Francisella tularensis causes the zoonotic disease tularemia. Arthropod vectors are important transmission routes for the disease, although it is not known how Francisella survives the efficient arthropod immune response. Here, we used Drosophila melanogaster as a model host for Francisella infections and investigated whether the bacteria are resistant to insect humoral immune responses, in particular to the antimicrobial peptides (AMPs) secreted into the insect hemolymph. Moreover, we asked to what extent such resistance might depend on lipopolysaccharide (LPS) structure and surface characteristics of the bacteria. We analyzed Francisella novicida mutant strains in genes, directly or indirectly involved in specific steps of LPS biosynthesis, for virulence in wild-type and RelishE20 immune-deficient flies, and tested selected mutants for sensitivity to AMPs in vitro. We demonstrate that Francisella is sensitive to specific fly AMPs, i.e. Attacin, Cecropin, Drosocin and Drosomycin. Furthermore, six bacterial genes, kpsF, manB, lpxF, slt, tolA and pal, were found to be required for resistance to Relish-dependent immune responses, illustrating the importance of structural details of Francisella lipid A and Kdo core for interactions with AMPs. Interestingly, a more negative surface charge and lack of O-antigen did not render mutant bacteria more sensitive to cationic AMPs and did not attenuate virulence in flies.

Keywords:
Francisella tularensis, Drosophila melanogaster, Antimicrobial peptides, Lipopolysaccharide, Host-pathogen interactions
1.
Larsson P, Elfsmark D, Svensson K, Wikstrom P, Forsman M, Brettin T, Keim P, Johansson A: Molecular evolutionary consequences of niche restriction in Francisella tularensis, a facultative intracellular pathogen. PLoS Pathog 2009;5:e1000472.
2.
Petersen JM, Mead PS, Schriefer ME: Francisella tularensis: an arthropod-borne pathogen. Vet Res 2009;40:7.
3.
Goethert HK, Telford SR 3rd: Nonrandom distribution of vector ticks (dermacentor variabilis) infected by Francisella tularensis. PLoS Pathog 2009;5:e1000319.
4.
Reese SM, Dietrich G, Dolan MC, Sheldon SW, Piesman J, Petersen JM, Eisen RJ: Transmission dynamics of Francisella tularensis subspecies and clades by nymphal Dermacentor variabilis (Acari: Ixodidae). Am J Trop Med Hyg 2010;83:645–652.
5.
Triebenbach AN, Vogl SJ, Lotspeich-Cole L, Sikes DS, Happ GM, Hueffer K: Detection of Francisella tularensis in alaskan mosquitoes (Diptera: Culicidae) and assessment of a laboratory model for transmission. J Med Entomol 2010;47:639–648.
6.
Lundström JO, Andersson A-C, Bäckman S, Schäfer ML, Forsman M, Thelaus J: Detection of Francisella tularensis holarctica in adult mosquitoes hatched from field collected larvae, suggest a novel transmission cycle originating in aquatic larval habitats. Emerg Infect Dis 2011;17:794–799.
7.
Vonkavaara M, Telepnev MV, Ryden P, Sjöstedt A, Stöven S: Drosophila melanogaster as a model for elucidating the pathogenicity of Francisella tularensis. Cell Microbiol 2008;10:1327–1338.
8.
Zasloff M: Antimicrobial peptides of multicellular organisms. Nature 2002;415:389–395.
9.
Hajjar AM, Harvey MD, Shaffer SA, Goodlett DR, Sjostedt A, Edebro H, Forsman M, Bystrom M, Pelletier M, Wilson CB, Miller SI, Skerrett SJ, Ernst RK: Lack of in vitro and in vivo recognition of Francisella tularensis subspecies lipopolysaccharide by toll-like receptors. Infect Immun 2006;74:6730–6738.
10.
Gunn JS, Ernst RK: The structure and function of Francisella lipopolysaccharide. Ann NY Acad Sci 2007;1105:202–218.
11.
Zhao J, Raetz CR: A two-component Kdo hydrolase in the inner membrane of Francisella novicida. Mol Microbiol 2010;78:820–836.
12.
Wang X, Ribeiro AA, Guan Z, Abraham SN, Raetz CR: Attenuated virulence of a Francisella mutant lacking the lipid a 4’-phosphatase. Proc Natl Acad Sci USA 2007;104:4136–4141.
13.
Gallagher LA, Ramage E, Jacobs MA, Kaul R, Brittnacher M, Manoil C: A comprehensive transposon mutant library of Francisella novicida, a bioweapon surrogate. Proc Natl Acad Sci USA 2007;104:1009–1014.
14.
Raynaud C, Meibom KL, Lety MA, Dubail I, Candela T, Frapy E, Charbit A: Role of the wbt locus of Francisella tularensis in lipopolysaccharide O-antigen biogenesis and pathogenicity. Infect Immun 2007;75:536–541.
15.
Lai XH, Shirley RL, Crosa L, Kanistanon D, Tempel R, Ernst RK, Gallagher LA, Manoil C, Heffron F: Mutations of Francisella novicida that alter the mechanism of its phagocytosis by murine macrophages. PLoS One 2010;5:e11857.
16.
Tzou P, Reichhart J-M, Lemaitre B: Constitutive expression of a single antimicrobial peptide can restore wild-type resistance to infection in immunodeficient drosophila mutants. Proc Natl Acad Sci USA 2002;99:2152–2157.
17.
Hedengren M, Åsling B, Dushay MS, Ando I, Ekengren S, Wihlborg M, Hultmark D: Relish, a central factor in the control of humoral, but not cellular immunity in Drosophila. Mol Cell 1999;4:827–837.
18.
Åhlund MK, Ryden P, Sjöstedt A, Stöven S: Directed screen of Francisella novicida virulence determinants using drosophila melanogaster. Infect Immun 2010;78:3118–3128.
19.
Hultmark D: Quantification of Antimicrobial Activity, Using the Inhibition Zone Assay. Fair Haven, SOS Publications, 1998.
20.
Boutros M, Agaisse H, Perrimon N: Sequential activation of signaling pathways during innate immune responses in Drosophila. Dev Cell 2002;3:711–722.
21.
De Gregorio E, Spellman PT, Tzou P, Rubin GM, Lemaitre B: The Toll and Imd pathways are the major regulators of the immune response in Drosophila. Embo J 2002;21:2568–2579.
22.
Pal S, Wu J, Wu LP: Microarray analyses reveal distinct roles for Rel proteins in the Drosophila immune response. Dev Comp Immunol 2008;32:50–60.
23.
Rohmer L, Fong C, Abmayr S, Wasnick M, Larson Freeman TJ, Radey M, Guina T, Svensson K, Hayden HS, Jacobs M, Gallagher LA, Manoil C, Ernst RK, Drees B, Buckley D, Haugen E, Bovee D, Zhou Y, Chang J, Levy R, Lim R, Gillett W, Guenthener D, Kang A, Shaffer SA, Taylor G, Chen J, Gallis B, D’Argenio DA, Forsman M, Olson MV, Goodlett DR, Kaul R, Miller SI, Brittnacher MJ: Comparison of Francisella tularensis genomes reveals evolutionary events associated with the emergence of human pathogenic strains. Genome Biol 2007;8:R102.
24.
Asare R, Akimana C, Jones S, Abu Kwaik Y: Molecular bases of proliferation of Francisella tularensis in arthropod vectors. Environ Microbiol 2010;12:2587–2612.
25.
Moule MG, Monack DM, Schneider DS: Reciprocal analysis of Francisella novicida infections of a drosophila melanogaster model reveal host-pathogen conflicts mediated by reactive oxygen and imd-regulated innate immune response. PLoS Pathog 2010;6:e1001065.
26.
Sebastian S, Dillon ST, Lynch JG, Blalock LT, Balon E, Lee KT, Comstock LE, Conlan JW, Rubin EJ, Tzianabos AO, Kasper DL: A defined O-antigen polysaccharide mutant of Francisella tularensis live vaccine strain has attenuated virulence while retaining its protective capacity. Infect Immun 2007;75:2591–2602.
27.
Lindemann SR, Peng K, Long ME, Hunt JR, Apicella MA, Monack DM, Allen LA, Jones BD: Francisella tularensis schu s4 O-antigen and capsule biosynthesis gene mutants induce early cell death in human macrophages. Infect Immun 2011;79:581–594.
28.
Tzeng YL, Datta A, Strole C, Kolli VS, Birck MR, Taylor WP, Carlson RW, Woodard RW, Stephens DS: KpsF is the arabinose-5-phosphate isomerase required for 3-deoxy-D-manno-octulosonic acid biosynthesis and for both lipooligosaccharide assembly and capsular polysaccharide expression in Neisseria meningitidis. J Biol Chem 2002;277:24103–24113.
29.
Godlewska R, Wisniewska K, Pietras Z, Jagusztyn-Krynicka EK: Peptidoglycan-associated lipoprotein (Pal) of Gram-negative bacteria: function, structure, role in pathogenesis and potential application in immunoprophylaxis. FEMS Microbiol Lett 2009;298:1–11.
30.
Vines ED, Marolda CL, Balachandran A, Valvano MA: Defective O-antigen polymerization in TolA and pal mutants of Escherichia coli in response to extracytoplasmic stress. J Bacteriol 2005;187:3359–3368.
31.
Kim TH, Sebastian S, Pinkham JT, Ross RA, Blalock LT, Kasper DL: Characterization of the O-antigen polymerase (Wzy) of Francisella tularensis. J Biol Chem 2010;285:27839–27849.
32.
Boman HG: Antibacterial peptides: basic facts and emerging concepts. J Intern Med 2003;254:197–215.
33.
Brogden KA: Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 2005;3:238–250.
34.
Silvestro L, Weiser JN, Axelsen PH: Antibacterial and antimembrane activities of cecropin A in Escherichia coli. Antimicrob Agents Chemother 2000;44:602–607.
35.
Otvos L Jr., Insug O, Rogers ME, Consolvo PJ, Condie BA, Lovas S, Bulet P, Blaszczyk-Thurin M: Interaction between heat shock proteins and antimicrobial peptides. Biochemistry 2000;39:14150–14159.
36.
Carlsson A, Nystrom T, de Cock H, Bennich H: Attacin – an insect immune protein – binds LPS and triggers the specific inhibition of bacterial outer-membrane protein synthesis. Microbiology 1998;144:2179–2188.
37.
Kanistanon D, Hajjar AM, Pelletier MR, Gallagher LA, Kalhorn T, Shaffer SA, Goodlett DR, Rohmer L, Brittnacher MJ, Skerrett SJ, Ernst RK: A Francisella mutant in lipid a carbohydrate modification elicits protective immunity. PLoS Pathog 2008;4:e24.
38.
Han S, Bishop BM, van Hoek ML: Antimicrobial activity of human beta-defensins and induction by Francisella. Biochem Biophys Res Commun 2008;371:670–674.
39.
Clay CD, Soni S, Gunn JS, Schlesinger LS: Evasion of complement-mediated lysis and complement C3 deposition are regulated by Francisella tularensis lipopolysaccharide O antigen. J Immunol 2008;181:5568–5578.
40.
Waterhouse RM, Kriventseva EV, Meister S, Xi Z, Alvarez KS, Bartholomay LC, Barillas-Mury C, Bian G, Blandin S, Christensen BM, Dong Y, Jiang H, Kanost MR, Koutsos AC, Levashina EA, Li J, Ligoxygakis P, Maccallum RM, Mayhew GF, Mendes A, Michel K, Osta MA, Paskewitz S, Shin SW, Vlachou D, Wang L, Wei W, Zheng L, Zou Z, Severson DW, Raikhel AS, Kafatos FC, Dimopoulos G, Zdobnov EM, Christophides GK: Evolutionary dynamics of immune-related genes and pathways in disease-vector mosquitoes. Science 2007;316:1738–1743.
© 2012 S. Karger AG, Basel
2012
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.