Vol. 56, No. 8INFECTION AND IMMUNITY, Aug. 1988, p. 2180-21880019-9567/88/082180-09$02.00/0Copyright X 1988, American Society for Microbiology

CS31A, a New K88-Related Fimbrial Antigen on BovineEnterotoxigenic and Septicemic Escherichia coli Strains

JEAN PIERRE GIRARDEAU,* MAURICE DER VARTANIAN, JEAN LUC OLLIER, AND MICHEL CONTREPOISLaboratoire de Microbiologie, Institut National de la Recherche AgronomiquelCentre de Recherche de

Clermont-Ferrand-Theix, 63122 Ceyrat, France

Received 28 December 1987/Accepted 25 April 1988

The nature of the common surface antigen of six hemagglutinating and adhesive piliated Escherichia colistrains isolated from diarrheic or septicemic calves was studied. By electron microscopy studies, the E. colisurface antigen designated CS31A was found on bacterial cells and in purified form to consist of thin (2-nm)"fibrillar" fimbriae. E. coli 31A, which was cured of a 105-megadalton plasmid, failed to express CS31Afimbriae, but retained the ability to hemagglutinate and to adhere in vitro on intestinal cells. Conversly, E. coliK-12, harboring the 105-megadalton plasmid originating from strain 31A, produced CS31A fimbriae but wasnot able to hemagglutinate or adhere on intestinal cells. A single fimbrial subunit of 29 kilodaltons was observedwhen purified fimbriae from the 105-megadalton plasmid-containing E. coli K-12 strain was subjected tosodium dodecyl sulfate-polyacrylamide gel electrophoresis or eluted by gel filtration after dissociation by 8.5 Mguanidium hydrochloride from an S300 Sephacryl column. Western immunoblot analysis and the N-terminalsequence and amino acid composition of CS31A indicate structural and immunological relatedness betweenCS31A and K88 protein subunits.

The role of Escherichia coli as a pathogen is well known,and many E. coli isolates have been associated with a widevariety of diseases in animals and humans. These pathogenicE. coli include enterotoxigenic E. coli (ETEC) and entero-pathogenic, enteroinvasive, and uropathogenic E. coli (30).Furthermore, E. coli is often the causative agent of oppor-tunistic infections in compromised hosts. Thus, septicemiccolibacillosis is a persistent problem in neonatal calves (8),especially when passive transfer of colostral immunoglob-ulins fails (14, 34). Epidemiological studies revealed that30% of newborn calves are hypogammaglobulinemic (14, 15)and are therefore susceptible to colisepticemia. The greatdiversity of potential pathogenic serotypes encountered incolisepticemia (12) and the failure of serotype-specific anti-body to cross-protect against a heterologous challenge inexperimental infection (46, 47) have made it difficult todevelop vaccines against septicemic colibacillosis. Investi-gations made to identify a common antigenic component thatcould elicit antibodies protective against a number of poten-tial septicemic E. coli strains found that passive transfer ofantibodies to the oligosaccharide core region of lipopolysac-charide led to a partial protection of colostrum-deprivedcalves against colisepticemia by neutralizing endotoxin (47).

Fimbriae from many bacterial pathogens have been exten-sively characterized and shown to be important for virulenceby promoting adherence of bacteria to the host epithelialcells (10, 13, 27, 31). An essential element of vaccinedevelopment is the detection of common fimbrial antigensoccurring among most pathogenic isolates and able to induceantibodies that block bacterial adhesion (27, 31). In ourprevious work with septicemic E. coli 31A we found asurface antigen referred to as 31a (1) on septicemic E. coliserotypes 08, 020, 078, 086, 0117, and 0153. However,initial attempts to characterize this common antigen hadfailed because of multiple surface appendages carried by thestudied strains.The purpose of this study was to investigate the possibility

* Corresponding author.

that the 31a antigen was a new fimbrial antigen. After theexistence of such fimbriae had been confirmed by immuno-electron microscopy, the next purpose of this study was topurify and characterize this fimbrial antigen. In agreementwith terminology proposed by Smyth (42), 31a antigen isreferred to as CS31A (for coli-surface-associated antigen).On the basis of amino acic composition, the N-terminalsequence, and Western immunoblot analysis, CS31A ap-pears to be a new fimbrial antigen belonging to the group ofK88-related proteins.

MATERIALS AND METHODSBacterial strains and media. Pertinent characteristics and

origins of E. coli strains are given in Table 1; included areRVC 330, a septicemic reference strain isolated from theblood of a septicemic calf, and five representative patho-genic bovine E. coli strains isolated from stools of diarrheiccalves. E. coli 31A expresses the surface antigen 31a andcauses experimental septicemia in calves (1). E. coli AY1expresses the fimbrial adhesin FY, previously described as anew fimbria occurring simultaneously with K99 and F41 onsome calf ETEC strains (2, 3). E. coli Orne 6 exhibitsadherence and hemagglutination mediated by two differentpili (type 1 and an unidentified pilus). Both strains 92b andB117 are bovine ETEC that produce heat-stable enterotoxin.B117 is also a reference strain, described by Hill (24) as acapsulated serum-resistant E. coli strain able to producesevere mastitis in cows. In addition, on the basis of experi-mental infections, B117 appears to be a ubiquitous patho-genic strain able to cause diarrhea (A. Bertin and M. DuchetSuchaux, Abstr. Int. Symp. Virulence Mech. Vet. BacterialPathogens 1987, p. 7) or septicemia (24). RVC 330, Orne 6,AY1, and the calf ETEC 92b were observed to produceaerobactin (unpublished results), which is characteristic ofE. coli strains that cause extraintestinal infections (8). E. colireference strains for K88 and F41 antigens are listed in Table1. Bacteria were grown at 37°C for 18 h on Minca agar (19)supplemented either with 0.2% glucose (MG2) or with 0.2%L-alanine (MA2).

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TABLE 1. Characteristics and sources of E. coli strains used in this study

Strain Source or reference Serotypea Fimbrial Aerobactinb Enterotoxincantigen

E68d Piglet diarrhea 0141:K85 K88ab -

G1253d Piglet diarrhea 0147:K89 K88ac -

56/190d Piglet diarrhea 08:K? K88ad -

B41M Spontaneous mutant from the K99 0101:K- F41 - +reference strain B41 (36)

B117 Diarrhea (24) 08:K85ab K99 - +92b Diarrhea, clinical isolate 08:K85ab:H2 K99 + +Orne 6 Diarrhea, clinical isolate 017:K7:H18 Type 1 +AY1 Diarrhea, clinical isolate 02:K7:H- FYRVC 330 Septicemia (25) 078:K80 +e +31A Diarrhea (1) 0153:K-:H- +e +31A/06 Laboratory derived; 105-MDa plasmid- 0153:K-:H- +e

cured variant of 31AXA Laboratory strain DB 6433 K12XA31A Strain XA with the 105-MDa plasmid from K12 This study +

strain 31Aa OKH serotypes were established by I. 0rskov.b Hydroxamate production.I Heat-stable enterotoxin detected by the assay of Dean et al. (5).d Reference strains were obtained from the World Health Organization Collaborative Centre for Reference and

Denmark.eRigid fimbriae detected by electron microscopy.

Plasmid analysis. Plasmid DNA was extracted by themethod of Kado and Liu (26). Plasmid-curing experimentswere carried out with ethidium bromide. For conjugationalplasmid transfer experiments, E. coli K-12 strain DB6433(referred to as XA) obtained from R. W. Davis, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., was therecipient strain and the membrane filter method was used.

Extraction and purification of CS31A. All CS31A naturallyoccurring strains simultaneously express several fimbriae.Therefore we purified CS31A from the E. coli K-12 trans-conjugant XA31A bearing he CS31A-encoding plasmid, orig-inating from strain 31A. Overnight MG2 cultures from 10Roux flasks were harvested in 70 ml of phosphate-bufferedsaline PBS (pH 7.2), and the suspension was blended for 2min with a Top Mix vortex blender at maximum speed.Bacterial cells were removed by centrifugation at 20,000 x gfor 15 min at 4°C. The supernatant was collected andcentrifuged at 50,000 x g for 50 min at 4°C to removemembrane vesicles. The resulting supernatant was thensubjected to 10 and 20% (of saturation) sequential ammo-nium sulfate precipitation. The 20% precipitate (5 h at 4°Cwithout shaking) was collected by centrifugation (10,000 x gfor 10 min) and suspended in 15 ml of PBS (pH 7.2). To thismaterial, solid crystallized sodium taurodeoxycholate wasadded to a final concentration of 0.5% (wt/vol), and themixture was stirred gently at 20°C for 2 h. Sodium tauro-deoxycholate-insoluble material was sedimented at 110,000x g for 200 min at 20°C. The crude antigen was incubated for2 h at 37°C with mild agitation in 3 ml of 5 mM Trishydrochloride (pH 7.8) in 8.5 M guanidine hydrochloride asdescribed by Eshdat et al. (9) to dissociate the CS31Afibrillar polymer into individual subunits. Subsequently, 5mM Tris hydrochloride (pH 7.8) was added to achieve 6 Mguanidine hydrochloride, and the preparation was allowed tostand at 20°C for equilibration. The 6 M guanidine hydro-chloride sample was subjected to chromatography on aSephacryl S300 (Pharmacia, Uppsala, Sweden) column (2.5by 64 cm) and eluted at a flow rate of 1.65 ml/min in 5 mMTris hydrochloride (pH 7.8) containing 6 M guanidine hydro-chloride. Fractions (5 ml) were collected, and the opticaldensity of the eluate was continuously monitored at 280 nm

Research on Escherichia, Copenhagen,

with a 0.5-ml flow cell inserted in a Uvikon spectrophotom-eter. Gel filtration on Sephacryl S300 in 5 mM Tris hydro-chloride (pH 7.8) containing 6 M guanidine hydrochlorideyielded three major peaks. Selected fractions were pooledand dialyzed against 5 mM Tris hydrochloride buffer (pH7.8) containing 10 mM EDTA to remove guanidine hydro-chloride. Fimbrial subunits were reassembled into polymersby the method of Eshdat et al. (9). Deaggregated fimbrialfractions were dialyzed at 4°C for 48 h against 5 mM Trishydrochloride buffer (pH 7.8) containing 10 mM MgCl2 andthen subjected to chromatography on a Sephacryl S300column (2.5 by 64 cm) equilibrated and eluted with 5 mMTris hydrochloride (pH 7.8). Reaggregated fimbrial subunitseluted in the void volume were dialyzed against distilledwater at 4°C for 12 h and lyophilized.PAGE and staining. Sodium dodecyl sulfate-polyacryl-

amide gel electrophoresis (SDS-PAGE; 10 or 15% polyacryl-amide) in slab gels (0.75 mm thick) and preparation ofsamples were carried out as described by Laemmli (29).Protein bands were visualized by a modified silver-stainingprocedure described by Oakley et al. (38). In brief, the gelwas fixed overnight in 50% (vol/vol) methanol with gentleshaking and then rinsed twice for 15 min in distilled water.The solution for the last rinse contained dithiothreitol (5 mg/liter), which resulted in more reproducible staining patterns(37). Without further rinsing, the gel was soaked for 15 minin an ammoniacal silver solution and rinsed twice in distilledwater for 2 and 5 min. After staining, the gel was washed for1 h in distilled water with three changes of water.

Preparation of antisera. Rabbit antisera against K99, F41,FY, and 31a were prepared by the procedure of Sojka (43) aspreviously described (1, 3, 36). Specific K88ab antiserumwas obtained by the procedure of Guinee et al. (20). Becauseall the CS31A E. coli strains produced various fimbrialproteins simultaneously (Table 1), purification of CS31Afrom these strains was difficult. Therefore, we undertookfirst to elaborate monospecific CS31A antiserum with adenatured antigen purified by a gel extraction procedure(21). In brief, protein was purified from a crude extract of the31A strain (sodium taurodeoxycholate insoluble) by prepar-ative SDS-PAGE (3 mm thick; 9% acrylamide). The CS31A

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FIG. 1. (A) Double gel immunodiffusion with anti-fimbriaeCS31A against 60 SM extracts from the following E. coli strains: 1,Orne 6; 2, AY1; 3, B117; 4, RVC 330; 5, 31A; 6, 31A/06; 7, 31Agrown at 18°C on MG2; 8, 31A grown at 37°C on Minca containing0.2% alanine. Extracts 1 to 6 were obtained from bacteria grown at37°C on MG2. (B to D) Double gel immunodiffusion with 60 SMextracts from the following E. coli strains: a, G1253; b, E68; c, 56/190; d, B41M; e, 31A; f, purified reaggregated CS31A fimbrialsubunits, against anti-F41 (panel B), anti-K88ab (panel C), andanti-reaggregated CS31A (panel D) subunits.

band was cut from unstained gel corresponding to a Coo-massie blue-stained part of the gel. Gel slices were homog-enized by being passed through a needle with incompleteFreund adjuvant and injected subcutaneously into rabbits(21). Each animal was injected five times at 5- to 6-dayintervals with 200 ,ug of protein. Rabbits were bled on day 8after the last injection. This antiserum was referred to asanti-denatured CS31A subunits.A second antiserum was produced against the native form

of CS31A by using the reaggregated fimbrial preparationpurified from the plasmid-encoded CS31A-producing strainXA31A. Rabbits were injected subcutaneously at multiplesites three times at 4-week intervals with 250, 250, and 500,ug of protein in incomplete Freund adjuvant (vol/vol). Tendays after, the last injection serum was collected; this wasreferred to as anti-reaggregated CS31A subunit. Alterna-tively, a third antiserum was produced by immunizing rab-bits by the method of Sojka (43) with the transconjugant E.coli XA31A grown at 37°C on MG2 and absorption with theisogenic plasmidless strain E. coli XA. This absorbed anti-serum was referred to as anti-fimbriae CS31A.

Immunodiffusion. Ouchterlony gel immunodiffusion (41)was conducted with 0.9% (wt/vol) agarose (E. Merck AG,Darmstadt, Federal Republic of Germany) made up in 0.05M Veronal (Winthrop Laboratories, Div. Sterling Drug Co.,New York, N.Y.) hydrochloride buffer (pH 8.2). Samples (15,ul) of 60°C-soluble extracts (60 SM extracts) or purifiedfractions were applied to the wells 6 h before the antiserawere.

Immunoblotting. The crude preparation (60 SM extracts)and purified CS31A were electrophoresed in duplicate: onegel was silver stained, and the other was electroblotted ontonitrocellulose. Western blots were developed as describedby Towbin et al. (45) with minor modifications. The nitro-cellulose filters were incubated overnight at room tempera-ture with antisera diluted in Tris-buffered saline (TBS; 20mM Tris base, 500 mM NaCl [pH 7.5]) and 1% (wt/vol)gelatin. Antisera against CS31A in native form (anti-fimbriaCS31A or anti-reaggregated CS31A subunits) were diluted 1:100, and serum against denatured CS31A subunits wasdiluted 1:50. Filters were washed three times for 20 min eachin TBS containing 0.05% (vol/vol) Tween 20 and thenincubated at room temperature for 1 h in 1:2,000 goat

anti-rabbit immunoglobulin G conjugated to horseradishperoxidase (Nordic Immunological Laboratories). Filterswere washed and developed with hydrogen peroxide sub-strate and 4-chloronaphthol chromogen.

Hemagglutination. Hemagglutination tests were performedas described by Evans et al. (11). All strains were examinedfor mannose-sensitive hemagglutination (MSHA) and man-nose-resistant hemagglutination (MRHA) against a 3% (vol/vol) suspension of erythrocytes from humans, sheep, cattle,guinea pigs, chickens, and horses.

In vitro adhesion and enterotoxin assays. Adhesion tests oncalf intestinal villi were made as previously described (16).Heat-stable enterotoxin activity was tested by the infant-mouse assay (5).

Electron microscopy. E. coli strains were grown at 37°C onMG2, and bacterial cells stained negatively with 1% (wt/vol)phosphotungstic acid on carbon-stabilized collodion-coated300-mesh copper grids were examined with an EM 400electron microscope (Philips Electronic Instruments, Inc.,Mahwah, N.J.) having an acceleration voltage of 80 kV.For immunoelectron microscopy, gold immunolabeling of

intact cells was performed as described by Levine et al. (32)with anti-fimbriae CS31A serum diluted 1:8 and 10-nmcolloidal gold-labeled goat anti-rabbit serum (Jansen Phar-maceutical, Piscataway, N.J.). Ferritin immunolabeling onsectioned cells was performed as described by 0rskov et al.(40). Normal rabbit serum and anti-fimbriae CS31A wereused diluted 1:9, and labeling was carried out with ferritin-conjugated goat anti-rabbit immunoglobulin G (Miles-YedaLtd., Rehovot, Israel) diluted 1:50 in PBS.

Preparation of 60 SM extracts. The 60 SM extracts wereprepared from bacterial cells grown on MG2 for 18 h at 37°C.Bacteria were scraped carefully from agar medium in petridishes (diameter, 13 cm), suspended in 4 ml ofPBS (pH 7.2),and heated at 60°C for 15 min. After centrifugation at 27,000x g for 10 min, the resulting supernatant was centrifuged at110,000 x g for 200 min, and the pellet was suspended in 400,ul of PBS. This suspension was used undiluted for immuno-diffusion and diluted 1:10 for SDS-PAGE.Chemical analysis. The protein content was determined by

the method of Markwell et al. (33) with bovine serum

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FIG. 2. SDS-PAGE (15% acrylamide) analysis of 60 SM extractsfrom the following E. coli strains: 1, 56/190; 2, G1253; 3, E68; 4,B41M; 5, 92b; 6, B117; 7, RVC330; 8, AY1; 9, Orne 6; 10, 31A; 11,31A/06; 12, XA31A; 13, XA; 14, purified reaggregated CS31Afimbrial subunit without heating before electrophoresis; 15, purifiedfraction with boiling before electrophoresis. Protein molecularweight standards (in thousands) were carbonic anhydrase (30.0),soybean trypsin inhibitor (20.1), and a-lactalbumin (14.4).

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FIG. 3. Western blot (SDS-PAGE; 10%o acrylamide) of 60 SMextracts from various E. coli strains (lanes 1 to 9) probed withanti-fimbriae CS31A. Lanes: 1, E. coli K12:XA; 2, XA31A; 3, 31A/06; 4, 31A; 5, Orne 6; 6, AY1; 7, RVC330; 8, B117; 9, 92b; 10,purified reaggregated CS31A fimbrial subunits.

albumin as the standard. Carbohydrate was measured by thephenol method (22) with glucose as the standard.Amino acid analysis. Amino acid analysis was performed

on a Kontron amino acid analyzer (Chromakon 500). Sam-ples were hydrolyzed in a nitrogen atmosphere for 24 h at110°C in 6 N HCI.

Deterniination of the N-terminal amino acid sequence. Theprimary structure of the N-terminal part of CS31A wasdetermined by automatic Edman degradation on a model470A amino acid sequenator (Applied Biosystems, FosterCity, Calif.) (23). Phenylthiohydantoin amino acid deriva-tives were identified by high-performance liquid chromatog-raphy as described by Zimmerman et al. (48).

RESULTS

Characterization of the CS31A E. coli isolates. The charac-teristics of the six CS31A strains studied are given in Table1. On the basis of electron microscopy studies, all thesestrains were heavily piliated when grown at 37°C on MG2.Slide agglutination assays with specific antisera against F41,K88, K99, and FY were performed to examine the serolog-ical characteristics of fimbriae. K99 was detected only oncalf ETEC 92b and B117. Only strain AY1 was agglutinatedby anti-FY serum, whereas specific anti-K88 and anti-F41sera did not react with any of the isolates. However, exceptfor the F41- and K88-producing reference strains, all thestrains reacted with the initial anti-31A antiserum. Theseresults suggest that these strains may share a previouslyunrecognized common antigen, originally detected on strain

31A. Confirmatory results obtained by slide agglutinationand immunodiffusion experiments indicate that these strainsexhibit a common antigen unrelated to F41 and K88 (Fig. 1).To examine a possible polypeptide nature of the common

antigen, crude 60 SM extracts of the studied strains were

subjected to SDS-PAGE and compared with those of theK88- and F41-producing strains. The protein-banding pat-terns were clearly unrelated, except for a common promi-nent 29-kilodalton (kDa) polypeptide band present on ex-

tracts of CS31A-, F41-, and K88ac-producing strains (Fig. 2).With E. coli 92b, two distinct polypeptide bands, of 29 and29.5 kDa, were revealed. In the Western immunoblottingstudies, anti-fimbriae CS31A reacted with a single 29-kDaband in crude cell preparations of all CS31A strains and withthe purified fimbriae (Fig. 3). However, with the 92b extract,both 29- and 29.5-kDa bands reacted with anti-CS31A,demonstrating that the polypeptide bands represent twoforms of CS31A subunits.

Since piliated bacteria cause the agglutination of erythro-cytes from various species, we tested the ability of the sixCS31A strains to hemagglutinate. All strains were observedto have MRHA, excepted strain Orne 6, which exhibitedonly MSHA of guinea pig and chicken erythrocytes, proba-bly resulting from the presence of type 1 pili (11). Fourdifferent patterns ofMRHA were found (Table 2), but all MRstrains caused agglutination of bovine erythrocytes. E. coli92b and B117 exhibited MRHA of bovine, horse, and sheeperythrocytes, indicating the presence of K99 (39). StrongMRHA of bovine erythrocytes by strain AY1 seemed to berelated to FY fimbriae (3).Adhesion to calf intestinal villi was tested to examine a

possible relationship with CS31A production. When bacteriawere grown at 37°C on MG2, all strains were observed tohave high adhesive capacity that was not reduced by serumanti-fimbriae CS31A.

Strains grown at 37°C on MA2 were not agglutinated byanti-fimbriae serum CS31A, and SDS-PAGE patterns lackedthe 29-kDa protein band (results not shown). However,piliation, hemagglutination, and adhesion were not affected,except for K99-producing ETEC (92b and B117), in whichthose K99-related surface properties were lost after growthon MA2 (17). After growth at 18°C on MG2 no strainexpressed any of the characteristics described above.Plasmid studies and characteristics of derivative strains.

Heterogeneous plasmid profiles were obtained with the sixrepresentative CS31A strains containing one to four plas-mids that range in size from 5 to 110 MDa (results notshown). Because strain 31A contained only one plasmid (105MDa), derivative strains were obtained from this strain. The31A/06 strain that was cured of the 105-MDa plasmid lacked

TABLE 2. Hemagglutination patterns of E. coli strains

Agglutination with following erythrocytesa:Strain

Guinea pig Chicken Horse Pig Sheep Bovine Human A

RVC 330 N N MR N MR MR NB117 N N MR N MR MRb MR92b N N MR N MR MR NOrne 6 MSb MSb N N N N NAY1 N N N N N MRb MR31A MR MR MR MR MR MRb MRb31A/06 MR MR MR MR MR MRb MRbXA31A N N N N N N N

a MR, Mannose resistant; MS, mannose sensitive; N, no agglutination.b Hemagglutination stable at 20'C.

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FIG. 4. Fractionation of partially purified CS31A fimbrial proteinon a Sephacryl S300 column (2.5 by 64 cm) by elution with 6 Mguanidine hydrochloride in 5 mM Tris hydrochloride (pH 7.8).Fractions of 5 ml were collected.

the 29-kDa polypeptide band (Fig. 2, lane 11) and did notreact by immunoblotting with anti-fimbriae CS31A (Fig. 3,lane 3). However, the plasmidless strain 31A/06 retainedpiliation, hemagglutination, and adhesion properties of theparental strain, indicating that these properties are notplasmid mediated. After conjugative transfer of the 105-MDaplasmid in E. coli XA, the resulting strain, XA31A, ex-pressed the 29-kDa subunits as revealed by SDS-PAGEanalysis (Fig. 2, lane 12) and reacted on immunoblotting withanti-fimbriae CS31A (Fig. 3, lane 2), but did not express anyother characteristics of the parental strain 31A (i.e., pilia-tion, hemagglutination, or adhesion). These results indicatedthat only genes encoding CS31A were located on the 105-MDa plasmid. Strain XA31A, like all the other isolates, didnot express CS31A antigen after growth at 37°C on MA2 orat 18°C on MG2.

Purification and chemical characterization of CS31A. Gelfiltration of sodium taurodeoxycholate-insoluble material ona Sephacryl S300 column in the presence of 6 M guanidinehydrochloride resulted in two major protein peaks (Fig. 4,peaks I and II). No protein could be detected in the minorpeak (peak III). On peak I, which emerged in the voidvolume of the column (8 x 105 Da), SDS-PAGE analysisrevealed only traces of the 29,000-molecular-weight peptideband. Peak II revealed only one polypeptide band with anapparent molecular weight of 29,000 and was free of high-molecular-weight contaminants (Fig. 2, lane 15). An appar-ent molecular weight of approximately 30,000 could beassigned to peak II on the basis of the elution volume on theSephacryl S300 column. In addition, after reaggregation, thepurified fraction eluted in peak II reacted with anti-fimbriaeCS31A in immunodiffusion experiments (Fig. 1), indicatingthat the purified fraction retained antigenic properties.

Chemical analysis of peak II after dialysis and lyophiliza-tion revealed that the purified fraction contained 92% proteinand 8% carbohydrate. Whether any carbohydrate is linked tothe polypeptide is not clear.The amino acid composition of CS31A differed from those

reported for F41, K99, colonization factor antigen I (CFA/I)

1 5 10

CS31A

TABLE 3. Amino acid composition of purified CS31A,K88ad, and K99 fimbriae

No. of residues/subunit of:Amino acid

CS31A K88ada K99a

Aspartic acid 25 30 23Threonine 22 25 19Serine 21 19 15Glutamic acid 22 21 10Proline 10 9 5Glycine 30 35 19Alanine 24 26 18Cysteine 0 0 4Valine 20 20 8Methionine 3 4 3Isoleucine 13 13 9Leucine 18 20 9Tyrosine 8 9 7Phenylalanine 10 10 9Lysine 12 10 9Histidine 1 2 3Arginine 4 7 6

a K99 and K88ad composition obtained from Gaastra et al. (13).

and type I pili, but was very similar to those of the three K88antigenic variants (Table 3). As for K88, the relative contentof hydrophobic amino acid residues was high (49%) andcysteine was absent. The number of residues of each aminoacid was estimated by assuming a molecular weight of 29,500for fimbrial subunits.The amino acid sequence of the 25 N-terminal residues of

the CS31A subunit is shown in Fig. 5 and compared withthose of K88, F41 and K99 fiuiibrial subunits. A high homol-ogy in sequence (66%) with conservative amino acid changesin positions 15 and 22 was observed with K88. Somehomology (22%) was also seen with F41, although they seemmore distantly related, with only the residues in positions 3,8, 14, 16, and 17 being identical.Immunoblot analysis. On the basis of slide agglutination

and double-diffusion tests, CS31A, K88, and F41 appearedto be antigenically unrelated (Fig. 1). This finding wasfurther investigated in Western immunoblotting experi-ments. All antibodies against CS31A (anti-fimbriae CS31A,anti-reaggregated and anti-denatured CS31A subunit) re-acted strongly with CS31A subunits as assayed by immuno-blot after SDS-PAGE of crude or purified preparations (Fig.3). Moreover, antibodies raised against the denatured formof CS31A reacted strongly with both the CS31A and the K88subunits (Fig. 6A). However, cross-reactivity with the K88subunit was markedly reduced with antiserum raised againstintact anti-fimbriae CS31A (Fig. 6B), and anti-K88ab fim-briae serum reacted only against the three K88 variants (Fig.6C). The F41 subunit did not react with any of the heterol-ogous antisera used for immunoblots. These results indicate

15 20 25

Gly-ThriThr-Gly-Asp-Phe-Asn-Gly-SerlPhelApTNet-AsnIGlyThrF Thr-a-AspA1aW Lys-AsplLYaIThr-K88 Trp-Met Thr-Gly-Asp-Phe-Asn-Gly-SeVal Ile-Gly-Gly|Se r Ie-Thr-Ala-Ap AsplJ rg-Gly4Trp-F41 Ala-Asp-Trp Thr *Glu-Gly-Gln-Pro Gly Asp-Ile-Leu-Ile-GlyJGly-Glu Ile-Thr x -Pro-Ser-Val-

K99 Asn-Thr-Gly -Thr Ile-Asn-Phe-Asn Gly Lys-Ile-Glu-Thr-Ala-Thr-Ser- x -Ile-Glu-Pro-Ala-Val-

FIG. 5. N-terminal amino acid sequence of the CS31A fimbrial subunit of E. coli XA31A. The N-terminal sequences of the K88, F41, andK99 fimbrial subunits of E. coli (13) have been included for comparison. Homologies between the amino acid sequences are enclosed in boxes.The bar signifies the K88 predicted antigenic determinant (28).

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1 2 3 4 5 6 1 2 3 4 5 6B C

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FIG. 6. Comparison of antigenic structures of CS31A, K88, andF41 subunits by Western blot analysis. Lanes: 1, 60 SM extract of E.coli 31A; 2, purified reaggregated CS31A fimbrial subunit; 3, 60 SMextract of E. coli E68 (K88ab); 4, E. coli G1253 (K88ac); 5, E. coli56/190 (K88ad); 6, E. coli B41M (F41). Extracts were probed withanti-denatured CS31A subunit (A), anti-fimbriae CS31A (B), oranti-fimbriae K88ab (C).

an immunological relatedness between CS31A and K88which was not detected by slide agglutination and double-diffusion tests.

Electron microscopy. Examination by electron microscopyof strain 31A negatively stained with 1% phosphotungsticacid revealed three morphologically distinct appendages.Flagella and long, rigid fimbriae (6 to 7 nm in diameter)appeared as prominent appendages. In addition, a thirdsurface material outside the bacterial cell wall was visible,presumably consisting of very fine (2 nm in diameter), wiry,fibrillar structures in which some individual filaments couldbe rarely resolved (see Fig. 8a). Purified CS31A appearedidentical to the structure observed on the surface of thebacteria. Moreover, fine individual filaments which could beeasily resolved in their purified form (Fig. 7b) confirmed thefimbrial structure of CS31A. Some bacteria showed thepresence of a wide, capsulelike zone around the cell whichalso appeared adhering to flagella and fimbriae. This abilityto coat rigid appendages was clearly observed with a crudeextract from strain 31A (Fig. 7a).A capsular organization of CS31A was suggested by

immunoelectron microscopy. With the gold immunolabelling

technique, anti-fimbriae CS31A coated the capsulelike zonearound the cell surface of E. coli 31A (Fig. 8b), confirmingthat this structure represents CS31A. No labeling occurredwith the plasmidless strain 31A/06 (Fig. 8c). CS31A wasapparently easily released from the bacterial cells and couldbe readily seen detached from the cells or localized on thebacterial appendages. Embedding and sectioning of immu-noferritin-labeled bacteria pretreated with anti-fimbriaeCS31A confirmed for both strains 31A (Fig. 8d) and XA31A(results not shown) the apparent capsulelike organization ofCS31A. Immunolabeling occurred neither with XA31A bac-teria preincubated with normal rabbit serum (not shown) norwith the cured derivative 31A/06 treated with anti-fimbriaeCS31A serum (Fig. 8e).

DISCUSSION

The present data show that the new plasmid-encodedfimbrial antigen CS31A, previously described as 31a (1),belongs to the K88-related protein group and is expressed byboth bovine septicemic E. coli and ETEC strains. Electronmicroscopy of negatively stained CS31A antigen showedthat the structure of the cell surface, such as on a purifiedfraction, consisted of very fine, wiry, fibrillar organelles 2nm in diameter. However, immunoelectron microscopy ofbacteria after incubation with specific antisera revealed awide capsulelike zone around the bacteria. This immunola-beled region was thus considered to consist of CS31Aantigen-antibody complexes, and the antigen was regardedas a protein capsule. With more reliability, an electronmicroscopy study of negatively stained bacteria indicatedthat the capsulelike zone most probably consisted of anabundance of very fine fibrils. Thus, CS31A seems to belongto the morphological class of fimbriae initially described forK88 (44) as fine flexible filaments (fibrillae) similar to CS3(32), Z antigen (40), F41 (7), and 2230 factor (4). Productionof CS31A fimbriae appeared to depend on the composition ofthe medium in the same way as observed for K99 and F41 (6,17, 18). Likewise, CS31A production was weaker in liquidmedium and was repressed by L-alanine, suggesting thatK99, F41, and CS31A might have a similar regulation basis.

... ..

..

r s .. ,. ^ <.s 9'F . . . *

4. , B

., @ :., t w ,. .

.. .; #, :. '... ... 's', .!: . e :s ,-4~~~~~~~~~~

FIG. 7. Electron micrograph of negatively stained preparations. (a) Crude extract of E. coli 31A; (b and c) partially purified CS31Afimbriae from E. coli XA31A before dissociation by guanidine hydrochloride. Magnification: x45,000 (panels a and b); x 130,000 (panel c).

1 2 3 4 5 6A

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FIG. 8. Electron micrograph of bacterial preparations. (a) Part of a 31A bacterium negatively stained with 1% phosphotungstic acid. Onlythin (2-nm), wiry CS31A fimbriae are seen. Magnification, x113,000. Bacterial cells were labeled by the immunogold technique withanti-fimbriae CS31A. Cells were negatively stained with ammonium molybdate. Magnification, x 113,000. (b) E. coli 31A negatively stainedwith ammonium molybdate after immunogold labeling with anti-fimbriae CS31A. The capsulelike zone consisting of the thin fimbriae ishighlighted by the CS31A immunolabel. Rigid fimbriae were not labeled by this gold probe. Magnification, x75,000. (c) Cured derivative 31A/06. Neither morphological type of surface organelle was immunolabeled. Magnification, x75,000. (d) E. coli 31A. Ferritin immunolabelingon sectioned cells with anti-fimbriae CS31A shows a wide capsulelike zone around the bacteria. This amorphous material consists ofantigen-antibody complexes, but probably consisted of an abundance of very fine fimbriae. Magnification, x67,000. (e) No labeling occurredwith the plasmidless strain 31A/06. Magnification, x97,000.

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NEW K88-RELATED FIMBRIAL ANTIGEN ON BOVINE E. COLI 2187

Our experiments demonstrate that CS31A fimbriae can bedissociated by 8.5 M guanidine hydrochloride in fimbrialsubunits as revealed by their apparent molecular weight,30,000, determined by gel filtration on Sephacryl S300. Thispurified fraction migrated as a single peptide band on SDS-PAGE with an apparent molecular weight of 29,000. Anti-bodies raised against the reaggregated 29,000-molecular-weight subunit reacted with the fimbrial subunits afterSDS-PAGE (Western blotting), as well as with intact fimbrialorganelles (immunodiffusion), indicating that reaggregated29-kDa subunits retained immunological characteristics ofintact CS31A fimbriae. CS31A from strain 92b appears onSDS-PAGE and Western blot as two distinct subunits. Wedo not propose to give a definitive explanation of thisobservation; however, this phenomenon was previouslyobserved with CS3, K99, 987P, and type I fimbriae (32). Assuggested for those fimbriae, two possible CS31A conforma-tions could be responsible for the particular banding ofCS31A subunits from 92b. On the basis of the amino acidcomposition, the CS31A subunit was shown to be composedof 243 amino acid residues, assuming a molecular weight of29,500. The amino acid composition of CS31A differed fromthose reported for F41, K99, CFAII, and type I pili (13, 27),but was closely related to that of K88, with a high relativecontent of hydrophobic amino acids (49%) and the absenceof cysteine residue. As observed with F41 and K88 fimbriae(7, 13), chemical analysis of the purified CS31A indicated thepresence of carbohydrates (8%). Whether any carbohydrateis linked to the protein is not known.

Unlike results obtained with slide agglutination and immu-nodiffusion experiments, immunoblot analysis revealed im-munological relatedness between CS31A and K88. Indeed,cross-reactivity between K88 and CS31A was observed byWestern bloting with antiserum raised against the denaturedform of CS31A. However, cross-reactivity was considerablyweaker when the K88 subunit reacted with antiserum raisedagainst the native form of CS31A. These results suggest thatthe fimbrial subunits of CS31A and K88 have common aminoacid sequences which do not form immunodominant epi-topes on the intact fimbrial organelles, but which are ex-posed and serve as imunogenic sites with the denaturedCS31A subunit (preparative electrophoresis). On the basis ofserological analysis, CS31A and F41 fimbrial proteins ap-peared immunologically unrelated.

Immunological relatedness between CS31A and K88 wasin accordance with amino acid sequence agreement ob-served for the first 25 N-terminal amino acids of the twoproteins. Sequences of the N-terminal part of the three K88antigenic variants (K88ab, K88ac, and K88ad) were con-served, probably by an evolutionary constraint on theseparts of the K88 protein (27, 35). It would be of interest tocompare amino acid sequences in other regions of proteinsto determine whether CS31A is more related to one of thethree K88 variants. The finding that amino acid changesbetween CS31A and K88 occur in a region located fromresidues 19 to 24, considered a probable antigenic determi-nant for K88 (28), suggests that this region may contribute tothe antigenic specificity of the two proteins. In agreementwith our results, genetic relatedness of K88 and CS31A hasbeen observed by hybridization of total DNA of repre-sentative CS31A strains with a K88 gene probe (H. W.Moon, personal communication).

Production of CS31A fimbriae is unable to confer hemag-glutinating or in vitro adhesive properties to the transconju-gant XA31A. Indeed, in strain 31A the above characteristicsare presumably mediated by a chromosome-encoded fim-

brial adhesin. On the basis of the finding, CS31A fimbriaediffer from the majority of fimbriae expressed by ETEC,which act as colonization factors by promoting adherence toepithelial cells. Experiments are under way to exploreCS31A as an adhesin. However, all studied CS31A isolatesexpressed known (K99, FY, and type 1 pili) or unknownfimbrial adhesins which could act as colonization factors. Itwould also be of interest to explore the effect of CS31A onphagocytosis, on complement binding, and on serum resis-tance, which are viewed as determinant factors in E. coli-mediated extraintestinal infections.

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2. Contrepois, M., and J. P. Girardeau. 1985. Additive protectiveeffects of colostral antipili antibodies in calves experimentallyinfected with enterotoxigenic Escherichia coli. Infect. Immun.50:947-949.

3. Contrepois, M., J. L. Martel, C. Bordas, F. Hayers, A. Millet, J.Ramisse, and R. Sendral. 1985. Frequence des pili FY et K99parmi des souches Escherichia coli isolees de veaux diarr-heiques en France. Ann. Rech. Vet. 16:25-28.

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8. De Rycke, J. 1984. Role des souches d'Escherichia coli nonen-terotoxinogenes dans la pathologie neonatale du veau. Ann.Rech. Vet. 15:75-95.

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