Vol. 59, No. 11

DNA Sequences of Three papA Genes from UropathogenicEscherichia coli Strains: Evidence of Structural

and Serological ConservationKENNETH DENICH,1 LAWRENCE B. BLYN,2 ABIE CRAIU,1 BRUCE A. BRAATEN,3 JONATHAN HARDY,'

DAVID A. LOW,3 AND PETER D. O'HANLEYl.4.5*Department of Medicine, Division of Infectious Diseases,4 and Department of Microbiology and Immunology,'Stanford University, Stanford, California 94305; Veterans Administration Hospital, Palo Alto, California 943045;

Division ofBiology, California Institute of Technology, Pasadena, California 911252; and Department ofPathology,University of Utah School of Medicine, Salt Lake City, Utah 841323

Received 16 May 1991/Accepted 11 August 1991

Pyelonephritis-associated pili (Pap) are important in the pathogenesis of ascending, unobstructive Esche-richia coli-caused renal infections because these surface bacterial organelles mediate digalactoside-specificbinding to host uroepithelial cells. Pap are composed of many different polypeptides, of which only the tipproteins mediate specific binding. The PapA moiety polymerizes to form the bulk of the pilus structure and hasbeen employed in vaccines despite its lack of Gala(1-4)Gal receptor specificity. Animal recipients of PapApilus-based vaccines are protected against experimental pyelonephritis caused by homologous and heterologousGal-Gal-binding uropathogenic E. coli strains. Specific PapA immunoglobulin G antibodies in urine are

correlated with protection in these infection models. The nucleotide sequences of the gene encoding PapA were

determined for three E. coli clones expressing F71, F72, and F9 pili and were compared with correspondingsequences for other F serotypes. Specific rabbit antisera were employed in enzyme-linked immunosorbentassays to study the cross-reactivity between Gal-Gal pili purified from recombinant strains expressing F71, F72,F9, or F13 pili and among 60 Gal-Gal-binding wild-type strains. We present data which corroborate theconcept thatpapA genes are highly homologous and encode proteins which exhibit >70% homology among piliof different serotypes. The differences primarily occur in the cysteine-cysteine loop and variable regions andconstitute the basis for serological diversity of these pili. Although there are differences in primary structuresamong these pili, antisera raised against pili of one serotype cross-reacted frequently with many other Gal-Galpili of different serotypes. Furthermore, antisera raised against pili of the F13 serotype cross-reacted stronglyor moderately with 52 (86%) of 60 wild-type Gal-Gal-binding E. coli strains. These data suggest that there arecommon immunogenic domains among these proteins. These additional data further support the hypothesisthat broadly cross-protective PapA pilus vaccines for the immunoprophylaxis of pyelonephritis might bedeveloped.

The adherence of Escherichia coli to uroepithelial cells isan important event in the pathogenesis of urinary tractinfections (63). Although afimbrial adhesins have been iden-tified (26), bacterium-to-host cell attachment is usually me-

diated by pili (also termed fimbriae or F antigens). There arethree adhesin classes (mannose, Gal-Gal, and X) expressedby uropathogenic E. coli strains. Mannose is the receptor forcommon type 1 pili (Fl serotype) (56). Gala(1-4)Gal is thereceptor for P blood group pili (also termed P or Gal-Gal pili)(23, 28, 29). This digalactose moiety is contained in neutralglycosphingolipids which are found on epithelial and eryth-rocyte surfaces. X adhesins are a heterogeneous group of piliand afimbrial adhesins which bind to receptors other thanmannose or Gal-Gal. The following receptors for X adhesinshave been identified: NeuAca(2-3)Gal (S-pilus adhesin),glycophorin A (M-pilus adhesin), Dra blood group antigen(AFA-I and AFA-III adhesins), and GalNAca(1-3)GalNAc(Forsmann binding pilus or F-pilus adhesin) (15, 36, 39, 40,48, 66).The role of these different adhesins in E. coli-caused

urinary tract infections has been addressed in part by a

number of recent epidemiologic surveys which utilize phe-

* Corresponding author.

notypic and/or genotypic assays (2-4, 13, 44, 52, 53). Man-nose adhesins are not readily expressed in the urinary tract(e.g., <40% of cystitis isolates express mannose pili in situ[30]). The frequency of X adhesins among uropathogenicstrains is greater than in normal fecal isolates (2, 3, 26).Certain X adhesins correlate with strains which cause spe-cific urinary tract syndromes (e.g., in one report, 50% ofcystitis isolates expressed the AFA-1 adhesin [3]). Gal-Galpili are frequently associated with pyelonephritis-causingstrains. Of isolates from patients with pediatric or adultepisodes of ascending, unobstructive pyelonephritis, 85 to100% harbor the Gal-Gal pilus operon or express the Gal-Gal-binding phenotype on subculture (44, 52, 62, 65). Also,>90% of the strains which cause cystitis or pyelonephritisexpress Gal-Gal pili in situ (30). In addition, Gal-Gal pilimediate upper urinary tract colonization by E. coli in simianand murine models of experimental pyelonephritis (16, 17,42, 50, 55). For these reasons, Gal-Gal pili are identified as

pyelonephritis-associated pili, or Pap.A number of pap operons have been cloned, and their

gene products have been extensively studied (3-5, 6, 9-11,20, 21, 25, 31, 33, 35, 38, 43, 52-54, 64, 68-71). The operonencodes at least 10 polypeptides involved in the biogenesisof receptor-binding pili. The fiber is composed of thousands

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3850 DENICH ET AL.

of major pilin subunits (PapA) and three minor tip-locatedproteins (PapE, PapF, and PapG) (34, 35, 38, 64). Transcom-plementation studies indicate that PapG is the Gal-Gal-specific adhesin, although PapF is also required for receptorspecificity (34). Development of an antiadhesive vaccine isproblematic, since PapG and other pilus tip proteins (PapEand PapF) are produced only in minute quantities and/or arepoorly immunogenic (35). In contrast, PapA is highly immu-nogenic and is the major constituent of the pilus fiber,accounting for >99.9% of its mass (35). PapA pilus-basedvaccines were assessed for their efficacy in preventingpyelonephritis in experimental murine and simian infectionmodels (42, 50, 55, 58). These data demonstrate the immu-noprophylactic value of the PapA antigen in mice in effec-tively clearing homologous and heterologous Gal-Gal-pili-ated E. coli strains from the urinary tract. These findings areinteresting for a number of reasons: (i) PapA is not requiredfor specific binding (38), (ii) anti-PapA immunoglobulin G(IgG) antibody does not prevent Gal-Gal binding (10, 50),and (iii) the evidence suggests that there are cross-reactingantibodies among the different PapA pili (10, 50).

Elucidation of the biochemistry and immunochemistry ofPapA pili is important for developing broadly protectivevaccines. The identification of epitopes which are uniformlyexpressed by uropathogenic strains is an important consid-eration for a potentially effective immunoprophylactic re-agent. To date, the complete nucleotide sequences of F71,F72, Fll, and F13 PapA pilins have been determined (5, 9,54, 69-71). On the basis of an analysis of deduced amino acidsequences of mature PapA pilins which contain 160 to 168amino acids residues (R), four regions are convenientlyidentified. They are the NH2-terminal region (Rl to R21), theCys-Cys loop (R22 to R61), the variable region (R62 toR153), and the COOH-terminal region (R154 to terminus).Analyses of these PapA pilins indicate (i) high homology (76to 87%) within the NH2 and COOH termini of the proteinsequences, (ii) decreased homology (50%) among proteinsequences within the Cys-Cys loop but conserved (75%)predicted secondary-structure attributes, and (iii) low ho-mology (<50%) in protein sequences and predicted second-ary-structure attributes within the variable region (69, 70).Gal-Gal pili associated with uropathogenic strains are cur-rently classified serologically among serotypes F7 to F13 (10,12, 46). In addition, uropathogenic strains can harbor multi-ple copy numbers of different pap operons (4, 33, 52). Theycan also simultaneously express many different F serotypes(43, 44, 47).The linear immunogenic and antigenic structures of the

PapA pilin from an F13 serotype organism were previouslyevaluated with polyclonal IgG and synthetic peptides (59).These terms are defined as follows. An immunogenic epitopeis a particular domain in the native protein which is recog-nized by the immune system and elicits antibodies able tobind synthetic peptides corresponding to this domain. Anantigenic epitope is a domain in the native protein which isrecognized by antibodies engendered by synthetic peptidescorresponding to that region of the protein. There have beensimilar efforts to elucidate the immunochemistry of F71, F72,and F9 serotypes (41). On the basis of these data, immuno-genic epitopes are localized to predicted hydrophilic beta-turns in the Cys-Cys loop and the variable region (59). Thedominant immunogenic epitope for the PapA pilin is locatedjust distal to the Cys-Cys loop, corresponding to R65 to R79in the variable region. Antigenic epitopes are localized to theNH2 terminus (R5 to R12) and span the variable region. The

ES H C S11SS I

B S SH S SI I 11 I I

H SK S SH SalI I1 I 11 I

CH SK S S B11 11 I I L

pp pBR322

S H C S H SEK S SE SI I I I I III I ILI I

pUCS

E' pDAL2O1BpBIt322 (C1212, pap-21)

C HBC HpDAL210B(C1212,pap.17)

pDAL200A(3669)

I I I I I II I III Kiloba pairs

FIG. 1. Physical maps of recombinant plasmids pDAL201B,pDAL210B, and pDAL200A. The construction of the plasmidsshown here is described in Materials and Methods. The locationsand sizes (in kilobase pairs) of the papA genes are shown by a thickline. Restriction sites abbreviations: E, EcoRI; S, SmaI; H, HindIll;C, ClaI; K, KpnI; B, BamHI; and Sal, Sall.

hydrophobic COOH-terminal region lacks immunogenic orantigenic potential.

In this report, the complete nucleotide sequence of thePapA pilin from an F9 serotype organism was determinedand confirmed, in part, by limited protein analyses. Thediversity of the nucleotide sequences within a given Fserotype was evaluated by sequencing the papA genes of F71and F72 serotype strains and then comparing these resultswith the previously published sequences for these F sero-types and for others derived from different strains. Theserological relatedness of F71, F72, F9, and F13 pili was alsoevaluated by rabbit anti-pilus sera in enzyme-linked im-munosorbent assays (ELISA). In addition, Gal-Gal-bindingclinical isolates from patients with well-defined urinary tractinfections or from stools from healthy patients were evalu-ated for reactivity to rabbit anti-F13 pilus IgG by competitiveELISAs. These data provide a basis for additional strategiesin developing broadly protective vaccines against E. coli-caused pyelonephritis.

MATERIALS AND METHODS

Plasmids for papA sequencing. Gal-Gal pilus recombinantplasmids pDAL201B, pDAL210B, and pDAL200A werepreviously described (33, 50). They were employed to deter-mine papA pilin DNA sequences. The source of chromo-somal DNA for pDAL201B and pDAL210B was E. coliC1212 (06:K2) (also termed AD111), originally isolated froma woman with acute cystitis (45, 47, 67). The source ofchromosomal DNA for pDAL200A was E. coli 3669(02:KS), originally isolated from a woman with acute pyelo-nephritis (59). pDAL201B, which harbored the pap-21 op-eron encoding the PapA F71 serotype, was constructed aspreviously described (33). pDAL210B, containing the pap-i 7operon encoding the PapA F72 serotype, was constructed bycloning a 13-kb BamHI DNA fragment from plasmidpDAL212B (33) into a BamHI site on plasmid vector pBR322(7). pDAL200A was derived from a Sau3AI digest of strain3669 DNA and ligated with BamHI-digested vector pUC8(72). pDAL200A encodes a PapA F9 serotype. The papAgene for each plasmid was previously located by TnS inser-tion mutagenesis. The strategy for sequencing the papAgenes involved sequencing the relevant fragments delineatedby SalI endonuclease digestion. Figure 1 depicts the physicalmaps of these three recombinant plasmids and the locationsof their papA genes.DNA sequencing and computer analyses. DNA restriction

fragments containing apapA gene (Fig. 1) were subcloned bystandard techniques into replicative forms of M13 phagevectors mpl8 and mpl9 (72). The inserts were sequenced

INFECT. IMMUN.

ANALYSIS OF papA GENES AND PapA SEROLOGY 3851

by using Taq DNA polymerase (Taq Track; Promega Biotec,La Jolla, Calif.) in the Sanger et al. dideoxynucleotide chaintermination method (57). Nucleotide and amino acid se-quences were aligned by using FASTA and TFASTA com-puter programs (32, 49). The deduced amino acids at theNH2 terminus were correlated with protein sequence dataobtained by subjecting purified pili to Edman degradationNH2-terminal sequencing (see below). In addition, standardcomputer program packages were used for DNA sequenceanalyses, including codon usage frequency (14).

Bacteria and culture conditions for Gal-Gal-binding as-says. Gal-Gal pilus recombinant strains DL130 [HB101(pDAL201B)], DL129 [HB101(pDAL210B)], DL450 [HB101(pDAL200A)], and HU849 [HB101(pHU845)] were used toproduce Gal-Gal pili. Recombinant strains were grown onLuria agar supplemented with ampicillin (100 jig/ml) toselect for the plasmids containing the different pap operons.E. coli HB101 was also employed as a nonpiliated controlstrain. A total of 60 Gal-Gal-binding wild-type E. coli strainswere used to assess their serological relatedness to F13antibody. Forty-three isolates were obtained from womenwith anatomically normal urinary tracts who had either acutepyelonephritis or cystitis (44) according to rigorous radiolog-ical studies and clinical definitions (44). Seventeen isolateswere from the stools of healthy women with no history ofurinary tract infection. The Gal-Gal-binding phenotype wasexpressed after three to six serial passages on Trypticase soyagar. The bacteria were grown for 18 h at 37°C, harvestedinto 0.1 M phosphate-buffered saline (PBS) (pH 7.4), andverified for Gal-Gal binding by latex agglutination (43).Lactose absorbed to latex was used as a negative control.Inhibition of Gal-Gal binding by recombinant Gal-Gal-pili-ated strains was determined after 106 bacteria were preincu-bated for 30 min at room temperature with various concen-trations of homologous hyperimmune sera (12). Controlsincluded HB101 bacteria and preimmune sera.

Purification of pili. Gal-Gal pili were purified from recom-binant strains by a modification of the method of Brinton (8).Briefly, each recombinant strain was grown for 20 h at 37°Con Trypticase soy agar, harvested into ice-cold 0.005 M Trisbuffer (pH 8.3), and homogenized for 15 min at 2,000 rpm ina Sorvall Omnimixer. The sheared bacteria were removed bycentrifugation (20,000 x g for 1 h). Pilus filaments were thenprecipitated in 0.1 M MgCl2-0.15 M NaCl-0.05 M Tris (pH7.0) and collected by centrifugation. The pellet was dis-solved in 0.005 M Tris buffer (pH 8.3). Six successive cyclesof precipitation and solubilization with intervening centrifu-gation steps resulted in pure pilus preparations as defined bythe presence of a single protein band on a Coomassie blue- orsilver (37)-stained polyacrylamide gel in which 100 ,ug ofprotein was electrophoresed according to the method ofLaemmli (27). The relative molecular weights for each PapApilin expressed by the recombinant strains were analyzed bysodium dodecyl sulfate-polyacrylamide gel electrophoresis(27) on 15% acrylamide gels. The HU849 PapA was 17.5kDa, the DL130 PapA was 21 kDa, the DL129 PapA was 17kDa, and the DL450 PapA was 19 kDa.Amino acid analysis. The amino acid composition of each

Gal-Gal pilus preparation was determined after performicacid oxidation (61) by 24 h of hydrolysis in 5.7 N HCI at110°C in vacuo on a Durrum model D500 amino acid ana-lyzer. The analysis was uncorrected for decomposition ofserine and destruction of threonine during hydrolysis or forincomplete hydrolysis of leucine, isoleucine, and valine.Cysteine and methionine were determined as cysteic acidand methionine sulfone, respectively.

Amino-terminal-sequence analysis. NH2-terminal aminoacid sequences of Gal-Gal pili were determined by auto-mated Edman degradation on a Beckman model 890-Cliquid-phase sequencer using a modified 0.1 M Quadral121078 program in combination with Polybrene. Each phen-ylthiohydantoin derivative was identified and quantitated byreverse-phase high-pressure liquid chromatography and con-firmed by back gas-liquid chromatography and/or thin-layerchromatography. Amino acids were assigned when peak-to-background ratios were .2:1.

Antibody production. Preimmune sera were obtained fromNew Zealand White rabbits via cardiac puncture, filtersterilized, heat treated, and stored at -20°C. Seventy-fivemicrograms of each Gal-Gal pilus protein in PBS was emul-sified with an equal volume of Freund's complete adjuvantand injected intramuscularly at multiple sites. Also, 108Formalin-treated, PBS-washed HB101 cells were also ad-ministered separately to a rabbit in a fashion similar to thatdescribed for Gal-Gal pilus immunization. After 6 weeks, abooster injection with Freund's incomplete adjuvant wasgiven. Ten days after the second immunization, the rabbitswere bled by cardiac puncture. The hyperimmune sera werefilter sterilized, heat treated, and stored at -20°C until usedin ELISA studies.ELISA. ELISAs were employed to screen the serological

relatedness of the F serotypes in the four recombinantstrains employed in this study and to determine the preva-lence of anti-HU849 (F13) pilus IgG reactivity among Gal-Gal-binding uropathogenic strains. Direct ELISAs wereused to screen for the serological relatedness of HU849,DL130, DL129, and DL450 pili. In brief, microtiter polysty-rene wells were sensitized with 100 ng of pili from each of thepreparations in 100 ,ul of 0.1 M sodium carbonate buffer (pH9.6) for 16 h at room temperature. Wells were washed threetimes with PBS containing 0.05% (vol/vol) Brij 35 (PBS-Brij)(Sigma Chemical, St. Louis, Mo.). Hyperimmune antipilisera were serially diluted in PBS-Brij (1:100, 1:200, 1:500,1:750, 1:1,000, 1:2,000, 1:5,000, 1:10,000, 1:25,000, 1:50,000,1:75,000, 1:100,000, 1:200,000, and 1:500,000), and 100-plIaliquots of each dilution were added to the wells. Theantibody preparations were incubated for 3 h at 37°C, andthen the plates were washed three times with PBS-Brij. Onehundred microliters of alkaline phosphatase-conjugated goatanti-rabbit IgG (Tago, Burlingame, Calif.) diluted 1:1,000 inPBS-Brij was added to each well and incubated for 1 h at37°C. The plates were then washed three times with PBS-Brij. Antibody bound to pili was detected by adding 100 pul ofp-nitrophenylphosphate (1 mg/ml) in 1.0 M diethanolaminebuffer (pH 9.8) to each well. The reaction was stopped after20 min by the addition of 2 N NaOH, and the A405 wasdetermined with a MicroELISA autoreader (Dynatech, Tor-rance, Calif.). All tests were performed in triplicate. Con-trols included nonsensitized wells and wells sensitized withalbumin. Pooled preimmune sera and hyperimmune serafrom HB101 administration were diluted 1:100 in PBS andemployed as negative serum controls. A positive result wasdefined as the mean A405 for a particular sample which wastwice the average background levels exhibited by negativeserum controls.

In addition, the reactivity of rabbit hyperimmune HU849pilus IgG among Gal-Gal-binding strains was assessed bycompetitive ELISA. First, the amount of anti-HU849 pilussera needed to produced a half-maximal A405 against homol-ogous pili in a direct ELISA was determined. Then, twicethis amount of specific antibody was mixed with an equalvolume of various concentrations of Gal-Gal-binding clinical

VOL. 59, 1991

3852 DENICH ET AL.

strains (viz., 108, 107, 106, and 105) in PBS supplementedwith 0.02% sodium azide or control substances. Thesesuspensions were continuously mixed and incubated at 37°Cfor 1 h. Then the suspensions were mixed at 4°C for another12 to 16 h. After centrifugation (12,000 x g for 30 min) topellet bacteria, the supernatants were screened for bindingto homologous HU849 pili in direct ELISAs as describedabove. Controls included replacement of a clinical strainwith HU849 or HB101 bacteria or with PBS alone in thepreabsorption step. In addition, the specific antibody wasreplaced with pooled preimmune sera and anti-HB101 sera.These sera were preincubated with HU849 bacteria to deter-mine baseline background levels. The final concentration ofthese negative control sera employed in the direct ELISAwas equivalent to a 1:100 dilution. All tests were performedin triplicate. Data on the controls and clinical isolates wereused to conveniently categorize characteristics of the anti-body reactivity according to the extent of homology ofanti-HU849 pilus IgG with the HU849 strain.

Nucleotide sequence accession numbers. The sequencesreported in this paper have been deposited in the GenBankdata base and were assigned the following accession num-bers: M68059 (pDAL200A), M68060 (pDAL210B), andM68061 (pDAL201B).

RESULTS

papA sequences and deduced PapA primary structures. ThepapA genes contained in the 9.5-kb pDAL201B, 11.5-kbpDAL210B, and 8.0-kb pDAL200A plasmids were se-quenced by the dideoxynucleotide chain terminationmethod. Sequence analysis of a 626-bp fragment ofpDAL200A (Fig. 2A) indicated one long open reading framebetween nucleotides 37 and 609. Sequence analysis of a776-bp fragment of pDAL201B (Fig. 2B) showed a singleopen reading frame between nucleotides 141 and 701. Se-quence analysis of a 778-bp fragment ofpDAL210B (Fig. 2C)showed a single open reading frame between nucleotides 130and 693.Analyses of these papA sequences revealed many typical

prokaryotic features for gene organization (Fig. 2). ThepapA gene sequences contained characteristic prokaryoticribosome-binding sites, ATG initiation codon signal se-quences, and TAA termination codons. The three papA genesequences contained a potential ribosome-binding site with asequence corresponding to -AAAGAGGT. It is located 16bases upstream from the putative ATG initiation codon (Fig.2). The deduced putative signal sequences for the papAgenes were located 25 and 21 codons upstream from theirterminal alanine residues in the pDAL200A and thepDAL201B or pDAL210B genes, respectively. The threesequences were highly conserved, viz., >80% homology(Table 1). Other features of these sequences were generallycharacteristic of most other bacterial signal sequences.These sequences contained a highly hydrophobic regioncomprising an amino acid stretch of Val-Val-Ser-Phe. Therewas a positively charge residue (viz., lysine) at the -23 or-20 position. The putative cleavage site for the signalsequence of pDAL201B and pDAL210B papA genes wascharacteristic of other bacterial genes. They included anAla-Asn-Ala (pDAL201B) or Ala-Tyr-Ala (pDAL210B) trip-let. The predicted cleavage site for the pDAL200A gene wasdifferent from those of most other bacterial genes. Thecleavage site for the pDAL200A papA gene was an Asp-Thr-Val triplet. The TAA terminator in pDAL201B andpDAL210B sequences was followed shortly thereafter by

dyad symmetry (Fig. 2B and C). This feature was consistentwith rho-dependent transcriptional termination. The pre-dicted PapA proteins were translated from an ATG triplet toan upstream TAA termination site. These predicted proteinswere encoded by 163 to 167 codons. The translated aminoacids predicted by these codons were confirmed by includinglimited NH2-terminal Edman degradation sequencing andamino acid compositions of the recombinant pili. The proteinsequences of the 31, 38, and 22 NH2-terminal amino acids ofDL450, DL130, and DL129 pili, respectively, correspondedto the predicted translation of codons in the open readingframe which started at the ATG codon. In addition, piluscompositions were the same as those predicted by the DNAsequences (data not shown). These data corroborate thePapA predicted amino acid sequences encoded by the papAgenes.Codon usage. The codon usage of the papA genes for

DL450, DL130, and DL129 pili were analyzed by using acodon frequency computer program (14). The pattern ofcodon utilization was not significantly different among thegenes. In addition, selected codons read by less-abundanttRNAs in one gene were less frequent in other genes, viz.,codons for Gly (GGA/G), Ile (ATA), Leu (CTA), Lys(AAG), and Pro (CCC) (data not shown).Comparison of papA nucleotide and PapA amino acid

sequences. LINEUP and GAP computer programs (14) wereused to calculate the overall percent homology of the pre-dicted mature PapA polypeptide at the nucleotide and aminoacid levels in a number of pilins (data not shown). Theoverall homology among DL450, DL130, and DL129 pili was72 to 75% at the nucleotide level and 73 to 74% at the aminoacid level. Compared with sequences of two other PapApilins corresponding to C1976 and HU849 pili (69), there was60 to 82% homology of nucleotide sequences and 57 to 83%homology of amino acids among the PapA pilins. Table 1indicates the percent homology of nucleotide sequences atspecific locations within the papA genes. Among the papAgenes, the signal sequences and the NH2- and COOH-terminal sequences exhibited the highest overall geneticconservation. In contrast, the variable region and the Cys-Cys loop exhibited the least overall sequence homologyamong the papA genes. Comparison of the genes indicatedthat papA genes from HU849, C1976, and DL129 strainswere highly homologous. Similar results were found whenthe deduced amino acids were evaluated. FASTA andTFASTA computer programs (32, 49) were employed toassess homology at the amino acid level between HU849PapA pilin and DL450, DL130, DL129, and C1976 PapApilins. Figure 3 summarizes these comparisons. The majorityof these differences occurred in two regions: the Cys-Cysloop and the variable region. The differences were assessedin terms of the type of amino acid substitutions which weremade. Overall, most of the amino acid differences observedwere nonconserved substitutions, except when HU849 andC1976 PapA pilins were compared. Comparisons of thesubstituted amino acid sequences between HU849 PapApilin and other PapA pilins from DL450, DL130, DL129, andC1976 strains (Fig. 3) exhibited the following frequencies ofnonconserved substitutions: 41 (68%) of 60 substituted res-idues in DL450, 32 (57%) of 56 substituted residues inDL130, 35 (67%) of 52 substituted residues in DL129, and 12(46%) of 26 substituted residues in C1976.

Characterization of hyperimmune anti-Gal-Gal pilus sera.Polyclonal rabbit hyperimmune sera to recombinant Gal-Galpili were elicited from DL130, DL129, DL450, and HU849strains. The reactivities of these sera were analyzed by an

INFECT. IMMUN.

CTGGTGTGTTCAGTAAICTGAAmAGATTA^ TCGGTTATTG;CCGGTGCGGTAGCTATGGCAGTG7GTGTCTTTTGGTGCAAATGCTGM I K S V I A G A V A M A V V S F G A N A A

-25

CTGATACCGTTGCGCCATCTCAGGGGSTCA;GGGCAGGTGAACTTTAAAGGACTGTTATTIGATGCCCCGTGTGGCATTGAAACGCAGTCAGCAAATCAGACD T V A P S QG S G Q V N F K G T V I D A P C G I E T Q S A N Q T

-1 +1

TATTGATTTTGCCAG;ATTTCGAAATTGTTTCTGGAGAATGGTGGAACCACCCAGCCGAAAGATCTTGATATTAAGCTGGATTGCGATATCACCAATI D F G Q I S K L F L E N G G T T Q P K D L D I K L V N C D I T N

KAAAAAGGCATCCAGGCGGTGCAGCTAAGACAGGTACGGTATCCV CTGA FCAGGTGTVCCTGCAGGAAACGCAAGTGACATGCTGAAGAF K K A A T P G G A A K T G T V S 'L T F A G V P A G N A S D M L K T

ccG T TA CTTAATCCACATGGCTCGCGTGTGAAGTGACGGTAAAACGTCAACGGGGCCTTCTAATCTGGTTGAV G E T N T A I V V L N P H G S R V K F D G K T S T G P S N L V D

TGGTAATAATAC CACTTACAACCTATGTAATGAAAGSTGATAGTGGCAACTCTGTA GATGCCTTCTCAGCAGTTGCAAATTTCAACCTGG N N T L H F T T Y V M K D D S G N S V K E G A F S A V A N F N L

601 ACTTATCAGTAATACTGATAATCCGGT Y Q *

100

200

300

400

500

600

626

TCAGTATTCTGTCGATAAATAACCTGCCCTGAAAATACGAGAATATTATTTGTATTGATCTGGTTATTAAAGAATGGGTCATTTTAAATTGCCAGAT

ATCTCTGGTTGTGTTCAGTAATAAGGGTTA TTATGATTAAGTCGGTTATTGCCGGTGC GGTAGCTATGGCAGTGGTGTCTTTTGGTGCAAATM I K S V I A G A V A M A V V S F G A N

-21

GCAGCTGCATCTATCCCTCA'AGGACAAGGTGAAGTAAGTTTTAAAGGGACTGTTGTTGACGCTCCGTGT'GTATTGAAACTCAGTCTGCAAAACAGGAAAA A A S I P QG Q G E V S F K G T V V D A P C G I E T Q S A K Q E I

-1 +1

301 TTATGCGTTCTAAATCCTTCCTGCAAGAGGCGGAGAGACTCAACCGAAAGATTTGAATATTAAGCTTGTGAATTGTGACATTACTAATTTD F G QI S K S F L Q E G G E T QP K D L N I K L V N C D I T N F

4 01 CAAACAGCTTCSAAGGcGGGGcAGcTAAAAAAGGTACAGTGTCATTGACATTTTCAGGTGTGCCTGCAGAAAATGCAGATGACATGCTGCAAACAGTTGGAK Q L Q G G A A K K G T V S L T F S G V P A E N A D D M L Q T V G

100

200

300

400

500

600D T N T A I V V T D S S G K R V K F D G A T E T G A S N L I N G D N

ATAkCAATTCATTTTACTGCATTTGTTAAGAAAGATAATAGTGGCAAGAATGTAGCTGAAGGTGCCTTCTCAGCAGTTGCGAATTTCAACCTGACTTATCA 70 0DTNTAIVVTDSSGKRVKFDGATETGASNLINGDNT I H F T A F V K K D N S G K N V A E G A F S A V A N F N L T Y Q

TAACAGTAATACGATAATCTGGTCCGGTAAAAGCGGAAATATTCCGCTGTTTATTTCTCAGGGTATTTATCATG; 77 6

1

101

GTCGATAA,ATAACCTGCCC'TGAAATAICGAGAATATTATTTGTATTGATCTAGTTATTAAAGGTAATCGGGTCATTTTAAATTGCCAGATATCTCTGGTGT

GTTCAGTAATGA AGW=lElATTTTATTAAGTCGGTTATTGCCGGTGCGGTAGCTATGGCAGTGTGTCTTTTGGTGCATATGCTGCTCCAACM I K S V I A G A V A M A V V S F G A Y A A P T

-21 -1 +1

201 TATTCCTCAGGGGCAGGTAAAGTAACTTTTAACGGAACTGTAGTAGATGCACCATGTGGTATTGATGCTCAGTCTGCTGATCAATCTATTGATTTTGGAI P QG Q G K V T F N G T V V D A P C G I D A Q S A D Q S I D F G

301 CAAGTATCAAAATTATTTCTGGAGAATGAInGGGGAAAGTCAACCCAAATCTTTTGATATTAAACTTATAAATTGTGATATTACAAACTTTAMAAAGCTGQ V S K L F L E N D G E S Q P K S F D I K L I N C D I T N F K K A A

401 CTGGCGGTGGTGGGGAAGACTGGCACAGTATCTCTGACTTTTTCGGGTGTCCCAAGTGGTCCGCAGAGTGACATGTTACAGACCGTCGGTGCAACAAAG G G G A K T G T V S L T F S G V P S G P Q S D M L Q T V G A T N

50 1 TACAG;CTATTGTTGTCACCGATCCACATGGAAAACGCGTAAAATTTGATGGTGCGACAGCAACAGGTGTTTCCTATTTAGTTGATGGTGATAACACAATTT A I V V T D P H G K R V K F D G A T A T G V S Y L V D G D N T I

60 1 CATTTTACTGCTGCCGTCAGAAAAGATGGTAGTGGCAACCCTGTAACAGAAGGGTTTCTCAGCAGTTGCGAATTTCAACCTGACTTATCAGTAACAGTH F T A A V R K D G S G N P V T E G A F S A V A N F N L T Y Q *

701

100

200

300

400

500

600

700

778

FIG. 2. DNA sequences of pDAL200A (A), pDAL201B (B), and pDAL210B (C) papA structural genes. The nontranscribed DNA strandfor each clone is shown. Numbering is from the 5' end. The deduced amino acid sequence for the correct frame is also shown below eachDNA sequence. Numbers at ends of lines refer to nucleotide positions, and numbers below lines refer to amino acid residues. The first aminoacid of the mature protein is numbered + 1. Stop codons are marked with asterisks. Putative ribosome-binding sites (19, 60) and possibletranscriptional termination sites (54) are underlined once and twice, respectively.

3853

A

1

101

201

301

401

501

B

1

101

201

501

601

701

C

GATACTAATAC=GATTGi rACTGAiTCGAGTGGAAAACGGGTGAAATTTGATGGAGCCACTGAr.ACCCX.=TTCAAATCTGATtAATGGCGATA

GI

AATACGATAATCTGGTCCGGTAAACAGCGGAAATATTCCGCTGTTTATTTCTCAGGGTATTTATCATGAGACTGCGAT

3854 DENICH ET AL.

-25 +1 2500 HU849 MIKSVIAGAV AMAWSFGVN NA APTIP QGQGKVTFNG TVVDAPCSIS

O t oO DL130 ---------- --------a- a-dtv--S --s-q-n- --I----G-eiroCrN DL129 ---------- --------a- a -AS-- ----e-S--- -------G-e

-4 |DL450 -------------------y a ----- -------G-d- ~~~~~~~~~~~~~~~C1976.----------a

u

50 75HU849 QKSADQSIDF GQLSKSFLEA GGVSKPMDLD IELVNCDITA FK GGNG

>tooo DL130 t---N-T--- --I--l---n --tTq-k--- -k-------- --kaatP-gAcn .4 6t O DL129 t-----e--- --I-----Qe --eTq-k--N -k-------- --q lq-gA0O O .O ir DL450 a--------- --V--1---n d-e-q-ksf- -k-I------ --kaa --g-

C1976 ---------- ------------.t------- ---------- --q -QA

100 125HU849 AKKGTVKLAF TG PIVNGHS DELDTNGGTG TAIW QGAG KNVVFDGS EDL130 --t---s-T- A---a gna- -m-k-v-e-n ------NPh- sr-k---k-s

U 00 00 00 00"o DL129 ------s-T- S-v-a Enad -m-Q-v-d-n ------DSS- -r-k---At-ON CO 0f C ON DL450 --t---s-T- S-v-s gpq- -m-Q-v-A-n ------DPh- -r-k---Ata

ONONX~ F-;C1976 --n-k-q-S- -- -q-t-qA E--a------ ----- -A-- ---s---T a

150 170HU849 GDANTLKDGE NVLHYTAWK KSSAVGAAVT EGAFSAVANF NLTYQ

0 tON DL130 TgPsn-v--n -t--F-Ty-m -ddS -nS-k ---------- -----

00ooDL129 Tg-sn-iN-D -tI-F--f-- -dnS -kn-A ---------- -----o

> t- r1 o DL450 Tgvsy-v--D -tI-F--a-R -dgS -nP-- ---------- -----

roio ,- o£ 8C1976 ---yP----D -------L-- - -n-GT-S ---------- --S-

FIG. 3. Comparison of deduced PapA amino acid sequencesfrom DL450, DL130, DL129, C1976, and HU849 strains. Thededuced amino acid sequences were aligned by using the FASTA

4o4 0 and TFASTA computer programs (32, 49). The first amino acid ofQ.U oo r- 0fO the leader sequence is numbered -25, and the first amino acid of the

.o v e' ON o o > mature protein is numnbered +1. Gaps, indicated by blank spaces,were introduced to obtain maximal similarity. Amino acid identitieswith the PapA sequence of HU849 strain (top line) are indicated by

ONDCZahyphen, and conserved amino acid substitutions (uppercasec; iE N letters) and nonconserved amino acid substitutions (lowercaseC ol, oo af oo R letters) are indicated by single-letter amino acid codes.

ELISA which employed anti-rabbit IgG enzyme conjugates.00 All hyperimmune sera reacted vigorously with their homol-

=,,iNN w w3 ogous pilus antigen (Table 2). The reciprocals of the IgGQ) 00OO QW O titers for homologous binding ranged between 25,0004) zi(anti-DL450) and 75,000 (anti-DL129). These antibody prep-

arations lacked the ability to inhibit Gal-Gal binding by the(U Srecombinant strain from which the antisera were elicited

(data not shown). Antisera were initially assessed for inhi-=i i N 0 0oO bition of Gal-Gal binding at a dilution equivalent to one-half

the reciprocal of their maximal-binding IgG titer in homolo-gous ELISA reactions. Further screening for inhibition ofGal-Gal binding by these sera included serial 10-fold dilu-

o0 tions up to a 1:100,000 dilution.O P4 xo0 1o " Co r-I The antipilus sera also reacted with all heterologous

00 'N> -< ONoGal-Gal recombinant pili. However, compared with homol-ogous binding, heterologous reactions were considerablyless reactive (Table 2). For example, there was 125-fold less

Q':=0> =<. anti-DL450 pilus IgG binding to HU849 pili than to thehomologous antigen. There were also pilus antigens which

CIi 6N O oio elicited antibodies that bound heterologous pili better than00i

0 r- r- ON, others. They included DL130 pili, which elicited high-titered.$arr >antibody against DL129 and HU849 pili; DL450 pili, which.9b7a>elicited high-titered antibody against DL130 pili; DL129 pili,0.o ° which elicited high-titered antibody against DL130 pili; and

HU849 pili, which elicited high-titered antibody against> DL130 and DL129 pili. None of the pooled preimmune sera

-b = D D or antisera raised against HB101 bacteria reacted with Gal-.o c Gal pilus antigens.

oI'- )i ~0 N0 | O - QResults from competitive ELISAs employing rabbit anti-. 0 la HU849 pilus sera with clinical strains are summarized in

'n .o u:O .=O ) :c....Table3. MeanA405s of specific IgG antibody reactivity afterCZ 61 E , *

preincubation with HU849 bacteria are representative ofzZ strong homology (positive control) (e.g., A405 < 0.10). Val-

c6 'V O' ....uesfor specific antibody reactivities after preincubation with

INFECT. IMMUN.

ANALYSIS OF papA GENES AND PapA SEROLOGY 3855

TABLE 2. Cross-reactivity of polyclonal rabbit hyperimmunesera among recombinant Gal-Gal pili

Strain IgG ELISA titera to Gal-Gal pili from strain(F serotype) (F serotype):providing DL130 DL129 DM50 HU849

antisera to pili (F71) (F72) (F2) (F13)

DL130 (F1) 4.6 4.0 3.0 3.6DL129 (F72) 4.3 4.8 3.3 4.0DL450 (F9) 3.3 2.6 4.3 2.3HU849 (F13) 3.0 4.0 2.6 4.6

aExpressed as the logarithm of the last reciprocal dilution of antiserumexhibiting an absorbance value twice that of background (see text).

buffer alone or with HB101 bacteria (negative controls) arerepresentative of weak or no homology (e.g., A405 s 0.75).On the basis of these results, we have arbitrarily definedhomology according to the following categories: strong(-90% ELISA inhibition), moderate (40 to 70% ELISAinhibition), and weak or none (<10% ELISA inhibition).Assignment of a strain to a particular homology categorywas based on A405s after preincubation with anti-HU849pilus sera and 108 CFU of the test strains. When fewer CFUof the test strain were used, strains that originally exhibitedstrong or moderate homology frequently elicited A405s abovetheir original A405s. Table 3 summarizes the pattern ofreactivity of anti-HU849 sera with the 60 Gal-Gal-bindingclinical isolates. The majority (87%) of the strains exhibitedstrong or moderate homology with this antibody prepara-tion. Only 8 (13%) of 60 Gal-Gal-binding strains lackedepitopes bound by this polyclonal rabbit anti-pilus IgGpreparation (Table 3).

DISCUSSION

Gal-Gal pilus and PapA pilus vaccines are consideredpotentially efficacious immunoprophylactic reagents in theprevention of ascending E. coli-caused pyelonephritis. Datafrom experimental pyelonephritis in simian and murine mod-els demonstrate that pili recipients are protected againstmost homologous and many heterologous Gal-Gal-piliatedchallenge strains (42, 50, 55, 58). These findings suggest thatthere are protective epitopes contained in PapA pilins andthat there might also be cross-protective epitopes in PapApilins. In this study, we sequenced three papA genes andevaluated the serological relatedness of four PapA pilinsfrom the recombinant strains used and from 60 Gal-Gal-binding wild-type E. coli strains to determine the diversity of

TABLE 3. Prevalence of anti-HU849 pili (F13) IgG reactivityamong Gal-Gal-binding uropathogenic E. coli strains as

determined by competitive ELISAs

Source of Gal- Extent of homology [no. positive/total no. (%)P'Gal-binding

strains Strong Moderate Little to none

Pyelonephritis 10/14 (71) 3/14 (21) 1/14 (7)Cystitis 19/29 (66) 5/29 (17) 5/29 (17)Normal stool 9/17 (53) 6/17 (35) 2/17 (12)

Overall 38/60 (63) 14/60 (23) 8/60 (13)a Homology was assessed as follows: strong, -90% ELISA inhibition;

moderate, 40 to 70%o ELISA inhibition; little to none, <10%o ELISA inhibition(see text).

these proteins in order to develop new insights for formulat-ing more-effective PapA vaccines.We sequenced papA genes from pDAL200A, pDAL201B,

and pDAL210B by the Sanger dideoxynucleotide chaintermination method. The putative translated structures ofthese mature PapA proteins were corroborated, in part, byNH2-terminal automated Edman degradation sequencingand the amino acid compositions of the expressed pili. Theprimary structures of pDAL201B and pDAL210B are repre-sentative of previously published F71 and F72 serotypes andwere 99.8 and 100% homologous, respectively, at the nucle-otide sequence level (data not shown) (54, 69, 70). Thepredicted PapA primary structure of pDAL200A was alsodetermined and was representative of the F9 serotype. Thereis high homology among these papA genes and other pub-lished papA sequences (5, 54, 69, 70). There is 60 to 82%conservation of nucleotide sequences among these genes.These data suggest that the genes are closely related in termsof evolution, as previously proposed (5, 54, 69, 70). Furtheranalyses of these genes (Fig. 2) support this hypothesis. Thegenetic organizations of the papA genes sequenced in thisreport and papA sequences published elsewhere are identi-cal (5, 22, 51, 54, 69-71). The putative ATG translationalstart codon for each papA sequence in this report is corrob-orated by NH2-terminal sequence data from the predictedtranslated mature PapA protein. The predicted polypeptidesequence of the mature PapA for all sequenced genesdemonstrates two cysteine residues at codons 22 and 61. TheNH2 and COOH termini are highly homologous (Table 1),and the terminal amino acid for all PapA proteins is glu-tamine. Codon usage for papA genes is similar to that ofother E. coli genes (1, 5, 73), indicating that this gene hasevolved within this species. In summary, these featuressuggest that pap genes diverged from a common ancestralpilus gene, as previously proposed (5, 69-71).There are a number of specific features regarding the

proposed PapA structure for these genes which are relevantfor vaccine development. The NH2-terminal amino acid R5through R12 of F72, F8, Fll, F12, and F13 serotype peptidesare characterized by a sequence corresponding to Pro-Gln-Gly-Gln-Gly-Lys-Val-Thr. According to in situ hybridizationand anti-synthetic-peptide antibody studies (13, 58, 59), thissequence or portions of it are found in 70 to 80% ofGal-Gal-binding uropathogenic strains. This sequence isnonimmunogenic within the native pili (59); however, thesynthetic peptide corresponding to this sequence has anti-genic properties (59) and can elicit protective antibody toprevent experimental pyelonephritis in a mouse model (58).Antibodies directed to this common R5-to-R12 NH2-terminalepitope do not react with F9 and F71 pili (58, 59). The F9PapA pilin from pDAL200A and the F71 PapA pilin frompDAL201B exhibit considerable differences in codons cor-responding to the common NH2-terminal amino acids atpositions 5 through 12 for F72, F8, Fll, F12, and F13.Compared with the prototypic R5-to-R12 PapA sequence,there are three of eight nonconserved amino acid substitu-tions in the F9 pilin from strain DL450 and two of eightnonconserved amino acid substitutions in the F71 pilin fromstrain DL130. The prevalence of NH2-terminal sequencesbetween R5 and R12 which correspond to F9 and F71sequences in uropathogenic strains is not known. Studieswith oligonucleotide probes and specific monoclonal anti-bodies are currently being pursued to assess the frequency ofthese sequences in uropathogenic strains. These potentiallyprotective epitopes within F9 and F71 serotype peptidesshould obviously be incorporated into pilus vaccines as

VOL. 59, 1991

3856 DENICH ET AL.

synthetic peptides if they are frequently harbored by uro-pathogenic strains. They might also be included in vaccinesto prevent the emergence of antigenic escape variants if theyare not in fact commonly found among uropathogenic E. colistrains which cause unobstructive, ascending pyelonephri-tis.The variable region encoded by codons corresponding to

R65 to R79 exhibits the greatest degree of heterogeneityamong the PapA pilin sequences (Fig. 3). R65 to R79 of F71,F72, F9, and F13 serotypes have both antigenic and immu-nogenic properties (41, 59). The R65-to-R79 linear sequencesconstitute the type-specific immunodominant PapA epitope.These epitopes also elicit protective IgG antibodies to pre-vent experimental pyelonephritis by homologous piliatedstrains in a murine model (41, 58). Nonetheless, because ofa number of other observations, it seems unlikely thatbroadly cross-reactive protective vaccines can be limited tothese peptide sequences from prototype F71, F72, F9, andF13 serotype strains. It appears that these sequences areserotype specific; i.e., anti-R65-to-R79-synthetic-peptide an-tibody reacts only with homologous sequences. In addition,the four epitopes corresponding to R65 to R79 or portions ofthem in DL450, DL130, DL129, and HU849 strains arecollectively detected in <3% of all Gal-Gal-binding uro-pathogenic strains (13, 58, 59). However, it is possible thatR65 to R79 corresponding to amino acid sequences in theother known F serotype strains associated with uropatho-genic strains (i.e., F8, F10, Fll, and F12) are more prevalentthan corresponding amino acid sequences in F9, F71, F72,and F13 serotype strains. However, only if these sequencesare found in a majority (>80%) of the uropathogenic strainsis it then plausible that these peptides can constitute aneffective polyvalent vaccine for broad immunoprophylacticeffect. To our knowledge, there has not been a systematicevaluation of the prevalence of specific F-serotype immuno-genic or antigenic epitopes among uropathogenic strains.These studies would obviously be useful for vaccine devel-opment.

Polyclonal hyperimmune pilus sera were used to assessthe serological properties or relatedness of four recombinantGal-Gal pili from DL450, DL130, DL129, and HU849 strainsand 60 Gal-Gal-binding wild-type E. coli strains. The hyper-immune sera raised against purified recombinant pili reactedwith homologous PapA pilin (Table 2) but also with the otherrecombinant PapA pilins. We are confident that the antibodyreactivity is directed to PapA pilin determinants because theELISA is not sensitive enough to detect minor pilin deter-minants (50), and none of these hyperimmune sera inhibitGal-Gal binding, as might be expected of an antiadhesinserum. These results are corroborated by other previouslyperformed serological studies employing specific rabbit,murine, and/or human antibodies (10-12, 50). There appearto be sufficient data to indicate that Gal-Gal pili have variousPapA pilin immunogenic determinants in common, as previ-ously observed by other investigators (10, 11, 58, 59). Incontrast, J. Hanley et al. determined that rabbit sera raisedto a limited number of pili from wild-type strains exhibitingmannose-resistant hemagglutination rarely cross-react withthe pili from clinical isolates exhibiting such a phenotype(18). One explanation for their finding is that wild-typestrains can simultaneously express different types of pili;e.g., C1210 can express F71, F72, Fl, and FlC pili (24). If thepili used by J. Hanley et al. were from different classes of pili(e.g., FlC and Gal-Gal pili), it is easy to understand whythere was no cross-reactivity of the immune sera among thepili.

In this report, the serological cross-reactivity of anti-piliIgG among the recombinant Gal-Gal pili can be categorizedaccording to relative antibody-binding reactivity. Gal-Galpili from the DL130 strain elicit high-titered binding antibodyto pili from DL129 and HU849 strains, DL129 pili elicithigh-titered antibody to pili from DL130 and DL450, andHU849 pili elicit high-titered antibody to pili from DL130and DL129 strains. These serological results and groupingsare compatible with the structural similarities and differ-ences found in the deduced amino acid sequences of pre-dicted immunogenic PapA epitopes of these genes (59).These results support the concept that there are commonimmunogenic determinants among Gal-Gal-binding pili. Onthe basis of these data, it should be possible to identify andassess the efficacy of common epitopes among the PapApilins for the prevention of E. coli-caused pyelonephritis.The results of anti-HU849 pilus IgG reactivity with 60Gal-Gal-binding strains support this conclusion. There is ahigh prevalence (86%) of strong to moderate antibody reac-tivity with these clinical strains (Table 3). Only 8 (13%) of the60 strains lack immunogenic epitopes recognized by thisantibody. Judging by the similarity of predicted immuno-genic amino acid sequences of F71, F72, F9, Fll, and F13PapA pilins, a likely location for a common immunogenicepitope for PapA pilins is at R108 to Rlll, corresponding tothe following HU849 PapA sequence: Ala-Ile-Val-Val (Fig.3). Although a synthetic peptide corresponding to this se-quence did not elicit protection against a homologous pili-ated E. coli strain in the BALB/c mouse model of pyelone-phritis (58), it might be reasonable to reevaluate theprotective capacity of this region if enhanced antibodyresponses are elicited in vaccine recipients.

ACKNOWLEDGMENTSWe thank Kenneth Vosti for a number of strains from patients

with well-defined clinical episodes of urinary tract infection.This work was supported by grants from the Veterans Adminis-

tration (P.D.O.) and by National Institutes of Health grants A122974(P.D.O.), A123435 (P.D.O.), A100881 (D.A.L.), and A123348(D.A.L.) and predoctoral training grant 5T32-6MO7464 (L.B.B.).

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