title: Pilin and sortase residues critical for endocarditis- and biofilm-associated pilus biogenesis in Enterococcus faecalis.
abstract: Enterococci commonly cause hospital-acquired infections, such as infective endocarditis and catheter-associated urinary tract infections. In animal models of these infections, a long hairlike extracellular protein fiber known as the endocarditis- and biofilm-associated (Ebp) pilus is an important virulence factor for Enterococcus faecalis. For Ebp and other sortase-assembled pili, the pilus-associated sortases are essential for fiber formation as they create covalent isopeptide bonds between the sortase recognition motif and the pilin-like motif of the pilus subunits. However, the molecular requirements governing the incorporation of the three pilus subunits (EbpA, EbpB, and EbpC) have not been investigated in E. faecalis. Here, we show that a Lys residue within the pilin-like motif of the EbpC subunit was necessary for EbpC polymerization. However, incorporation of EbpA into the pilus fiber only required its sortase recognition motif (LPXTG), while incorporation of EbpB only required its pilin-like motif. Only the sortase recognition motif would be required for incorporation of the pilus tip subunit, while incorporation of the base subunit would only require the pilin recognition motif. Thus, these data support a model with EbpA at the tip and EbpB at the base of an EbpC polymer. In addition, the housekeeping sortase, SrtA, was found to process EbpB and its predicted catalytic Cys residue was required for efficient cell wall anchoring of mature Ebp pili. Thus, we have defined molecular interactions involved in fiber polymerization, minor subunit organization, and pilus subcellular compartmentalization in the E. faecalis Ebp pilus system. These studies advance our understanding of unique molecular mechanisms of sortase-assembled pilus biogenesis.
authors.affiliation: Department of Molecular Microbiology and Center for Women's Infectious Disease Research, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: Hailyn V
authors.initials: HV
authors.lastname: Nielsen
authors.forename: Ana L
authors.initials: AL
authors.lastname: Flores-Mireles
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Kau
authors.forename: Kimberly A
authors.initials: KA
authors.lastname: Kline
authors.forename: Jerome S
authors.initials: JS
authors.lastname: Pinkner
authors.forename: Fabrice
authors.initials: F
authors.lastname: Neiers
authors.forename: Staffan
authors.initials: S
authors.lastname: Normark
authors.forename: Birgitta
authors.initials: B
authors.lastname: Henriques-Normark
authors.forename: Michael G
authors.initials: MG
authors.lastname: Caparon
authors.forename: Scott J
authors.initials: SJ
authors.lastname: Hultgren
mesh_terms: D019881:Aminoacyltransferases / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D018441:Biofilms / Q000254:growth & development; D002473:Cell Wall; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D013293:Enterococcus faecalis / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D036781:Fimbriae Proteins / Q000235:genetics / Q000378:metabolism*; D010861:Fimbriae, Bacterial / Q000502:physiology*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D009154:Mutation
chemical_list: D001426:Bacterial Proteins; C509822:sortase C; D036781:Fimbriae Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases
references.citation: J Biol Chem. 2002 May 3;277(18):16241-8
references.pmid: 11856734
references.citation: Nat Commun. 2011 Sep 06;2:462
references.pmid: 21897373
references.citation: Infect Immun. 2009 Sep;77(9):3626-38
references.pmid: 19528211
references.citation: Microbiology (Reading). 2004 Jul;150(Pt 7):2069-2078
references.pmid: 15256550
references.citation: J Biol Chem. 2008 Dec 26;283(52):36676-86
references.pmid: 18940793
references.citation: Clin Microbiol Rev. 1994 Oct;7(4):462-78
references.pmid: 7834601
references.citation: Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):2081-5
references.pmid: 7892228
references.citation: PLoS One. 2011;6(7):e22095
references.pmid: 21760959
references.citation: mBio. 2012 Jul 24;3(4):e00177-12
references.pmid: 22829678
references.citation: Infect Immun. 2007 Nov;75(11):5399-404
references.pmid: 17785477
references.citation: Trends Microbiol. 2008 Jan;16(1):33-40
references.pmid: 18083568
references.citation: Virulence. 2010 Jul-Aug;1(4):236-46
references.pmid: 20676385
references.citation: Biotechniques. 1990 May;8(5):528-35
references.pmid: 2357375
references.citation: Mol Microbiol. 2007 Nov;66(4):961-74
references.pmid: 17919283
references.citation: Mol Microbiol. 2003 Nov;50(4):1429-38
references.pmid: 14622427
references.citation: Proc Natl Acad Sci U S A. 2008 Sep 16;105(37):14147-52
references.pmid: 18779588
references.citation: J Bacteriol. 2009 May;191(10):3237-47
references.pmid: 19286802
references.citation: Proc Natl Acad Sci U S A. 2008 Jul 22;105(29):10215-20
references.pmid: 18621716
references.citation: Science. 1999 Jul 30;285(5428):760-3
references.pmid: 10427003
references.citation: J Cell Biol. 2008 Jan 28;180(2):389-402
references.pmid: 18209100
references.citation: Mol Microbiol. 2008 Nov;70(3):595-607
references.pmid: 18761697
references.citation: Pharmacol Rev. 2008 Mar;60(1):128-41
references.pmid: 18321961
references.citation: Mol Microbiol. 2006 Jun;60(6):1401-13
references.pmid: 16796677
references.citation: PLoS One. 2011;6(10):e25300
references.pmid: 21991306
references.citation: Biochem J. 2012 Feb 1;441(3):833-41
references.pmid: 22013894
references.citation: Nat Rev Microbiol. 2012 Mar 16;10(4):266-78
references.pmid: 22421879
references.citation: PLoS One. 2011 Jan 11;6(1):e15969
references.pmid: 21264317
references.citation: Science. 2003 Mar 28;299(5615):2071-4
references.pmid: 12663927
references.citation: Methods Mol Biol. 1995;47:217-26
references.pmid: 7550738
references.citation: Structure. 2008 Dec 10;16(12):1838-48
references.pmid: 19081060
references.citation: Trends Microbiol. 2012 Jan;20(1):40-9
references.pmid: 22169461
references.citation: Microbiol Mol Biol Rev. 2006 Mar;70(1):192-221
references.pmid: 16524923
references.citation: PLoS One. 2011 Apr 15;6(4):e18747
references.pmid: 21525979
references.citation: Appl Environ Microbiol. 2012 Apr;78(7):2337-44
references.pmid: 22247175
references.citation: Proc Natl Acad Sci U S A. 2006 Feb 21;103(8):2857-62
references.pmid: 16481624
references.citation: Appl Environ Microbiol. 2005 Oct;71(10):5837-49
references.pmid: 16204495
references.citation: J Biol Chem. 2009 May 8;284(19):12989-97
references.pmid: 19269972
references.citation: J Bacteriol. 2007 Feb;189(4):1426-34
references.pmid: 17012387
references.citation: J Biol Chem. 2010 Jun 25;285(26):20381-9
references.pmid: 20427291
references.citation: FASEB J. 2008 Jun;22(6):1715-24
references.pmid: 18198218
references.citation: Nat Rev Microbiol. 2011 Mar;9(3):166-76
references.pmid: 21326273
references.citation: J Bacteriol. 2007 Apr;189(8):3156-65
references.pmid: 17277070
references.citation: J Infect Dis. 2007 Jun 1;195(11):1671-7
references.pmid: 17471437
references.citation: Mol Microbiol. 2001 Jun;40(5):1049-57
references.pmid: 11401711
references.citation: Mol Microbiol. 2004 Jul;53(1):251-61
references.pmid: 15225319
references.citation: Biopolymers. 2009 Dec;91(12):1126-34
references.pmid: 19226623
references.citation: Mol Microbiol. 2011 Dec;82(5):1044-59
references.pmid: 22026821
references.citation: Infect Immun. 2010 Oct;78(10):4166-75
references.pmid: 20696830
references.citation: Infect Control Hosp Epidemiol. 2008 Nov;29(11):996-1011
references.pmid: 18947320
references.citation: Infect Immun. 2011 Jul;79(7):2901-10
references.pmid: 21505082
references.citation: Genome Biol. 2008;9(7):R110
references.pmid: 18611278
references.citation: J Clin Invest. 2006 Oct;116(10):2799-807
references.pmid: 17016560
references.citation: Mol Microbiol. 2012 Mar;83(5):1035-47
references.pmid: 22320452
references.citation: J Bacteriol. 2008 Sep;190(17):6002-13
references.pmid: 18606733
references.citation: J Med Microbiol. 2007 Dec;56(Pt 12):1581-1588
references.pmid: 18033823
references.citation: Infect Immun. 2008 Aug;76(8):3550-60
references.pmid: 18541657