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1996, 64(9):3555.

Infect. Immun.

M C Méchin, E Rousset and J P Girardeau

and antipeptide antibodies.

Escherichia coli by using overlapping peptides epitopes of the nonfimbrial adhesin CS31A of Identification of surface-exposed linear B-cell

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INFECTION ANDIMMUNITY, Sept. 1996, p. 3555–3564 Vol. 64, No. 9 0019-9567/96/$04.0010

Copyrightq 1996, American Society for Microbiology

Identification of Surface-Exposed Linear B-Cell Epitopes of the Nonfimbrial Adhesin CS31A of Escherichia coli by Using

Overlapping Peptides and Antipeptide Antibodies

MARIE-CLAIRE ME´ CHIN,† ELODIE ROUSSET,‡ANDJEAN-PIERRE GIRARDEAU*

Laboratoire de Microbiologie, Institut National de la Recherche Agronomique, Centre de Recherches de Clermont-Ferrand-Theix, 63122 Saint-Gene`s-Champanelle, France

Received 18 December 1995/Returned for modification 17 January 1996/Accepted 29 May 1996

As a first step toward the design of an epitope vaccine, by using the nonfimbrial adhesin CS31A of Escherichia coli as a carrier, a low-resolution topological and epitope map of the CS31A subunit was developed by using solid-phase peptide synthesis and polyclonal rabbit antibodies raised against both native and denatured proteins. Peptides constituting antigenic epitopes on the major subunit (ClpG) of the multimeric CS31A anti- gen were identified by examining the binding of the antibodies to 249 overlapping nonapeptides covering the amino acid sequence of ClpG. With antibodies raised against denatured ClpG subunit, seven major epitope regions, corresponding to residues 10 to 18, 45 to 58, 88 to 107, 148 to 172, 187 to 196, 212 to 219, and 235 to 241, were located. Most of the epitopes were hydrophilic and were located in variable regions, residing largely in loop regions at the boundaries of secondary structural elements of ClpG. In contrast, antibodies raised against native CS31A antigen reacted only with the peptide AVNPNA (positions 179 to 184), demonstrating that this peptide was the only linear B-cell epitope of the native protein. The different immunogenic profiles of native CS31A antigen and denatured ClpG indicated that the denaturation process resulted in marked conformational changes in the protein, which could expose epitopes hidden or absent in native CS31A. To identify the surface-exposed epitopes, nine peptides covering the dominant antigenic regions of ClpG were synthesized and used to prepare site-specific antibodies. Antipeptide antibodies were tested, in a competitive enzyme-linked immunosorbent assay (ELISA), for cross-reactivity with native CS31A and denatured ClpG subunit. Four of these antipeptide antibodies bound to the native protein in an accessibility ELISA, indicating that residues 44 to 56, 174 to 190, 185 to 199, and 235 to 249 were surface exposed on CS31A. These data indicate that an immunodominant surface-exposed linear epitope was present in the region from positions 179 to 184 of ClpG in the native CS31A antigen on intact bacterial cells and suggest that the four surface-exposed epitopes constitute potential sites for insertions or substitutions with heterologous peptides.

Enterotoxigenic Escherichia coli and septicemic E. coli bear- ing the CS31A antigen are associated with diarrhea and sep- ticemia in both humans and animals (6, 8, 19). Although CS31A has been described as a K88-related antigen (the major fimbrial adhesin found on porcine enterotoxigenic E. coli), it appears as a capsule-like structure, surrounding the bacterial cells (15), and its structure is reminiscent of that of the non- fimbrial adhesin described for uropathogenic E. coli (18). Un- like K88 fimbriae, CS31A fails to bind on enterocytes of various animal species (15) but has been reported to promote adhesion to cultured polarized epithelial cells MDCK (24) and Caco-2 (19).

The K88 fimbriae are composed mainly of a repeating single 27-kDa monomer (major subunits), designated FaeG, and of minor subunits FaeH, FaeI, and FaeJ required for the initia- tion and formation of the K88 fimbriae (3). The CS31A antigen has genetic and functional organizations similar to those of K88 (26) and is composed mainly of the repeating major subunit ClpG (14). Comparisons of amino acid sequences of ClpG

and FaeG have revealed that the major subunit has four regions of amino acid variability (called V1, V2, V3, and HV) interspersed among five conserved hydrophobic clusters (P1 to P5) of up to 20 residues (14). Despite the extensive changes found within the four variable regions, a common overall fold for ClpG and FaeG was deduced from their sequences by hydrophobic cluster analysis and secondary structure predictions (27). Using information obtained from the multiple sequence alignments of major and minor K88-related subunits, we developed a schematic two-dimensional model of the major subunits that closely matches the results of site-directed mu- tagenesis experiments (5, 9, 29).

By using monofactorial antisera obtained by absorbing polyclonal antisera with bacteria of heterologous subtypes, three major K88 subtypes (K88ab, -ac, and -ad) have been estab- lished (21). The antigenic variations have been found to reside in minor differences in the corresponding primary structures of the major subunits of the three K88 subtypes. These differences are clustered within variable hot spots (10, 22) that mostly correspond to the four regions of variability identified within major subunits ClpG and FaeG (14). Antigenic determinants on FaeG have been predicted on the basis of their variability, flexibility, and hydrophobicity (25). However, only antibodies directed to a peptide of the variable region V3 reacted with native K88. Using fused FaeG peptides, Thiry et al. (34) have identified four antigenic peptides located within two variable regions, but it is not known whether these peptides are surface exposed. Other studies (2, 36), using a panel

* Corresponding author. Phone: (33) 73 62 42 42. Fax: (33) 73 62 45 81.

† Present address: Hormone and Metabolic Research Unit, Univer- sity of Louvain, Medical School and International Institute of Cellular and Molecular Pathology, 12000 Brussels, Belgium.

‡ Present address: Department of Pathology and Microbiology, Faculty of Veterinary Medicine, University of Montreal, Sainte- Hyacinthe, Que´bec, Canada.

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of K88-specific monoclonal antibodies (MAbs), have revealed the conformational nature of the subtype-specific antigenic determinants and indicate a clear correlation between the re- ceptor-binding site and the subtype-specific antigenic determinants. However, attempts to map linear epitopes on FaeG subunits failed when K88-specific MAbs were used (36).

Recent findings demonstrate that CS31A could represent an efficient carrier for epitope presentation and oral vaccine design: it can be expressed at a high level (5), and it may be easily released from the bacterial surface and then purified by a simple affinity procedure (15). CS31A is strongly immunogenic, and high antibody titers have been obtained against foreign peptidic inserts (5, 9). This protein has been extensively studied at the genetic level (26), permissive sites for insertion and deletion (indel) have been identified (9), and a two-dimensional model of the subunit has been proposed (27). In addition, preliminary results indicated that CS31A is synthesized at high levels by live E. coli strains in the intestinal lumens of mice, lambs, and calves and is able to stimulate good secretory immunoglobulin A (secretory IgA) antibody responses in mu- cosal tissue after oral immunization (13). Different B-cell epitopes, derived from the S protein of the transmissible gas- troenteritis virus, have been successfully introduced in the variable region V3 (5, 9); high antibody titers against the foreign epitopes were obtained, and viruses were seroneutralized.

However, although the foreign epitopes fused in ClpG V3 demonstrate their essential antigenic and immunogenic properties, the biogenesis of the recombinant antigens appeared greatly influenced by the amino acid sequence of the fused peptides. Therefore, to identify new ClpG regions likely to be immunopotent if substituted with foreign epitopes and to ex- tend the information on CS31A beyond its structural organi- zation, we wished to define the natural surface epitopes of CS31A. Moreover, in the absence of three-dimensional structure data on the fimbrial subunit, the location of the antigenic peptides on the native protein could provide useful structure- function information.

In this study, we first employed solid-phase peptide synthesis, as described by Geysen et al. (11, 12), to comprehensively map antigenic sites on ClpG with polyclonal antibodies raised against both native and denatured CS31A. Subsequently, we wished to identify regions of CS31A that might be accessible to antibodies. Our approach was to generate a panel of site- specific antipeptide antibodies, raised against the dominant antigenic determinants of ClpG, to identify the surface-exposed epitopes on native CS31A. The positions of these epitopes, in relation to accessibility and secondary structure predictions, are also presented.

MATERIALS AND METHODS

Bacterial strains and media.The recombinant E. coli strain C600(pFM205), carrying the gene cluster for K88ab fimbriae and provided by F. K. de Graaf, is described elsewhere (28). The E. coli recombinant strain HB101(pEH524), car- rying the gene cluster for CS31A antigen, was available from previous work (26), and the recipient strain was E. coli HB101. Strains were grown on Minca medium (16), supplemented with 2 g of glucose per liter and appropriate antibiotics, at 378C for 18 h.

Purification of antigens.The major structural subunit ClpG was purified by Sephacryl S300 chromatography after exposure of CS31A antigen to 6.5 M guanidium hydrochloride as described elsewhere (15). Similarly, the major structural subunit FaeG was purified from the K88ab-producing strain C600 (pFM205). Native polymeric CS31A antigen was purified from E. coli HB101 (pEH524) as described for the ClpG subunit, with the following modifications: to the 20% ammonium sulfate fraction dissolved in 5 mM Tris HCl (pH 7.8), solid NaCl was added to achieve a concentration of 2 M and the solution was then kept for 1 h at room temperature to equilibrate. A 4-mg protein sample of the preparation was then applied to a phenyl Sepharose CL4B column (1.5 by 7 cm;

Pharmacia, Uppsala, Sweden) equilibrated with 2 M NaCl in 5 mM Tris HCl.

After a rinsing with 50 ml of 2 M NaCl in 5 mM Tris HCl, elution was continued

by application of a discontinuous gradient of decreasing NaCl concentration (from 1 to 0.065 M). Native CS31A antigen bound to phenyl Sepharose was eluted by application of 5 mM Tris HCl (pH 7.8). Protein analysis of the eluate was monitored by sodium dodecyl sulfate (SDS)-polyacrylamide gel electro- phoresis (PAGE) of selected fractions, and the protein content of fractions was determined by the bicinchoninic acid method (Pierce, Chicago, Ill.).

PAbs.Six rabbits were immunized by three subcutaneous injections of 200mg of purified ClpG subunit in incomplete Freund’s adjuvant at 3-week intervals, and sera (RNs1 to RNs6) were collected 2 weeks after the last injection and identified as anti-ClpG polyclonal antibodies (PAbs). Similarly, two other rabbit antisera (RFs1 and RFs2) were prepared against purified FaeG (major subunit of the K88ab subtype) and identified as anti-FaeG PAbs. By the same method, two rabbit antisera (RHn1 and RHn2) were obtained against native CS31A antigen and identified as anti-CS31A PAbs.

Peptide synthesis and peptide-pin ELISA (PEPscan).Duplicate sets of 249 overlapping nonapeptides covering the entire sequence of the ClpG subunit protein were synthesized on solid polypropylene rods by using a commercial kit (Cambridge Research Biochemicals, Northwich, United Kingdom) according to the manufacturer’s instructions. This is a modification of the technique of multiple peptide synthesis, using 9-fluorenylmethoxycarbonyl-amino acid esters on polypropylene rods, as described originally by Geysen et al. (11). Successful peptide synthesis was monitored by the simultaneous synthesis of positive and negative control peptides and subsequent testing of their binding to the supplied MAb. The synthesized peptides, on block pins configured to a 96-well microtiter plate, were then tested for binding with the different antisera in a peptide-pin- based enzyme-linked immunosorbent assay (ELISA). Rods carrying the peptides were coated with 1% ovalbumin–1% bovine serum albumin (BSA)–0.1% Tween 20 in phosphate-buffered saline (PBS) for 1 h at room temperature, to prevent nonspecific absorption of antibodies. The pins were then incubated overnight at 48C with 100 ml of an appropriate dilution of serum per well. The pins were thoroughly washed with PBS-Tween and incubated with 100ml of peroxidase- labeled anti-rabbit IgG (1:2,000 dilution; Nordic Immunological Laboratories, Tilburg, The Netherlands) per well for 2 h at room temperature. After being washed with PBS-Tween, the pins were placed in wells containing 100ml of ABTS substrate (2,29-azino-di-3-ethyl-benzthiazoline-sulfonate–hydrogen perox- ide in phosphate-citrate buffer) for 20 min in the dark. Plates were read at 405 nm in a Dynatech MR5000 reader. The solid-phase peptides were reused after the stripping of antibodies by sonication for 30 min at 608C in a solution containing 1% SDS, 0.1% 2-mercaptoethanol, and 0.1 M sodium phosphate (pH 7.2). A second identical peptide set was synthesized, and immunoreactivities of antisera were compared for the two peptide sets.

Peptide-carrier conjugation and production of rabbit antipeptide antisera.

Nine peptides, 14 to 20 residues long, covering immunoreactive sites identified by epitope scanning or selected for their critical positions in the ClpG sequence, were synthesized by Altergen (Strasbourg, France). The quality of the peptide was evaluated by reverse-phase high-performance liquid chromatography. All sequences contained the desired peptide at the expected purity (.80%). The ClpG peptides (designated CLP1 to CLP9), identified in Table 1, were conjugated to BSA before injection to produce antipeptide antibodies. Except for peptides CLP2 and CLP4, all peptides were conjugated to BSA with glutaralde- hyde, at a 10:1 molar ratio of peptide to BSA, according to the method described by Van Regenmortel et al. (35). Peptides CLP2 and CLP4, which contain inter- nal lysine, were synthesized with an additional tyrosine at their N termini to facilitate the coupling to BSA with bis diazobenzidine (BDB). Peptides were conjugated to BSA with BDB at a 30:1 molar ratio of peptide to carrier protein after amino groups of peptide had been blocked by citraconic anhydride acid, as described by Van Regenmortel et al. (35). Two rabbits per peptide conjugate were immunized subcutaneously first with peptide-BSA conjugates (500mg) emulsified in complete Freund’s adjuvant and then with two booster doses (250 mg) in incomplete Freund’s adjuvant at 2-week intervals, and antisera were

TABLE 1. Sequences of synthetic peptides

Peptide^a Residues Sequence^b

CLP1* 5–18 DFNGSFDMNGTITA

CLP2* 44–56 (Y)GDSKLLTITQSEPA

CLP3* 82–96 AFSDYEGNGVALQSS

CLP4* 97–109 (Y)GDNGKGFFELPMKD

CLP5 132–146 SEISTGLYGITSVAS

CLP6* 148–162 DNTSIYYGGLVSPAI

CLP7 174–190 KFGNYNHTQLLGQLQAV

CLP8* 185–199 GQLQAVNPNAGNRGQ

CLP9* 235–249 TFTNPVVSTTQWSAP

aPeptides covering the dominant antigenic determinant delineated by peptide scanning are indicated by an asterisk. Peptides CLP2 and CLP4 were synthesized with an additional tyrosine at their N termini for conjugation to BSA with BDB.

bResidues are listed in single-letter code starting from the N-terminal end.

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collected 2 weeks after the last injection. IgGs were then isolated from each antipeptide antiserum by protein A column chromatography, according to the manufacturer’s (Pierce) instructions. After dialysis, the concentration of IgG was adjusted to that of the total IgG fraction of antipeptide antiserum.

ELISAs.Microtiter wells (polysorp; Nunc) were coated with 250 ng of purified ClpG subunit in 100ml of coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) at 48C for 16 h. The plates were then blocked with blocking buffer (0.5% goat serum albumin, 1% nonfat dry milk in PBS) for 2 h at room temperature. Serially diluted antisera were added to the wells, and plates were incubated for 2 h at 378C. The wells were washed twice with PBS-Tween and then incubated for 2 h at 378C with goat anti-rabbit horseradish peroxidase conjugate (1:2,000 dilution) and processed as described above.

To determine whether the antipeptide antibodies recognized antigenic peptide on the native antigen, an indirect ELISA was performed on plates precoated with purified native CS31A. Wells of microtiter plates (Immulon 2; Dynatech) were coated with purified native CS31A (10mg/ml) in 100 ml of coating buffer for 16 h at 48C. The plates were washed in PBS-Tween and then blocked and processed as described above. Specific antipeptide titers were subtracted from the titers obtained with pooled preimmune rabbit antisera. Endpoint titrations were the highest dilutions of sera giving an A405of 0.3 optical density units.

Peptide-specific ELISA.Immulon 2 microtiter wells (Dynatech Laboratories, Inc.) were coated with 100ml of a solution of each synthetic peptide (20 mg/ml) in coating buffer containing 10 mM dithiothreitol and incubated at 48C for 16 h.

After being washed four times with PBS-Tween, wells were incubated for a minimum of 16 h at 48C with 200 ml of the blocking buffer and then washed with PBS-Tween. A 100-ml volume of a serial dilution of the antibody was then added, and the plates were incubated at 48C for 16 h. After being washed, the plates were developed and read at 405 nm as described above. Irrelevant peptides derived from capsid protein VP1 of foot-and-mouth disease virus (51-RYKQKI IAPAQKGG) and from capsid protein VP1 of poliovirus (91-YDNPASTT NKDKLFA) were used, as previously described (5), as negative controls in the peptide-specific ELISA. Assays were performed in duplicate, and the reactive titer of each antiserum was defined as the dilution that showed a twofold increase in optical density over that obtained with the negative control.

Competitive ELISA.The specificity of antipeptide antibody binding to the respective peptide on native CS31A was confirmed by a competition assay with free synthetic peptides. Duplicate aliquots of a pretitered antipeptide antibody concentration (protein A-purified IgG), capable of 70% of the maximum binding to native CS31A coated wells, were mixed in polypropylene tubes with various concentrations (ranging from 3.9 to 125mg/ml) of the relevant synthetic peptide and incubated for 16 h at 48C. Similarly, as a control, each antipeptide antibody was incubated with a mixture of five irrelevant CLP peptides. The antibodies were then applied to the microtiter plate precoated with purified native CS31A.

The residual activity of antibody binding to purified native CS31A antigen-coated wells was assayed with goat anti-rabbit horseradish peroxidase conjugate, as described above. Percent inhibition of antipeptide binding to native CS31A by synthetic peptides was calculated for each antipeptide by comparing assay values with those for control antipeptides incubated with irrelevant peptides. Each assay was run in duplicate, and antibody was assayed twice.

Accessibility ELISA.The ability of antipeptide antibodies to recognize their epitopes on the intact bacteria was determined with an accessibility ELISA. A test was designed to determine the ability of native CS31A antigen to compete for antibody with denatured ClpG subunit absorbed on the plate. Antipeptide antibodies were adjusted to a concentration corresponding to their 70% maximum binding to the denatured ClpG subunit and preincubated for 4 h at 48C with increasing amounts of bacterial cells (0.13 10⁸to 23 10⁸cells per ml), HB101 (pEH524). Similarly, aliquots of antipeptides were preincubated with the recipient strain HB101 and used as controls. Microtiter wells (Immulon-2) were coated with 500 ng of ClpG subunit in coating buffer for 16 h at 48C. Uncoated sites were blocked with the blocking buffer for 16 h at 48C. After centrifugation to remove bacteria, residual antipeptide antibodies were transferred to the wells, the plates were incubated for 4 h at 48C, and the bound antibodies were detected as described above. The difference between the A405of the individual antipeptide incubated with the CS31A-positive strain (specific binding) and the value obtained when the antipeptide was incubated with the CS31A-negative strain (nonspecific binding) was measured and expressed as percent reduction of the absorbance. The experiments were repeated twice.

RESULTS AND DISCUSSION

B-cell epitope mapping with anti-ClpG subunit antibodies.

A duplicate series of 249 overlapping nonapeptides, covering the entire sequence of the ClpG subunit, was synthesized on polypropylene pins as described by Geysen et al. (11) and used to identify immunoreactive determinants in an ELISA with different specific polyclonal antibodies. To better differentiate adjacent antigenic determinants, we used six individual antisera to map overlapping nonapeptide sets. Since adjacent nonapeptides completely overlap with a single amino acid shift, we

were able to determine critical residues for each antigenic site.

By the approach of Zhong et al. (37), the ClpG peptides were classified according to their reactivities with antibodies: peptides with high frequencies and high titers (optical densities of .1.2) represent the immunodominant sequences, while peptides with low-frequency, low-titer or high-frequency, low-titer binding patterns may represent immunorecessive regions.

Representative examples of nonapeptide immunoreactivity patterns are shown in Fig. 1. Critical residues of antigenic determinants identified with each of the 10 rabbit polyclonal antibodies are detailed in Fig. 2. Different patterns of antigenic profile emerged, depending on the particular rabbit immune serum tested. However, the PEPscan assay showed that individual antisera gave almost identical reactivity patterns, suggesting that the denatured proteins have a stabilized conformational state with a well-defined tertiary structure and thus the same immunogenic epitopes. Higher peaks of reactivity were found in six major areas capable of binding with antibod-

FIG. 1. Reactivities of representative rabbit polyclonal antibodies with overlapping nonapeptides covering the amino acid sequence of the ClpG subunit.

Each vertical bar represents the absorbance of a peptide in the peptide-pin ELISA. The peptide number corresponds to the position of the amino-terminal residue of the peptide in the ClpG sequence. (A, B, and C) Scans using 1:500 dilutions of three different anti-ClpG PAbs (RNs1, RNs2, and RNs3); (D) scan using a 1:300 dilution of an anti-CS31A PAb (RHn2); (E) scan using a 1:300 dilution of an anti-FaeG PAb (RFs1); (F) scan using 1:300 dilutions of five pooled rabbit preimmune antisera used as a background control. Variable (V1, V2, V3, and HV) and conserved (P1 to P5) regions of the ClpG subunit are indicated above the scans.

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ies present in all antisera: region A (positions 10 to 18), region B (positions 45 to 58), region C (positions 88 to 107), region D (positions 148 to 172), region E (positions 187 to 196), and region F (positions 212 to 219). Additionally, the C-terminal end of the ClpG subunit appeared as a major immunoreactive area in which peptide scanning failed to identify individual epitopes. However, the peptide encompassing positions 235 to 241 (region G), which reacted with antibodies at both high titer and high frequency, can be considered immunodominant. Con- sistent with the K88ac epitope mapping using fused FaeG peptides (34), the three reactive regions C, D, and F corresponded in position to the immunoreactive FaeG peptides.

This finding suggests that regions from positions 88 to 107, 148 to 172, and 212 to 219 are immunodominant in both K88 and CS31A. Analysis of all peptide scans obtained with antibodies to denatured ClpG subunit revealed that most of the detect- able antigenic sites were localized within variable regions of ClpG. Three of the major antigenic areas (regions B, E, and F) were centered within the two highly variable regions V1 (located at positions 28 to 58) and V3 (located at positions 193 to 221). In addition, the N- and C-terminal antigenic determinants (regions A and G) were centered within two limited variable regions located, respectively, at positions 10 to 15 and 237 to 243. Only variable region V2 formed an exception, since nonapeptides derived from this area did not react at high titers with antibody. No peptide-antibody binding was detected with peptides derived from amino acid sequences in the conserved regions. These comprised four of the five hydrophobic clusters associated with conserved proline (P1, P2, P3, and P5) which are thought to form the hydrophobic core of the ClpG subunit.

Only the conserved hydrophobic cluster P4 (151-SIYYGG LVSP) represents an exception, since nonapeptides derived from this region were mostly immunoreactive with rabbit antisera to the denatured ClpG subunit (Fig. 2). Our finding that conserved cluster P4 is a dominant epitope of ClpG is therefore consistent with the results obtained by Thiry et al. (34) which showed that the corresponding FaeG peptide (positions 147 to 160) was immunoreactive with anti-K88ac antibodies (Fig. 2).

In the first reactive group of antigenic areas (regions A, E, F, and G), the six antisera gave almost identical peptide reactivity patterns over a small number of nonapeptides (three to six) covering a sequence of five to six critical residues which probably represent a single B-cell epitope. In region A, individual antisera identified a single B-cell epitope, but fine mapping revealed that each antiserum bound to a different epitope.

These epitopes overlapped with a single amino acid shift but had NGT residues in common, suggesting that these residues are structurally important and critical for antibody binding.

This finding was consistent with the conservation of a glycyl residue at position 14 among all major and minor subunits of the K88-related antigens (27), suggesting that this glycyl residue is subject to conformational constraints in the native protein. In region E, located near the N-terminal end of V3, rabbit

antibody required residues VNPNAG or QAVNPN for binding to the overlapping nonapeptides. However, only NPN residues, which coincide with the highest probability of b-turn occurrence (14), are critical for antibody binding.

At the C-terminal end of V3, nonapeptides 211 to 216 (covering region F) (the number identifying each nonapeptide corresponds to the ClpG residue at the amino terminus of the peptide) required residues TTGD for antibody binding, and scanning data showed that peptide 213-TTGDVI was an immunodominant epitope among rabbits immunized against the denatured ClpG subunit. Although the amino acid sequences of ClpG and FaeG are highly variable in this region (14), two FaeG peptides (211-YREDMEY and 217-YTDGTVVSAA YAL) have been identified as antigenic determinants of K88 fimbriae (25, 34). Our findings are consistent with these obser- vations and indicate that the cluster of residues 211 to 221 is variable and immunoreactive in both K88 and CS31A antigens.

At the carboxy-terminal end of ClpG, many nonapeptides gave strong positive signals with rabbit antisera, indicating that this area is immunodominant in rabbits and contains overlapping epitopes. However, nonapeptides 235 to 241 from region G exhibited strong reactions with the six antisera. As identified by peptide scanning, residues TNPVV centered within a limited variable region (positions 236 to 243) were critical for rabbit antibody binding.

In a second group of antigenic areas, individual rabbit antisera gave different peptide reactivity patterns. Three strong antigenic clusters consisting of peptides from positions 45 to 58 (region B), 88 to 107 (region C), and 148 to 172 (region D) were found in the central part of ClpG. The first cluster (region B), centered within the variable region V1, probably contains at least two epitopes in close proximity, as suggested by the epitope profile with rabbit antisera (Fig. 1). As shown in Fig. 2, antisera RNs1, RNs3, and RNs5 required KLLTI residues for recognition, while RNs2 and RNs4 antisera required TQSEPA residues. Both antigenic determinants (KLLTI and TQSEPA), which probably represent single B-cell epitopes, are contiguous in sequence (KLLTITQSEPA) and encompass the conserved motif TIT, which was predicted with high potential to fold in a b-strand conformation (27).

The second immunoreactive cluster (region C) was found within the moderately variable region located at positions 88 to 105. Analysis of all the peptide scans obtained with the six antisera raised against the denatured ClpG subunit revealed that antibodies recognized at least two linear immunodominant epitopes in this area. Different patterns of antigenic profile emerged, depending on the particular serum tested (Fig.

2). RNs1, RNs2, and RNs4 antisera bound with two groups of nonapeptides (nonapeptides 86 to 100 and 97 to 105) whose members all covered the respective amino acid sequences GVALQ and GKGFFE. However, RNs6 did not recognize nonapeptides derived from amino acid sequences covering region C.

The third antigenic cluster (region D) was centered on the

FIG. 2. Antigenic determinants identified in the ClpG subunit sequence with different rabbit antibodies raised against denatured ClpG subunit (RNs), denatured FaeG subunit (RFs), and native CS31A antigen (RHn). The amino acid sequence of the ClpG subunit is presented in single-letter code. Residues composing the minimum antigenic determinant identified with individual rabbit antibodies are indicated, and dominant antigenic determinants (high titer, high frequency) are in boldface type. Sequences for RNs1 to RNs6 are antigenic determinants identified with six individual anti-denatured ClpG subunit antibodies. Sequences for RHn1 and RHn2 are antigenic determinants identified with two individual anti-native CS31A antibodies. Sequences for RFs1 and RFs2 are antigenic determinants identified with two individual anti-denatured FaeG subunit antibodies. Sequences for RNs1(2) are antigenic determinants obtained with RNs1 in the second peptide set synthesized.

The seven dominant antigenic regions (A to G) are shown boxed. Variable (h) and conserved (■) regions of major subunits are shown above the ClpG sequence.

In PHDsec lines,b-strand (E), a-helix (H), and loop (L) potentials were predicted by using the PHD program (31), as previously reported (27). Only secondary structures with high prediction potential are reported. The three antigenic FaeG peptides identified by Thiry et al. (34) are indicated (K88ac) for comparison. At the bottom, positions of the synthetic peptides used as antigens to generate the nine site-specific antipeptide antibodies are shown as boxes. The residues required for recognition of epitopes by antibodies to CLP8 are indicated. The shaded boxes indicate antipeptide antibodies which bind to native CS31A antigen.

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conserved cluster P4 (IYYGGLVSP). Although this region is hydrophobic and has a low probability of topographic protru- sion, the scanning data showed that peptide IYYGGL was immunodominant in rabbits. Five of the six antisera required residues YYG for antibody binding, as identified by overlapping nonapeptides. With RNs2 antiserum, loss of the first tyrosyl residue in the epitope significantly decreased binding of antibodies (30%) and loss of the two tyrosyl residues completely eliminated binding. Similarly, with RNs3, RNs4, RNs5, and RNs6, loss of tyrosyl residues altered the ability of the antibody to recognize the nonapeptides, indicating that the two tyrosyl residues are critical in the antigenic determinant. The second epitope (GKDAASA) identified in this area with RNs2, RNs4, and RNs5 corresponded in position to the hypervariable region identified on amino acid sequences of the three FaeG subtypes (10).

Identification of common linear epitope between FaeG and ClpG. To investigate the antigenic cross-reactivity previously observed by Western blot (immunoblot) analysis between ClpG and FaeG subunits (15), we sought to identify the putative common linear epitope responsible for cross-reactivity by scanning the 249 nonapeptides derived from the ClpG sequence with rabbit antisera raised against the denatured FaeG subunit (RFs1 and RFs2). The two sera tested gave compara- ble high background reactivity patterns (Fig. 1E), but four nonapeptides (nonapeptides 232 to 235) gave clearly stronger signals, suggesting that these peptides contained part of the putative common linear epitopes. This sequence shared only a 3-amino-acid identity (underlined) with the corresponding amino acid sequence of FaeG, 240-TFNQAVTTS; however, recognition of the ClpG peptide (235-TFTNPVVST) by anti- FaeG antibodies may be correlated with a possible common folding of the two peptides (27). However, an alternative ex- planation of this antigenic recognition may be that the continuous epitopes of the ClpG subunit are part of a discontinuous

epitope recognized by anti-FaeG PAbs. This hypothesis is supported by the high background of reactivity found with rabbit sera, which may be due to reactivity with peptides that contained only part of the epitope. No linear antigenic determinant was found within the two highly conserved clusters P1 (58-PILLGRTKEAFA) and P2 (80-IPLIAFSDYEG) predicted, on the basis of their hydrophobicity or estimated surface accessibility, to have a high probability of antigenicity (12, 22, 25). However, the putative binding of the anti-FaeG antibodies to nonapeptides derived from these conserved clusters may be masked by the high background reactivity.

Epitope mapping with anti-native CS31A antibodies. To define the linear epitopes of the native CS31A antigen, we tested by ELISA the binding of anti-CS31A antibodies (RHn1 and RHn2) to the set of synthetic nonapeptides covering the ClpG sequence. The scan shown in Fig. 1D revealed that antisera against native CS31A reacted strongly with nonapeptides covering a single region (188-QAVNPNAG). RHn1 also reacted with the adjacent nonapeptide 180 (TQLLGQLQA), suggesting that antibodies may recognize a discontinuous epitope incorporating contact sites on both peptides which can juxtapose through a potential b-turn centered on the NPN residues. Therefore, anti-ClpG PAbs, anti-CS31A PAbs, and antibodies to peptide CLP8, which contained the sequence QAVNPNAG, gave almost identical reactivity patterns centered on the NPN sequence (Fig. 3). The fine map of the epitope QAVNPNAG showed that the seven nonapeptides, 185 to 191, that contained the motif NPN produced much stronger signals with antibodies generated against the three conformational states of the peptide antigen (synthetic-peptide, denatured-subunit, and native multimeric antigen). In addition, a dramatic decrease in binding of antibody to the peptides was observed when the prolyl residue was lost, indicating that proline is obviously important for antibody binding.

Thus, antibodies both to native CS31A and to peptide CLP8

FIG. 3. ELISA reactivities of the seven nonapeptides, 185 to 191, containing the motif NPN. The entire set of nonapeptides was subjected to epitope scanning with antibodies to peptides 185 to 199 (anti-CLP8) (A), antibodies to denatured ClpG subunit (B), and antibodies to native CS31A antigen (C). Only nonapeptides covering the sequence AVNPNAGNR are shown. The number that identifies each nonapeptide corresponds to the ClpG residue at the amino end of a given peptide. The epitope scanning showed that all antisera produced much higher signals with nonapeptides containing the NPN motif. OD, optical density.

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find the two asparagyl residues (positions 191 and 193) essential for binding and required a prolyl residue at position 192.

Our findings in epitope analyses, with antibodies generated against both native and denatured CS31A, indicate that antibodies elicited by the two forms of antigen recognize different linear epitopes. The process of denaturation of CS31A into denatured ClpG subunit probably resulted in important conformational changes in the protein which might have exposed epitopes hidden or absent in native and multimeric CS31A.

However, although other studies (2, 36) suggest that all epitopes of K88 fimbriae are conformational epitopes, our findings demonstrate that peptide QAVNPNAG is an immunogenic linear epitope of the native CS31A antigen that also appeared as an immunodominant epitope among immunized rabbits.

Determination of surface-exposed epitopes on native CS31A.

The quaternary assembly of the ClpG subunit into multimeric CS31A is thought to involve a relatively large contact surface area as described for other protein-protein interactions (23, 30). Since a substantial portion of the subunit surface can be inaccessible to other proteins, antibody recognition regions in CS31A were assumed to be associated with highly exposed areas. Our approach, to examine whether antigenic peptides identified by the PEPscan method are surface exposed on native CS31A, was to generate a panel of site-specific antipeptide antibodies covering the regions containing the immunodominant epitopes to identify those epitopes that are surface exposed by competitive ELISAs.

(i) Characterization of antipeptide conjugate antibodies.

Nine synthetic peptides corresponding to the immunodominant epitopes of the ClpG subunit (see positions on Fig. 2) were conjugated to BSA and injected into rabbits. Table 2 summarizes the characteristics of each of the nine antipeptide conjugate antibodies (protein A-purified IgG). As determined in a peptide-specific ELISA, all antipeptide conjugate antibodies reacted with the relevant unconjugated peptide, showing mean titers ranging from 4,096 to 65,500. Nonspecific cross- reactivity of antipeptides with irrelevant peptides was observed only at a low range (,500), and protein A-purified normal rabbit IgG did not react in an ELISA using wells coated with free peptides. The reactivity of the antipeptide antibodies with

surface-bound synthetic peptides was highly reduced (80%) by incubation with the relevant peptide, demonstrating that peptide recognition was specific. All antipeptide antibodies reacted by ELISA with the ClpG subunit (Table 2), and recognition of the denatured ClpG subunit by all antipeptide antibodies was confirmed by immunoblotting of ClpG after SDS-PAGE (data not shown). In contrast, only antibodies to peptides 44 to 56, 174 to 190, 185 to 199, and 235 to 249 (anti-CLP2, -CLP7, -CLP8, and -CLP9 antibodies) bound clearly to native CS31A. Antipeptide antibodies raised against residues 148 to 162 (CLP6) showed poor recognition of the native antigen, suggesting that residues recognized within the epitope are not fully accessible or are maintained in a config- uration that decreases antibody binding.

(ii) Antibody probes to determine accessible surface epi- topes.Two methods were used to determine whether epitopes were accessible to antibodies in the native CS31A antigen.

First, a competitive ELISA was employed to determine the inhibition of antipeptide antibodies binding to native CS31A by synthetic peptides to study the proportion of antipeptide antibodies that bind to their epitope and to determine the specificity of this binding. A representative result of our antibody-binding experiment is shown in Fig. 4. The ability of peptides 44 to 56, 174 to 190, 185 to 199, and 235 to 249 to block binding of their respective antipeptide antibodies (anti- CLP2, -CLP7, -CLP8, and -CLP9) to native CS31A in a competitive ELISA confirmed the specificity of epitope recognition on the native protein. The three peptides CLP2, CLP7, and

FIG. 4. Inhibition by synthetic peptides of antibody binding to native CS31A by competitive ELISA. Subsaturating amounts of five antipeptide antibodies to peptides corresponding to residues 44 to 56, 148 to 162, 174 to 190, 185 to 199, and 235 to 249 whose titers had previously been adjusted (70% of the maximum binding to CS31A) were preincubated with increasing concentrations of their cognate synthetic peptides (CLP2 [■], CLP6 [å], CLP7 [Ç], CLP8 [h], and CLP9 [F]). The antiserum was then applied to an ELISA plate coated with native CS31A antigen. The binding of residual antibodies to immobilized CS31A was assayed as described in Materials and Methods. Percent inhibition of antipeptide binding to CS31A antigen by the respective synthetic peptide was calculated for each antipeptide by comparing assay values with those for controls incubated with irrelevant peptides. Percent inhibition is represented as the mean6 standard deviation (error bars) from three experiments.

TABLE 2. Immunological properties of rabbit antibodies raised against CLPG-peptide conjugates

Antibody (IgG fraction)

Reactive titer of given coated antigen as determined by ELISA Homologous

peptide^a

Denatured subunit (ClpG)

Native CS31A^b

Anti-CLP1 8,200^c 4,096 32

Anti-CLP2 65,500 4,096 2,048

Anti-CLP3 4,096 4,096 32

Anti-CLP4 65,500 8,200 128

Anti-CLP5 8,200 4,096 128

Anti-CLP6 65,500 8,200 612

Anti-CLP7 16,400 8,200 16,400

Anti-CLP8 65,500 8,200 16,400

Anti-CLP9 8,200 4,096 4,096

aSpecific antipeptide titers were subtracted from those of irrelevant synthetic peptides (foot-and-mouth disease virus residues 51 to 65 and poliovirus residues 91 to 105) used as negative controls.

bAs determined by ELISA with HB101(pEH524) intact bacterial cells. Spe- cific titers were subtracted from the titer obtained with the recipient strain HB101.

cTwo rabbits per group were immunized. The data shown are those obtained with the rabbit having the highest antibody titers.

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CLP9 demonstrated similar dose-dependent inhibitions of binding of their respective antipeptide antibodies (,30% reduction); only peptide CLP8 showed a higher inhibitory effect (40%) at the highest concentration tested. As suggested by Tan et al. (33), the inability of the remaining 60% of the antibodies to bind to the peptides may have resulted from a lower affinity for short synthetic peptides. Peptide CLP6 had no effect on antibody binding (Fig. 5), demonstrating that the limited recognition of native CS31A by anti-CLP6 antibodies seems to be nonspecific and may depend on conformational changes in- duced by adsorption of antigen to polystyrene (1), rather than on binding to accessible epitopes on native CS31A.

To prevent these possible perturbations of protein structure occurring on adsorption to the polystyrene surface, an accessibility ELISA was used to determine the effectiveness of native CS31A, on intact bacterial cells, in competing for antibody with the denatured ClpG subunit adsorbed on plates. The results of the accessibility ELISA are presented (Fig. 5) as percent inhibition of the ELISA reaction resulting from preincubation of a subsaturating concentration of an individual antipeptide antiserum; whose titer had been previously adjusted, with a suspension of intact bacterial cells of strain HB101(pEH524) at various dilutions (0.23 10⁸to 2.6 3 10⁸ cells per ml). Three classes of site-specific antibodies were ap- parent. Antipeptide antibodies to residues 185 to 199 (CLP8) and 235 to 249 (CLP9) were clearly neutralized by native CS31A, antipeptide antibodies to peptides 44 to 56 (CLP2) and 174 to 190 (CLP7) showed intermediate responses, and the remaining antipeptide antibodies (anti-CLP1, -CLP3, -CLP4, and

-CLP5 antibodies) showed no or low titer reductions. Thus, the accessibility ELISA method provided information in accord with other results reported in this paper. Finally, both competitive and accessibility ELISAs resolved the antipeptide antibodies into three classes: (i) antibodies to residues 185 to 199 (CLP8) and 235 to 249 (CLP9) clearly recognized epitopes exposed on the surface of native CS31A; (ii) antibodies to peptides 5 to 18, 82 to 96, 97 to 109, 132 to 146, and 148 to 162 reacted only with the denatured ClpG subunit and recognized epitopes that are either inaccessible or in an unreactive conformation in native CS31A; and (iii) antibodies to residues 44 to 56 (CLP2) and 174 to 190 (CLP7) recognized epitopes that are probably exposed on the surface but had intermediate reactivity with native protein, which could result from a complex response of the antibody probe to the peptide antigen.

Our results agree with those of Krogfelt et al. (25) and have revealed on native CS31A three new surface-exposed linear epitopes centered on peptides 44 to 56, 174 to 190, and 235 to 249. The present findings with ClpG indicate that synthetic peptides and antipeptide antibodies based upon continuous B-cell epitopes of the denatured subunit could provide useful information on accessibility of peptides on the multimeric CS31A antigen and suggest that our topological epitope-mapping strategies may have general applicability.

Location of the antigenic peptides in relation to prediction of secondary structure and predictive antigenic determinant parameters.Predicting antigenicity of epitopes on the basis of information provided by the primary structure of the protein is a controversial issue. However, previous studies (4, 7, 33) have shown that amino acid segments having access to the periphery of a molecular structure, a tendency to variability, and seg- mented mobility have an increased likelihood of representing immunological determinants. Except for region D, computer analysis of all epitope regions of the ClpG subunit revealed similar secondary structures (Fig. 6). All are variable, have a high level of predicted flexibility, contain stretches of amino acids likely to be surface exposed, and have a high potential to reside in loop regions at the boundaries of secondary structural elements (Fig. 2). These findings indicate that variability, frag- ment mobility, and hydrophilicity are important features of these antigenic epitopes, at least in the denatured ClpG subunit. This is in sharp contrast to the features of the areas between antigenic regions (residues 23 to 35, 68 to 85, 119 to 148, and 170 to 186), which are mainly hydrophobic, have a low level of predicted flexibility, contain stretches of conserved amino acids predicted to be buried within the hydrophobic core of the subunit, and are mostly predicted to reside in well- ordered secondary structures (b-sheet or a-helix elements).

Only the variable region V2 (residues 121 to 153) formed an exception to the general features that determined the immunogenicity of peptides on the ClpG subunit. Although a previous study (2) demonstrated that residues 134, 136, and 147, which reside in V2, contribute with other residues (located within the hypervariable region) to the subtype-specific epitopes of FaeG, our epitope mapping demonstrated the absence of a dominant linear epitope within the corresponding regions of ClpG. However, the conformational nature of subtype-specific epitopes (2) could explain why the PEPscan method failed to detect epitopes within the V2 region of ClpG. In the light of previous secondary structure predictions (27) and DNA mu- tagenesis experiments (5), which showed that region V2 probably has a highly ordered secondary structure (b-sheet ele- ment) essential for protein stability, it is likely that the epitopes of this region are defined by tertiary structures.

Our purpose was to delineate the dominant surface-exposed linear epitope of ClpG for subsequent substitution or insertion

FIG. 5. Accessibility ELISA analysis of antipeptide antibodies. Amount of antipeptide antibodies whose titers had previously been adjusted (70% of maximum binding to denatured ClpG) were preincubated with increasing amounts (0.13 10⁸to 23 10⁸cells per ml) of HB101(pEH524) bacterial cells. The residual antibody binding was then tested on an ELISA plate coated with the denatured ClpG subunit. The results are presented as percent inhibition of maximum binding resulting from preincubation with native antigen on intact cells. Only results for clearly neutralized antibodies (.10% inhibition) are shown. ■, anti-CLP2; Ç, anti-CLP7; h, anti-CLP8; F, anti-CLP9. Percent inhibition is represented as the mean6 standard deviation (error bars) from three experiments.

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of foreign epitopes, and our topological and epitope-mapping strategies have proven useful to evaluate a region of ClpG that could be surface oriented in the native CS31A antigen. At- tempts at this substitution have been successfully made in V3 (residues 190 to 221), where a range of foreign epitopes (22 residues in size) can be introduced at different positions with- out interfering with secretion and assembly of CS31A (5). The implication of surface-oriented segments for residues 45 to 58, 174 to 190, and 235 to 249 (epitope regions B, E, and G, respectively) is a new finding. As segments from residues 45 to 58 and 235 to 249 are both variable, have a high level of predicted flexibility, and are surface oriented in the native CS31A antigen, the choice of these two sites is encouraging in regard to the possibilities for future insertions or substitutions by heterologous peptides. Insertions and mutations are known to affect stability, secretion, and assembly of the fimbrial subunit, and each of the new sites can be tested to determine which best allows assembly of the antigen at the cell surface

and retains the immunogenic properties of the foreign epitope.

Thus, given that the insertion of a linker (48 bp) between positions 52 and 53 did not greatly interfere with CS31A biogenesis (9), we assume that the size of region B (residues 45 to 58) is not critical for the secretion and assembly of the ClpG subunit. These results further support the idea that region B can serve as a new site for presenting heterologous peptide at the surface of the assembled ClpG subunit on the native CS31A antigen. Whether the three sites (B, V3, and G) can concert to correctly present different B-cell epitopes of a complex antigen requires more detailed investigation.

Our original purpose in mapping the B-cell antigenic determinant of ClpG was to identify immunoaccessible epitopes as a first step toward the rational design of vaccine using CS31A as a carrier, and our study demonstrates that regions from residues 45 to 58, 174 to 190, 185 to 199, and 235 to 249 are possible candidates. Our findings confirm the immunodomi- nance of the variable region V3 and demonstrate that peptide

FIG. 6. Locations of the seven antigenic regions of the ClpG subunit in relation to secondary structure predictions and antigenic determinant prediction parameters.

The previously identified (14) variable regions (V1, V2, V3, and HV) are indicated by open bars, and the conserved regions containing the five hydrophobic clusters (P1 to P5) are indicated by solid bars. The ClpG sequence was analyzed by using prediction parameters for possible antigenic determinants, hydropathy profiles (17), and chain flexibility (20).b-Strand ( ),a-helix ( ), and loop ( ) potentials as determined following secondary structure prediction by the program PHDsec (31), by using information from multiple sequence alignments as previously reported (27), are indicated. Regions predicted to be exposed (;) or buried ( ), as determined following predictions of solvent accessibility by the program PHDacc (32), are reported. The positions at which insertion of linkers or foreign peptides did not interfere with biogenesis of K88 (^V) or CS31A (^M) and positions at which insertion greatly interfered with biogenesis of K88 (^v) or CS31A (^f) are indicated (DNA exp.) as reported elsewhere (5, 9, 29). Antigenic determinants of ClpG derived from PEPscan analysis with antibodies generated with both the denatured subunit (d) and native CS31A antigen (N) are indicated. The locations of the binding sites for the antipeptide antibodies are depicted at the bottom, with the accessible regions shown as open boxes. For comparison, the locations on FaeG of the antigenic sites that are exposed at the surface of the intact K88 fimbriae (25) are also shown (open boxes). The filled boxes represent the locations of antigenic sites which are not accessible. Positions of the three antigenic FaeG peptides identified by Thiry et al. (34) are also shown (FaeG antigenic peptides).

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189-AVNPNAG is immunogenic on the native CS31A antigen.

In addition, the present study provides a low-resolution topo- graphical map of CS31A epitopes that will enable the design of mutant forms with which to study the role of the surface- oriented segments of ClpG in the conformation and function of CS31A.

ACKNOWLEDGMENT

This work was supported by EU grant AGRE-008-C (ECLAIR program).

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Editor: J. R. McGhee