Immunology Letters 101 (2005) 117–122

Review

Salmonella flagellin, a microbial target of the innateand adaptive immune system

Rosa Maria Salazar-Gonzalez, Stephen J. McSorley∗

Department of Medicine, Division of Immunology, University of Connecticut Health Center, Farmington, CT 06030-1319, USA

Received 9 May 2005; accepted 13 May 2005Available online 6 June 2005

Abstract

Bacterial flagellins are important components of the motility apparatus used by many microbial pathogens. These proteins are also targetsof the innate and adaptive immune response of the host during infection and autoimmune disease. Flagellin interacts with TLR-5 and leads tothe generation of a pro-inflammatory response and activation of host dendritic cells in vivo. Furthermore, flagellin is recognized by antibodyand CD4 T cells responses duringSalmonella infection. Here, we review recent developments in the understanding of flagellin interactionswith the host immune system.©

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2005 Elsevier B.V. All rights reserved.

eywords: Flagellin;Salmonella; Innate immunity

ontents

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2. Structure and function of flagellin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3. The role of flagella inSalmonella pathogenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4. Structural interactions between flagellin and TLR-5. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5. Pro-inflammatory activity of flagellin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6. Flagellin and inflammatory disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7. Adjuvant activity of flagellin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8. Adaptive immune response to flagellin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. Introduction

Bacterial flagellins have been studied for decades, due tohe importance of these proteins in bacterial motility, andhe complex nature of flagellar gene expression. However,enewed interest in flagellins arises from the recent under-tanding that these molecules are specifically recognized by

∗ Corresponding author. Tel.: +1 860 679 8785; fax: +1 860 679 1868.E-mail address: mcsorley@uchc.edu (S.J. McSorley).

the innate immune system. Therefore, these microbial pucts are members of a growing family of pathogen-associmolecule patterns (PAMPs), molecules that are used bymammalian host to detect invasion by microbial pathogRecent work in our laboratory, and others, points to thethat Salmonella flagellin is a target of both the innate aadaptive immune response during murine typhoid. Herediscuss the interaction of bacterial flagellins with the innand adaptive immune system, placing particular emphasthe role of this interaction duringSalmonella infection.

165-2478/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.imlet.2005.05.004

118 R.M. Salazar-Gonzalez, S.J. McSorley / Immunology Letters 101 (2005) 117–122

2. Structure and function of flagellin

Flagellin is the major protein constituent of bacterial flag-ella, complex surface appendages that are involved in bacte-rial locomotion. More than 50 genes are known to be involvedin the regulated expression and function of the flagellum,implying that motility and chemotaxis are critically impor-tant for bacterial survival[1]. Flagella are part of the sensorymachinery that allow bacteria to either respond to chemo-tactic stimuli, or simply avoid an unfavorable environment,such as extreme pH or saline concentrations. The structureis a self-assembling sub-system, consisting of a long helicalfilament emerging from a flexible hook, connected to a basalbody that is anchored in the inner and outer cell membrane[1]. When in use, the flagellum exhibits a random patternof movement characterized by two different turns, a coun-terclockwise rotation, when the bacteria packs all filamentsinto one structure, and an alternative clockwise rotation thatcauses separation of the filaments and chaotic movement ofthe bacteria[2].

Polymers of a single protein, flagellin, compose the 11 fila-ments of each flagellum.Salmonella flagellin is comprised of494 amino acids and distinct domains have been described,based on homology between differentSalmonella serovars[3–5]. Both the amino- and carboxy-terminus are well con-served amongSalmonella serovars, while the central portiond medt stals att thea losep fil-a ting3 lini plee to bet

3

eti-c aved eriala ma-t llin-d es strain[ thatfl i, inc ringw rm per-s[ llai eria

to the cell surface, biofilm formation and ultimate lethality[11,12].

Salmonella infection of inbred mouse strains is the bestavailable laboratory model to study human typhoid fever[13]and flagellin expression is not normally considered to be avirulence factor[14,15]. However, recent experiments usingrabbits uncovered a potential role for flagella in the initialinteraction ofSalmonella with M cells of the appendix[16].Furthermore, flagellin expression is required forSalmonellainvasiveness in a cell culture model, and for the inductionof polymorphonuclear leukocyte infiltration using the calfintestine model of infection[15]. Therefore, although flag-ellin appears to be dispensable forSalmonella virulence in themouse model, there are multiple lines of evidence to suggestthat it is critically important for establishment ofSalmonellainfection in other species. The importance of flagellin expres-sion to human typhoid fever remains to be established, butthe available data from the models above suggest that it couldplay an important role inSalmonella typhi pathogenesis.

4. Structural interactions between flagellin andTLR-5

TLR-5 is expressed by epithelial cells, monocytes, andimmature dendritic cells[17,18]. Initial identification of flag-e teds tantp ti-viT bothG sim-p ia.

ousn truc-t ntg fterp strat-i ento onalm g-e esr d ind ini oft ored vitywT ellinc

llini enti ogni-t ctureo areu uta-

isplays more diversity, one particular region being terhe “hypervariable region”. Recent analysis of the crytructure of aSalmonella flagellin fragment revealed thhe protein filament is folded back upon itself such thatmino- and carboxy-termini are physically located in croximity to each other, within the central portion of thement, and are likely involved in determining the repea-D structure[6,7]. In contrast, the central portion of flagel

s exposed on the outside of the filament, providing a simxplanation for the fact that antibody responses tendargeted to this region[8].

. The role of flagella in Salmonella pathogenesis

Mucosal infection with bacterial strains that are genally deficient in flagellin or flagellar-associated genes hemonstrated an obligatory role for the flagella in bactdhesion to epithelial surfaces, colonization, biofilm for

ion, and invasion of host tissues. For example, a flageeficient strain ofPseudomonas aeruginosa was found to beverely attenuated compared to the flagellated parental9]. Histological analysis in this model demonstratedagellin-deficient strains cause a localized infection focontrast to the typical spreading of infection observed duild-type Pseudomonas pulmonary infection[9]. In anotheodel system, expression of flagella is required for the

istence ofHelicobacter pylori following infection of piglets10]. For infection withVibrio species, production of flages known to contribute to initial attachment of the bact

llin as the ligand for TLR-5 came from a study that isolatimulatory components from Listeria culture supernaroteins by HPLC[19]. Parallel studies examining the acation of epithelial cells in response toSalmonella alsodentified flagellin as a stimulatory ligand for TLR-5[20].hus, TLR-5 can recognize flagellin that is produced byram-positive and Gram-negative bacteria, providing ale strategy for the host to respond to flagellated bacter

In contrast to many other TLR ligands, the proteinaceature of flagellin has enabled a detailed study of the s

ural basis of flagellin–TLR-5 interactions. Two differeroups have documented bioactivity of flagellin even aroduction in a eukaryotic expression system, demon

ng that the stimulatory capacity of flagellin is independf other bacterial proteins or prokaryotic post-translatiodifications[21,22]. Using defined deletion mutants of flallin, two different groups initially identified two distinct sitequired for bioactivity, the hypervariable region localizeomain 3, and the conserved amino- and carboxy-term

he protein[23,24]. This discrepancy was resolved by metailed mapping, which defined TLR-5 stimulatory actiithin N-amino terminal residues 79–117 and 408–439[22].hus, TLR-5 most likely recognizes a spatial area of flagomprising areas of the amino- and carboxy-terminus.

The observation that the TLR-5 binding site of flages most likely located within the central base of the filams interesting since the residues needed for TLR-5 recion are the same residues essential for the native struf the flagellum and bacterial motility. Thus, the bacterianable to avoid host recognition by flagellin sequence m

R.M. Salazar-Gonzalez, S.J. McSorley / Immunology Letters 101 (2005) 117–122 119

Fig. 1. Unresolved issues with regard to flagellin interactions with theimmune system. Flagellin induces pro-inflammatory cytokine productionand dendritic cell (DC) activation, eventually leading to CD4 T cell acti-vation. However, several key aspects of this activity are unclear and arehighlighted in the figure numerically. (1) What is the bioactive form of flag-ellin (polymer or oligomers) that activates pro-inflammatory activity? (2)Does flagellin induce DC maturation directly, via TLR-5 expressed on theDC, or (3) indirectly through cytokines produced by other cell types? (4)What type of adaptive immune response is induced by the adjuvant effect offlagellin, TH1, or TH2?

tion without compromising the beneficial aspects of bacterialmotility. However, it is not yet clear how the internal structureof the flagellum filament would actually come in contact withTLR-5 in order to mediate pro-inflammatory activity in vivo(Fig. 1)? One possibility is that monomers or oligomers offlagellin are released from the filament in the hostile environ-ment of the phagosome, or may be exposed during bacterialreplication[25,26].

5. Pro-inflammatory activity of flagellin

Initial studies using human blood mononuclear cellsdemonstrated that flagellin from different bacterial speciesstimulated the production of cytokines, such as TNF-�and IL-1-� [25,27]. Furthermore, flagellin produced byS.typhi was found to induce synthesis of IL-6 and the anti-inflammatory cytokine IL-10[25,27–29].

More recent studies by Gewirtz et al. identified flagellinas the bacterial factor inducing IL-8 production followingthe interaction ofSalmonella with human intestinal epithelialcells [30]. Interestingly, the activation of this inflammatoryresponse was dependent upon the translocation of flagellinto the basolateral surface of the epithelial cells, where TLR-5 is expressed[20,30]. Presumably, this requirement limitsepithelial cell activation to pathogens that can translocatefl or-m ellina tedt uentm mu-

lus[31]. In contrast to other studies, this report suggested thatapical expression of TLR-5 was responsible for chemokinesecretion[31]. In agreement with this notion, constitutiveexpression of TLR-5 has been reported on the apical sur-face of human primary IECs[17], and another recent studydemonstrated that flagellin from commensal bacteria caninduce TLR-5 activation on the apical surface of freshly iso-lated murine IECs[32]. Future studies are obviously requiredto resolve the issue of TLR-5 expression on the surface ofepithelial cells and flagellin translocation (Fig. 1). If TLR-5is expressed on the apical surface of epithelial cells, it remainsunclear why a permanent inflammatory state is not induced inthe intestinal mucosa in response to flagellated commensalbacterial. One possibility is that commensal microbes mayalso induce anti-inflammatory mediators or block inflamma-tory signaling pathways of epithelial cells[33,34].

TLR-5 is also highly expressed in the lung[35] and seemsto play an important role in the defense against pathogensof the respiratory tract[36,37]. Interestingly, there is a cor-relation between a common human TLR-5 polymorphismand susceptibility to Legionnaires disease[38]. A single-point mutation to a stop codon at position 392 resulted inimpaired responsiveness to flagellin in these patients[38].Taken together, the available evidence suggests that expres-sion of TLR-5 in the intestinal and respiratory mucosa is animportant innate immune sensor for flagellated pathogens.

6

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agellin, and therefore, avoids TLR-5 ligation by the nal intestinal flora. Other studies have examined flagctivation of human intestinal epithelial cell lines and no

he production of the chemokine CCL-20, and the subseqigration of immature human DC in response to this sti

. Flagellin and inflammatory disease

Flagellin can induce expression of numerousnflammatory mediators, such as TNF-�, IL-1, IL-6, MIP-3�,nd iNOS, and therefore, may be involved in the deveent of bacterial-associated pathology. In support ofypothesis, direct intratracheal instillation of 1�g of flag-llin to mice resulted in an acute inflammatory process in

ung, involving neutrophil and macrophage infiltration ahe detection of inflammatory cytokines in the bronchoalar lavage (BAL) fluid[39]. Surprisingly, the administratiof flagellin was more potent than LPS in the developmf a lung inflammatory response[39], which could be du

o low levels of TLR-4 in the lung, as described for inteal epithelium[17]. It is possible, therefore, that chronic luathology caused by flagellated pathogens likePseudomonasr Legionella is due in part to host responses to flage

37,38].Analogous to endotoxin, flagellin is an effective med

or of systemic inflammation. Intravenous injection of mith flagellin rapidly produced the typical pattern of cytokihemokine, and NO production, as well causing the cigns of septic shock; hypotension, respiratory distyanosis, organ injury, and death[26,40]. Although it is nolear whether LPS or flagellin is more effective at cauoxic shock in mice[26,40,41], these data demonstrate tagellin could be an important contributor to the inflammory processes during bacterial sepsis.

120 R.M. Salazar-Gonzalez, S.J. McSorley / Immunology Letters 101 (2005) 117–122

Crohn’s disease and ulcerative colitis are intestinal inflam-matory disorders mediated by an abnormal immune responseto commensal microbes. The identification of potential tar-get antigens of the immune response has been a particularfocus of research in this field. Recently, two different groupsidentified bacterial flagellin as an immunodominant anti-gen in Crohn’s disease[42,43]. High titers of anti-flagellinantibodies were detected in Crohn’s patients and coliticmice. Although these studies identified flagellin as a tar-get of the adaptive immune response, it is also likely thatthe innate pro-inflammatory activity of bacterial flagellinscan contribute to the pathology associated with this intestinaldisease.

7. Adjuvant activity of flagellin

As with other PAMPs, the function of flagellin as anadjuvant has been examined in detail. An initial report byour laboratory demonstrated thatSalmonella flagellin wascapable of enhancing antigen specific CD4+ T cell expan-sion and memory development in vivo[21]. This adjuvanteffect of flagellin was dependent upon the ability to acti-vate CD80/86 molecules expression and was completelyblocked by administration of CTLA-4-Ig. Another interest-ing report noted that bacterial flagellin can directly activateh tingt ectae otherr en-d a-t ciesb atorys den-d andf sue(

mento n ofIo on-t ineS ngfl of aT calTo rob-o thata nti-b ne vantm ffl in-i esed

8. Adaptive immune response to flagellin

As already noted above, bacterial flagellins are targets ofthe adaptive immune response during Crohn’s disease, andrecent reports also suggest that flagellins may be importantT and B cell targets during bacterial infection. Indeed, bac-terial flagellins have been used for decades as target antigensto study the immune response in vivo. Elegant studies exam-ining tolerance induction in rats and B cell antigen receptorrecognition, made use of bacterial flagellin as a target antigen[51–53]. Furthermore, the polymeric structure and abundanceof protein expressed by bacteria led to the use of flagellinas a carrier protein for B and T cell epitopes[54,55]. Morerecently, flagellin has been identified as a major target antigenof CD4+ T cell cells during murine and humanSalmonellainfection[56–58]. The poor definition of other targets of theadaptive immune response toSalmonella make evaluation ofthe importance of the flagellin-specific response difficult[59].However, we do know that T cell responses toSalmonellaflagellin occur rapidly after oral infection, and may con-tribute to vaccine-induced immunity[58,60]. Therefore, itseems likely that adaptive immune responses to flagellin areimportant in defense against bacterial infection.

9. Conclusion

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uman but not murine dendritic cells in vitro, suggeshat any adjuvant effect of flagellin in mice may be indirnd involve TLR-5 ligation on other cell types[44]. How-ver, this latter observation has been challenged by aneport that described direct flagellin activation of murine dritic cells in vitro[45]. Differences in dendritic cell matur

ion or flagellin purification may explain these discrepanetween these studies. Preliminary data in our laboruggest that flagellin does not directly activate murineritic cells (Salazar and McSorley, unpublished data),

uture experiments will hopefully resolve this important isFig. 1).

In our hands, and others, flagellin induced the developf antigen-specific Th1 response involving the productio

FN-� and not IL-4 upon recall stimulation[21,46,47]. Thesebservations are consistent with a role for flagellin in c

ributing to the normal development of Th1 during muralmonella infection[48,49]. However, another report usiagellin as an adjuvant has described the generationh2 CD4+ T cell response, finding IL-4, IL-13, and a typih2 antibody response after immunization[45]. This abilityf flagellin to generate a Th2 response in vivo was corrated by other group, who demonstrated convincinglydministration of soluble flagellin caused a Th2-like aody response[50]. It remains unclear why flagellin cavoke such different T cell responses in different adjuodel systems (Fig. 1). It seems likely that the purity o

agellin preparation, extent of polymerization, dose admstered, and/or route of administration will explain thifferences.

Bacterial flagellins are interesting microbial proteinsre targeted by both the innate and adaptive immune resp

t seems likely that these proteins are critical to the depment of protective immunity to bacterial infection, a

he development of inflammatory conditions due to baial infection. The use of flagellins as part of vaccinedjuvant preparations may lead to the development of n

ive or sub-unit prophylactic treatments for several infectiseases.

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