Flagellin and Lung Infection
• JID 2007:196 (15 July) • 289
M A J O R A R T I C L E
The Role of Flagellin versus Motility in Acute
Lung Disease Caused by Pseudomonas aeruginosa
Viviane Balloy,1Amrisha Verma,2Sudha Kuravi,2Mustapha Si-Tahar,1Michel Chignard,1and Reuben Ramphal1
1Institut Pasteur, Unite ´ de De ´fense Inne ´e et Inflammation, and INSERM U874, Paris, France;2Department of Medicine, University of Florida, Gainesville
The flagellum of Pseudomonas aeruginosa has been implicated in acute pneumonia, and its flagellin is known
to cause lung inflammation. However, its proinflammatory role, versus its motility function, as a cause of
death by a whole bacterium has not been demonstrated. This issue was examined in a lung model of acute
infection using different flagellar mutants. We found that the absence of motility does not significantly alter
the LD50, whereas the production of excess amounts of flagellin lowers it and results in early death. Next, we
found that the absence of the Toll-like receptor 5 (TLR5) ligand, flagellin, results in slower clearance of this
organism from the lungs and a delay in the time to death. These findings demonstrate the dual role of flagellin
in host defense and in disease and suggest that the death in this model may be biphasic with flagellin playing
a role early in the disease.
Pneumonia caused by Pseudomonas aeruginosa is a ma-
jor cause of morbidity and mortality among patients
who require mechanical ventilation . Death in these
patients has been ascribed to a number of virulence
factors, including the type III secreted exotoxins [2–4],
phospholipase C , proteases , exotoxin A , and
Pseudomonas lipopolysaccharide . The flagellum has
also been implicated in death and virulence in a neo-
natal lung model of infection , but whether it is
flagellar mediated motility that confers virulence or the
proinflammatory activity of flagellin  has not been
demonstrated. The paradigm concerning the role of
flagellin is that as a pathogen-associated molecularpat-
tern (PAMP) sensed by Toll-like receptor 5 (TLR5), it
induces an inflammatory host defense response aimed
at the eradication of the pathogen. The best relevant
Received 11 September 2006; accepted 24 January 2007; electronically pub-
lished 11 June 2007.
Potential conflicts of interest: none reported.
Financial support: National Institutes of Health (grants AI 45014 and AI 47852
Reprints or correspondence: Dr. Reuben Ramphal, Dept. of Medicine, College
of Medicine, 1600 SW Archer Rd., Gainesville, FL 32610 (ramphr@medicine
The Journal of Infectious Diseases
? 2007 by the Infectious Diseases Society of America. All rights reserved.
support is that a common dominant TLR5 stop codon
polymorphism is associated with susceptibility to Le-
gionnaires disease . Additionally, the instillation of
in a marked inflammatory response [9, 12], implying
that, during infections in which living bacteria are in-
volved, Pseudomonas flagellin may be involved in trig-
gering host defenses or inflammatory damage. Thus, to
examine the role of Pseudomonas flagellin and motility
in acute lung infection, mice were infected with wild-
type and flagellar mutants of P. aeruginosa. The LD50
values of these strains were determined, survival ex-
periments were performed, and bacterial clearance and
the inflammatory response after challenges were mea-
sured. The results are consistent with a biphasic mode
of death of acute lung infections, with flagellin pos-
sibly being involved in the early mortality. Further-
more, the loss of motility itself does not attenuate
virulence as measured by the LD50, implying that it
is the absence of flagellin that attenuated virulence
in previous studies .
MATERIALS AND METHODS
1. Flagellar mutants were previously constructed or
The strains used are shown in table
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296 • JID 2007:196 (15 July) • Balloy et al.
the experiments with sublethal doses may be overwhelmed
when lethal doses are used. We have also introducedanartificial
situation, by use of mutants that release large amounts of mo-
nomeric flagellin, to demonstrate a role for flagellin. Whether
there is an actual in vivo counterpart—that is, clinical strains
that hyperproduce monomeric flagellin—is unknown, but it is
conceivable that strains with mutations in promoters or reg-
ulators may exist and can overproduce flagellin. Besides shed-
ding light on the probable role of flagellin versus motility in
disease and bacterial clearance in anacutelunginfectionmodel,
these studies demonstrate the general roles of TLR5-flagellin
interactions in a whole organism because of the ability to gen-
erate specific flagellin mutants.
1. Chastre J, Fagon FY. Ventilator-associated pneumonia. Am J Respir
Crit Care Med 2002;165:867–903.
2. Hauser AR, Cobb E, Bodi M, et al. Type III protein secretion is as-
sociated with poor clinical outcomes in patients with ventilator-as-
sociated pneumonia caused by Pseudomonas aeruginosa. Crit CareMed
3. Roy-Burman A, Savel RH, Racine S, et al. Type III protein secretion
is associated with death in lower respiratory and systemicPseudomonas
aeruginosa infections. J Infect Dis 2001;183:1767–74.
4. Schulert GS, Feltman H, Rabin SD, et al. Secretion of the toxin ExoU
from patients with hospital-acquired pneumonia. J Infect Dis 2003;
5. Wieland C, Siegmund WB, Senaldi G, Vasil ML, DinarelloCA,Fantuzzi
G. Pulmonary inflammation induced by Pseudomonas aeruginosa li-
popolysaccharide, phospholipase C, and exotoxin A: role of interferon
regulatory factor 1. Infect Immun 2002;70:1352–8.
6. Tang HB, DiMango E, Bryan R, et al. Contribution of specific Pseu-
domonas aeruginosa virulence factors to pathogenesis of pneumonia in
a neonatal mouse model of infection. Infect Immun 1996;64:37–43.
7. Hirakata Y, Furuya N, Tateda K, Kaku M, Yamaguchi K. In vivo pro-
duction of exotoxin A and its role in endogenous Pseudomonas aeru-
ginosa septicemia in mice. Infect Immun 1993;61:2468–73.
8. Skerrett SJ, Martin TR, Chi EY, Peschon JJ, Mohler KM, Wilson CB.
Role of the type 1 TNF receptor in lung inflammation after inhalation
of endotoxin of Pseudomonas aeruginosa. Am J Physiol 1999;276:
9. Feldman M, Bryan R, Rajan S, et al. Role of flagella in pathogenesis
of Pseudomonas aeruginosa pulmonary infection. Infect Immun 1998;
10. Hayashi F, Smith KD, Ozinsky A, et al. The innate immune response
to bacterial flagellin is mediated by Toll-like receptor 5. Nature 2001;
11. Hawn TR, Verbon A, Lettinga KD, et al. A common dominant TLR5
stop codon polymorphism abolishesflagellinsignalingandisassociated
12. Honko AN, Mizel SB. Mucosal administration of flagellin induces in-
nate immunity in the mouse lung. Infect Immun 2004;72:6676–9.
13. Dasgupta N, Wolfgang MC, Goodman AL, et al. A four-tiered tran-
scriptional regulatory circuit controls flagellar biogenesis in Pseudo-
monas aeruginosa. Mol Microbiol 2003;50:809–24.
14. Arora SK, Ritchings BW, Almira EC, Lory S, Ramphal R. Pseudomonas
Infect Immun 1998;66:1000–7.
15. Arora SK, Neely AN, Blair B, Lory S, Ramphal R. Role of motility and
flagellin glycosylation in the pathogenesis of Pseudomonas aeruginosa
burn wound infections. Infect Immun 2005;73:4395–8.
16. Homma M, Fujita H, Yamaguchi S, Iino T. Excretion of unassembled
flagellin by Salmonella typhimurium mutants deficient in hook-asso-
ciated proteins. J Bacteriol 1984;159:1056–9.
17. Verma A, Arora SK, Kuravi S, Ramphal R. Roles of specific aminoacids
in the N terminus of Pseudomonas aeruginosa flagellin and of flagellin
glycosylation in the innate immune response. Infect Immun 2005;73:
18. Totten PA, Lory S. Characterization of the type a flagellin gene from
Pseudomonas aeruginosa PAK. J Bacteriol 1990;172:7188–99.
19. HoangTT, KutchmaAJ, BecherA,SchweizerHP.Integration-proficient
plasmids for Pseudomonas aeruginosa: site-specific integration and use
for engineering of reporter and expression strains. Plasmid 2000;43:
20. Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP. A
broad-host-range Flp-FRT recombination system for site-specific ex-
cision of chromosomally-located DNA sequences: application for iso-
lation of unmarked Pseudomonas aeruginosa mutants. Gene 1998;212:
21. Ramphal R, Balloy V, Huerre M, Si-Tahar M, Chignard M. TLRs 2 and
4 are not involved in hypersusceptibility to acute Pseudomonas aeru-
ginosa lung infections. J Immunol 2005;175:3927–34.
22. Hirano S. Migratory responses of PMN after intraperitoneal and in-
tratracheal administration of lipopolysaccharide. Am J Physiol 1996;
23. Montie TC, Doyle-Huntzinger D, Craven RC, Holder IA. Loss of vir-
ulence associated with absence of flagellum in an isogenic mutant of
Pseudomonas aeruginosa in the burned-mouse model. Infect Immun
24. Hybiske K, Ichikawa JK, Huang S, Lory SJ, Machen TE. Cystic fibrosis
airway epithelial cell polarity and bacterial flagellin determine host
response to Pseudomonas aeruginosa. Cell Microbiol 2004;6:49–63.
25. Shimizu R, Taguchi F, Marutani M, et al. The delta fliD mutant of
Pseudomonas syringae pv. tabaci, which secretes flagellin monomers,
induces a strong hypersensitive reaction (HR) in non-hosttomatocells.
Mol Genet Genomics 2003;269:21–30.
26. Ren T, Zamboni DS, Roy CR, Dietrich W, Vance RE. Flagellin-deficient
Legionella mutants evade caspase-1 and Naip5-mediated macrophage
immunity. PLoS Pathog 2006;2:e18.
27. Molofsky AB, Byrne BG, Whitfield NN, et al. Cytosolic recognition of
flagellin by mouse macrophages restricts Legionella pneumophila in-
fection. J Exp Med 2006;203:1093–104.
28. Feuillet V, Medjane S, Mondor I, et al. InvolvementofToll-likereceptor
5 in the recognition of flagellated bacteria. Proc Natl Acad Sci USA
29. Skerrett SJ, Wilson CB, Liggitt HD, Hajjar AM. Redundant Toll-like
receptor signaling in the pulmonary host response to Pseudomonas
aeruginosa. Am J Physiol Lung Cell Mol Physiol 2007;292:L312–22.
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