appl Environ Microbiol. 2006 Aug;72(8):5414-20
references.pmid: 16885293
references.citation: FEMS Microbiol Lett. 2009 Aug;297(2):150-6
references.pmid: 19548894
references.citation: J Infect Dis. 1989 Feb;159(2):195-204
references.pmid: 2492587
references.citation: Infect Immun. 2003 Dec;71(12):6844-9
references.pmid: 14638771
references.citation: EMBO J. 2004 Nov 24;23(23):4538-49
references.pmid: 15549136
references.citation: Emerg Infect Dis. 2013 Apr;19(4):566-73
references.pmid: 23628376
references.citation: Mol Microbiol. 2011 Dec;82(5):1129-49
references.pmid: 22050461
references.citation: J Biol Chem. 1972 Jun 25;247(12):3962-72
references.pmid: 4555955