Vol. 56, No. 2INFECTION AND IMMUNITY, Feb. 1988, p. 513-5170019-9567/88/020513-05$02.00/0Copyright C 1988, American Society for Microbiology

A Block of Urovirulence Genes Encoding Multiple Fimbriae andHemolysin in Escherichia coli 04:K12:H-

NICOLA J. HIGH,' BARBARA A. HALES,' KLAUS JANN,2 AND GRAHAM J. BOULNOISl*Department of Microbiology, University of Leicester, Leicester, United Kingdom,' and Max Planck Institute for

Immunobiology, Freiburg, Federal Republic of Germany2

Received 23 June 1987/Accepted 4 November 1987

Cosmid gene libraries were constructed from a uropathogenic isolate of Escherichia coli 04:K12:H- thatsecretes alpha-hemolysin and produces the F14, F12-rel, F1C, and F13 fimbrial antigens. A series ofoverlapping clones was generated, and individual cosmid clones were found to express various combinations offimbriae and hemolysin, suggesting that the genes for these potential virulence factors are closely linked. Byusing Southern hybridization analysis and restriction endonuclease mapping, it was demonstrated that thecosmid clones carried a nested set of overlapping, cloned, genomic DNA fragments. A comparison of thephenotypic properties of individual cosmid clones and subclones allowed the order of the gene clusters encodingthese factors to be deduced. The cloning also revealed the presence of a fifth fimbria that had P-adhesinspecificity.

Escherichia coli is the major pathogen in upper and lowerurinary tract infections in humans. Its success as a uro-pathogen is, to an extent, dependent on its ability to producecell surface filaments, termed fimbriae (7), or F antigens (29),which are thought to mediate adhesion to uroepithelial cells(11). The success of E. coli as a uropathogen is also probablydependent on the secretion of a toxin, alpha-hemolysin (41,42).The majority of E. coli isolates have common, or type 1,

fimbriae (serotype Fl), which promote agglutination oferythrocytes and yeast by a process sensitive to mannose(28). These fimbriae have been postulated to facilitate colo-nization of the bladder (10, 27). A second heterogenousgroup of fimbriae exhibits mannose-resistant hemagglutina-tion (MRHA) via adhesion to a variety of surface receptors(21, 30, 38). Included within this group are the P fimbriae ofpyelonephritic E. coli, which adhere to the a-D-Gal(1-4)-P-D-Gal moiety (Gal, galactose) of the P blood group antigenand related molecules on epithelial cells (18, 23). Manyisolates of urovirulent E. coli elaborate multiple fimbriae(17), the expression of which is subject to phase variation(33). This process dictates the fimbrial type expressed on thebacterial cell surface.

Epidemiological evidence indicates a role for alpha-hemo-lysin in urovirulence, although its precise function has notbeen defined. It may act as a direct tissue-damaging agent(41) or as a mediator of indirect damage via interaction withleukocytes (35). It has been demonstrated in vitro thatnanogram amounts of alpha-hemolysin are cytolytic forhuman peripheral leukocytes and monocytes (4, 5, 9). He-molysin has also been suggested to provide essential iron forbacterial growth via hemolysis (24).To study the molecular basis for phased expression of

multiple fimbriae, a pyelonephritic isolate of E. coli 2002504:K12:H- was chosen for study since it expresses at leastfour readily distinguishable fimbriae (1). Strain 20025 elabo-rates two P-related fimbriae: one is the F13 fimbrial antigen,and the other is F12-rel, which is serologically related to butdistinct from the F12 fimbrial antigen. This strain also

* Corresponding author.

513

expresses the F14 fimbrial antigen, which has unknownreceptor specificity, and the FlC antigen, which does notexhibit adhesive properties. These individual fimbriae can bedistinguished from each other on the basis of the molecularweight of the appropriate fimbrial subunits and with the useof monoclonal antibodies to the F14, F12-rel, and FlCfimbrial subunits, which have been isolated and character-ized (1, 36). Additionally, 20025 secretes alpha-hemolysin.

This paper describes the identification of a contiguous 60-to 70-kilobase (kb) region of DNA which encompasses thegene blocks required for expression of the F14, F12-rel,FlC, and F13 fimbrial antigens and alpha-hemolysin. A fifthP-like fimbria expressed by 20025 has also been discovered.

MATERIALS AND METHODS

Bacterial strains and cultivation. the E. coli K-12 deriva-tive LE392 (hsdR514 supE supF lacY galT galK metB trpR)was used as the host for all recombinant plasmids. Bacteriawere routinely grown for 16 to 18 h at 37°C in Luria broth oragar supplemented with ampicillin (100 ,ug/ml) as required.Strain 20025 04:K12:H- has been described elsewhere (17).

Plasmids. The plasmids used in this study were cos4 (34)and pUC8 (40).DNA procedures. Restriction endonucleases and T4 DNA

ligase were obtained from GIBCO Laboratories, GrandIsland, N.Y., Bethesda Research Laboratories, Inc., Gai-thersburg, Md., and Amersham International, respectively,and used according to the recommendations of the manufac-turers. Plasmid DNA was prepared by the alkaline lysismethod (3). Gene libraries were created from strain 20025 bycloning size-selected 45-kb fragments of chromosomal DNAgenerated by the restriction endonuclease Sau3A into theBamHI site of the cosmid vector cos4 as described previ-ously (34). Recombinant molecules were packaged intobacteriophage X particles and introduced into E. coli LE392by infection. Clones were selected on Luria agar platescontaining 100 p.g of ampicillin per ml after a 30-min incu-bation in broth to allow expression of the drug marker.DNA-DNA hybridization. Restriction endonuclease-gener-

ated DNA fragments were transferred to nitrocellulose as

514 HIGH ET AL.

previously described (37). Radiolabeled DNA probes weregenerated as described elsewhere (8).

Hemagglutination assays. To detect clones showingMRHA, agar-grown bacteria were suspended in phosphate-buffered saline (PBS) containing ot-D-mannoside (50 mM)supplied by Sigma Chemical Co., Poole, Dorset, UnitedKingdom, and mixed with PBS-washed human erythrocytes.When required, a-D-Gal(1-4)-p-D-Gal (50 mM), obtainedfrom Sokerbolaget, Arlov, Sweden, was used in place ofa-D-mannoside.

Secretion of alpha-hemolysin. Clones secreting alpha-he-molysin were detected by plating the cosmid clones on Luriaagar containing 100 jig of ampicillin and 5% PBS-washedsheep erythrocytes and by searching for zones of beta-hemolysis after overnight growth at 37°C.

Fimbrial purification. Fimbriae were purified essentially asdescribed previously (20).

Immunological methods. The reaction of clones withmonoclonal antibodies was determined by using a solid-phase immunosorbent assay. Bacteria in PBS were spottedonto a nitrocellulose filter, and nonspecific protein-bindingsites were blocked by treating the filter with 3% bovineserum albumin in Tris saline (150 mM NaCl, 10 mM Trishydrochloride [pH 7.5]). Filters were incubated at 37°C withthe appropriate monoclonal antibody diluted in the bovineserum albumin solution described above. Unbound mono-clonal antibody was removed by extensive washing in Trissaline, and bound monoclonal antibody was detected byincubating the filters (3 h, 37°C) with horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulins. After beingwashed, the filters were incubated with the enzyme substrate(0.5 mg of 3-chloronapthol per ml, 6% [vol/vol] methanol,and 0.05% [vol/vol] 20-volume hydrogen peroxide in Trissaline).

Electron microscopy. Bacteria grown on agar plates wereprepared for electron microscopy by suspension in PBScontaining 4% gluteraldehyde before the bacteria were ap-plied to 300-mesh copper grids coated with 2% celloidin.After being washed to remove unbound cells, the grids wereair dried and gold shadowed in a vacuum coating unit.

RESULTSConstruction and screening of cosmid libraries. Gene librar-

ies of strain 20025 were constructed in E. coli LE392(phenotypically nonfimbriated and nonhemolytic) by usingthe cosmid cloning procedure (6) and the vector cos4.Preparation of cos4 involved cleavage of the vector with theendonuclease PvuII and dephosphorylation of the resultingblunt ends. Linear molecules produced in this manner werethen purified and cleaved with the endonuclease BamHI,resulting in the formation of two arms, both carrying cossites. DNA fragments were generated by partial digestionwith the endonuclease Sau3A. Fragments of about 45 kbwere purified from agarose gels. The prepared cosmid armswere then ligated to these 45-kb fragments of chromosomalDNA. Recombinant molecules were packaged into phageheads (13) and used subsequently to infect LE392.Presumptive fimbriated clones were isolated from the gene

library by their ability to promote MRHA of human eryth-rocytes or by their reaction with monoclonal antibodies in asolid-phase immunosorbent assay. Confirmation that theclones detected by these two procedures were indeed ex-pressing fimbriae on the cell surface was obtained by directvisualization of gold-shadowed bacteria by electron micros-copy. Clones expressing alpha-hemolysin were identified bythe appearance of zones of hemolysis surrounding individual

TABLE 1. Phenotypic properties of cosmid clones

MRHA of MRHA ofClone F12-rela F141 Hly F1C' F13a HRBCb SRBCc

pGB750 + + + + - + +pGB751 + + + - - + +pGB752 + + - - - + +pGB753 - - - + + + -pGB754 - - - - + + -pGB755 - - - _ _ + _

a Reaction of whole cells with the appropriate monoclonal antibody.b Human erythrocytes.c Sheep erythrocytes.

colonies after overnight growth on blood agar. These proce-dures identified six phenotypically distinct groups of cosmidclones, each expressing different combinations of the fourfimbriae and alpha-hemolysin (Table 1). Since more than onefactor was being expressed by individual clones, this resultsuggested that in strain 20025, the genes encoding theindividual fimbrial antigens and alpha-hemolysin wereclosely linked.

Characterization of the cosmid clones. The cosmid cloneLE392(pGB750) carries genes for the fimbrial antigens F14,F12-rel, and FlC and for alpha-hemolysin. Four other cos-mid clones have been identified which are phenotypic sub-sets of LE392(pGB750), expressing one or more of the sameproperties (Table 1).A fifth group of clones was identified which promoted

MRHA of human erythrocytes but failed to react with any ofthe monoclonal antibodies (Table 1). On the basis of restric-tion endonuclease cleavage patterns of cosmids isolatedfrom these clones, two classes were identified. Serologicalanalysis revealed that one class of cosmid clone, exemplifiedby LE392(pGB754), expressed the F13 fimbria. By usingfimbriae from this clone as immunogens, monoclonal anti-bodies to F13 were subsequently generated (1). In immuno-blots (data not shown), this antibody did not react withfimbriae from the second class of cosmid clone describedabove. This second class, of which LE392(pGB755) is rep-resentative, probably expressed a previously undefined fim-bria, indicating that strain 20025 must express a fifth fimbriawhich was not identified previously in fimbrial preparationsof 20025. The receptor specificity of this fifth fimbria wascharacterized by performing hemagglutination assays in thepresence of a-D-Gal(1-4)-p-D-Gal. No hemagglutination wasobserved when assays were performed in the presence ofot-D-Gal(1-4)-13-D-Gal. Thus, this fifth fimbria has the samecell surface receptor specificity as P fimbriae. The serotypeof this fimbria awaits elucidation.By reaction with anti-F13 monoclonal antibodies in a

solid-phase immunosorbent assay, it has been shown that inaddition to expressing the FlC fimbrial antigen, LE392-(pGB753) also elaborates the F13 fimbrial antigen (Table 1),indicating that the genes encoding the FlC and F13 fimbrialantigens are closely linked. The isolation of LE392(pGB753)also allowed the position of the gene encoding the F13fimbrial antigen in relation to the cluster of genes on pGB750(F14, F12-rel, Hly, and F1G) to be deduced (Fig. 1). Byexamining and comparing the combinations of determinantsexpressed by each cosmid clone, it was apparent that thesegenes must occur in one oftwo possible orders: F14, F12-rel,Hly, F1C, F13 or F12-rel, F14, Hly, F1C, F13 (Fig. 1). Thegene cluster encoding the new P-fimbria-like adhesin ex-pressed by LE392(pGB755) have not been shown to belinked to any of the other fimbriae or to alpha-hemolysin

INFECT. IMMUN.

A BLOCK OF UROVIRULENCE GENES 515

F12erel | F14 |

| F14 | I F12reL I HLY FIC F| f13pGB 750pG B751pGB752

pGB753pGB754

FIG. 1. Determination of the possible orders in which the fimbrial and alpha-hemolysin gene clusters occur on the strain 20025chromosome. Horizontal lines represent DNA present in each plasmid. Boxes labeled F12, F13, F14, F1C, and Hly represent the relativepositions of the gene clusters encoding these determinants.

genes. Localization of the genes encoding this fimbria awaitsfurther analysis.

Restriction endonuclease analysis of the cosmid clones.Comparison of the restriction endonuclease patterns of theindividual cosmid clones revealed that plasmids which en-coded similar combinations of fimbriae and alpha-hemolysinshared common fragments of DNA. Since the cosmids wereconstructed from 40-kb size-selected DNA fragments, thisfact demonstrates that isolated plasmids were all derivedfrom the same segment of DNA and that an overlappingseries ofDNA fragments had been cloned. Alignment of therestriction endonuclease maps of individual cosmids on thebasis of common restriction endonuclease fragments furtheremphasized that this series of cosmid clones represented anested set of cloned DNA fragments (Fig. 2). Confirmationthat each clone contained one continuous fragment of DNAwas established by Southern hybridization experiments. Aseries of restriction endonuclease digests of 20025 total DNAand pGB750 DNA were probed with pGB750. The resultinghybridization patterns were identical, apart from the twofragments carrying cos4 vector sequences. Thus, the organ-ization of the cloned DNA in pGB750 was the same as in20025 total DNA. The F14, F12-rel, and FlC fimbrial anti-gens and alpha-hemolysin are thus encoded by a contiguoussegment of DNA.

Since cosmid clones which direct the expression of boththe FlC and F13 fimbrial antigens have been isolated, it ishighly likely that the genes encoding production of the F13fimbrial antigen are also linked to this region. Since adetailed genetic analysis of pGB754 (FlC and F13) has notbeen conducted, the distance between the FlC and F13 geneclusters has not yet been established.

Analysis of subclones. A series of subclones was con-structed to determine which of the two predicted gene orders

(see above) was correct (Table 2). A subclone, pGB760, wascreated from the 14-kb BamHI fragment (coordinates 1 to 35kb) derived from pGB751, which carries cos4 vector se-quences. This subclone failed to confer on LE392 the abilityto promote hemagglutination, and LE392(pGB760) showedno reaction to the three antifimbriae monoclonal antibodiesand exhibited no hemolytic activity. The 19-kb ClaI frag-ment from pGB751 (coordinates 30 to 49 kb), which alsocarries vector sequences, was used to generate pGB761.When LE392(pGB761) was grown on blood agar plates,beta-hemolysis was observed around individual colonies.Since approximately 7.5 kb of DNA is required for thesynthesis and secretion of alpha-hemolysin (2), the alpha-hemolysin gene cluster cannot be encompassed by the 5 kbof DNA between the ClaI and BamHI sites (coordinates 30to 35 kb) and therefore must span the BamHI site atcoordinate 35 kb.The 14-kb BamHI fragment (coordinates 21.5 to 35 kb)

was cloned into pUC8, creating pGB762. When pGB762 wasintroduced into LE392, it conferred the ability to provokeMRHA of human and sheep erythrocytes. Purified fimbriaefrom LE392(pGB762) reacted with anti-F14 monoclonal an-tibodies in solid-phase immunosorbent assays, demonstrat-ing that pGB762 directed the synthesis of the F14 fimbrialantigen. The genes encoding the F14 fimbrial antigen musttherefore lie within the 14-kb BamHI fragment (21.5 to 35kb). Since this fragment lies next to the BamHI site atcoordinate 36 kb, which bisects the alpha-hemolysin genecluster, the F14 and alpha-hemolysin gene must therefore lieadjacent to each other.The 10.5-kb BamHI fragment (coordinates 12 to 21.5 kb)

was cloned into pUC8 to yield pGB763. LE392(pGB763) didnot promote hemagglutination, nor did it react with theantifimbriae monoclonal antibodies. To determine if a fim-

B B K C B

c

B

C

B K B, C

B B

B B

B B K c 8

pGB751

pGB760

pGB761

pGB752

pGB762

pGB763

p6B764

BK C. KQ B,C, pGB750

2kb * C p6B765FIG. 2. Physical maps of the recombinant plasmids carrying fimbrial and alpha-hemolysin gene clusters. The restriction endonuclease

cleavage maps were aligned on the basis of common patterns of cleavage sites. Restriction endonuclease target sites: B, BamHI; C, CMaI; K,KpnI. Solid horizontal lines denote DNA present in each plasmid. Open boxes denote vector sequences. Plasmids pGB760 and pGB761 arederivatives of pGB751. pGB762, pGB763, and pGB764 are subclones of pGB752. pGB765 is a subclone of pGB750.

CB K

B

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516 HIGH ET AL.

TABLE 2. Phenotypic properties of subclones

Subclone F12-rela F141 Hly FlC' F131 MRHABCf MRHA of

pGB760 - - - - - - -

pGB761 - - + - - - -pGB762 - + - - - + +pGB763 - - - - - - -pGB764 + - - - - + -pGB765 - _ - +d _pGB766 - - - - + + -

a Reaction of whole cells with the appropriate monoclonal antibody.b Human erythrocytes.c Sheep erythrocytes.d Reaction of cell extracts with the appropriate monoclonal antibody.

brial gene cluster was bisected by the BamHI sites atcoordinates 21.5 kb and 13 kb, a 30-kb ClaI fragment(coordinates 43 to 30 kb) derived from pGB752 was circu-larized to generate pGB764. When introduced intoLE392(pGB764), it promoted MRHA and reacted with anti-F12-rel monoclonal antibodies. The F12-rel gene clustermust therefore span the BamHI site at either coordinate 13kb or coordinate 21.5 kb and must lie next to the F14 fimbrialgene cluster.The position of the FlC genes was determined by subclon-

ing a 6.5-kb ClaI fragment (coordinates 45.5 to 52 kb) frompGB750 into pUC8 to give pGB765. Strains harboring thisplasmid were found to be producing the FlC fimbrial subunitprotein intracellularly, since sonic cell extracts reacted withanti-FlC monoclonal antibodies in a solid-phase immunosor-bent assay. A 12-kb BamHI fragment derived from pGB754(data not shown) was cloned into pUC8 to yield pGB766.LE392(pGB766) promoted MRHA of human erythrocytesand reacted in immunoblots (Fig. 2) with anti-F13 monoclo-nal antibodies. Thus, the F13 fimbrial gene cluster was foundto be contained within this 12-kb BamHI fragment.From the properties of the various subclones, it can be

unequivocally established that the order F12-rel, F14, Hly,F1C, F13 is the order of the genes encoding this group ofvirulence determinants.

DISCUSSION

In strain 20025, a region of DNA was defined whichencompasses genes necessary for the expression of determi-nants thought to be important in the successful colonizationof the urinary tract. The strain used in this study, 20025,shows a marked similarity to the multifimbriate strain KS71(31). Genetic analysis of the fimbrial antigens expressed bythis strain has not indicated linkage of fimbrial gene clusters(32). Additionally, Hoekstra et al. (12) examined five multi-fimbriate strains of E. coli and concluded that in each casethe gene clusters were unlinked (9). To date, linkage offimbrial gene clusters has only been demonstrated betweenG fimbriae and M agglutinin (30). Linkage between P fim-briae and alpha-hemolysin gene clusters has been reportedpreviously (25). Approximately 4 to 6 kb was found toseparate the genes encoding MRHA and Hly in certain 06strains of E. coli. Similar studies with an E. coli 04 strainindicated that the gene clusters were separated by 14 to 16kb. Linkage between alpha-hemolysin and S-fimbria geneclusters has also been observed, since spontaneous chromo-somal deletions in E. coli 06 strain 536 which led to the lossof hemolysin production also eliminated the expression of anS-fimbria antigen (14, 19). The block of genes described in

the present study represents a far more extensive clusteringof determinants associated with urovirulence than that de-scribed for any other isolate of uropathogenic E. coli.P fimbriae are encoded by nine closely linked genes which

are encompassed by approximately 9.5 kb of DNA (26).Such gene clusters have been demonstrated to encode thefimbrial subunit protein, adhesin-associated proteins, fim-brial assembly functions, and regulatory elements. In multi-fimbriated isolates of E. coli, each subunit gene has beenfound to be associated with a complete set of fimbrialassembly, adhesin, and regulatory genes (39). Since we havebeen able to clone DNA fragments which direct the synthe-sis of the F13 and F14 fimbrial antigens, it can be assumedthat in strain 20025, each of the five fimbrial subunit genes isassociated with its own assembly and adhesin functions. Onthis basis, the DNA segment defined in this work must spana region of about 60 kb. In other P-fimbriated uropathogenicE. coli isolates, P fimbriae and alpha-hemolysin gene clustershave been mapped to specific regions of the E. coli chromo-some (15, 16). It is therefore highly likely that this uroviru-lence gene block is present in the 20025 chromosome,although we have no direct evidence to substantiate this.A major criterion for the successful establishment of a

urinary tract infection is the synergistic interaction of mul-tiple virulence factors. It may therefore be logical for thesegenes to become linked on the chromosome and may serveto provide a selective advantage for such strains. Linkage ofthese potential virulence factors may have occurred bytranspositional or recombinational events between plasmidsor other regions of the chromosome. An analogous situationto that reported here has previously been described forSalmonella ordenz, which exhibits linkage of multiple anti-biotic resistance genes on the chromosome that are sepa-rated by directly repeated insertion sequence-like se-quences. It has been suggested that linkage occurred bytranslocation of resistance genes from plasmids to the chro-mosome, an arrangement which has been suggested toincrease the stability of these genes (22).The segment ofDNA that we have defined carries multiple

virulence factor genes that are thought to act synergisticallyto promote a urinary tract infection. Such clustering ofurovirulence determinants may allow programmed expres-sion of factors required for the colonization of differentanatomical sites to facilitate ascending infection of theurinary tract.

ACKNOWLEDGMENTS

This work was supported by grants from the Medical ResearchCouncil of Great Britain (to G.J.B.). N.J.H. is supported by astudentship from the Medical Research Council of Great Britain.G.J.B. is a Lister Institute-Jenner Research Fellow.

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