sticky fibers and uropathogenesis: bacterial adhesins in...

10.2217/17460913.1.1.75 © 2006 Future Medicine Ltd ISSN 1746-0913 Future Microbiol. (2006) 1(1), 75–87 75

REVIEW

Sticky fibers and uropathogenesis: bacterial adhesins in the urinary tractKelly J Wright & Scott J Hultgren†

†Author for correspondenceWashington University School of Medicine, Department of Molecular Microbiology, Box 8230, 660 S. Euclid Avenue, MO 63110, USATel.: +1 314 362 6772;Fax: +1 314 362 1998;[email protected]

Keywords: adhesins, chaperone–usher systems, fimbriae, pilus biogenesis, urinary tract infection, uropathogenicEscherichia coli

Adhesins mediate the introduction of bacteria to the host in the sometimes life-long relationship of uropathogenic Esherichia coli (UPEC) and the human urinary tract. As a class of extracellular proteins, adhesins enable bacteria to adhere to and, in some cases, invade host tissue; adhesins render UPEC virulent and permit host colonization. Adhesin receptor interactions at the host interface determine tissue tropism and disease progression in that niche, with each adhesin preferring unique sites within the urinary tract. This review focuses on known adhesins implicated in uropathogenesis, the structural basis of tissue tropism, postinvasion intracellular replication, current therapeutic design strategies, and newly discovered fimbrial gene clusters that may play a role in urinary tract infections.

Urinary tract infectionsIn the USA, more than 8 million urinary tractinfections (UTIs) occur each year in otherwisehealthy young women [1,2]. Approximately onein four women will experience a UTI withintheir lifetime and, of those, 30% will experi-ence a recurrent infection within 3–6 monthsof the initial infection [3]. This incidence rateimposes an economic burden of approximatelyUS$1.6 billion annually. The high costs oftreatment and the high incidence rate empha-size UTI as a significant infectious diseaseprocess, which requires greater attention tobetter understand the underlying, complexpathogenic mechanisms [1,2].

An individual’s susceptibility to UTIsdepends on behavioral, biological and geneticfactors. The ultimate infectious outcome isdetermined by the balance between these fac-tors and the bacteria’s arsenal of defenses [4].Escherichia coli is the predominant isolaterecovered from afflicted women and is respon-sible for 80–90% of uncomplicated UTIs.Staphylococcus saprophyticus is the next mostpredominant organism responsible for UTIsand accounts for the remaining 10–15% ofinfections in young, healthy women. Addi-tionally, Klebsiella, Proteus, Enterobacter andEnterococcus species can cause uncomplicatedUTIs, but are often associated with compli-cated, nosocomial infections in immuno-compromised, diabetic, elderly and urinarycatheterized patients [5]. To date, uro-pathogenic E. coli (UPEC) is the best charac-terized UTI pathogen and serves as anappropriate model organism for targetedtherapeutic design.

UPEC bacterial virulence factorsBacterial genomes have evolved from less viru-lent ancestors to include a variety of genes orvirulence factors necessary to overcome hostdefenses and subsequently establish diseasestates. Virulence gene acquisition is thought tohave occurred by horizontal gene transfer in thecase of pathogenicity islands (PAI), which con-tain gene clusters of a given functional category,or by selective environmental pressure. Viru-lence genes may reside on the chromosome ormay be episomal and the degree of virulence ofan organism is roughly proportional to thenumber of known virulence factors present inthe genome [6,7]. Virulence gene families identi-fied thus far include proteinaceous adhesins,toxins, proteases, iron acquisition systems andthe carbohydrate-based lipopolysaccharide andcapsule [8].

The toxins hemolysin (hlyA) and the secretedautotransporter protein (SAT) are associatedwith eukaryotic plasma membrane permeabili-zation and vacuolization, respectively. hlyA is amember of the repeats-in-toxin family andhlyA-induced pore formation in the plasmamembrane leads to dose-dependent calciumoscillations and the triggering of an inflamma-tory response in rat renal proximal tubulecells [9,10]. SAT, the secreted autotransportertoxin of UPEC, induces severe vacuolizationand compromises gap junctions of bladder andkidney epithelium in vitro. In vivo, SAT pro-duces significant renal histological changes in amouse model of UTI, including dissolution ofthe glomerular membrane and vacuolation ofproximal tubule cells [11]. UPEC may alsoencode for other toxins, such as cytotoxic

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necrotizing factor type 1 (CNF-1), which has amore controversial role in UTI. CNF-1 constitu-tively activates rho GTPases and has beenreported to inhibit phagocytosis by polymorpho-nuclear cells [12], modulate polymorphonuclearcell function [12] and kill bladder epithelial cellsin vitro [13]. Whereas Rippere-Lampe and col-leagues demonstrate significantly attenuatedbladder and urine colonization with cnf-deficientUPEC and a greater recovery of cnf-positiveUPEC in co-challenge experiments [14], Johnsonand colleagues report no attenuation in host col-onization or histological changes with cnfmutants [15]. Siderophore systems are probablyexploited by UPEC to scavenge iron andenhance their survival in nutrient-limited envi-ronments, such as urine and the bladder [8,16,17].Finally, most UPEC produce capsule, which isknown to inhibit complement activation andphagocytosis by human polymorphonuclear cells.These actions protect UPEC from the innateimmune system [18]. Bahrani-Mougeot and col-leagues recently identified capsule as a pre-emi-nent virulence determinant in a signature-taggedmutagenesis screen for genes essential for survivalin a mouse model of ascending UTI [19]. Never-theless, of all the virulence factors, adhesins arethe best studied owing to their crucial role in ini-tiating bacterial–host contact. Interception of thisintimate acquaintance is an ideal strategy to com-bat initial and recurrent host colonization eventsand to improve the lives of millions of peopleeach year who suffer from UTIs.

Adhesin structure & functionInitiation of disease requires intimate contact ofthe pathogen with the host. As their nameimplies, adhesins perform this function and ena-ble bacteria to adhere to abiotic and living tissues,a process that results in biofilm formation andinitiation of host colonization events, respec-tively. Biofilms formed on abiotic surfaces aredynamic bacterial communities that exhibit anti-biotic resistance, a growing concern in the treat-ment of bacterial infections [20]. In vivo, adhesinsfacilitate host colonization by mediating the firstand crucial interaction with host tissue. In thecase of UTIs, binding not only protects the bacte-rium from clearance by innate host defenses, suchas the shear forces encountered during urination,but may also mediate invasion, whereby replica-tion can occur in a protected, intracellularniche [21]. There are multiple families of adhesins,including type 4 pili, outer membrane proteins,curli, filamentous hemagglutinins and adhesive

pili, also termed fimbriae, assembled by the chap-erone–usher pathway. This review will focus onthe latter group.

Adhesins assembled by the chaperone–usherpathway are nearly ubiquitous in Gram-negativeorganisms [22], which often contain severaladhesin gene clusters [23,24]. Of importance in theurinary tract are the type 1, P, F1C, S and Afa/Drfamilies of adhesins. As a group, the adhesinsshare common genomic organization, assemblyand, in the case of the fimbrial adhesins or pili,they share similar quaternary structural traits.Adhesin regulatory genes precede the major subu-nit gene, which is followed by the periplasmicchaperone, outer membrane usher and, finally, theoperon terminates with the minor and adhesinsubunit genes (Figure 1). Organization of the papoperon differs by having the positions of the usherand chaperone inverted.

In addition to conserved organization of chap-erone–usher adhesin gene clusters, conservationof subunit structure is apparent at the tertiarylevel despite primary sequence differences. Thethree dimensional structures of theadhesin–chaperone [25], subunit–chaperone [26]

and adhesin–receptor complexes [27] have beensolved for type 1 and P pili, respectively, anddemonstrate that subunits contain a single pilindomain with an incomplete immunoglobulin(Ig)-fold topology. The adhesins contain twodomains: a pilin domain connected by a linker tothe adhesin domain and an elongated11-stranded β-barrel with a jelly roll-like topol-ogy in the case of FimH [25]. The adhesindomain confers receptor specificity and hence,tissue tropism to the pilus.

Crystallography and modeling studies haverevealed that adhesin–receptor interactions arestrikingly different among the fimbrialadhesins [28]. Thus, investigations of structurehave provided insights into adhesin function, tis-sue tropism and the basis for pathogenesis.Formed by residues invariant among more than200 UPEC strains, the type 1 pili adhesin, FimH,contains an acidic pocket at the distal tip thataccommodates mono-mannose oligosaccharides[25,29], whereas PapG, the P pilus adhesin, bindsα-D-galactopyranosyl(1-4)-β-D-galactopyranoside(GbO4)-containing receptors on the side usingan elongated eight-stranded β-sandwich [27].Structures of the F1C and S pili adhesins are notavailable but are expected to reveal unique bind-ing sites for lactosylceramide-containing glycolip-ids and sialylated glycoproteins, respectively.Modeling of DraE, the Dr adhesin, onto Caf1,

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Sticky fibers and uropathogenesis: bacterial adhesins in the urinary tract – REVIEW

the prototypic Yersinia pestis F1 antigen subunit,revealed two opposite binding sites. Daf-bindingsite residues are posited to cluster on a surfaceformed by DraE and an adjacent subunit [28].The type IV collagen-binding site is located onthe opposite face of the protein [30].

Assembly of the fimbrial adhesins or pili bythe highly conserved chaperone–usher pathwayoccurs via donor-strand exchange in which theIg-fold of each mature subunit is completed bythe N-terminal extension of the adjacent subunit(Figure 2) [31,32]. Pilus biogenesis has been com-prehensively reviewed elsewhere [33]. High reso-lution electron micrographs indicated thattype 1, P, F1C and S pili are composite hair-like

fibers that radiate peritrichously from the bacte-rial surface [28]. Pili consist of a right handed,rigid, hollow, helical rod comprised of thousandsof repeating major subunits joined to a distal,flexible tip fibrillum containing adapter proteinsand terminates with the cognate adhesin [34].The type 1 pili tip fibrillum is short and stubbyrelative to the longer and flexible P, F1C and Stip fibrillum. Dr adhesins were initially classifiedas ‘afimbrial’ but may, in fact, be fine fibers thatare sensitive to electron microscopy fixationtechniques [35].

Of importance in the urinary tract are thetype 1, P, F1C, S and Afa/Dr families of adhes-ins. Each adhesin recognizes a specific receptor

Figure 1. Schematic view of uropathogenic fimbrial adhesin gene clusters.

Genes of similar function are shown in the same color. Black: Regulatory proteins; purple: Major subunit; dark blue: Unknown function; light blue: Periplasmic chaperone; green: OM usher; yellow and orange: Minor subunits; red: Adhesin.OM: Outer membrane.

Regulators Majorsubunit

Chaperone OM usher Terminator/adaptor/initiator

?Regulators Majorsubunit


Mannose-binding adhesin

?

Tip fibrillum

B E A I C D F G H

ChaperoneRegulators Majorsubunit

OM usher Adaptor Adaptor AdhesinRod terminator

Tip fibrillum

I B A H C D J K E F G

Regulators Majorsubunit

Chaperone OM usher Terminator/adaptor/initiator adhesin

Adhesin??

Tip fibrillum

C B DA E F G S H

fim

pap

sfa

F1C

dra

Chaperone OM usher Adhesin

I B C D EA

Majorsubunit


Adhesin?

Tip fibrillum

I C D F G HA



in the urinary tract (Table 1). Type 1 pili areessential in mediating cystitis and they recog-nize mono- and tri-mannose oligosaccharidespresent on the luminal surface of bladder epi-thelium [29], Tamm-Horsfall protein [36], type 1and type IV collagens [37], laminin [38] andfibronectin [39]. The minimum PapG receptor

isotype is globotriasylceramide (GbO3). Recep-tor family members differ by addition of a sin-gle N-acetylgalactosamine (GalNAc) moiety toform globoside (GbO4), addition of twoGal-NAc moieties to create the Forssman anti-gen (GbO5), or by the addition of sialic acidsto form more complex receptors [27,40,41]. Three

Figure 2. Donor strand complementation and pilus biogenesis.

(A) Schematic diagram of donor-strand complementation in which the N-terminal extension of an adjacent pilus subunit complements the incomplete Ig-fold of its neighbor. (B) Ribbon diagram of the FimH adhesin domain stabilized by the chaperone, FimC G1 strand, prior to complementation by FimG during pilus biogenesis. (C) Pilus biogenesis. The periplasmic chaperone, FimC, stabilizes subunits prior to pilus biogenesis initiated at the usher, FimD, located on the inner leaflet of the outer membrane. Excess or misfolded subunits go off pathway and are degraded by periplasmic proteases/chaperones such as DegP. C (inset) High resolution electron micrograph of type 1 pili.Ig: Immunoglobulin.

D DD

H

H

G

G

F

H

G

F

F

A

A

A

A

A

C

C

C

C

C

C CC

C

CC

A

A

YE

G

YE

G

DegP

Outer membrane

Cytoplasmic membrane

Periplasm

COOH

NH2

A F

A

C

D E B A F C

D E B A F C

N-t

erm

inal

exte

nsio

nN

-ter

min

alex

tens

ion Fi

mC

G1

β st

rand

B



PapG alleles exist (class I, II and III) and differ-entially bind to receptor family members. Theclass II allele is primarily associated withhuman pyelonephritis and the class III alleleassociated with human cystitis [42–44]. F1C andS pili are thought to play a role in ascendingUTI. F1C pili recognize lactosylceramide-con-taining receptors [45,46] whereas S pili recognizeα-sialyl-2,3-β-galactoside (NeuNAcα[2-3]Gal)-containing receptors [47,48]. Dr adhesins do notrecognize carbohydrate-linked receptors butrather specifically bind the Dr(a+) blood groupantigen present on the complement cascaderegulatory factor, decay-accelerating factor,(DAF) or CD55 [49]. Dr adhesins are alsoknown to bind type IV collagen [50] and α5β1integrin [51]. Clinical isolate analysis suggestspregnant women to be most at risk for UTIscaused by UPEC that harbor Dr adhesins [52,53].

Adhesins & experimental models of UTIThe type of pili produced by different strains ofUPEC can determine the site of disease in theurinary tract. Type 1 piliated UPEC specificallybind mannosylated receptors on bladder epithe-lium and are an essential cystitis determinant;isogenic phase ‘lock off ’ and adhesin-deficienttype 1 piliated UPEC cannot bind to urothelialcells and colonize them [Wright et al. Unpublished

Data] [54]. F1C and P pili target glycosphingolipidreceptors in the kidney to preferentially causepyelonephritis. F1C pili may play a role inmediating mucosal inflammation as evidencedby renal epithelial cell interleukin-8 productionin response to interaction with F1C positivebacteria [45]. P piliation provided UPEC with a

fitness advantage in a murine model of ascend-ing UTI by enhancing colonization and reduc-ing the local secretory antibody immuneresponse [55]. Similarly, pyelonephritis did notdevelop in primates challenged with PapG-neg-ative pili [56]. Although S pili are predominantlycorrelated with neonatal meningitis and are har-bored by sepsis clinical isolates, a formal role inUTI has yet to be demonstrated. However,in situ hybridization experiments demonstratedthat S pili bind to human bladder and kidneyepithelium [57–59]. Dr family adhesins recognizeDAF, a complement regulatory protein foundon most mammalian cells, which protects cellsfrom autologous complement-mediateddamage [60]. Although Dr family adhesins areassociated with cystitis, these adhesins predomi-nantly cause chronic pyelonephritis in mice [61]

and human gestational pyelonephritis [53], andare implicated in mortality in a pregnant ratmodel of intrauterine infection [62]. While all ofthese pili–tissue interactions appear importantin UTI pathogenesis, the type 1 pili interactionwith bladder epithelium is best characterized.

Intracellular replication in bladder epitheliumHistorically, UTIs were considered luminalinfections. However, intracellular niches in theform of UPEC ‘pods’ were discovered as a newreservoir [63,64]. Uroplakin proteins (UPIa, Ib, IIand III) line the bladder lumen in the form ofhexameric, crystalline plaques, which form animpermeable barrier necessary for the preventionof solute and toxin retrograde absorption [65,66].Uropathogenic bacteria breach this barrier by

Table 1. Adhesin sugar specificity.

Adhesin Sugar/receptor specificity

Sugar structure Method for receptor Ref.

Type 1 Mono- and tri-mannose Man- and (Man)3- Hemagglutionation withcompetitive inhibitor X-ray crystallography

[30]

[26]

P pili Globotriasylceramide (GbO3)Globoside (GbO4)Forssman antigen (GbO5)

GalB1-4GalX1-4GlcCerGalNaCX1-3GalB1-4GalX1-4GlcCerGalNacB1-3GalNaCX1-3GalB1-4GalX1-4GlcCer

Hemagglutionation withcompetitive inhibitor X-ray crystallography

[87]

[28]

F1C pili Lactosylceramide-containingglycolipids*

GalX1-4GlcX1-Cer TLC overlay [46,47]

S pili B-sialyl-2,3-X-galactoside NeuNAcB(2-3)Gal- Neuramindase-sensitivehemagglutination

[48,49]

Dr/Afa Decay-accelerating factor – – –

*TLC overlay studies identified multiple F1C ligands; lactosylceramide was the common backbone of this family of ligands and the degree of F1C binding depended on fatty acid chain composition.TLC: Thin layer chromatography.



production of type 1 pili. FimH, the type 1 pilusadhesin, binds mannosylated uroplakins Ia andIb, initiating invasion via a partial zipperingmechanism [64]. Interestingly, UPIII is reportedto contain the S pilus receptor and S pili havebeen demonstrated to bind murine and humanbladder epithelium. Thus, the S pili–UPIIIinteraction may result in a protective bindingevent as well, although this hypothesis has notbeen tested formally in vivo.

FimH-mediated invasion into the cytoplasmicmilieu activates a complex developmentalprogram (Figure 3), whereby the bacteria form intra-cellular bacterial communities (IBC), a niche withbiofilm-like properties protected from innate hostdefenses and antibiotic treatment [63,64]. The cellu-lar details of the IBC cascade have been visualizedby high resolution time-lapse video microscopyusing green fluorescent protein-positive UPEC-infected, ex vivo bladder tissue [67]. The IBC matu-ration cascade is multifaceted and includes distinctevents (Figure 3). A loose collection of rod-shapedbacteria matures into a dense, globular shaped bio-mass containing more than 104 organisms withinsuperficial umbrella cells. The bacteria within earlyIBCs undergo a reversible rod to cocci morpholog-ical change to allow greater bacterial packingwithin the IBC to form a three-dimensional struc-ture reminiscent with biofilm-like properties [63,68].Realtime observation of IBC maturation suggestsactive aggregation mechanisms at work to maintainthe dense, compact IBC ultrastructure. Althoughthese aggregative factors are currently unknown,deepetch freeze fracture electron micrographsreveal fibers radiating from the bacterial surface,fibers similar to those presenting adhesins [63].

Middle, or midstage, IBCs contain only coc-coid bacteria with delayed doubling times ofgreater than 45 min. IBCs eventually reach matu-rity and the large intracellular biofilm-like masstakes over much of the facet cell cytoplasm. Oncemature, IBC dispersal begins and disseminationof IBC progeny to naïve superficial umbrella cellsinitiates subsequent rounds of IBC formation,presumably by type 1 pili FimH-mediated inva-sion. Upon IBC dispersal, a subpopulation ofUPEC has a filamentous morphology. Filamen-tous bacteria are more resistant to neutrophilattack, suggesting that filamentation may serve toprotect against host attack and facilitate bacterialsurvival in the bladder [67]. Cycles of IBC forma-tion continue at progressively slower rates untileventually, small clusters of intracellular bacteriaare left, which form a silent, quiescent reservoir.The events that follow are not well understood,

but are the focus of intense scientific inquiry: inresponse to unknown signals, bacteria within thisreservoir re-activate and re-enter the IBC cascadeto initiate recurrent infection [67].

Overall, properly timed and coordinated geneexpression undoubtedly functions to promoteIBC maturation, to maintain bacterial and IBCviability, and to protect against innate hostdefenses. The identification of factors that conferthe ability of UPEC to undergo the IBC cascadewill highlight novel virulence factors inuropathogenesis for targeted therapeutic devel-opment. Moreover, IBC-like communities havealso been described for Helicobactor pylori andPseudomonas aeruginosa in a chronic atrophicgastritis model [69] and tracheal epithelial cellmodel [70], respectively. It is an exciting andintriguing possibility that the IBC developmen-tal cascade is universal and represents a paradigmin pathogen persistence and resistance againsthost defenses.

Newly discovered adhesin gene clustersAll pathogens are not created equal in their abil-ity to cause disease. In addition to the fim andpap operons, virulent uropathogenic strainscontain other chaperone–usher pilus systemscomplete with putative adhesins. Most of thesenewly identified systems have yet to be charac-terized in urinary tract pathogenesis. The pres-ence of multiple pilus systems likely confersniche-adaptive advantages that, for example,enable a bacterium to access the urethra via thegenitourinary tract or to establish infectionthroughout the urinary tract, or to disseminatesystemically in extreme infectious cases. As eachsite within these routes is unique, the combina-tion of receptor specificity and tissue-specificreceptor production will ultimately determinethe site of action for a given pilus. Additionally,the newly discovered pilus systems may playroles in non-UTI disease processes.

Advent of the genomic era has allowedphylogenetic classification of uropathogens andhas refined our understanding of disease pheno-types associated with a particular clinical iso-late. Genome sequencing of a prototypiccystitis strain, UTI89, revealed ten differentchaperone–usher (adhesin systems) or pilus sys-tems [23] whose identity and locations are illus-trated in Figure 4. UTI89 contains the fim, pap,sfa, F17-like, auf, yad, yfc, yqi, yeh and fml oper-ons, of which fim and pap have been best char-acterized for their roles in pathogenesis, asdiscussed previously.



Although most commonly correlated withneonatal meningitis strains, the S-pili sfa genecluster is present and expressed among 25–30%of UPEC clinical isolates [71,72]. S pili have beenshown to bind the extracellular matrix compo-nents fibronectin and laminin [73,74] andsialoglycoproteins on brain microvascularendothelial cells [75], an interaction that mayexplain migration across physiological barriers.S pili also bind to human bladder and kidney

epithelium [57,76], suggesting relevance in UTI,although a direct correlation or role in diseasehas not been formally examined. Interestingly,pyelonephritic strain CFT073 contains theF1C (foc) operon in the same chromosomallocation as the UTI89 sfa operon, further corre-lating S-pili and uropathogenesis, since F1Cpili are suggested to be associated with UPECisolates [71,72,77,78]. The F17-like operon wasannotated based upon its similarity to the F17

Figure 3. Intracellular bacterial community developmental cascade.

Binding and invasion of a single bacterium may lead to intracellular replication, ultimately forming an intracellular bacterial community with biofilm-like properties. Upon IBC maturation, uropathogenic Escherichia coli (UPEC) disseminate for additional rounds of type 1 pili-mediated IBC formation in naïve superficial umbrella cells. Reservoir formation events are unclear at this time; however, reservoir UPEC are believed to reactivate and enter into the IBC cascade, resulting in recurrent urinary tract infection. Representative micrographs depict major events during the IBC cascade.Images modified from: *Mulvey and colleagues [64,86]; ‡Anderson and colleagues [63]; §Justice and colleagues [67].IBC: Intracellular bacterial communities.

?

Fluxing/filamentation

Binding Invasion

Reservoir formation

Early IBC

Middle IBCLate IBC

* *

‡

§



pilus system found on PAI I in pyelonephriticisolate, 536 [79]. The prevalence of this genecluster among UPEC isolates has yet to bedetermined and the F17 and F17-like pilus sys-tems remain largely uncharacterized in UTI.However, a role in UTI pathogenesis is possi-ble, given the conservation among the twoUPEC isolates.

Recently, a novel UPEC associated operon,auf, was identified in CFT073. Although thisoperon is highly prevalent in UPEC clinical iso-lates, deletion of this operon did not impactvirulence in a murine model of UTI [80]. InUTI89, this operon is nonfunctional owing to aframe-shift mutation in aufG, the Auf fimbrialadhesin, and probably will not play a role inUTI89. To date, the other pilus operons yad,yfc, yqi, yeh and fml remain uncharacterized. Acomprehensive analysis of each unknownchaperone–usher pilus in UTI pathogenesis iswarranted and may provide insight into themechanisms underlying disease initiation andprogression. Characterization of their roles inUTI pathogenesis will also provide new targetsfor therapeutic intervention in the treatmentand prevention of UTI.

Current & future therapiesThe clinical paradigm for UTI managementrelies upon antibiotic treatment of infection.However, antibiotic resistance and intracellularreservoirs often defeat this standard treatment.The ongoing advances in our understanding ofUTIs drive the development of several rationaldrug design approaches. One obvious strategy toinhibit UPEC colonization of the urinary tract isto inhibit pilus biogenesis. Another strategy, withparticular promise to prevent recurrent UTI, is totarget steps critical in the IBC developmental cas-cade. For example, one might inhibit the aggrega-tion of UPEC into the dense biofilm-likecollection of cells that renders protection fromantibiotic therapy. Or, one might target the proc-esses that lead to filamentation, which rendersprotection from host defenses. Alternatively, andperhaps most attractive, the ability to interceptthe crucial adhesin-mediated binding of bacteriato host tissue may be more feasible than abolish-ing pilus biogenesis and is a target that can pre-vent initial and recurrent UTI.

To date, several lines of evidence support pilusbiogenesis and adhesin inhibition as worthwhileapproaches. Kihlberg and colleagues have

Figure 4. Relative location and operon structure of known and putative adhesin gene clusters in cystitis strain, UTI89.

Gene cluster names are in italics. Genes of similar function are shown in the same color. Inset, type 1 piliated UTI89 bacterium.

nt 1

F17

pap

auf

yqi

yfc yeh

fml

sfa

yad

fim

UTI89 adhesingene clusters



described the development of P pili adhesininhibitors that are 30-fold more potent than thenatural ligand at blocking wild type PapG-GbO4interactions [81]. Although promising, these drugsonly target one pilus system and have yet to betested in vivo in experimental models of UTI.FimC and PapD chaperone functions are essen-tial for pilus biogenesis [82] and represent anothertarget for inhibition. Compounds that interferewith chaperone activity are termed ‘pilicides’ andhave proved efficient at significantly reducingboth type 1 and P pilus biogenesis [83]. Similar tothe PapG inhibitors, the pilicides are in need ofin vivo analyses. Traditional approaches in com-bating infectious disease have also proved promis-ing. Vaccination of mice and cynomolgusmonkeys with FimCH demonstrates a greaterthan 99% reduction in mucosal colonization [84]

and 75% reduction in both colonization andinflammation [85], respectively. Ultimately, acombinatorial treatment approach should beconsidered that addresses both intra- and extra-cellular bacterial niches and the multitude ofadhesins produced by the bacterium.

ConclusionUropathogenic bacteria navigate the complexlandscape of the genitourinary tract and ascendthe urethra to cause UTI via an arsenal ofadhesins and virulence factors. The data

reviewed here highlight the diversity of bacte-rial adhesins, the structural basis for tissue tro-pism, the consequences of type 1 pili-mediatedinvasion and the extensive number of pilusoperons within UPEC genomes. Presumably,the additional pilus operons confer niche-adap-tive adherence capabilities to the bacteriumthroughout the genito-urinary tract to aid inthe establishment of UTI. The additional pilussystems may also play roles elsewhere in otherphysiological (e.g., gastrointestinal) and naturalenvironments. Currently, type 1, P and Dradhesins are best characterized for their directroles in UTI. Nevertheless, the newly identifiedpilus gene clusters in UPEC genomes may pro-vide additional adhesive resources for efficienthost colonization.

Future perspectiveThe ever evolving use of niche-specific genes bybacteria illustrates the need for intelligent thera-peutic design in treating acute and recurrentUTI. Given the crucial nature of tissue bindingas the first step in the infectious process, theknown adhesins and yet to be characterizedadhesins provide rational targets for drug devel-opment. The substantial incidence rate and theaging population’s risk of UTI demand ourattention and necessitate the development ofmore efficacious UTI therapeutics.

Executive summary

• Urinary tract infections (UTIs) constitute one of the most common bacterial infections, with an incidence rate of more than 8 million infections per year.

• Uropathogenic Escherichia coli adhesins mediate adherence to and colonization of epithelial tissues throughout the urinary tract.

• Adhesin receptor specificity dictates tissue tropism and the subsequent urinary tract disease.

• Type1 pili bind mannosylated uroplakins in bladder epithelium and are essential cystitis virulence determinants.

• P pili bind α-D-galactopyranosol(1–4)-β-D-galactopyranoside (GbO4)-containing receptors in the upper urinary tract and initiate pyelonephritis.

• F1C pili recognize lactosylceramide receptors and are thought to play a role in ascending UTI.

• S pili recognize α-sialyl-2,3-β-galactoside (NeuNAcα(2–3)Gal)-containing receptors and are thought to play a role in ascending UTI.

• Dr family adhesins are associated with cystitis and are more commonly implicated in chronic and gestational pyelonephritis.

• Type 1 pili- mediated invasion leads to protected, intracellular replication in the form of intracellular biofilm communities.

• The current clinical paradigm must be dynamic and incorporate the newly discovered intracellular UPEC reservoir.

• This protected niche challenges clinicians and researchers in the treatment of UTI and the development of new therapeutics.

• Uropathogenic isolates contain multiple fimbrial systems for niche-adaptive adhesion during the establishment of UTI.

• Multiple adhesins provide additional targets for drug development to treat acute and recurrent UTIs using a combinatorial approach.



AcknowledgementsThe authors are grateful for helpful discussions with,advice from and critical review of this manuscript byLynette Cegelski, Karen Dodson, Sheryl Justice, IndiraMysorekar and Eric Miller. Figure 2B is courtesy of Craig

Smith PhD. This work was supported by NIH GrantR01DK51406, AI 48689, AI 29549 and ORWH SCORP50DK64540 with the FDA (S Hultgren) and partiallysupported as a Lucille P Markey Special Pathways Fellow(K Wright).

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• Cytotoxic necrotizing factor type 1 (CNF-1) is demonstrated to be necessary for optimal virulence relative to wild-type bacteria using single and co-infection studies in a murine model of urinary tract infection (UTI). Human neutrophil resistance is suggested to be the underlying mechanism of persistent infection, since CNF-1 mutants were more sensitive to human neutrophil killing.

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19. Bahrani-Mougeot FK, Buckles EL, Lockatell CV et al.: Type 1 fimbriae and extracellular polysaccharides are preeminent uropathogenic Escherichia coli virulence determinants in the murine urinary tract. Mol. Microbiol. 45, 1079–1093 (2002).

20. Donlan RM, Costerton JW: Biofilms: survival mechanisms of clinically relevant microorganisms. Clin. Microbiol. Rev. 15, 167–193 (2002).

21. Anderson GG, Martin SM, Hultgren SJ: Host subversion by formation of intracellular bacterial communities in the urinary tract. Microbes. Infect. 6, 1094–1101 (2004).

22. Hung DL, Hultgren SJ: Pilus biogenesis via the chaperone/usher pathway: an integration of structure and function. J. Struct. Biol. 124, 201–220 (1998).

23. Chen SL, Hung CS, Xu J et al.: Identification of genes subject to positive selection in uropathogenic strains of Escherichia coli: a comparative genomics approach. Proc. Natl. Acad. Sci. USA 103, 5977–5982 (2006).

•• The sequence of UTI89, a cystitis isolate, was determined and used with six other Escherichia coli genome sequences in a likelihood-based, genome-wide search for genes under positive selection. The authors propose that genes contributing to virulence in pathogenic organisms are under positive selection and present an analysis pipeline that can be applied to other organisms and pathogens.

24. Welch RA, Burland V, Plunkett G 3rd et al.: Extensive mosaic structure revealed by the complete genome sequence of uropathogenic Escherichia coli. Proc. Natl. Acad. Sci. USA 99, 17020–1704 (2002).

• Presents the complete genome sequence of pyelonephritis strain, CFT073, and compares it to genomes of other pathogenic and laboratory strains of E. coli to assess common and different virulence traits. This study highlights the power of genomics in providing insight into ancestral lineages among uropathogens.

25. Choudhury D, Thompson A, Stojanoff V et al.: X-ray structure of the FimC-FimH chaperone–adhesin complex from uropathogenic Escherichia coli. Science 285, 1061–1066 (1999).

•• X-ray crystallography techniques elucidate the three-dimensional structure of FimH and its carbohydrate ligand, α-D-mannose, to provide a structural understanding of receptor tropism for type 1 pili. The binding site for this sugar is formed by an acidic pocket at the distal tip of the adhesin domain.



26. Sauer FG, Futterer K, Pinkner JS, Dodson KW, Hultgren SJ, Waksman G: Structural basis of chaperone function and pilus biogenesis. Science 285, 1058–1061 (1999).

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•• 3D structure of the P pilus adhesin, PapG, in complex with its sugar receptor ligand is described in this study. Mutagenesis studies of the binding domain explore the specific functions of residues comprising the binding site to complement their examination of the differential binding specificities of the PapG alleles. Results reported here further highlight the structural basis for unique sugar specificity among uropathogenic Escherichia Coli (UPEC) adhesins.

28. Berglund J, Knight SD: Structural basis for bacterial adhesion in the urinary tract. Adv. Exp. Med. Biol. 535, 33–52 (2003).

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31. Sauer FG, Pinkner JS, Waksman G, Hultgren SJ: Chaperone priming of pilus subunits facilitates a topological transition that drives fiber formation. Cell 111, 543–551 (2002).

32. Barnhart MM, Sauer FG, Pinkner JS, Hultgren SJ: Chaperone-subunit-usher interactions required for donor strand exchange during bacterial pilus assembly. J. Bacteriol. 185, 2723–2730 (2003).

33. Sauer FG, Remaut H, Hultgren SJ, Waksman G: Fiber assembly by the chaperone–usher pathway. Biochim. Biophys. Acta. 1694, 259–267 (2004).

• Excellent background on pilus biogenesis, including detailed discussions of donor-strand exchange and donor-strand complementation.

34. Schilling JD, Mulvey MA, Hultgren SJ: Structure and function of Escherichia coli type 1 pili: new insight into the pathogenesis of urinary tract infections. J. Infect. Dis. 183(Suppl. 1), S36–S40 (2001).

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36. Pak J, Pu Y, Zhang ZT, Hasty DL, Wu XR: Tamm-Horsfall protein binds to type 1 fimbriated Escherichia coli and prevents E. coli from binding to uroplakin Ia and Ib receptors. J. Biol. Chem. 276, 9924–9930 (2001).

37. Pouttu R, Puustinen T, Virkola R et al.: Amino acid residue Ala-62 in the FimH fimbrial adhesin is critical for the adhesiveness of meningitis-associated Escherichia coli to collagens. Mol. Microbiol. 31, 1747–1757 (1999).

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44. Johanson IM, Plos K, Marklund BI, Svanborg C: Pap, papG and prsG DNA sequences in Escherichia coli from the fecal flora and the urinary tract. Microb. Pathog. 15, 121–129 (1993).

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• Site-directed mutagenesis studies confirm and map the Dr adhesin binding site to the third short consensus repeat domain in decay-accelerating factor cell–cell interaction assays.

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• Phase variation of the invertible element controlling Type 1 pili expression is demonstrated to significantly contribute to host colonization in a murine model of UTI. Phase-locked off E. coli CFTO73 mutants were reduced in their ability to colonize the urinary tract in single and co-infection studies whereas phase-locked on mutants out-performed wild type CFT073.



55. Rice JC, Peng T, Spence JS et al.: Pyelonephritic Escherichia coli expressing P fimbriae decrease immune response of the mouse kidney. J. Am. Soc. Nephrol. 16, 3583–3591 (2005).

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63. Anderson GG, Palermo JJ, Schilling JD, Roth R, Heuser J, Hultgren SJ: Intracellular bacterial biofilm-like pods in urinary tract infections. Science 301, 105–107 (2003).

• Describes intracellular collections of uropathogenic E. coli termed ‘pods’ that possess biofilm-like qualities. These dense pods contain bacteria encased in a polysaccharide-containing matrix protected by an uroplakin coating. Intracellular ‘pod’ formation may provide a protected niche to the bacteria for evasion of host defenses and establishment of persistent infection.

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•• The authors of this study utilized high resolution time-lapse video microscopy for imaging of ex vivo, uropathogenic E. coli-infected bladders. The fine details of intracellular bacterial community formation and maturation were observed, in addition to discovering filamentation as a novel defense mechanism for intracellular bacterial community-disseminated, extracellular bacteria.

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Affiliations• Kelly J Wright

Washington University School of Medicine, Department of Molecular Microbiology, Box 8230, Washington University School of Medicine, 660 S. Euclid Avenue, MO 63110, USATel.: +1 314 747 3627;Fax: +1 314 362 3230;[email protected]

• Scott J HultgrenWashington University School of Medicine, Department of Molecular Microbiology, Box 8230, Washington University School of Medicine, 660 S. Euclid Avenue, MO 63110, USATel.: +1 314 362 6772;Fax: +1 314 362 1998;[email protected]

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