title: Functional and structural analysis of HicA3-HicB3, a novel toxin-antitoxin system of Yersinia pestis.
abstract: The mechanisms involved in the virulence of Yersinia pestis, the plague pathogen, are not fully understood. In previous research, we found that a Y. pestis mutant lacking the HicB3 (YPO3369) putative orphan antitoxin was attenuated for virulence in a murine model of bubonic plague. Toxin-antitoxin systems (TASs) are widespread in prokaryotes. Most bacterial species possess many TASs of several types. In type II TASs, the toxin protein is bound and neutralized by its cognate antitoxin protein in the cytoplasm. Here we identify the hicA3 gene encoding the toxin neutralized by HicB3 and show that HicA3-HicB3 constitutes a new functional type II TAS in Y. pestis. Using biochemical and mutagenesis-based approaches, we demonstrate that the HicA3 toxin is an RNase with a catalytic histidine residue. HicB3 has two functions: it sequesters and neutralizes HicA3 by blocking its active site, and it represses transcription of the hicA3B3 operon. Gel shift assays and reporter fusion experiments indicate that the HicB3 antitoxin binds to two operators in the hicA3B3 promoter region. We solved the X-ray structures of HicB3 and the HicA3-HicB3 complex; thus, we present the first crystal structure of a TA complex from the HicAB family. HicB3 forms a tetramer that can bind two HicA3 toxin molecules. HicA3 is monomeric and folds as a double-stranded-RNA-binding domain. The HicB3 N-terminal domain occludes the HicA3 active site, whereas its C-terminal domain folds as a ribbon-helix-helix DNA-binding motif.
authors.affiliation: Equipe Peste et Yersinia pestis, INSERM U1019, Lille, France CNRS UMR 8204, Lille, France Institut Pasteur de Lille, Centre d'Infection et d'Immunité, Lille, France Université Lille Nord de France, Lille, France UDSL, Lille, France.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Bibi-Triki
authors.affiliation: Equipe Fonction et Architecture des Assemblages MacroMoléculaires, IBBMC, Université Paris-Sud, CNRS UMR 8619, Orsay, France.
authors.forename: Inès
authors.initials: I
authors.lastname: Li de la Sierra-Gallay
authors.affiliation: Equipe Fonction et Architecture des Assemblages MacroMoléculaires, IBBMC, Université Paris-Sud, CNRS UMR 8619, Orsay, France.
authors.forename: Noureddine
authors.initials: N
authors.lastname: Lazar
authors.affiliation: EA 4529, UFR de Pharmacie, Université Paris-Sud, Châtenay-Malabry, France.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Leroy
authors.affiliation: Equipe Fonction et Architecture des Assemblages MacroMoléculaires, IBBMC, Université Paris-Sud, CNRS UMR 8619, Orsay, France.
authors.forename: Herman
authors.initials: H
authors.lastname: Van Tilbeurgh
authors.affiliation: Equipe Peste et Yersinia pestis, INSERM U1019, Lille, France CNRS UMR 8204, Lille, France Institut Pasteur de Lille, Centre d'Infection et d'Immunité, Lille, France Université Lille Nord de France, Lille, France UDSL, Lille, France florent.sebbane@inserm.fr elizabeth.pradel@inserm.fr.
authors.forename: Florent
authors.initials: F
authors.lastname: Sebbane
authors.affiliation: Equipe Peste et Yersinia pestis, INSERM U1019, Lille, France CNRS UMR 8204, Lille, France Institut Pasteur de Lille, Centre d'Infection et d'Immunité, Lille, France Université Lille Nord de France, Lille, France UDSL, Lille, France florent.sebbane@inserm.fr elizabeth.pradel@inserm.fr.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Pradel
mesh_terms: D000818:Animals; D000992:Antitoxins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D001482:Base Composition; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010930:Plague / Q000382:microbiology; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D014774:Virulence; D015010:Yersinia pestis / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity
chemical_list: D000992:Antitoxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins
references.citation: Genome Biol. 2003;4(12):R81
references.pmid: 14659018
references.citation: J Bacteriol. 2009 Feb;191(4):1191-9
references.pmid: 19060138
references.citation: Proc Natl Acad Sci U S A. 1990 Jun;87(12):4645-9
references.pmid: 2162051
references.citation: J Bacteriol. 2005 Sep;187(17):6094-105
references.pmid: 16109951
references.citation: J Bacteriol. 2006 Dec;188(24):8360-7
references.pmid: 17041039
references.citation: Nat Rev Microbiol. 2011 Sep 19;9(11):779-90
references.pmid: 21927020
references.citation: PLoS Pathog. 2014 Mar 27;10(3):e1004029
references.pmid: 24675805
references.citation: Methods Mol Biol. 2007;363:21-37
references.pmid: 17272835
references.citation: J Bacteriol. 2013 Sep;195(17):3947-56
references.pmid: 23813728
references.citation: J Mol Biol. 1999 Jan 29;285(4):1667-77
references.pmid: 9917404
references.citation: Proc Natl Acad Sci U S A. 2006 Aug 1;103(31):11766-71
references.pmid: 16864791
references.citation: Biochemistry. 1990 Dec 25;29(51):11189-95
references.pmid: 2271706
references.citation: J Mol Biol. 1986 May 5;189(1):113-30
references.pmid: 3537305
references.citation: Biochem J. 2014 Apr 15;459(2):333-44
references.pmid: 24502667
references.citation: Nucleic Acids Res. 2012 May;40(10):4347-57
references.pmid: 22287572
references.citation: Infect Immun. 1987 Mar;55(3):572-8
references.pmid: 3818085
references.citation: Mol Cell. 2013 Oct 24;52(2):248-54
references.pmid: 24095282
references.citation: Acta Crystallogr D Biol Crystallogr. 2011 Apr;67(Pt 4):235-42
references.pmid: 21460441
references.citation: Cold Spring Harb Symp Quant Biol. 1981;45 Pt 1:135-40
references.pmid: 6271456
references.citation: Protein Pept Lett. 2007;14(2):113-24
references.pmid: 17305597
references.citation: J Mol Biol. 1993 Dec 5;234(3):534-41
references.pmid: 8254658
references.citation: Proc Natl Acad Sci U S A. 2009 Jan 20;106(3):894-9
references.pmid: 19124776
references.citation: J Bacteriol. 1990 Nov;172(11):6557-67
references.pmid: 2172216
references.citation: Microbiol Mol Biol Rev. 2008 Dec;72(4):579-89, Table of Contents
references.pmid: 19052321
references.citation: Proc Natl Acad Sci U S A. 2005 Dec 6;102(49):17786-91
references.pmid: 16306265
references.citation: Curr Biol. 2001 Jun 26;11(12):941-50
references.pmid: 11448770
references.citation: Mol Microbiol. 2008 Oct;70(1):258-70
references.pmid: 18761622
references.citation: PLoS One. 2012;7(12):e52503
references.pmid: 23285069
references.citation: Science. 1999 Jul 2;285(5424):110-3
references.pmid: 10390360
references.citation: Mol Microbiol. 2008 Aug;69(4):841-57
references.pmid: 18532983
references.citation: Mol Microbiol. 2011 Mar;79(6):1418-29
references.pmid: 21323758
references.citation: Mol Cell. 2003 Apr;11(4):875-84
references.pmid: 12718874
references.citation: Mol Microbiol. 2010 May;76(3):719-32
references.pmid: 20345661
references.citation: PLoS Pathog. 2013;9(12):e1003827
references.pmid: 24385907
references.citation: J Biol Chem. 2012 Feb 17;287(8):5340-56
references.pmid: 22199354
references.citation: J Mol Biol. 2009 Nov 27;394(2):183-96
references.pmid: 19747491
references.citation: Biochim Biophys Acta. 2013 Jun-Jul;1829(6-7):523-31
references.pmid: 23454553
references.citation: PLoS Genet. 2009 Dec;5(12):e1000767
references.pmid: 20011113
references.citation: J Bacteriol. 2009 Feb;191(4):1258-67
references.pmid: 19060153
references.citation: Mol Microbiol. 1992 Sep;6(18):2693-704
references.pmid: 1447977
references.citation: J Biol Chem. 1999 Jun 11;274(24):16813-8
references.pmid: 10358024
references.citation: Biochim Biophys Acta. 2013 Jun;1834(6):1155-67
references.pmid: 23459128
references.citation: J Bacteriol. 2010 Jul;192(14):3669-77
references.pmid: 20472800
references.citation: Gene. 1997 Mar 25;188(1):69-75
references.pmid: 9099861
references.citation: Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5
references.pmid: 10829079
references.citation: Annu Rev Genet. 2011;45:61-79
references.pmid: 22060041
references.citation: Structure. 2012 Oct 10;20(10):1641-8
references.pmid: 22981948
references.citation: Am J Trop Med Hyg. 2013 Oct;89(4):788-93
references.pmid: 24043686
references.citation: J Biol Chem. 2013 Jan 11;288(2):1286-94
references.pmid: 23172222
references.citation: Crit Rev Biochem Mol Biol. 2011 Oct;46(5):386-408
references.pmid: 21819231
references.citation: Mol Microbiol. 1999 Sep;33(6):1232-43
references.pmid: 10510237
references.citation: Nucleic Acids Res. 2005 Feb 17;33(3):966-76
references.pmid: 15718296
references.citation: Gene. 1998 May 28;212(1):77-86
references.pmid: 9661666
references.citation: BMC Microbiol. 2012 Nov 19;12:263
references.pmid: 23157645
references.citation: Yeast. 2000 Jan 30;16(2):177-87
references.pmid: 10641039
references.citation: Am J Trop Med Hyg. 1994 Jul;51(1):109-14
references.pmid: 8059908
references.citation: J Bacteriol. 1995 Jul;177(14):4121-30
references.pmid: 7608087
references.citation: Cell. 2010 Jul 9;142(1):101-11
references.pmid: 20603017