title: Kdo hydrolase is required for Francisella tularensis virulence and evasion of TLR2-mediated innate immunity.
abstract: The highly virulent Francisella tularensis subsp. tularensis has been classified as a category A bioterrorism agent. A live vaccine strain (LVS) has been developed but remains unlicensed in the United States because of an incomplete understanding of its attenuation. Lipopolysaccharide (LPS) modification is a common strategy employed by bacterial pathogens to avoid innate immunity. A novel modification enzyme has recently been identified in F. tularensis and Helicobacter pylori. This enzyme, a two-component Kdo (3-deoxy-d-manno-octulosonic acid) hydrolase, catalyzes the removal of a side chain Kdo sugar from LPS precursors. The biological significance of this modification has not yet been studied. To address the role of the two-component Kdo hydrolase KdhAB in F. tularensis pathogenesis, a ΔkdhAB deletion mutant was constructed from the LVS strain. In intranasal infection of mice, the ΔkdhAB mutant strain had a 50% lethal dose (LD(50)) 2 log(10) units higher than that of the parental LVS strain. The levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) in bronchoalveolar lavage fluid were significantly higher (2-fold) in mice infected with the ΔkdhAB mutant than in mice infected with LVS. In vitro stimulation of bone marrow-derived macrophages with the ΔkdhAB mutant induced higher levels of TNF-α and IL-1β in a TLR2-dependent manner. In addition, TLR2(-/-) mice were more susceptible than wild-type mice to ΔkdhAB bacterial infection. Finally, immunization of mice with ΔkdhAB bacteria elicited a high level of protection against the highly virulent F. tularensis subsp. tularensis strain Schu S4. These findings suggest an important role for the Francisella Kdo hydrolase system in virulence and offer a novel mutant as a candidate vaccine.
  
  
  OBJECTIVE
  The first line of defense against a bacterial pathogen is innate immunity, which slows the progress of infection and allows time for adaptive immunity to develop. Some bacterial pathogens, such as Francisella tularensis, suppress the early innate immune response, killing the host before adaptive immunity can mature. To avoid an innate immune response, F. tularensis enzymatically modifies its lipopolysaccharide (LPS). A novel LPS modification-Kdo (3-deoxy-d-manno-octulosonic acid) saccharide removal--has recently been reported in F. tularensis. We found that the kdhAB mutant was significantly attenuated in mice. Additionally, the mutant strain induced an early innate immune response in mice both in vitro and in vivo. Immunization of mice with this mutant provided protection against the highly virulent F. tularensis strain Schu S4. Thus, our study has identified a novel LPS modification important for microbial virulence. A mutant lacking this modification may be used as a live attenuated vaccine against tularemia.
authors: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.
authors: Nihal A
authors: NA
authors: Okan
authors: Sabina
authors: S
authors: Chalabaev
authors: Tae-Hyun
authors: TH
authors: Kim
authors: Avner
authors: A
authors: Fink
authors: Robin A
authors: RA
authors: Ross
authors: Dennis L
authors: DL
authors: Kasper
mesh_terms: D000818:Animals; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry; D016207:Cytokines / Q000032:analysis; D004195:Disease Models, Animal; D005604:Francisella tularensis / Q000201:enzymology* / Q000235:genetics / Q000276:immunology / Q000472:pathogenicity*; D017353:Gene Deletion; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D057131:Immune Evasion; D007928:Lethal Dose 50; D008264:Macrophages / Q000276:immunology / Q000382:microbiology; D051379:Mice; D018345:Mice, Knockout; D016019:Survival Analysis; D051195:Toll-Like Receptor 2 / Q000172:deficiency / Q000276:immunology*; D014406:Tularemia / Q000382:microbiology / Q000473:pathology; D037521:Virulence Factors / Q000378:metabolism*
chemical_list: D016207:Cytokines; C493486:Tlr2 protein, mouse; D051195:Toll-Like Receptor 2; D037521:Virulence Factors; D006026:Glycoside Hydrolases
references: J Biol Chem. 2004 Nov 19;279(47):49470-8
references: 15339914
references: PLoS Pathog. 2008 Feb 8;4(2):e24
references: 18266468
references: Infect Immun. 2006 May;74(5):2809-16
references: 16622218
references: J Immunol. 2007 Apr 1;178(7):4538-47
references: 17372012
references: Immunol Rev. 2008 Oct;225:244-55
references: 18837786
references: Infect Immun. 2002 Aug;70(8):4092-8
references: 12117916
references: Methods. 2001 Dec;25(4):402-8
references: 11846609
references: J Biol Chem. 1999 Feb 19;274(8):5114-9
references: 9988760
references: FEMS Microbiol Lett. 2003 Dec 12;229(2):195-202
references: 14680699
references: J Bacteriol. 2006 Jan;188(1):191-201
references: 16352835
references: PLoS One. 2011;6(7):e22335
references: 21799828
references: Infect Immun. 2007 May;75(5):2591-602
references: 17296751
references: Proc Natl Acad Sci U S A. 2007 Apr 3;104(14):6037-42
references: 17389372
references: J Infect Dis. 2012 Apr 1;205(7):1056-65
references: 21969334
references: J Biol Chem. 2010 Sep 3;285(36):27839-49
references: 20605777
references: Mol Microbiol. 2010 Nov;78(4):837-52
references: 20659292
references: Biochemistry. 2006 Dec 5;45(48):14427-40
references: 17128982
references: Vaccine. 2009 Jan 14;27(3):388-95
references: 19014988
references: Infect Immun. 2008 Dec;76(12):5843-52
references: 18852251
references: Carbohydr Res. 2003 Nov 14;338(23):2477-89
references: 14670710
references: J Leukoc Biol. 2011 Oct;90(4):787-97
references: 21750122
references: J Biol Chem. 2010 Sep 17;285(38):29375-86
references: 20634284
references: FEMS Microbiol Immunol. 1992 Oct;5(4):201-10
references: 1419118
references: J Biol Chem. 2004 May 7;279(19):20044-8
references: 15014080
references: Infect Immun. 2004 Jan;72(1):371-80
references: 14688118
references: J Bacteriol. 2005 May;187(10):3374-83
references: 15866922
references: Immunol Res. 2008;41(3):188-202
references: 18592144
references: J Biol Chem. 2010 Nov 5;285(45):34330-6
references: 20801884
references: Biochemistry. 1999 Aug 17;38(33):10758-67
references: 10451371
references: Future Microbiol. 2011 Apr;6(4):391-405
references: 21526941
references: Proc Natl Acad Sci U S A. 2007 Mar 6;104(10):4136-41
references: 17360489
references: Infect Immun. 2009 Jan;77(1):152-61
references: 18981253
references: Annu Rev Biochem. 2002;71:635-700
references: 12045108
references: Infect Immun. 2006 Jun;74(6):3657-62
references: 16714598
references: Eur J Biochem. 2002 Dec;269(24):6112-8
references: 12473106
references: Infect Immun. 2009 Feb;77(2):615-21
references: 19015249
references: Microbes Infect. 2008 Jun;10(7):773-80
references: 18539500
references: J Leukoc Biol. 2009 Sep;86(3):469-70
references: 19720615
references: Eur J Biochem. 1985 Apr 1;148(1):1-5
references: 2579812
references: J Biol Chem. 2008 Feb 15;283(7):3751-60
references: 18079113
references: Microbiology (Reading). 2011 Nov;157(Pt 11):3172-3179
references: 21852349
references: Clin Vaccine Immunol. 2009 Mar;16(3):414-22
references: 19176692
references: Infect Immun. 2008 Jul;76(7):3001-10
references: 18411294
references: Infect Immun. 2010 Apr;78(4):1700-10
references: 20123717
references: J Immune Based Ther Vaccines. 2010 Feb 05;8(1):2
references: 20181102
references: Eur J Biochem. 2002 Mar;269(6):1746-52
references: 11895445
references: Infect Immun. 2002 Nov;70(11):6043-7
references: 12379680
references: Biochem Biophys Res Commun. 2004 Jan 23;313(4):856-62
references: 14706621
references: Am J Physiol Lung Cell Mol Physiol. 2009 Jun;296(6):L1076-84
references: 19346432
references: Infect Immun. 2007 Jan;75(1):536-41
references: 17030571
references: Mol Microbiol. 2010 Nov;78(4):820-36
references: 20662782
references: Trends Microbiol. 2007 May;15(5):233-40
references: 17398100