coli B Polysaccharide Cross-Reacting with Salmonella typhi Vi ... - NCBI

INFECTION AND IMMUNITY, JUlY 1983, p. 224-231 0019-9567/83/070224-08$02.00/0

Vol. 41, No. 1

Purification and Immunochemical Properties of Escherichia coli B Polysaccharide Cross-Reacting with Salmonella typhi Vi Antigen: Preliminary Evidence for Cross-Reaction of the Polysaccharide with Escherichia coli Ki Antigen BOGUS4AW SZEWCZYK AND ALINA TAYLOR* Department of Biochemistry, University of Gdanisk, PL-80-822 Gdanisk, Poland

Received 29 July 1982/Accepted 25 April 1983

An acidic polysaccharide of Escherichia coli B was isolated by a mild procedure and purified to homogeneity. The polysaccharide was found to react in Salmonella typhi Vi antisera and E. coli Kl antisera. Serological analysis and preliminary chemical characterization of the polysaccharide indicated that it is an aminouronic acid polymer which, although not structurally identical to either Vi or Kl, appears more like the Vi antigen, both immunochemically and chemically.

Escherichia coli Kl antigen and Salmonella typhi Vi antigen are linear homopolymers in which the basic sugar unit contains the carboxyl group and the acetylated amino group. Both polysaccharides may exist either in O-acetylated (OAc+) or in non-O-acetylated (OAc) form (18, 34). The Kl polysaccharide consists of linear chains of a-2-8-linked N-acetylneuraminic acid, and its structure has been found to be identical with group B meningococcal capsular polysaccharide (1, 4, 5). Vi antigen is composed of Nacetylated galactosaminouronic acid units connected through a-1-4 linkages (10). There has been widespread interest and intensive research to obtain information about these polysaccharides since they contribute to the capsules of highly pathogenic bacteria. Vi antigen is present in the outermost layer of S. typhi, a primary etiological agent of typhoid fever, which remains a serious clinical and public health problem in many parts of the world. Its role as an agent conferring virulence on S. typhi has been questioned (3, 38) but never entirely abandoned (6, 15). E. coli Kl is associated with invasive diseases in humans and in domesticated animals. Kl isolates account for over 80% of E. coli neonatal meningitis and comprise the majority of capsular types in upper urinary tract infections in infants (11, 25). Several reports suggest that the Kl capsular polysaccharide confers invasiveness upon E. coli (24, 27), and there are indications that the polysaccharide exerts an anti-phagocytic effect similar to that observed with other encapsulated bacteria (32, 41). Both polysaccharides may be produced by the respective bacterial strains as two populations of

molecules, one with OAc+ and OAc- sugar units and another in which the homopolymer is only OAc-. Serological studies have shown two major antigenic determinants in the OAc+ form of the Kl and Vi polysaccharides (18, 33). The first determinant results from the OAc- polysaccharide. The second is presumably due to the 0acetylation modifying the OAc- determinants. Previously, we showed that E. coli B contains a polysaccharide which precipitates antibodies from Vi antisera (34). In this paper we characterize the polysaccharide serologically and physiochemically and present evidence that it also reacts in E. coli Kl antisera. MATERIALS AND METHODS Bacterial strains and growth conditions. S. typhi Ty2 was obtained from the National Laboratory for Enteric Phage Typing in Gdanisk, Poland. E. coli F11119/41 (016:K1:H-) was kindly donated by F. 0rskov (Statens Seruminstitut, Copenhagen, Denmark). E. coli B and S. typhi Ty2 were cultivated on 1% agar (Oxoid Ltd., London, England) supplemented with 1% peptone and 0.35% NaCI. E. coli 016:K1:H- was grown in a liquid medium (aerated casein hydrolysate broth containing 0.1% glucose). Harvested cells were dried with acetone. Isolation of E. coli B polysaccharide. Acetone-dried cells were shaken with O.9o NaCi for 1 h (1 g of dried cells in 50 ml of saline), and the bacteria were centrifuged off. The supernatant was fractionated with cetavion (Koch-Light, Colnbrook, England) by the method of Scott (29). After extensive dialysis, the nondialyzable material was lyophilized and dissolved in 50 mM sodium acetate buffer, pH 4.0. To that solution, bovine serum albumin (2 mg/ml in the same buffer) was added until no further precipitate was found. The precipitate was collected by sedimentation, dissolved in 100 mM phosphate buffer, pH 7.5, and digested 224

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Vi-RELATED E. COLI B POLYSACCHARIDE

consecutively with RNase (Koch-Light), DNase (E. Merck AG, Darmstadt, West Germany), and pronase (Serva Feinbiochemica, Heidelberg, West Germany) as previously described (14). The solution was dialyzed against 50 mM phosphate buffer, pH 7.0, loaded onto a DEAE-Sepharose CL-6B (Pharmacia Fine Chemicals, Uppsala, Sweden) column (1.6 by 60 cm), and fractionated with a 0 to 2 M NaCl gradient (in 50 mM phosphate buffer, pH 7.0) as an eluant. Isolation of other antigens. Vi antigen was purified by the method of Taylor (36) with modifications as previously described (33). OAc- E. coli Kl antigen was extracted by a cetavlon precipitation procedure previously described (29), digested with RNase, DNase, and pronase (14), and fractionated on a DEAESepharose CL-6B column (1.6 by 60 cm) with a 0 to 2 M NaCl gradient (in 50 mM phosphate buffer, pH 7.0) as an eluant. 2,3-Di-O-acetylpolygalacturonic acid was synthesized as described previously (33). Authentic OAc+ and OAc- Kl polysaccharide samples were generously donated by J. B. Robbins (Bureau of Biologics, Food and Drug Administration, Bethesda, Md.). Analytical methods. Hydrolysis of polysaccharides was done either with 6 N HCI at 100°C for 6 h for the release of aminouronic acids (37) or with 0.5 M trifluoroacetic acid at 100°C for 12 h for the release of neutral monosaccharides or uronic acids. Thin-layer chromatography of the former hydrolysate was performed on Silica Gel or cellulose plates (Merck) in the n-butan-1ol-acetic acid-water (4:1:5 [vol/vol/vol]) solvent system. Thin-layer chromatography plates were sprayed with 0.2% ninhydrin in acetone. The latter hydrolysate was chromatographed on cellulose plates in the ethyl acetate-pyridine-water (2:1:2 [vol/vol/vol], upper phase) solvent system. Neutral monosaccharides were detected with 100 mM anisidine phthalate reagent in ethanol. Analysis of charged sugars was performed by electrophoresis by the method of Miyamoto and Nagase (16) on cellulose acetate plates (Serva Feinbiochemica) with 100 mM zinc acetate as the electrolyte. The plates were stained with silver nitrate (16). The polysaccharides were also hydrolyzed in optimum conditions for the release of sialic acid (0.1 N H2SO4, 80°C, 30 min [4]). The effect of neuraminidase treatment was determined with Vibrio cholerae neuraminidase (Serva Feinbiochemica) in sodium acetate buffer, pH 5.5, containing 9 mM CaC12. Sialic acid was determined by the method of Warren (39), and total uronic acids were determined by the method of Bitter and Muir (2). Determinations of O-acyl content were performed by the method of Snyder and Stephens (31); pyruvate determinations were carried out as described by Sloneker and Orentas (30). Nucleic acids were determined by measuring absorption at 260 nm assuming an extinction coefficient of 20 for a solution of 1.0 mg of DNA per ml in a 1-cm cuvette. Acidic polysaccharides in eluates from columns were determined by the method of Webster et al. (40). The standard curve was prepared for Vi antigen solutions. The relation was linear in the range of 40 to 200 F±g of Vi antigen (total volume of the polysaccharide and albumin solutions, 2.5 ml). Agarose electrophoresis for testing the purity of polysaccharides was performed in 50 mM barbital buffer, pH 8.6, and in 50 mM barium acetate, pH 5.8, at 10 to 15 V/cm with cresol red used as a tracking dye. The polysaccharides were stained by a

225

method in which the ability of bovine serum albumin to precipitate acidic polysaccharides (40) was employed. Briefly, an agarose gel was immersed for 1 h in 50 mM sodium acetate, pH 4.0, containing 2 mg of albumin per ml. The excess albumin was removed by repeated washings (three times for 1 h with stirring) in 50 mM sodium acetate, pH 4.0. The gels were stained with 0.1% Coomassie brilliant blue R-250 (Fluka AG, Buchs, Switzerland) in acetic acid-methanol-water (1:5:5 [vol/vol/vol]) for 1 h and were destained with the same solution without the dye. Antisera. Antisera against S. typhi 21802 prepared by intravenous injections of rabbits with acetonekilled bacteria were obtained from the National Reference Laboratory for Enteric Phage Typing. These antisera reacted with the native Vi antigen (OAc+) but not with the OAc- Vi antigen, in contrast with the majority of rabbit Vi antisera, which react with both forms of the antigen (OAc+ and OAc) (33). E. coli Kl antiserum (rabbit 5466) prepared by intravenous injections with formalinized bacteria (strain D6980Ac+, derived from parent E. coli O1:K1:H7 [18]) was donated by F. Orskov. The antiserum reacted with the OAc' form of Kl antigen but not with OAc- Kl antigen (18). The immunoglobulin fraction of equine (horse 46) antigroup B meningococcal serum was kindly provided by J. B. Robbins. The antiserum reacted with both forms (OAc+ and OAc-) of Kl

antigen. Serological techniques. Precipitin reactions in agarose gels were performed by the Ouchterlony technique (21). One-dimensional immunoelectrophoresis was carried out by the method of Scheidegger (26); crossed immunoelectrophoresis was performed by the method of Laurell (13). The dried gels were stained with 0.2% solution of Coomassie brilliant blue in acetic acidmethanol-water (1:5:5 [vol/vol/vol]) for 20 min at room temperature. The excess stain was removed by washing the gels with the same solution without the dye. Indirect hemagglutination (HA) was performed with formolized sheep erythrocytes (SRBC). To sensitize SRBC with antigens, 50 F.g of antigenic material was added to 10 ml of 1% washed SRBC suspension in saline. After incubation at 37°C for 30 min, the excess antigen was washed off, and the sensitized SRBC were suspended in saline to give a 1% suspension. The SRBC suspension was added to series of antiserum dilutions in microtiter plates as described previously

(17).

To measure the inhibiting capacity of a substance, we also used the HA inhibition (HAI) test. Serial dilutions (0.2 ml) of the inhibitor were incubated at 37°C for 1 h with 0.2 ml of an antiserum dilution corresponding to 2 HA units. Then, 0.2 ml of the sensitized SRBC was added, and the plates were incubated again for the same length of time. The lowest inhibitor concentration giving a total inhibition of HA was recorded after 2 h at room temperature. Quantitative precipitin assays were performed essentially as described previously (23). Portions of antisera (0.1 ml) and polysaccharide solutions (0.2 ml) were added to Eppendorf tubes and capped. The tubes were incubated at 37°C for 1 h and at 4°C for 3 days and were occasionally agitated gently. The mixtures were centrifuged at 4°C, the supernatants were poured off, and the precipitates were washed three times with 0.5 ml of chilled saline. The washed precipi-

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INFECT. IMMUN.

tates were dissolved in 0.5 ml of 0.8% sodium lauryl sulfate (Sigma Chemical Co., St. Louis, Mo.), and the absorbance was measured at 280 nm in a Pye-Unicam SP8-100 spectrophotometer with 1-cm cuvettes for a total volume of 0.5 ml. The contents of precipitated antibody were determined assuming an extinction coefficient of 1.4 for 1.0 mg of immunoglobulin G solution per ml at 280 nm in a 1-cm cuvette. Antisera were absorbed by incubating them for 1 h at 37°C and for 3 days at 4°C with amounts of polysaccharides determined from the zone of maximum precipitation (equivalence) and by centrifuging off the resulting precipitate.

RESULTS Purification of E. coli B polysaccharide. The E. coli B polysaccharide extracted from bacterial cells with 0.9% NaCl was purified by a procedure which included catavlon precipitation, bovine serum albumin precipitation, and DEAESepharose chromatography. One prominent polysaccharide peak eluted from the DEAESepharose column in the range 0.6 to 1.0 M NaCl contained the material which, when examined by double immunodiffusion against the Vi antiserum, yielded the precipitin line fusing with that of the Vi antigen isolated from S. typhi. The polysaccharide was virtually free of protein (below 0.5%) and nucleic acids (below 0.1%). On a crossed immunoelectrophoresis pattern, two distinct peaks were observed (Fig. 1), both of them with a lower electrophoretic mobility than Vi antigen. The polysaccharide gave two badly resolved spots (corresponding to immunoelectrophoretic peaks) on electrophoresis at pH 8.6 and at pH 5.8 in the presence of divalent cations. The yield of the polysaccharide amounted to

about 0.05% of bacterial dry weight.

FIG. 1. Antigen-antibody crossed immunoelectrophoresis of 30 ad (30 F.g) of the main acidic polysaccharide fraction from the DEAE-Sepharose separation. Rabbit Vi antiserum (25 ,ul/ml) was used in the seconddimension gel. Technical details: dimensions of the plate, 10 by 8.5 cm; 1% (wt/vol) agarose gel in barbital buffer, pH 8.6; ionic strength, 0.05; thickness of gel, 1 mm; first-dimension electrophoresis, 10 V/cm for 1 h at 5°C, anode to the right; second-dimension electrophoresis, 2 V/cm for 24 h at 5°C, anode at the top; staining, Coomassie brilliant blue.

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r

i~

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FIG. 2. One-dimensional immunoelectrophoresis of purified E. coli B polysaccharide (100 Rg) showing the precipitation pattern in S. typhi antiserum (300 ,ul) (A) and equine meningococcal group antiserum (500 ,ul) (B). Technical details: dimensions of the plate, 10 by 5 cm; 1% (wt/vol) agarose gel in barbital buffer, pH 8.6; electrophoresis conditions, 10 V/cm for 30 min at 5°C, anode at the top.

Reaction of E. coli B polysaccharide in K1 antisera. The purified E. coli B polysaccharide was examined by immunoelectrophoresis against Vi and Kl antisera (Fig. 2). The precipitin bands produced in both antisera appeared to originate from the same antigen since their positions on immunoelectrophoretograms were identical. Therefore, the possibility that the spur on the side of the Kl antiserum is produced by an antigen contaminating the E. coli B polysaccharide is unlikely. The pattern of double imtnunodiffusion precipitation lines produced by the E. coli B polysaccharide when reacting with equine anti-group B meningococcal serum was similar to that observed for OAc+ Kl antigen (18). Two precipitation lines were visible, one prominent at high polysaccharide concentrations and one at low polysaccharide concentrations. E. coli B polysaccharide also precipitated antibodies from rabbit Kl antiserum (rabbit 5466 antiserum obtained from Statens Seruminstitut, Copenhagen) which reacts with OAc+ Kl antigen, but not with OAc- Kl antigen, in contrast to horse 46 antiserum, which reacts with both forms of the Kl antigen (OAc+ and OAc) (18). E. coli B polysaccharide was compared by the double immunodiffusion test with the Kl polysaccharide. Their precipitation lines did not intersect, which suggests that E. coli B polysac-


VOL. 41, 1983

charide precipitates Kl antibodies. however, the complete fusion of precipitin lines was not observed, and the precipitation arc of Kl antigen (OAc) extended over the point where these two precipitin lines met. Quantitative precipitation tests. Quantitative precipitation of antibodies from the rabbit Vi antiserum and equine Kl antiserum by the E. coli B polysaccharide, Vi antigen, and Kl antigens is shown in Fig. 3. The following conclusions can be drawn from this set of experiments: (i) E. coli B polysaccharide precipitates almost the same amount of antibodies from the Vi antiserum as the Vi antigen does, although at the higher antigen concentration; (ii) neither the OAc- nor the OAc+ variant of Kl antigen precipitates antibodies in the Vi antiserum; (iii) the behavior of E. coli B polysaccharide in the Kl antiserum resembles that of the OAc+ variant of Kl antigen, although more E. coli B polysaccharide than OAc+ Kl antigen is necessary to precipitate the same amou'if of antibodies; and (iv) Vi antigen does not precipitate antibodies in the Kl antiserum. Reactions in absorbed antisera. Absorption of the horse 46 meningococcal antiserum with Kl antigen (OAc- type) and absorption of rabbit Vi antiserum with Vi antigen yielded antisera which did not react with E. coli B polysaccharide in either case. This result indicates that E. coli B polysaccharide reacts with both Vi and Kl antibodies. Absorption of Vi antiserum with E. coli

3O00

227

B polysaccharide removed all Vi antibodies from this antiserum. Attempts were also made to remove all Kl antibodies from horse 46 antiserum by repeated absorptions (1 mg of polysaccharide per 0.1 ml of antiserum) with E. coli B polysaccharide. In this case, the resulting antiserum still reacted with Kl antigen (OAcform). This observation does not contradict the postulated cross-reactivity of E. coli B polysaccharide and the Kl polysaccharide since the data reported by 0rskov et al. (18) suggest that the latter antigen may possess more than one antigenic determinant. E. coli B polysaccharide would react with populations of antibodies with a specificity for some, but not all, antigenic determinants of the Kl polysaccharide. Passive HA and HAI of erythrocytes. The OAc- form of the Kl antigen does not coat SRBC. Erythrocytes are, however, sensitized with the OAc+ Kl antigen, so this polysaccharide was used in HA studies along with Vi antigen and E. coli B polysaccharide, both of which are active in sensitizing formolized SRBC. No cross-reaction was observed for Kl and Vi antigens, although on the other hand, the E. coli B polysaccharide-coated SRBC were agglutinated in both antisera. The reciprocal titers of the Vi antiserum for Vi and E. coli B polysaccharides were equal, whereas with horse 46 antiserum, the reciprocal titer for Kl antigen was about 20 times that for E. coli B polysaccharide.

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1 2 4 8 16 32 64 128 256 8 16 32 64 128 256 POLYSACCHARIDE / pg 1 POLYSACCHARIDE / pg / FIG. 3. Quantitative precipitin curves obtained with rabbit Vi antiserum (A) and equine (horse 46) antiserum to N. meningitidis group B (B). Symbols: 0, E. coli B polysaccharide; *, Vi antigen; x, Kl antigen OAc- form; A, Kl antigen OAc+ form.

1

2

4

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SZEWCZYK AND TAYLOR

INFECT. IMMUN.

HAI tests were performed with equine meningococcal antiserum and rabbit S. typhi antiserum in dilutions corresponding to 2 HA units, and the SRBC were coated with Vi antigen and Kl antigen (OAc+ form). 2,3-Di-O-acetylpolygalacturonic acid, a polysaccharide which inhibits the HA of Vi-coated erythrocytes with Vi antisera (33), was also included in HAI studies. E. coli B polysaccharide inhibited HA in both systems, namely Vi-sensitized SRBC with S. typhi antiserum and Kl-sensitized SRBC with horse 46 meningococcal antiserum (Table 1). The HAI of Kl-coated erythrocytes by Vi antigen deserves particular attention since it may reflect the structural similarity between Kl and Vi antigens. Preliminary chemical characterization of E. coli B polysaccharide. Table 2 and Fig. 4 show the results of assays used for the preliminary characterization of E. coli B polysaccharide. The primary aim of the assays was to determine the common constituents in E. coli B polysaccharide and the other two polysaccharides. The chromatographic patterns of E. coli B polysaccharide and Vi antigen hydrolysates indicate that both polysaccharides may contain the same aminouronic acid (i.e., galactosaminouronic acid) or that the E. coli B polysaccharide may contain an aminouronic acid closely related to galactosaminouronic acid. The structure of E. coli B polysaccharide appears to be more complex than that of the Vi antigen (homopolymer of N-acetylgalactosaminouronic acid) since a uronic acid was also detected in the hydrolysis products of E. coli B polysaccharide (Table 2). The electrophoretic mobility of this uronic acid corresponded to that of glucuronolactone when the electrophoresis was carried out on cellulose acetate plates in 0.1 M zinc acetate as an electrolyte. TABLE 1. Serological activity of E. coli B polysaccharide and related antigens in HAI' Inhibitory amt in HAI

(ju.g/ml)

Polysaccharide

Kl-coated

Vi-coated

SRBC/ horse 46

typhi anti-

SRBC/S.

antiserum

serum

E. coli B polysaccharide

1.6

0.8

Vi antigen

3.2

0.2

Kl antigen OAcOAc+

0.2 0.2

0.8 250.0 2,3-Di-O-acetylpolygalacturonic acid a Maximum concentration of antigen in HAI was 1 mg/ml.

1

2

3

4

FIG. 4. Thin-layer chromatogram on cellulose showing the hydrolysis products of E. coli B polysaccharide and the Vi antigen. Lane 1, E. coli B polysaccharide hydrolyzed with 0.5 M trifluoroacetic acid at 100°C for 12 h; lane 2, E. coli B polysaccharide hydrolyzed with 6 N HCI at 100°C for 6 h; lane 3, Vi antigen hydrolyzed with 0.5 M trifluoroacetic acid at 100°C for 12 h; lane 4, Vi antigen hydrolyzed with 6 N HCI at 100°C for 6 h. Detection, 0.2% ninhydrin in acetone. The arrow indicates the position of a monomer of galactosaminouronic acid. In the case of the Vi antigen hydrolysate, the spots probably corfespond to oligomers of galactosaminouronic acid.

DISCUSSION Despite the wide use of E. coli B in many laboratories, the serological properties of this bacterium have not been fully determined. It is an R mutant of which the S ancestors are not known (22); it is, however, considered to be a smooth strain (35). The smoothness of an E. coli strain does not always indicate the presence of a complete 0-specific polysaccharide attached to the lipopolysaccharide core, and it may be caused merely by the presence of K antigen (20). The polysaccharide described in this report may be one of the macromolecules contributing to the smoothness of E. coli B since the method of its detachment from bacteria (shaking with 0.9% NaCl) indicates that it is loosely bound to the bacterial surface. The high negative charge of E. coli B polysaccharide and the absence of neutral sugars in its chemical composition exclude the possibility that it may be identical to the cell wall lipopolysaccharide of E. coli B, the structure of which is known (22). The data described in this report indicate that E. coli B polysaccharide reacts with the specific Vi antibodies from the S. typhi antiserum. For


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229

TABLE 2. Comparison of chemical compositions of E. coli B polysaccharide and Ki and Vi antigens Neutral NeurSialic(%)acids Uronic monoPyruvate aminidase acids sacchaPolysacchanide sensitivity rides'

90 Sensitive 8d ND ND Kl antigen (OAc) a Detected on thin-layer chromatography plates after hydrolysis with 0.5 M trifluoroacetic acid at 100°C for 12 h. b Expressed as number of O-acetyl groups per sugar residue (molecular weight of sugar residue assumed to be equal to 220). c ND, Not detectable. d Formed as the result of degradation of the labile sialic acid to N-acetylmannosamine and pyruvate.

clarity of results, the Vi antiserum chosen for our studies was unusual in that it did not contain antibodies reactive with OAc- Vi antigen, generally present in most Vi antisera. When antisera containing both types of Vi antibodies (OAc+ and OAc-) were examined (data not shown), then the amount of antibodies precipitated by E. coli B polysaccharide was lower than that precipitated by the native Vi antigen, and E. coli B polysaccharide antigenically resembled 2,3-diO-acetylpolygalacturonic acid (33). This polysaccharide precipitates antibodies reactive with the Vi determinant in which O-acetyl plays a dominant role and inhibits antibodies reacting with OAc- Vi antigen. The data on the behavior of E. coli B polysaccharide in the Kl antiserum (horse 46 antiserum) suggest that this polysaccharide reacts only with some antibody types (recognizing one or more, but not all, Kl antigenic determinants) since the complete absorption of anti-Kl antibodies by the addition of-E. coli B polysaccharide could not be achieved. Extensive cross-reactions have been found among many bacterial capsular polysaccharides, including those of E. coli. For example, E. coli Kl antigen cross-reacts with Neisseria meningitidis group B polysaccharide (8, 12), and E. coli K92 antigen also cross-reacts with N. meningitidis group B polysaccharide (7). Heidelberger et al. (9) reported that E. coli K30, K42, and K85 induce antibodies reactive with pneumococcal types 1, 4, 8, 12, and 19; Schneerson et al. (28) described an E. coli strain with a polysaccharide K antigen (presently designated as K100 [19]) closely related to Haemophilus influenzae type b capsule antigen. Cross-reactivity in these cases is often due to the complete identity of polysaccharides or to the occurrence of a common immunodominant sugar (or sugars) within the antigen molecule (20). No explanation of the cross-reactivity of E. coli B polysaccharide with two nonidentical homopolymers, Vi and Kl antigens, is afforded by any of these simple models. Cross-precipitation of Vi and Kl anti-

gens in the respective antisera was not detected, although, as the results of HAI testing show, these two antigens exhibit some relationship. E. coli B polysaccharide appears to be structurally closer to Vi antigen than to Kl antigen since it precipitates most of the Vi antibodies in the

antiserum studied at a concentration only slightly higher than that of the native Vi antigen. The structural basis for the cross-reactivity of E. coli B polysaccharide cannot be inferred from the preliminary chemical analyses performed for the polysaccharide. However, some valuable conclusions can be drawn from this set of experiments. E. coli B polysaccharide is much more susceptible to acid hydrolysis than Vi antigen. The acid hydrolysates of the polysaccharide and Vi antigen contain the same or very closely related aminouronic acids; no sialic acid is detected in the hydrolysates of E. coli B polysaccharide. Neither Vi nor Kl antigen reacts with carbazole-sulfuric acid to give a characteristic reaction for uronic acids. Hence, E. coli B polysaccharide which gives a characteristic product in this reaction is likely to have a uronic acid as the second element of the chemical structure. The O-acyl content of E. coli B polysaccharide is low. Assuming for calculations that O-acyl represents O-acetyl, then less than every tenth sugar moiety is OAc+. OAc- E. coli B polysaccharide is not capable of sensitizing SRBC, although the other antigenic properties, including precipitation of antibodies in Vi antiserum and HAI (systems, E. coli B polysaccharide-coated SRBC with Vi antiserum or Vi antigen-coated SRBC with Vi antiserum) are not markedly altered. These results are surprising because, as mentioned earlier, OAc- Vi antigen fails to react in the Vi antiserum chosen for our studies, and the usual treatment for O-deacylation of sugars (0.1 N NaOH, 12 h, ambient temperature) renders the Vi antigen incapable of reacting in this antiserum. One possible explanation of this phenomenon is that the E. coli B polysaccharide attains the conformation of the

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SZEWCZYK AND TAYLOR

native Vi antigen as the result of the presence of the second sugar moiety besides the N-acetylgalactosaminouronic acid. To obtain an unequivocal answer to this suggestion, as well as to account for the immunochemical similarity of E. coli B polysaccharide to the Kl antigen, thorough,structural studies are necessary, including 13C nuclear magnetic resonance, methylation, and perhaps crystallographic analyses. These studies are in progress. ACKNOWLEDGMENTS The authors thank J. B. Robbins for the authentic samples of KI polysaccharides and for horse 46 antiserum, and we thank F. 0rskov for rabbit 5466 antiserum and E. coli F11119/ 41. The technical assistance of Bogusjawa Malankowska and Jadwiga Pomorska is gratefully acknowledged. This work was supported by the Polish Academy of Sciences under project no. 09.7.

LITERATURE CITED 1. Bhattacharjee, A. K., H. J. Jennings, C. P. Kenny, A. Martin, and I. C. P. Smith. 1975. Structural determination of the sialic acid polysaccharide antigens of Neisseria meningitidis serogroups B and C with carbon 13 nuclear magnetic resonance. J. Biol. Chem. 250:1926-1932. 2. Bitter, T., and H. M. Muir. 1962. A modified uronic acidcarbazole reaction. Anal. Biochem. 4:330-334. 3. Collins, F. M. 1979. Cellular antimicrobial immunity. Crit. Rev. Microbiol. 7:27-91. 4. Dewitt, C. W., and J. A. Rowe. 1961. Sialic acids (N,7-0diacetylneuraminic acid and N-acetylneuraminic acid) in Escherichia coli. I. Isolation and identification. J. Bacteriol. 82:838-848. 5. Dewitt, C. W., and E. A. Zell. 1961. Sialic acids (N,7-0diacetylneuraminic acid and N-acetylneuraminic acid) in Escherichia coli. II. Their presence on the cell wall surface and relationship to K antigen. J. Bacteriol. 82:849-856. 6. Felix, A., and R. M. Pitt. 1934. Virulence of B. typhosus and resistance to 0 antibody. J. Pathol. Bacteriol. 38:409420. 7. Glode, M. P., J. B. Robbins, T.-Y. Liu, E. C. Gotschlich, I. Orskov, and F. Orskov. 1977. Cross-antigenicity and immunogenicity between capsular polysaccharides of group C Neisseria meningitidis and of Escherichia coli K92. J. Infect. Dis. 135:94-102. 8. Grados, O., and W. H. Ewing. 1970. Antigenic relationship between Eseherichia coli and Neisseria meningitidis. J. Infect, Dis. 122:100-103. 9. Heidelberger, M., K. Jann, B. Jann, F. 0rskov, I. 0rskov, and 0. Westphal. 1968. Relations between structures of three K polysaccharides of Escherichia coli and crossreactivity in antipneumococcal sera. J. Bacteriol. 95:2415-2417. 10. Heyns, K., and G. Kiessling. 1967. Strukturaufklarung des Vi-Antigens aus Citrobacterfreundii 5396/38. Carbohydr. Res. 3:340-353. 11. Kaliser, B., L. A. Hanson, V. Jodal, G. Linden-Janson, and J. B. Robbins. 1977. Frequency of E. coli K antigens in urinary tract infections in children. Lancet i:663-664. 12. Kasper, D. L., J. L. Winkelhage, W. D. Zollinger, B. Brandt, and M. S. Artenstein. 1973. Immunochemical similarity between polysaccharide antigens of Escherichia coli 07:K1(L):NM and group B Neisseria meningitidis. J. Immunol. 110:262-268. 13. Laurell, C. B. 1965. Antigen-antibody crossed electrophoresis. Anal. Biochem. 10:358-361. 14. Lee, C.-J., and K. Koizuml. 1981. Immunochemical relations between pneumococcal group 19 and Klebsiella capsular polysaccharides. J. Immunol. 127:1619-1623.

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