Proc. Natl. Acad. Sci. USAVol. 93, pp. 9827-9832, September 1996Medical Sciences

Type 1 fimbrial expression enhances Escherichia coli virulencefor the urinary tract

(urinary tract infection/inflammation/adherence/neutrophil influx)

H. CONNELL*tt, W. AGACE*t, P. KLEMM§, M. SCHEMBRI§, S. MARILD$, AND C. SVANBORG**Department of Medical Microbiology, Division of Clinical Immunology, Lund University, S-22362 Lund, Sweden; §Department of Microbiology, DanishTechnical University, DK-2800 Lyngby, Denmark; and IDepartment of Pediatrics, University of Goteborg, S-41685 Goteborg, Sweden

Communicated by Emil C. Gotschlich, The Rockefeller University, New York, NY April 19, 1996 (received for review October 25, 1995)

ABSTRACT Type 1 fimbriae are adhesion organelles ex-pressed by many Gram-negative bacteria. They facilitateadherence to mucosal surfaces and inflammatory cells in vitro,but their contribution to virulence has not been defined. Thisstudy presents evidence that type 1 fimbriae increase thevirulence ofEscherichia coli for the urinary tract by promotingbacterial persistence and enhancing the inflammatory re-sponse to infection. In a clinical study, we observed thatdisease severity was greater in children infected with E. coliO1:Kl:H7 isolates expressing type 1 fimbriae than in thoseinfected with type 1 negative isolates of the same serotype. TheE. coli O1:Kl:H7 isolates had the same electrophoretic type,were hemolysin-negative, expressed P fimbriae, and carriedthefim DNA sequences. When tested in a mouse urinary tractinfection model, the type 1-positive E. coli O1:Kl:H7 isolatessurvived in higher numbers, and induced a greater neutrophilinflux into the urine, than O1:Kl:H7 type 1-negative isolates.To confirm a role of type 1 fimbriae, a flmH null mutant(CN1016) was constructed from an O1:Kl:H7 type 1-positiveparent. E. coli CN1016 had reduced survival and inflammato-genicity in the mouse urinary tract infection model. E. coliCN1016 reconstituted with type 1 fimbriae (E. coli CN1018)had restored virulence similar to that of the wild-type parentstrain. These results show that type 1 fimbriae in the geneticbackground of a uropathogenic strain contribute to thepathogenesis of E. coli in the urinary tract.

Fimbriae-mediated adherence is important for the virulence ofEscherichia coli in the urinary tract (1). Uropathogenic E. colistrains may express a variety of fimbrial adherence factors,such as P, S, Dr, and type 1 fimbriae (2). It has been establishedthat P fimbriae enhance the virulence of uropathogenic strainsthrough specific adherence and increased induction of mucosalinflammation (1), but the role of type 1 fimbriae in virulenceremains undefined (3). Studies in animal models have sug-gested that type 1 fimbriation increases the survival of E. coliin the urinary tract (4-7); however, epidemiological studieshave failed to reveal a correlation between type 1 fimbriationand virulence (8).Type 1 fimbriae are encoded by the chromosomally located

fim gene cluster. The fimbriae consist of a major structuralsubunit (FimA) and several minor components, including theadhesin (FimH). The FimH adhesin is located at the fimbrialtip and interspersed along the shaft of the fimbriae (9). FimHrecognizes terminally located D-mannose moieties on cell-bound and secreted glycoproteins (10, 11). It was also recentlyshown to mediate mannose-sensitive binding to nonglycosy-lated peptide epitopes (12). Interactions with such receptorsenable type 1-fimbriated bacteria to bind to a range of cells,including erythrocytes, epithelial cells, granulocytes, macro-phages, and mast cells (13-16). This interaction can result in

the activation of the respiratory burst in granulocytes, degran-ulation of mast cells, and cytokine release from epithelial cellsin vitro (16-18).

In a recent study of acute pyelonephritis in children, E. coliO1:Kl:H7 emerged as the most common serotype (19). The 14children infected with E. coli O1:Kl:H7 isolates showed a morerapid onset of infection, higher fever, longer fever duration,and higher blood leukocyte counts when compared withchildren infected with other E. coli serotypes. The most severedisease occurred in a subset of the children with O1:Kl:H7strains that expressed type 1 fimbriae. The aim of this study wasto examine the role of type 1 fimbrial expression for thevirulence of E. coli O1:Kl:H7 in these children.

MATERIALS AND METHODSPatients. Children with febrile urinary tract infection (UTI)

were enrolled in a prospective study of symptomatic bacteri-uria in childhood (19). Fever was defined as a rectal temper-ature >38.50C within 48 h of enrollment. Bacteriuria wasdefined as homogenous growth of > 105 colony-formingunits/ml in a mid-stream urine sample, or as any growth inurine obtained by suprapubic bladder aspiration. The 88children (65 girls and 23 boys) experienced their first knownUTI episode. Their ages ranged from 2 months to 6 years(median age was 0.8 year). Several parameters were recordedto assess the host response to infection. These parametersincluded (i) the duration of symptoms before the start ofantibacterial treatment, assessed by an interview of the parentsat enrollment; (ii) the body temperature, defined as the meanof the recordings obtained during the first 24 h of hospitaladmission; (iii) the duration of fever, defined as the time fromthe start of antibiotic treatment until the first of two consec-utive recordings of a temperature <37.80C; and (iv) leukocytecounts, determined from blood and uncentrifuged urine. Forother diagnostic criteria and laboratory parameters, see ref. 19.

Bacteria. Urine was cultured by routine laboratory tech-niques. Of the 88 children, 84 were infected with E. coli (19).The E. coli isolates were serotyped (20), and 14 isolates wereidentified as O1:Kl:H7. The clonal identity of these 14 isolateswas determined by multilocus enzyme electrophoresis (21) formalate dehydrogenase, ,B-galactosidase, 6-phosphogluconatedehydrogenase, adenylate kinase, phenylalanyl-leucine pepti-dase, leucyl-glycyl-glycine peptidase, isocitrate dehydrogenase,phosphoglucose isomerase, aconitase, glucose-6-phosphatedehydrogenase, and glutamate oxaloacetate transaminase.Each isolate was assigned an electrophoretic type based onthese 11 electromorphs.Hemolysin production was assessed by plating the strains on

nutrient agar containing 5% washed horse erythrocytes and

Abbreviations: UTI, urinary tract infection; RFLP, restriction frag-ment length polymorphism; IL, interleukin.

I tH.C. and W.A. contributed equally to this work.ITo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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observation of a hemolytic zone larger than the overlyingcolony.

Expression of P fimbriae was analyzed after culture ontryptic soy agar (Difco) for 16 h at 37°C. Strains agglutinatinghuman erythrocytes of the AP1 blood group in the presence ofD-mannose, and which failed to agglutinate Ap erythrocytes,were considered P-fimbriated (22). The expression of type 1fimbriae was analyzed after overnight culture in Luria brothcontaining 0.1% CaCl2. Strains agglutinating guinea pig eryth-rocytes in the absence but not in the presence of D-mannosewere considered type 1-fimbriated (13).DNA Probes and DNA-DNA Hybridizations. A 10-kb Hin-

dIll-Styl fragment, containing the fim gene cluster from therecombinant plasmid pPKLO4 (23), was used as a probe for thefim DNA sequences. A 1.0-kb HindIII-EcoRI fragment, con-taining thefimH gene from the recombinant plasmid pLPA22(24), was used as a probe for the fimH DNA sequences. A1.9-kb HincII fragment from the recombinant plasmid pCK155(25) was used as a probe for the neomycin phosphotransferasegene (npt). A 12.3-kb BamHI fragment, containing the papgene cluster from the recombinant plasmid pDC1 (26), wasused as a probe for the pap DNA sequences.Chromosomal DNA isolated from each E. coli strain (27)

was cut withAval (Boehringer Mannheim) to probe for thefimsequences and with HindIII (Boehringer Mannheim) to probefor the pap sequences. The cleaved DNA was electrophoresedon 0.7% agarose (Boehringer Mannheim) gels and transferredto nylon membranes (Hybond-N, Amersham) by alkali blottingusing a Vacuum blotting apparatus (Bio-Rad). The blots wereprehybridized at 62°C for 2-4 h in 2x standard saline citrate(SSC; lx SSC = 0.15 M sodium chloride/0.015 M sodiumcitrate, pH 7), 1% SDS, 1% skim milk (Difco), and 0.1 mg/mlfreshly denatured herring sperm DNA (Sigma). The DNAprobes were labeled with [a-32P]dCTP using the RediPrimeDNA labeling kit (Amersham), and 0.1-0.5 ,ug/ml freshlydenatured probe DNA was added to the prehybridizationsolution. Hybridization was carried out at 62°C for 16-18 h.Posthybridization filters were washed twice in 2x SSC/0.1%(wt/vol) SDS for 5 min at room temperature, followed by twowashes in 0.1 x SSC/0.1% (wt/vol) SDS for 15 min at 62°C, andexposed to Kodak X-Omatic film at -70°C for 24-48 h. Thefragment sizes were determined by comparison to a 1-kb DNAladder (GIBCO/BRL).

Construction of E. coli CN1016 (E. coli 1177AfImH). ThefimH sequences were deleted from E. coli 1177 by P1 trans-duction from afimH null mutant, E. coli MS4 (28). The cloningstrategy used to construct the strain E. coli MS4 (E. coli PC31fimH null mutant) is shown in Fig. 1. E. coli MS4 wastransfected with bacteriophage P1 and a chloroform bacterio-phage lysate was prepared (33). This lysate was then used totransfect E. coli 1177 and an E. coli 1177fimH null mutant (E.coli CN1016) was selected by kanamycin resistance (30 gg/ml)and loss of mannose-sensitive agglutination of guinea pigerythrocytes.

Construction of E. coli CN1018. E. coli CN1016 was elec-trotransformed as previously described (34) with the plasmidpPKL04 (26). Transformants were selected by kanamycin (30,ug/ml) and ampicillin (100 ,ug/ml) resistance. Transformantsexpressing both antibiotic resistances were also tested for therestoration of mannose-sensitive agglutination of guinea pigerythrocytes.

Experimental UTI. C3H/HeN mice were bred in the animalfacilities, Department of Medical Microbiology, Lund Univer-sity. Female mice were used at 8-14 weeks of age. The E. colistrains were grown in Luria broth overnight, harvested bycentrifugation, and resuspended in 0.01 M PBS (pH 7.2) to-109 bacteria per ml. Bacteria (0.1 ml) were inoculated intothe mice by urethral catheterization under ether anesthesia asdescribed (35).

Cm{fi( cII

BamHl

pPKL52 npt

fmD'

27 np

fimEcoRV HicII

KpnI

Sanl EcoRVAm r R6K

pGP7O4

Cm

SaiI EcoRV

pCH102 hinDD'

~ ~ m

KpnIIHincIII

\ EcoRVAmp fimD'

rimF

A1mGpCH103 DrmH'

orl R6 npt

Sall ~ flmH'

FIG. 1. Plasmids used in the construction of the E. coli 1177fimHnull mutant E. coli CN1016. A neomycin phosphatase gene (npt; the1.9-kb HincII from pCK155) (25) conferring kanamycin resistance wasinserted into the KpnI site of thefimH gene on plasmid pPKL52 (29).The resulting plasmid (pCH102) was used to transform E. coli MC1000(30). Transformants that were kanamycin- and chloramphenicol-resistant were selected. The plasmid pGP704 (31) isolated from E. coliCC118(Apir) (32) and the plasmid pCH102 isolated from E. coliMC1000 were digested with SalI and EcoRV. The 3.7-kb SalI-EcoRVfragment of pGP704 and the 7.1-kb SalI-EcoRV fragment frompCH102 were ligated to form pCH103, which was used to transformE. coli CC118(Apir). Transformants that were kanamycin- and ampi-cillin-resistant were selected. Plasmid pCH103 was isolated from E.coli CC118(Apir) and used to transform E. coli PC31 (23). Afterallowing double crossover and recombination to occur at 370C,ampicillin-sensitive and kanamycin-resistant recombinants were se-lected (E. coli MS4) (28). The oi R6K origin of replication isApir-dependent and temperature-sensitive (30°C). E. coli PC31 doesnot contain the lysogenic phage Apir. ThefimH sequences were deletedfrom E. coli 1177 by Pl transduction of DNA from E. coli MS4 to E.coli 1177.

Bacteria in kidneys and bladders were quantitated by viablecounts on tissue homogenates-obtained at the time the micewere killed, 24 h after infection (35). Leukocyte numbers inurine samples taken at 0, 2, 6, and 24 h after inoculation werecounted using a Burker chamber (36). Urine samples weretaken from individual mice before each experiment and ex-amined for the presence of neutrophils. Mice with a preexistingneutrophil response were excluded.Immunoelectromicroscopy. Immunoelectronmicroscopy

was carried out as described (37). Briefly, cells from anovernight culture were harvested, washed in PBS, and incu-bated at room temperature for 1 h with rabbit anti-type-i-fimbrial anti-serum (1:5) (38). After washing, bacteria wereincubated at room temperature for 1 h with 15 nm gold-labeledgoat anti-rabbit IgG (H+L; 1:10; Amersham). A 15-,ul aliquotof washed bacteria was adsorbed to a carbon-coated, glow-discharged copper grid for 30 s and then negatively stainedwith 2% sodium phosphotungstenate (pH 7.0). The grids wereobserved at 60 kV in a Philips CM10 Transmission ElectronMicroscope (Philips, Eindhoven, The Netherlands).

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Statistics. The Mann-Whitney U test and Pearson Corre-lation were used where applicable.

RESULTS

Characterization of the O1:Kl:H7 Isolates. The E. coliO1:Kl:H7 isolates were examined for variation in electro-phoretic type, hemolysin expression, and fimbrial phenotype.The 14 isolates had the same electrophoretic type type, asdefined by the 11 electromorphs. Two isolates (E. coli 3116 andE. coli 1106) had a null allele for phenylalanyl-leucine. All theisolates expressed P fimbriae and tested negative for hemolyticactivity. Eight of the Ol:Kl:H7 isolates expressed type 1fimbriae, whereas four did not. The remaining two isolateswere not tested for type 1 expression at the time of isolationand were removed from the study.Host Response to E. coli O1:Kl:H7 in Relation to Type

1-Fimbrial Expression. The inflammatory response was com-pared among the 8 children infected with the type 1-positive E.coli O1:Kl:H7 isolates, the 4 children infected with the type1-negative E. coli O1:Kl:H7 isolates, and the 54 childreninfected with P-fimbriatedE. coli strains other than O1:K1:H7.Children infected with type 1-positive E. coli O1:Kl:H7 iso-lates had more rapid onset of symptoms before antibiotictreatment, higher fever, longer fever duration, and higherleukocyte counts than children infected with other E. colistrains. The inflammatory responses did not differ betweenpatients infected with the type 1-negative E. coli O1:Kl:H7isolates and patients infected with other E. coli strains (Table1). The host response in children with type 1-positive or type1-negative E. coli could not be compared due to the smallnumbers.

Fimbrial Genotype. The O1:Kl:H7 isolates carried the fimandpap DNA sequences. Threefim restriction fragment lengthpolymorphisms (RFLPs) were observed after genomic diges-tion with AvaI. A single 23-kb fragment was found in twostrains (both type 1-negative). Three fragments of 8.8, 3.6, and3.0 kb were found in 2 strains (one type 1-positive; one type1-negative). A third group of 7 strains (six type 1-positive; onetype 1-negative) exhibited an RFLP pattern similar to that ofE. coli PC31 (8.8/6/1.6/1.3/1.1 kb) (24). Twopap RFLPs wereobserved after genomic digestion with HindIII. Three frag-ments of 10, 3.8, and 2.2 kb were shared by all strains. Inaddition, two strains (both type 1-negative) had extra bands of7.5 and 1.5 kb. There was no correlation between the RFLPpatterns and type 1-fimbrial phenotype.

Experimental UTI. Three E. coli O1:Kl:H7 isolates wereselected for in vivo experiments in the mouse UTI model: E.coli 1177 expressing type 1-fimbriae, E. coli 1106 with a weaktype 1-fimbrial expression, and E. coli 845 that did not expresstype 1 fimbriae.

Higher numbers of E. coli 1177 were recovered from themouse bladders and kidneys than those of E. coli 845 (P <0.0001 andP < 0.0001, respectively) andE. coli 1106 (P < 0.007andP < 0.04, respectively) 24 h after infection (Table 2). E. coli

Table 2. Bacterial recovery after experimental UTI in mice

E. coli No. of Type 1 Bacterial recovery*strain mice expression Bladder Kidneys

1177 38 +++ 4,770 (21) 9,798 (22)1106 12 154 (68) 699 (55)845 32 - 2 (4.3) 19 (10.6)

1177 31 +++ 16,648 (4) 8,519 (7)CN1016 23 - 5.2 (15) 395 (5)CN1018 12 +++ 11,731 (6) 16,482 (12)*Geometric mean of bacterial counts on homogenized tissue, 24 hafter infection; SD of geometric mean is in parentheses.

tE. coli 1106 was recently shown to express a FimH protein withdifferent isoreceptor specificity to the FimH ofE. coli 1177 (D. Hasty,personal communication).

1106 persisted in greater numbers than E. coli 845 in mousebladders (P < 0.0004) and kidneys (P < 0.009).The inflammatory response to infection was greater in those

animals infected with the type 1-expressing O1:Kl:H7 isolates.Urinary neutrophil numbers were higher at 2 h (P < 0.0005),6 h (P < 0.01), and 24 h (P < 0.0001) after infection with E.coli 1177 than after infection with E. coli 845 (Fig. 2A). Urinaryneutrophil numbers were greater at 2 h (P < 0.017) and 24 h(P < 0.002) after infection withE. coli 1106 than after infectionwith E. coli 845.

Verification of the AflmH Construction in E. coli CN1O16and of the Restored Type 1 Phenotype in E. coli CN1018. ThefimH null sequences were successfully P1-transduced from E.coli MS4 toE. coli 1177 with the resultingfimH null phenotype.The fimH null mutant constructions in E. coli MS4 andCN1016 were verified by Southern blot analysis. The Southernblot patterns of E. coli PC31, MS4 (E. coli PC31AfimH), 1177,and CN1016 (E. coli 1177AfimH) are shown in Fig. 3. E. coliCN1016 hybridized with thefimH probe (Fig. 3A) and the nptprobe (Fig. 3B). The fragment sizes in Fig. 3 A and B wereconsistent with those predicted for PvuII digestion of E. coliCN1016. E. coli CN1016 agglutinated human AP1 erythrocytesin the presence of D-mannose (P fimbriae-positive) and failedto agglutinate guinea pig erythrocytes in the absence ofD-mannose (type 1 fimbriae-negative). P1 transduction and theresulting recombination in E. coli CN1016 did not result in thedisturbance of any genes 9.0 kb upstream or 5.0 kb downstreamoffimH as verified by Southern blot analysis (data not shown).The plasmids pCH102 and pCH103 were constructed for use

in the derivation of an E. coli 1177fimH null mutant (see Fig.1). E. coli 1177was transformed with these plasmids but doublecrossover recombinant transformants were not isolated. Theseplasmids were used to construct a fimH null mutant in theintestinal isolate E. coli PC31 (E. coli MS4).The transformation of E. coli CN1018 with pPKLO4 was

verified by plasmid extraction and Southern blot analysis (datanot shown) using the 10-kb HindIII-Styl fragment from therecombinant plasmid pPKLO4 (23). E. coli CN1018 carried a

Table 1. Host response* to infection with E. coli O1:K1:H7 or other E. coli serotypes

Symptoms,t Fever - White blood celldays Fever, °C duration, h serum, 109/liter Urine, mm3

O1:K1:H7 E. coliType 1+, n = 8 1.0t (1.9) 39.1§ (39.1) 36t (36) 20.0§ (24) 1050 (5254)Type 1-, n = 4 2.5 (2.5) 38.3 (38.4) 22 (23) 18.5 (20) 760 (818)

Other E. coli 4.0 (5.0) 37.9 (38.1) 17 (20) 15.0 (16) 800 (1463)Sixty-eight of the 84 E. coli strains expressed P fimbriae; 14 were E. coli O1:K1:H7, leaving 54 E. coli strains for comparison.

*Host response variables are given as medians with means in parentheses.tSymptom duration before diagnosis.*P < 0.02.§P < 0.004 for difference to other P-fimbriated E. coli isolates.

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A 300

200262

2000

Co

Z 100

00 2 6 24

Time I HoursE

jE 200-0

C

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00 2 6 24

Time /HoursFIG. 2. Urinary neutrophil influx in response to intravesical infec-

tion. Each mouse received 108 bacteria. (A) Wild-type isolates. *, E.coli 1177 (type 1-positive); *, E. coli 1106 (weak type 1-positive); 0, E.coli 845 (type 1-negative). Mean of 12-38 mice with standard error. (B)*, E. coli 1177 (type 1-positive); El, E. coli CN1016 (1177 fimH nullmutant; type 1-negative); o, E. coli CN1018 (E. coli CN1016/pPKL04;type 1-positive). Mean of 14 mice with standard error.

plasmid of the correct molecular weight which hybridized withthe 10-kb HindIII-StyI fragment from pPKLO4.Immunoelectronmicroscopy. Cells of E. coli 1177, CN1016,

and CN1018 were stained with anti-type-1-fimbrial antibody(Fig. 4). E. coli HB101, HB101/pPKLO4 (fim+) (23), andHB101/pDC1 (pap+) (26) were used as controls for nonfim-brial, type 1-fimbrial, and P-fimbrial expression, respectively.E. coli HB101/pPKLO4, 1177, and CN1018 all stained positivefor type 1 fimbriae (Fig. 4 B, D, and F), whereas E. coli HB101,HB101/pDC1, and CN1016 did not (Fig. 4 A, C, and E). Itappeared that the deletion offimH from E. coli 1177 resultedin the loss of type 1 fimbriae, as evidenced by the lack ofimmunogold staining on E. coli CN1016 (Fig. 4E). Expressionwas restored following the addition of the fim sequences (E.coli CN1018) (Fig. 4F). The type 1 antiserum used for theimmunogold staining was specific for type 1 fimbriae as it didnot react with E. coli HB101 (Fig. 4A) orE. coli HB101/pDC1expressing P fimbriae (Fig. 4C).

Virulence of E. coli 1177, CN1016, and CN1018. The role oftype 1 fimbriae for bacterial survival and neutrophil influx wasfurther examined by comparing E. coli 1177 with E. coliCN1016 and E. coli CN1018 in the mouse UTI model. E. coli1177 and E. coli CN1018 survived in higher numbers in boththe kidneys (P < 0.0001) and bladders (P < 0.0001) comparedwith E. coli CN1016 24 h after infection (Table 2).A significantdifference in bacterial numbers in the kidneys and bladders ofmice infected with E. coli CN1018 or E. coli 1177 was notobserved.

Urinary neutrophil numbers were higher at 2 h (P < 0.0001),6 h (P < 0.0001), and 24 h (P < 0.0001) after infection with E.coli 1177 or E. coli CN1018 than after infection with E. coliCN1016 (Fig. 2B). Urinary neutrophil numbers were higher at2 h (P < 0.013) in mice infected with E. coli 1177 than E. coli

A B

1.8 -

1.4-1.8

* -1.1

* ---0.4

1 2 3 4 1 2 3 4

C(i) 1177

(ii) CN1016

0.1 4.3 1.1 1.4it T t

LJ-J I 1 LAJ WX J11 LJ_I E JL H

0.1

I4.3 1.1 1.1 0.4 1.8

L t 1 t'WBf I F. LJ W LLJI: 11 1 i

H

IOkb

FIG. 3. The Southern blots of PvuII-cleaved chromosomal DNAfrom E. coli PC31 (lane 1), MS4 (lane 2), 1177 (lane 3), and CN1016(lane 4) were probed with the 1.0-kb fimH (A) and the 1.9-kb nptfragments (B). (C) The PvuII restriction sites (*) for E. coli 1177 andCN1016 (drawn to scale).

CN1018. A significant difference in urinary neutrophil num-bers after infection with E. coli 1177 or E. coli CN1018 at 6 and24 h was not observed.

DISCUSSIONThis study was prompted by a clinical observation in childrenwith febrile UTI. Out of 88 children with acute pyelonephritis,14 carried E. coli of serotype O1:K1:H7 (19). These childrenhad a shorter duration of symptoms before antibiotic treat-ment, indicating a more rapid onset of symptoms. They alsohad higher fever, longer fever duration, and higher white bloodcell counts than children infected with other E. coli isolates.We examined the properties of the E. coli O1:K1:H7 isolatesin an attempt to explain the increased virulence associatedwith this serotype. The O1:K1:H7 E. coli strains were shown tobe members of the same clone, and they were all P-fimbriatedbut differed in their expression of type 1 fimbriae. Childreninfected with type 1-positive O1:K1:H7 strains showed ashorter duration of symptoms before antibiotic treatment,higher fever, longer fever duration, and higher leukocytecounts than children infected with type 1-negative O1:K1:H7isolates or other E. coli strains. The results suggested that it wasthe expression of type 1 fimbriae by the E. coli O1:K1:H7isolates that led to the increased severity of infection in thechildren.The role of type 1-fimbrial expression for the virulence of

the E. coli O1:K1:H7 clone was further examined in a mouseUTI model. Infections were performed with three clinical E.coli O1:K1:H7 isolates that differed in type 1-fimbrial expres-sion. Type 1-positive isolates showed increased persistence inthe kidneys and bladders of the mice and induced a greaterinflammatory response than type 1-negative isolates. A role fortype 1-mediated adhesion in the virulence of the E. coliO1:K1:H7 isolate was confirmed with the construction of afimH null mutant of a type 1-positive E. coli O1:K1:H7 isolate.

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B

D E

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F

'lk

FIG. 4. Immunoelectronmicrographs of E. coli HB101 (A); E. coli HB101/pPKLO4 (B); E. coli HB101/pDC1 (C); E. coli 1177 (D); E. coliCN1016 (E); and E. coli CN1018 (F) stained with rabbit anti-type-1-fimbrial anti-serum raised against purified type 1 fimbriae and detected with15 nm gold-labeled goat anti-rabbit IgG. (Bar = 0.5 ,um.)

The fimH null mutation abolished D-mannose sensitive ad-herence. This mutant survived in significantly lower numbersin mouse kidneys and bladders and induced a significantlylower neutrophil influx compared with the fimH-positiveparent. To fulfill Koch's molecular postulates, the fim DNAsequences were returned to thefimH null mutant to restore thetype 1-fimbrial phenotype. This type 1-complemented mutantsurvived in similar numbers in mouse kidneys and bladderswhen compared with the wild-type type 1-positive parent strainand in significantly higher numbers than thefimH null mutant.It induced a neutrophil response similar to the wild-type type1-positive parent strain and induced a significantly higherneutrophil response compared with the fimH null mutant.Together, these results suggest that type 1 fimbriae play an

important role in the virulence of the uropathogenicO1:Kl:H7 clone.Immunoelectromicroscopy was carried out to examine fim-

brial expression of the wild-type type 1-positive parent strain,the fimH null mutant, and the fim-complemented fimH nullmutant. The wild-type parent and thefim-complementedfimHnull mutant had immunoreactive fimbriae on their surface,whereas the fimH null mutant did not. In addition, the threestrains had fimbriae on the surface that were not immunore-active (most likely P fimbriae). The absence of immunoreactivefimbriae on thefimH null mutant was expected since Schembriet al. recently observed that deletion offimH fromE. coli PC31caused a substantive reduction in fimbrial expression (28).FimH has also been shown to be involved in the initiation offimbrial biogenesis and fimbrial assembly (24, 29).The presence offim DNA sequences is common among E.

coli strains. In fact the majority of clinical isolates, bothvirulent and avirulent, can be induced to express type 1fimbriae. Consequently, there has been no evidence fromepidemiological studies of an association between type 1fimbriae and the severity of infection (8). Several studies inexperimental UTI models have indicated that type 1 fimbriae

can aid in the persistence of E. coli in the urinary tract.Aronson et al. (4) showed that the addition of a FimH receptoranalogue to an inoculum of type 1-fimbriated E. coli signifi-cantly reduced bacteriuria in mice. Similarly, immune seradirected against type 1 fimbriae prevented colonization of thekidneys in a rat model of E. coli-induced pyelonephritis (39).Hagberg et al. (5) showed that a type 1-negative mutant of awild-type uropathogenic strain, produced by chemical mu-tagenesis with nitrosoguanidine, survived in lower numbers inthe mouse bladder than the type 1-positive parent. In contrast,survival in the human urinary tract was reduced after trans-formation of a wild-type E. coli strain with a plasmid encodingthe fim sequences (40).

This is the first study that we know of to take a fully virulentwild-type clinical isolate from a patient with acute pyelone-phritis and attempt to delete the fimH adhesin. It is also thefirst study that bases the choice of that strain on an observeddifference in severity of infection in patients related to ex-pression of type 1 fimbriae in the same clonal background.Type 1-mediated adherence has been proposed to play a role

in the induction of inflammation. Early in vitro studies showedthat type 1-fimbriated bacteria bind to phagocytic cells andinduce a respiratory burst (41). Further studies have shownthat type 1 fimbriae and FimH induce many of the inflamma-tory effects associated with type 1-fimbriated E. coli, includingan oxidative burst in neutrophils (17, 42) and proliferation anddifferentiation of human B cells (43, 44). More recently, type1-fimbriated E. coli and FimH-coated latex beads were shownto induce mast cell degranulation and histamine release inmice (16). Despite these observations, the role of type 1fimbriae as inducers of inflammation in UTI remains unde-fined. We have recently shown that E. coli stimulate urinarytract epithelial cells to secrete interleukin 8 (IL-8, an achemokine with chemotactic activity for neutrophils) and IL-6(an endogenous pyrogen and hepatocyte activator) (18, 45,46). Type 1-fimbriated strains induced higher levels of IL-8 and

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IL-6 secretion than nonfimbriated isogens. In the presentstudy, mice infected with a type 1-positive O1:Kl:H7 isolateshowed a higher urinary neutrophil influx into the urine thantype 1-negative isolates. These results demonstrate that type1-fimbrial adhesion is involved in the induction of the inflam-matory response in vivo. Type 1 fimbriae may thus contributeto the increased inflammatory response in patients infectedwith the type 1-positive E. coli O1:Kl:H7.

Several properties of uropathogenic E. coli strains are rareor absent in other E. coli strains. These properties have beencalled virulence factors by inference, but the cellular mecha-nisms by which they act are not well understood. The mostextensive information available is for E. coli P fimbriae. Pfimbriae are expressed by most strains causing acute pyelone-phritis ("90%) but by few strains causing asymptomaticbacteriuria ("20%). P fimbriae mediate attachment to urinarytract epithelial cells and enhance cytokine responses in vitroand in vivo (46). Mutations in thepap gene cluster encoding Pfimbriae reduced bacterial persistence in the mouse urinarytract (5). Mutational inactivation of the papG adhesin in aurinary tract pathogen was recently shown to dramaticallydecrease colonization and inflammation in the kidneys ofmonkeys (47). The results presented here show that thevirulence of a P-fimbriated uropathogenic E. coli strain can bereduced by inactivation of a second fimbrial type. This illus-trates how the different fimbriae, and probably other virulencefactors, act in concert to achieve the virulent phenotype.

We thank Erik Carlemalm (Department of Electronmicroscopy,Jubileum Institute, Lund University) for his technical expertise withthe electronmicroscopy; Lars Pallesen (Department of Research andDevelopment, Division of Microbiology, State Serum Institute,Copenhagen S, Denmark) for generously supplying us with the plasmidpLPA22; and Claus Kristensen (Department of Microbiology, DanishTechnical University, Lyngby, Denmark) for generously supplying uswith the plasmid pCK155. These studies were supported by TheSwedish Medical Association; the Medical Faculty, Lund University;Swedish Medical Research Council Grant 7934; the Royal Physi-ographical Society of Lund; and the Osterlund, Nordic ResearchEducation Academy, and Crafoord foundations. W.A. is supported bya Swedish Medical Research Council Scholarship.

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