Type 1 Fimbrial Expression Enhances Escherichia Coli Virulence for the Urinary Tract (Urinary Tract Infection/Inflammation/Adherence/Neutrophil Influx) H

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

Type 1 fimbrial expression enhances Escherichia coli virulence for 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, Danish Technical 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- the activation of the respiratory burst in granulocytes, degran- pressed by many Gram-negative bacteria. They facilitate ulation of mast cells, and cytokine release from epithelial cells adherence to mucosal surfaces and inflammatory cells in vitro, in vitro (16-18). but their contribution to virulence has not been defined. This In a recent study of acute pyelonephritis in children, E. coli study presents evidence that type 1 fimbriae increase the O1:Kl:H7 emerged as the most common serotype (19). The 14 virulence ofEscherichia coli for the urinary tract by promoting children infected with E. coli O1:Kl:H7 isolates showed a more bacterial persistence and enhancing the inflammatory re- rapid onset of infection, higher fever, longer fever duration, sponse to infection. In a clinical study, we observed that and higher blood leukocyte counts when compared with disease severity was greater in children infected with E. coli children infected with other E. coli serotypes. The most severe O1:Kl:H7 isolates expressing type 1 fimbriae than in those disease occurred in a subset of the children with O1:Kl:H7 infected with type 1 negative isolates ofthe same serotype. The strains that expressed type 1 fimbriae. The aim of this study was E. coli O1:Kl:H7 isolates had the same electrophoretic type, to examine the role of type 1 fimbrial expression for the were hemolysin-negative, expressed P fimbriae, and carried virulence of E. coli O1:Kl:H7 in these children. thefim DNA sequences. When tested in a mouse urinary tract infection model, the type 1-positive E. coli O1:Kl:H7 isolates survived in higher numbers, and induced a greater neutrophil MATERIALS AND METHODS influx into the urine, than O1:Kl:H7 type 1-negative isolates. Patients. Children with febrile urinary tract infection (UTI) To confirm a role of type 1 fimbriae, a flmH null mutant were enrolled in a prospective study of symptomatic bacteri- (CN1016) was constructed from an O1:Kl:H7 type 1-positive uria in childhood (19). Fever was defined as a rectal temper- parent. E. coli CN1016 had reduced survival and inflammato- ature >38.50C within 48 h of enrollment. Bacteriuria was genicity in the mouse urinary tract infection model. E. coli defined as homogenous growth of > 105 colony-forming CN1016 reconstituted with type 1 fimbriae (E. coli CN1018) units/ml in a mid-stream urine sample, or as any growth in had restored virulence similar to that of the wild-type parent urine obtained by suprapubic bladder aspiration. The 88 strain. These results show that type 1 fimbriae in the genetic children (65 girls and 23 boys) experienced their first known background of a uropathogenic strain contribute to the UTI episode. Their ages ranged from 2 months to 6 years pathogenesis of E. coli in the urinary tract. (median age was 0.8 year). Several parameters were recorded to assess the host response to infection. These parameters Fimbriae-mediated adherence is important for the virulence of included (i) the duration of symptoms before the start of Escherichia coli in the urinary tract (1). Uropathogenic E. coli antibacterial treatment, assessed by an interview of the parents strains may express a variety of fimbrial adherence factors, at enrollment; (ii) the body temperature, defined as the mean such as P, S, Dr, and type 1 fimbriae (2). It has been established of the recordings obtained during the first 24 h of hospital that P fimbriae enhance the virulence of uropathogenic strains admission; (iii) the duration of fever, defined as the time from through specific adherence and increased induction of mucosal the start of antibiotic treatment until the first of two consec- inflammation (1), but the role of type 1 fimbriae in virulence utive recordings of a temperature <37.80C; and (iv) leukocyte remains undefined (3). Studies in animal models have sug- counts, determined from blood and uncentrifuged urine. For gested that type 1 fimbriation increases the survival of E. coli other diagnostic criteria and laboratory parameters, see ref. 19. in the urinary tract (4-7); however, epidemiological studies Bacteria. Urine was cultured by routine laboratory tech- have failed to reveal a correlation between type 1 fimbriation niques. Of the 88 children, 84 were infected with E. coli (19). and virulence (8). The E. coli isolates were serotyped (20), and 14 isolates were Type 1 fimbriae are encoded by the chromosomally located identified as O1:Kl:H7. The clonal identity of these 14 isolates fim gene cluster. The fimbriae consist of a major structural was determined by multilocus enzyme electrophoresis (21) for subunit (FimA) and several minor components, including the malate dehydrogenase, ,B-galactosidase, 6-phosphogluconate adhesin (FimH). The FimH adhesin is located at the fimbrial dehydrogenase, adenylate kinase, phenylalanyl-leucine pepti- tip and interspersed along the shaft of the fimbriae (9). FimH dase, leucyl-glycyl-glycine peptidase, isocitrate dehydrogenase, recognizes terminally located D-mannose moieties on cell- phosphoglucose isomerase, aconitase, glucose-6-phosphate bound and secreted glycoproteins (10, 11). It was also recently dehydrogenase, and glutamate oxaloacetate transaminase. shown to mediate mannose-sensitive binding to nonglycosy- Each isolate was assigned an electrophoretic type based on lated peptide epitopes (12). Interactions with such receptors these 11 electromorphs. enable type 1-fimbriated bacteria to bind to a range of cells, Hemolysin production was assessed by plating the strains on including erythrocytes, epithelial cells, granulocytes, macro- nutrient agar containing 5% washed horse erythrocytes and phages, and mast cells (13-16). This interaction can result in Abbreviations: UTI, urinary tract infection; RFLP, restriction frag- The publication costs of this article were defrayed in part by page charge ment length polymorphism; IL, interleukin. payment. This article must therefore be hereby marked "advertisement" inI tH.C. and W.A. contributed equally to this work. accordance with 18 U.S.C. §1734 solely to indicate this fact. ITo whom reprint requests should be addressed. 9827 Downloaded by guest on September 28, 2021 9828 Medical Sciences: Connell et al. Proc. Natl. Acad. Sci. USA 93 (1996)

observation of a hemolytic zone larger than the overlying Cm colony. {fi( cII Expression of P fimbriae was analyzed after culture on BamHl tryptic soy agar (Difco) for 16 h at 37°C. Strains agglutinating pPKL52 npt

human erythrocytes of the AP1 blood group in the presence of D ' fm 27 np D-mannose, and which failed to agglutinate Ap erythrocytes, EcoRV were considered P-fimbriated (22). The expression of type 1 fi m HicII fimbriae was analyzed after overnight culture in Luria broth KpnI containing 0.1% CaCl2. Strains agglutinating guinea pig erythrocytes in the absence but not in the presence of D-mannose were considered type 1-fimbriated (13). Cm DNA Probes and DNA-DNA Hybridizations. A 10-kb Hin- Sanl EcoRV SaiI dIll-Styl fragment, containing the fim gene cluster from the Am r R6K EcoRV recombinant plasmid pPKLO4 (23), was used as a probe for the pCH102 hinDD' fim DNA sequences. A 1.0-kb HindIII-EcoRI fragment, containing thefimH gene from the recombinant plasmid pLPA22 pGP7O4 (24), was used as a probe for the fimH DNA sequences. A ~ ~ m 1.9-kb HincII fragment from the recombinant plasmid pCK155 KpnIIHincII (25) was used as a probe for the neomycin phosphotransferase I gene (npt). A 12.3-kb BamHI fragment, containing the pap gene cluster from the recombinant plasmid pDC1 (26), was \ EcoRV used as a probe for the pap DNA sequences. Amp fimD' Chromosomal DNA isolated from each E. coli strain (27) rimF was cut withAval (Boehringer Mannheim) to probe for thefim A1mG sequences and with HindIII (Boehringer Mannheim) to probe pCH103 DrmH' for the pap sequences. The cleaved DNA was electrophoresed on 0.7% agarose (Boehringer Mannheim) gels and transferred orl R6 npt to nylon membranes (Hybond-N, Amersham) by alkali blotting Sall ~ flmH' using a Vacuum blotting apparatus (Bio-Rad). The blots were prehybridized at 62°C for 2-4 h in 2x standard saline citrate FIG. 1. Plasmids used in the construction of the E. coli 1177fimH (SSC; lx SSC = 0.15 M sodium chloride/0.015 M sodium null mutant E. coli CN1016. A neomycin phosphatase gene (npt; the citrate, pH 7), 1% SDS, 1% skim milk (Difco), and 0.1 mg/ml 1.9-kb HincII from pCK155) (25) conferring kanamycin resistance was freshly denatured herring sperm DNA (Sigma). The DNA inserted into the KpnI site of thefimH gene on plasmid pPKL52 (29). The resulting plasmid (pCH102) was used to transform E. coli MC1000 probes were labeled with [a-32P]dCTP using the RediPrime (30). Transformants that were kanamycin- and chloramphenicol- DNA labeling kit (Amersham), and 0.1-0.5 ,ug/ml freshly resistant were selected. The plasmid pGP704 (31) isolated from E. coli denatured probe DNA was added to the prehybridization CC118(Apir) (32) and the plasmid pCH102 isolated from E. coli solution. Hybridization was carried out at 62°C for 16-18 h. MC1000 were digested with SalI and EcoRV. The 3.7-kb SalI-EcoRV Posthybridization filters were washed twice in 2x SSC/0.1% fragment of pGP704 and the 7.1-kb SalI-EcoRV fragment from (wt/vol) SDS for 5 min at room temperature, followed by two pCH102 were ligated to form pCH103, which was used to transform washes in 0.1 x SSC/0.1% (wt/vol) SDS for 15 min at 62°C, and E. coli CC118(Apir). Transformants that were kanamycin- and ampi- exposed to Kodak X-Omatic film at -70°C for 24-48 h. The cillin-resistant were selected. Plasmid pCH103 was isolated from E. coli CC118(Apir) and used to transform E. coli PC31 (23). After fragment sizes were determined by comparison to a 1-kb DNA allowing double crossover and recombination to occur at 370C, ladder (GIBCO/BRL). ampicillin-sensitive and kanamycin-resistant recombinants were se- Construction of E. coli CN1016 (E. coli 1177AfImH). The lected (E. coli MS4) (28). The oi R6K origin of replication is fimH sequences were deleted from E. coli 1177 by P1 trans- Apir-dependent and temperature-sensitive (30°C). E. coli PC31 does duction from afimH null mutant, E. coli MS4 (28). The cloning not contain the lysogenic phage Apir. ThefimH sequences were deleted strategy used to construct the strain E. coli MS4 (E. coli PC31 from E. coli 1177 by Pl transduction of DNA from E. coli MS4 to E. fimH null mutant) is shown in Fig. 1. E. coli MS4 was coli 1177. transfected with bacteriophage P1 and a chloroform bacteriophage lysate was prepared (33). This lysate was then used to Bacteria in kidneys and bladders were quantitated by viable transfect E. coli 1177 and an E. coli 1177fimH null mutant (E. counts on tissue homogenates-obtained at the time the mice coli CN1016) was selected by kanamycin resistance (30 gg/ml) were killed, 24 h after infection (35). Leukocyte numbers in and loss of mannose-sensitive agglutination of guinea pig urine samples taken at 0, 2, 6, and 24 h after inoculation were erythrocytes. counted using a Burker chamber (36). Urine samples were Construction of E. coli CN1018. E. coli CN1016 was elec- taken from individual mice before each experiment and ex- trotransformed as previously described (34) with the plasmid amined for the presence ofneutrophils. Mice with a preexisting pPKL04 (26). Transformants were selected by kanamycin (30 neutrophil response were excluded. ,ug/ml) and ampicillin (100 ,ug/ml) resistance. Transformants Immunoelectromicroscopy. Immunoelectronmicroscopy expressing both antibiotic resistances were also tested for the was carried out as described (37). Briefly, cells from an restoration of mannose-sensitive agglutination of guinea pig overnight culture were harvested, washed in PBS, and incu- erythrocytes. bated at room temperature for 1 h with rabbit anti-type-i- Experimental UTI. C3H/HeN mice were bred in the animal fimbrial anti-serum (1:5) (38). After washing, bacteria were facilities, Department of Medical Microbiology, Lund Univer- incubated at room temperature for 1 h with 15 nm gold-labeled sity. Female mice were used at 8-14 weeks of age. The E. coli goat anti-rabbit IgG (H+L; 1:10; Amersham). A 15-,ul aliquot strains were grown in Luria broth overnight, harvested by of washed bacteria was adsorbed to a carbon-coated, glow- centrifugation, and resuspended in 0.01 M PBS (pH 7.2) to discharged copper grid for 30 s and then negatively stained -109 bacteria per ml. Bacteria (0.1 ml) were inoculated into with 2% sodium phosphotungstenate (pH 7.0). The grids were the mice by urethral catheterization under ether anesthesia as observed at 60 kV in a Philips CM10 Transmission Electron described (35). Microscope (Philips, Eindhoven, The Netherlands). Downloaded by guest on September 28, 2021 Medical Sciences: Connell et aL Proc. Natl. Acad. Sci. USA 93 (1996) 9829

Statistics. The Mann-Whitney U test and Pearson Corre- Table 2. Bacterial recovery after experimental UTI in mice lation were used where applicable. E. coli No. of Type 1 Bacterial recovery* strain mice expression Bladder Kidneys RESULTS 1177 38 +++ 4,770 (21) 9,798 (22) Characterization of the O1:Kl:H7 Isolates. The E. coli 1106 12 154 (68) 699 (55) O1:Kl:H7 isolates were examined for variation in electro- 845 32 - 2 (4.3) 19 (10.6) phoretic type, hemolysin expression, and fimbrial phenotype. The 14 isolates had the same electrophoretic type type, as 1177 31 +++ 16,648 (4) 8,519 (7) defined by the 11 electromorphs. Two isolates (E. coli 3116 and CN1016 23 - 5.2 (15) 395 (5) E. coli 1106) had a null allele for phenylalanyl-leucine. All the CN1018 12 +++ 11,731 (6) 16,482 (12) isolates expressed P fimbriae and tested negative for hemolytic *Geometric mean of bacterial counts on homogenized tissue, 24 h activity. Eight of the Ol:Kl:H7 isolates expressed type 1 after infection; SD of geometric mean is in parentheses. fimbriae, whereas four did not. The remaining two isolates tE. coli 1106 was recently shown to express a FimH protein with were not tested for type 1 expression at the time of isolation different isoreceptor specificity to the FimH ofE. coli 1177 (D. Hasty, and were removed from the study. personal communication). Host Response to E. coli O1:Kl:H7 in Relation to Type 1106 persisted in greater numbers than E. coli 845 in mouse 1-Fimbrial Expression. The inflammatory response was com- bladders (P < 0.0004) and kidneys (P < 0.009). pared among the 8 children infected with the type 1-positive E. coli O1:Kl:H7 isolates, the 4 children infected with the type The inflammatory response to infection was greater in those 1-negative E. coli O1:Kl:H7 isolates, and the 54 children animals infected with the type 1-expressing O1:Kl:H7 isolates. infected with P-fimbriatedE. strains other than O1:K1:H7. Urinary neutrophil numbers were higher at 2 h (P < 0.0005), coli < < Children infected with type 1-positive E. coli O1:Kl:H7 iso- 6 h (P 0.01), and 24 h (P 0.0001) after infection with E. coli 1177 than after infection withE. coli 845 (Fig. 2A). Urinary lates had more rapid onset of symptoms before antibiotic < treatment, higher fever, longer fever duration, and higher neutrophil numbers were greater at 2 h (P 0.017) and 24 h leukocyte counts than children infected with other E. coli (P < 0.002) after infection withE. coli 1106 than after infection strains. The inflammatory responses did not differ between with E. coli 845. patients infected with the type 1-negative E. coli O1:Kl:H7 Verification of the AflmH Construction in E. coli CN1O16 isolates and patients infected with other E. coli strains (Table and of the Restored Type 1 Phenotype in E. coli CN1018. The 1). The host response in children with type 1-positive or type fimH null sequences were successfully P1-transduced from E. 1-negative E. coli could not be compared due to the small coli MS4 toE. coli 1177 with the resultingfimH null phenotype. numbers. The fimH null mutant constructions in E. coli MS4 and Fimbrial Genotype. The O1:Kl:H7 isolates carried the fim CN1016 were verified by Southern blot analysis. The Southern andpap DNA sequences. Threefim restriction fragment length blot patterns ofE. coli PC31, MS4 (E. coli PC31AfimH), 1177, polymorphisms (RFLPs) were observed after genomic diges- and CN1016 (E. coli 1177AfimH) are shown in Fig. 3. E. coli tion with AvaI. A single 23-kb fragment was found in two CN1016 hybridized with thefimH probe (Fig. 3A) and the npt strains (both type 1-negative). Three fragments of 8.8, 3.6, and probe (Fig. 3B). The fragment sizes in Fig. 3 A and B were 3.0 kb were found in 2 strains (one type 1-positive; one type consistent with those predicted for PvuII digestion of E. coli 1-negative). A third group of 7 strains (six type 1-positive; one CN1016. E. coli CN1016 agglutinated human AP1 erythrocytes type 1-negative) exhibited an RFLP pattern similar to that of in the presence of D-mannose (P fimbriae-positive) and failed E. coli PC31 (8.8/6/1.6/1.3/1.1 kb) (24). Twopap RFLPs were to agglutinate guinea pig erythrocytes in the absence of observed after genomic digestion with HindIII. Three frag- D-mannose (type 1 fimbriae-negative). P1 transduction and the ments of 10, 3.8, and 2.2 kb were shared by all strains. In resulting recombination in E. coli CN1016 did not result in the addition, two strains (both type 1-negative) had extra bands of disturbance of any genes 9.0 kb upstream or 5.0 kb downstream 7.5 and 1.5 kb. There was no correlation between the RFLP offimH as verified by Southern blot analysis (data not shown). patterns and type 1-fimbrial phenotype. The plasmids pCH102 and pCH103 were constructed for use Experimental UTI. Three E. coli O1:Kl:H7 isolates were in the derivation of an E. coli 1177fimH null mutant (see Fig. selected for in vivo experiments in the mouse UTI model: E. 1). E. coli 1177was transformedwith these plasmids but double coli 1177 expressing type 1-fimbriae, E. coli 1106 with a weak crossover recombinant transformants were not isolated. These type 1-fimbrial expression, and E. coli 845 that did not express plasmids were used to construct a fimH null mutant in the type 1 fimbriae. intestinal isolate E. coli PC31 (E. coli MS4). Higher numbers of E. coli 1177 were recovered from the The transformation of E. coli CN1018 with pPKLO4 was mouse bladders and kidneys than those of E. coli 845 (P < verified by plasmid extraction and Southern blot analysis (data 0.0001 andP < 0.0001, respectively) andE. coli 1106 (P < 0.007 not shown) using the 10-kb HindIII-Styl fragment from the andP < 0.04, respectively) 24 h after infection (Table 2). E. coli recombinant 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 cell days Fever, °C duration, h serum, 109/liter Urine, mm3 O1:K1:H7 E. coli Type 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. Downloaded by guest on September 28, 2021 9830 Medical Sciences: Connell et al. Proc. Natl. Acad. Sci. USA 93 (1996) A 300 A B

200 1.8 - 1.8 0 1.4- * -1.1 Z 1002 00262

0 * ---0.4 0 2 6 24 ...... Time I Hours 1 2 3 4 1 2 3 4 E Co C 0.1 4.3 1.1 1.4 jE 200- it T t 0 (i) 1177 LJ-J I 1 LAJX 11W J LJ_I E JL H

C oo 0.1 4.3 1.1 1.1 0.4 1.8

,I t '1 t I II - . .... - L-i1 (ii) CN1016 WBf I F. LJ W LLJI: 11 1 11,0ITi Ul H 0 0 2 6 24 IOkb Time /Hours FIG. 3. The Southern blots of PvuII-cleaved chromosomal DNA from E. coli PC31 (lane 1), MS4 (lane 2), 1177 (lane 3), and CN1016 FIG. 2. Urinary neutrophil influx in response to intravesical infec- (lane 4) were probed with the 1.0-kb fimH (A) and the 1.9-kb npt tion. Each mouse received 108 bacteria. (A) Wild-type isolates. *, E. fragments (B). (C) The PvuII restriction sites (*) for E. coli 1177 and coli 1177 (type 1-positive); *, E. coli 1106 (weak type 1-positive); 0, E. CN1016 (drawn to scale). 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 null CN1018. A significant difference in urinary neutrophil num- mutant; type 1-negative); o, E. coli CN1018 (E. coli CN1016/pPKL04; bers after infection with E. coli 1177 or E. coli CN1018 at 6 and type 1-positive). Mean of 14 mice with standard error. 24 h was not observed. plasmid of the correct molecular weight which hybridized with the 10-kb HindIII-StyI fragment from pPKLO4. DISCUSSION Immunoelectronmicroscopy. Cells of E. coli 1177, CN1016, and CN1018 were stained with anti-type-1-fimbrial antibody This study was prompted by a clinical observation in children (Fig. 4). E. coli HB101, HB101/pPKLO4 (fim+) (23), and with febrile UTI. Out of 88 children with acute pyelonephritis, HB101/pDC1 (pap+) (26) were used as controls for nonfim- 14 carried E. coli of serotype O1:K1:H7 (19). These children brial, type 1-fimbrial, and P-fimbrial expression, respectively. had a shorter duration of symptoms before antibiotic treat- E. coli HB101/pPKLO4, 1177, and CN1018 all stained positive ment, indicating a more rapid onset of symptoms. They also for type 1 fimbriae (Fig. 4 B, D, and F), whereas E. coli HB101, had higher fever, longer fever duration, and higher white blood HB101/pDC1, and CN1016 did not (Fig. 4 A, C, and E). It cell counts than children infected with other E. coli isolates. appeared that the deletion offimH from E. coli 1177 resulted We examined the properties of the E. coli O1:K1:H7 isolates in the loss of type 1 fimbriae, as evidenced by the lack of in an attempt to explain the increased virulence associated immunogold staining on E. coli CN1016 (Fig. 4E). Expression with this serotype. The O1:K1:H7 E. coli strains were shown to was restored following the addition of the fim sequences (E. be members of the same clone, and they were all P-fimbriated coli CN1018) (Fig. 4F). The type 1 antiserum used for the but differed in their expression of type 1 fimbriae. Children immunogold staining was specific for type 1 fimbriae as it did infected with type 1-positive O1:K1:H7 strains showed a not react with E. coli HB101 (Fig. 4A) orE. coli HB101/pDC1 shorter duration of symptoms before antibiotic treatment, expressing P fimbriae (Fig. 4C). higher fever, longer fever duration, and higher leukocyte Virulence ofE. coli 1177, CN1016, and CN1018. The role of counts than children infected with type 1-negative O1:K1:H7 type 1 fimbriae for bacterial survival and neutrophil influx was isolates or other E. coli strains. The results suggested that it was further examined by comparing E. coli 1177 with E. coli the expression of type 1 fimbriae by the E. coli O1:K1:H7 CN1016 and E. coli CN1018 in the mouse UTI model. E. coli isolates that led to the increased severity of infection in the 1177 and E. coli CN1018 survived in higher numbers in both children. the kidneys (P < 0.0001) and bladders (P < 0.0001) compared The role of type 1-fimbrial expression for the virulence of with E. coli CN1016 24 h after infection (Table 2). A significant the E. coli O1:K1:H7 clone was further examined in a mouse difference in bacterial numbers in the kidneys and bladders of UTI model. Infections were performed with three clinical E. mice infected with E. coli CN1018 or E. coli 1177 was not coli O1:K1:H7 isolates that differed in type 1-fimbrial expres- observed. sion. Type 1-positive isolates showed increased persistence in Urinary neutrophil numbers were higher at 2 h (P < 0.0001), the kidneys and bladders of the mice and induced a greater 6 h (P < 0.0001), and 24 h (P < 0.0001) after infection with E. inflammatory response than type 1-negative isolates. A role for coli 1177 or E. coli CN1018 than after infection with E. coli type 1-mediated adhesion in the virulence of the E. coli CN1016 (Fig. 2B). Urinary neutrophil numbers were higher at O1:K1:H7 isolate was confirmed with the construction of a 2 h (P < 0.013) in mice infected with E. coli 1177 than E. coli fimH null mutant of a type 1-positive E. coli O1:K1:H7 isolate. Downloaded by guest on September 28, 2021 Medical Sciences: Connell et aL Proc. Natl. Acad. Sci. USA 93 (1996) 9831 B C

D E F

.1.

'lk FIG. 4. Immunoelectronmicrographs of E. coli HB101 (A); E. coli HB101/pPKLO4 (B); E. coli HB101/pDC1 (C); E. coli 1177 (D); E. coli CN1016 (E); and E. coli CN1018 (F) stained with rabbit anti-type-1-fimbrial anti-serum raised against purified type 1 fimbriae and detected with 15 nm gold-labeled goat anti-rabbit IgG. (Bar = 0.5 ,um.)

The fimH null mutation abolished D-mannose sensitive ad- can aid in the persistence of E. coli in the urinary tract. herence. This mutant survived in significantly lower numbers Aronson et al. (4) showed that the addition of a FimH receptor in mouse kidneys and bladders and induced a significantly analogue to an inoculum of type 1-fimbriated E. coli signifi- lower neutrophil influx compared with the fimH-positive cantly reduced bacteriuria in mice. Similarly, immune sera parent. To fulfill Koch's molecular postulates, the fim DNA directed against type 1 fimbriae prevented colonization of the sequences were returned to thefimH null mutant to restore the kidneys in a rat model of E. coli-induced pyelonephritis (39). type 1-fimbrial phenotype. This type 1-complemented mutant Hagberg et al. (5) showed that a type 1-negative mutant of a survived in similar numbers in mouse kidneys and bladders wild-type uropathogenic strain, produced by chemical mu- when compared with the wild-type type 1-positive parent strain tagenesis with nitrosoguanidine, survived in lower numbers in and in significantly higher numbers than thefimH null mutant. the mouse bladder than the type 1-positive parent. In contrast, It induced a neutrophil response similar to the wild-type type survival in the human urinary tract was reduced after trans- 1-positive parent strain and induced a significantly higher formation of a wild-type E. coli strain with a plasmid encoding neutrophil response compared with the fimH null mutant. the fim sequences (40). Together, these results suggest that type 1 fimbriae play an This is the first study that we know of to take a fully virulent important role in the virulence of the uropathogenic wild-type clinical isolate from a patient with acute pyelone- O1:Kl:H7 clone. phritis and attempt to delete the fimH adhesin. It is also the Immunoelectromicroscopy was carried out to examine fim- first study that bases the choice of that strain on an observed brial expression of the wild-type type 1-positive parent strain, difference in severity of infection in patients related to ex- the fimH null mutant, and the fim-complemented fimH null pression of type 1 fimbriae in the same clonal background. mutant. The wild-type parent and thefim-complementedfimH Type 1-mediated adherence has been proposed to play a role null mutant had immunoreactive fimbriae on their surface, in the induction of inflammation. Early in vitro studies showed whereas the fimH null mutant did not. In addition, the three that type 1-fimbriated bacteria bind to phagocytic cells and strains had fimbriae on the surface that were not immunore- induce a respiratory burst (41). Further studies have shown active (most likely P fimbriae). The absence of immunoreactive that type 1 fimbriae and FimH induce many of the inflamma- fimbriae on thefimH null mutant was expected since Schembri tory effects associated with type 1-fimbriated E. coli, including et al. recently observed that deletion offimH fromE. coli PC31 an oxidative burst in neutrophils (17, 42) and proliferation and caused a substantive reduction in fimbrial expression (28). differentiation of human B cells (43, 44). More recently, type FimH has also been shown to be involved in the initiation of 1-fimbriated E. coli and FimH-coated latex beads were shown fimbrial biogenesis and fimbrial assembly (24, 29). to induce mast cell degranulation and histamine release in The presence offim DNA sequences is common among E. mice (16). Despite these observations, the role of type 1 coli strains. In fact the majority of clinical isolates, both fimbriae as inducers of inflammation in UTI remains unde- virulent and avirulent, can be induced to express type 1 fined. We have recently shown that E. coli stimulate urinary fimbriae. Consequently, there has been no evidence from tract epithelial cells to secrete interleukin 8 (IL-8, an a epidemiological studies of an association between type 1 chemokine with chemotactic activity for neutrophils) and IL-6 fimbriae and the severity of infection (8). Several studies in (an endogenous pyrogen and hepatocyte activator) (18, 45, experimental UTI models have indicated that type 1 fimbriae 46). Type 1-fimbriated strains induced higher levels of IL-8 and Downloaded by guest on September 28, 2021 9832 Medical Sciences: Connell et al. Proc. Natl. Acad. Sci. USA 93 (1996) IL-6 secretion than nonfimbriated isogens. In the present 13. Duguid, J., Smith, I., Dempster, G. & Edmunds, P. (1955) study, mice infected with a type 1-positive O1:Kl:H7 isolate J. Pathol. Bacteriol. 70, 335-348. showed a higher urinary neutrophil influx into the urine than 14. Bar-Shavit, Z., Ofek, I., Goldman, R., Mirelman, D. & Sharon, type 1-negative isolates. These results demonstrate that type N. (1977) Biochem. Biophys. Res. Commun. 78, 455-460. 1-fimbrial adhesion is involved in the induction of the inflam- 15. Ofek, I., Mirelman, D. & Sharon, N. (1977) Nature (London) 265, matory response in vivo. Type 1 fimbriae may thus contribute 623-625. to the increased 16. Malaviya, R., Ross, E., Jaksfhik, B. & Abraham, S. (1994) J. Clin. inflammatory response in patients infected Invest. 93, 1645-1653. with the type 1-positive E. coli O1:Kl:H7. 17. Steadman, R., Topley, N., Jenner, D. E., Davies, M. & Williams, Several properties of uropathogenic E. coli strains are rare J. (1988) Infect. Immun. 56, 815-822. or absent in other E. coli strains. These properties have been 18. Agace, W., Hedges, S., Ceska, M. & Svanborg, C. (1993) J. Clin. called virulence factors by inference, but the cellular mecha- Invest. 92, 780-785. nisms by which they act are not well understood. The most 19. Marild, S., Jodal, U., 0rskov, I., 0rskov, F. & Svanborg-Eddn, C. extensive information available is for E. coli P fimbriae. P (1989) J. Pediatr. 115, 40-45. fimbriae are expressed by most strains causing acute pyelone- 20. 0rskov, I. & 0rskov, F. (1984) in Methods in Microbiology, eds. phritis ("90%) but by few strains causing asymptomatic Bergen, T. & Norris, J. (Academic, New York), pp. 43-112. bacteriuria ("20%). P fimbriae mediate attachment to urinary 21. Caugant, D., Levin, B., 0rskov, I., 0rskov, F., Svanborg-Eden, C. tract epithelial cells and enhance cytokine responses in vitro & Selander, R. (1985) Infect. Immun. 49, 407-413. and in vivo (46). Mutations in thepap gene cluster encoding P 22. Leffler, H. & Svanborg-Eden, C. (1980) FEMS Microbiol. Lett. 8, fimbriae reduced bacterial persistence in the mouse urinary 127-134. tract Mutational of the in a 23. Klemm, P., J0rgensen, B., van Die, I., de Ree, H. & Bergmans, (5). inactivation papG adhesin H. (1985) Mol. Gen. Genet. 199, 410-414. urinary tract pathogen was recently shown to dramatically 24. Pallesen, L., Poulsen, L., Christiansen, G. & Klemm, P. (1995) decrease colonization and inflammation in the kidneys of Microbiology 141, 2839-2848. monkeys (47). The results presented here show that the 25. Eberl, L., Kristensen, C., Givskov, M., Grohmann, E., Gerlitz, M. virulence of a P-fimbriated uropathogenic E. coli strain can be & Schwab, H. (1994) Mol. Microbiol. 12, 131-141. reduced by inactivation of a second fimbrial type. This illus- 26. Clegg, S. (1982) Infect. Immun. 38, 739-744. trates how the different fimbriae, and probably other virulence 27. Grimberg, J., Maguire, S. & Belluscio, L. (1989) Nucleic Acids factors, act in concert to achieve the virulent phenotype. Res. 17, 8893. 28. Schembri, M., Pallesen, L., Connell, H., Hasty, D. & Klemm, P. We thank Erik Carlemalm (Department of Electronmicroscopy, (1995) FEMS Microbiol. Lett. 137, 257-264. Jubileum Institute, Lund University) for his technical expertise with 29. Klemm, P. & Christiansen, G. (1987) Mol. Gen. Genet. 208, the electronmicroscopy; Lars Pallesen (Department of Research and 439-445. Development, Division of Microbiology, State Serum Institute, 30. Casabadan, M. & Cohen, S. (1980) J. Mol. Biol. 138, 179-207. Copenhagen S, Denmark) for generously supplying us with the plasmid 31. Miller, V. & Mekalanos, J. (1988) J. Bacteriol. 170, 2575-2583. pLPA22; and Claus Kristensen (Department of Microbiology, Danish 32. Herrero, M., de Lorenzo, V. & Timmis, K. (1990) J. Bacteriol. Technical University, Lyngby, Denmark) for generously supplying us 172, 6557-6567. with the plasmid pCK155. These studies were supported by The 33. Miller, J. (1992) A Short Course in Bacterial Genetics: A Labo- Swedish Medical Association; the Medical Faculty, Lund University; ratory Manual and Handbook for Escherichia coli and Related Swedish Medical Research Council Grant 7934; the Royal Physi- Bacteria (Cold Spring Harbor Lab. Press, Plainview, NY). ographical Society of Lund; and the Osterlund, Nordic Research 34. Dower, W., Miller, J. & Ragsdale, C. (1988) NucleicAcids Res. 16, Education Academy, and Crafoord foundations. W.A. is supported by 6127-6145. a Swedish Medical Research Council Scholarship. 35. Hagberg, L., Engberg, I., Freter, R., Lam, J., Olling, S. & Svanborg-Eden, C. (1983) Infect. Immun. 40, 273-283. 1. Svanborg, C., 0rskov, F. & 0rskov, I. (1994) in Fimbriae: 36. Shahin, R., Engberg, I., Hagberg, L. & Svanborg-Eden, C. (1987) Adhesion, Genetics, Biogenesis, and Vaccines, ed. Klemm, P. J. Immunol. 10, 3475-3480. (CRC, Boca Raton, FL), pp. 239-254. 37. Klemm, P., Christiansen, G., Kreft, B., Marre, R. & Bergmans, 2. Johnson, J. (1991) Clin. Microbiol. Rev. 4, 80-128. H. (1994) J. Bacteriol. 176, 2227-2234. 3. Klemm, P. & Krogfelt, K. (1994) in Fimbriae:Adhesion, Genetics, 38. Krogfelt, K. & Klemm, P. (1988) Microb. Pathog. 4, 231-238. Biogenesis, and Vaccines, ed. Klemm, P. (CRC, Boca Raton, FL), 39. Silverblatt, F. & Cohen, L. (1979) J. Clin. Invest. 64, 333-336. pp. 9-26. 40. 4. Aronson, M., Medalia, O., Schori, L., Mirelman, D., Sharon, N. Anderson, P., Engberg, I., Lidin-Janson, G., Lincoln, K., Hull, R., & Ofek, I. (1979) J. Infect. Dis. 139, 329-332. Hull, S. & Svanborg-Eden, C. (1991) Infect. Immun. 59, 2915- 5. Hagberg, L., Hull, R., Hull, S., Falkow, S., Freter, R. & Svanborg- 2921. Eden, C. (1983) Infect. Immun. 40, 265-272. 41. Sharon, N. & Ofek, I. (1986) in Microbial Lectins andAgglutinins: 6. Abraham, S., Babu, J., Giampapa, C., Hasty, D., Simpson, W. & Properties and Biological Activity, ed. Mirelman, D. (Wiley, New Beachey, E. (1985) Infect. Immun. 48, 625-628. York), pp. 55-82. 7. Keith, B., Maurer, L., Spears, P. & Orndorff, P. (1986) Infect. 42. Tewari, R., MacGregor, J., Ikeda, T., Little, R., Hultgren, S. & Immun. 53, 693-696. Abraham, S. (1993) J. Biol. Chem. 268, 3009-3015. 8. Hagberg, L., Jodal, U., Korhonen, T., Lidin-Janson, G., Lindberg, 43. Ponniah, S., Abraham, S., Docktor, M., Wall, C. & Endres, R. U. & Svanborg-Eddn, C. (1981) Infect. Immun. 31, 564-570. (1989) J. Immunol. 142, 992-998. 9. Krogfelt, K., Bergmans, H. & Klemm, P. (1990) Infect. Immun. 44. Ponniah, S., Abraham, S. & Endres, R. (1992) Infect. Immun. 60, 58, 1995-1998. 5197-5203. 10. Giampapa, C., Abraham, S., Chiang, T. & Beachey, E. (1988) 45. Hedges, S., Svensson, M. & Svanborg, C. (1992) Infect. Immun. J. Biol. Chem. 263, 5362-5367. 60, 1295-1301. 11. Wold, A., Mestecky, J., Tomana, M., Kobata, A., Ohbayashi, H., 46. Hedges, S, Agace, W. & Svanborg, C. (1995) Trends Microbiol. 3, Endo, T. & Svanborg-Eden, C. (1990) Infect. Immun. 58, 3073- 266-270. 3077. 47. Roberts, J., Marklund, B., Ilver, D., Haslam, D., Kaak, M., 12. Sokurenko, E., Courtney, H., Ohman, D., Klemm, P. & Hasty, D. Baskin, B., Louis, M., M6llby, R., Winberg, K. & Normark, S. (1994) J. Bacteriol. 176, 748-755. (1994) Proc. Natl. Acad. Sci. USA 91, 11889-11893. Downloaded by guest on September 28, 2021