INFECTION
AND IMMUNITY,
Nov. 1990,
p.
Vol. 58, No. 11
3698-3705
0019-9567/90/113698-08$02.00/0 Copyright C) 1990, American Society for Microbiology
Purification and Characterization of a Novel Hemagglutinin from Vibrio cholerae K. K.
BANERJEE,1* A. N. GHOSH,2
K. DUTTA-ROY,' S. C. PAL,' AND A. C. GHOSE't
Division of Immunology' and Division of Electron Microscopy,2 National Institute of Cholera and Enteric Diseases, Calcutta-700 010, India Received 3 April 1990/Accepted 8 August 1990
A lectin with strong hemagglutinating activity toward erythrocytes of several animal species was isolated from an 18-h culture supernatant of a diarrheagenic strain, V2, of non-O1 Vibrio cholerae. The hemagglutinin (HA) was purified free of lipopolysaccharide by salt fractionation followed by gel filtration, hydrophobic interaction chromatography, and, finally, gel filtration in the presence of urea and deoxycholate. The purification procedure resulted in an HA preparation with 80-fold enhancement of specific activity. The HA consisted of noncovalently bound subunits of Mr 62,000 and behaved essentially as a single component with pI 6.0. Nonpolar and acidic amino acids contributed 46 and 24%, respectively, to the total amino acid residues. Electron micrographs of the HA showed it to consist of large, nonstoichiometric aggregates of disklike molecules of 10-nm diameter. Inhibition of the HA by the glycoproteins fetuin, asialofetuin, and mucin, but not by ovalbumin and simple sugars, suggested the specific requirement of complex carbohydrates for binding. Rabbit antisera to the purified HA inhibited the hemagglutinating activities of the crude cell-free HA preparations, but not cell-associated HA activities of the parent (V2) or of other 01 and non-Ol V. cholerae strains. This suggested that the released and cell-associated HA activities were mediated by antigenically distinct components. Immunoblotting experiments showed that the antisera recognized a polypeptide component of Mr 62,000 in the cell envelope preparations of the parent and several other V. cholerae 01 and non-Ol strains. These data suggested that the HA was a nonfimbrial lectin of somatic origin with no protease activity and was apparently distinct from V. cholerae HAs described so far.
Bacterial lectins are known to mediate attachment of microorganisms to host tissue by specific, noncovalent binding to carbohydrate receptors and are, thus, designed to play a potentially significant role in microbial pathogenicity (8, 25). However, in contrast to the extensive study of the structure and function of plant lectins (22), the bacterial lectins have been subjected to systematic study only recently. Hemagglutinating or lectinlike activities are associated with fimbrial as well as nonfimbrial surface structures of gram-negative bacteria and also with apparently soluble substances released in the culture medium (25). While a number of fimbrial hemagglutinins (HA) have been purified to homogeneity, characterized, and recognized as critical virulence determinants (22, 25), the nonfimbrial HAs have been much less explored. Vibrio cholerae, a gram-negative bacterium associated with cholera, expresses more than one cell-associated HA with distinct carbohydrate-binding specificities during in vitro growth (15, 21). The organism is also known to elaborate apparently soluble or cell-free HA in the culture medium (13). Recently, several groups of investigators (5, 12, 28) have attempted to isolate and characterize cholera HAs. However, no clear picture has emerged, presumably due to the multiplicity of HAs and to the lack of availability of purified materials sufficiently free of lipopolysaccharide (LPS). In particular, Finkelstein and Hanne (12) reported the purification and characterization of a soluble HA with protease activity from the culture supernatant of V. cholerae. This HA was initially described as a lectin (12), but Booth et
*
al. (4) later concluded that its hemagglutination activity could be due to protease modification of erythrocyte surface rather than carbohydrate receptor-mediated interaction. Despite the possible involvement of cholera HAs in the attachment of the organisms to the intestinal epithelium (4, 18), the molecular identity of the true cholera lectin remained elusive. The present communication describes the purification and properties of a novel HA from the culture supernatant of a diarrheagenic strain of V. cholerae non-O1. The HA was of envelope origin and shared by several 01 and non-O1 V. cholerae strains.
MATERIALS AND METHODS Bacterial strains and media. Non-O1 V. cholerae strain V2 used in this study was a clinical isolate kindly made available to us by R. Sakazaki, Tokyo, Japan. This strain evoked cholera toxin-mediated fluid accumulation in the rabbit ligated ileal loop assay, as described previously (10). Brain heart infusion (BHI) broth and nutrient agar were obtained as dehydrated media from Difco Laboratories, Detroit, Mich. HA assay. Twofold serial dilutions of 100-,ul samples of HA preparations in 10 mM sodium phosphate buffer, pH 7.2, containing 150 mM NaCl, (phosphate-buffered saline), were mixed with a 100-pul suspension of erythrocytes of rabbit or other animal species in the same buffer in glass tubes (10 by 1 cm). The mixture was incubated for 1 h at 25°C and agglutination was monitored visually. The HA titer was defined as the reciprocal of the highest dilution of the sample causing visible agglutination of erythrocytes. The specific activity was expressed as the number of minimum HA doses per milligram of the sample. The cell-associated HA activity of the whole organism was determined by the same procedure, using a bacterial suspension adjusted to a turbidity
Corresponding author.
t Present address: Department of Microbiology, Bose Institute, Calcutta-700 009, India. 3698
HEMAGGLUTININ FROM V. CHOLERAE VNOVEL VOL. 58, 1990
corresponding to 200 Klett units against a green filter (KlettSummerson photoelectric colorimeter). Inhibition of hemagglutination by simple sugars and glycoconjugates was performed as described elsewhere (2). Protease assay. The protease activity of the purified HA preparation was examined by using azoalbumin or azocasein as substrate (12). Protein estimation. Protein was estimated by a modified Folin-Ciocalteu method (24), with bovine serum albumin as the standard. Preparation and estimation of LPS. The LPS was extracted, by the phenol-water extraction method (30), from acetone-dried cells of V. cholerae V2 grown overnight in BHI broth at 37°C with shaking. Contaminating protein was removed by digestion with pronase and subsequent dialysis, and the LPS was further purified by ultracentrifugation at 100,000 x g for 1 h. Total carbohydrate content of the LPS was estimated by the phenol-sulfuric acid method (7). Heptose, the LPS-specific sugar, was independently determined by the cysteine-sulfuric acid method (31). Quantitative determinations of LPS in different HA preparations were carried out by estimation of total carbohydrate as well as heptose and comparison of these values with those determined from a weighed sample of lyophilized LPS. Purification of HA. All chromatographic procedures were performed at 25°C. Protein concentrations of the column eluents were monitored by A280. (i) Step 1. Bacteria were grown in BHI broth with shaking at 37°C for 18 h. Cells were removed by centrifugation at 12,000 x g for 10 min. The supernatant was made 50% saturated with solid ammonium sulfate. The precipitate, collected by centrifugation, was dissolved in and dialyzed against 50 mM Tris hydrochloride buffer, pH 7.4, containing 50 mM NaCl and 1 mM EDTA. Any precipitate formed during dialysis was discarded, and the supernatant (fraction A) was retained. (ii) Step 2. A 20-ml amount (360 mg of protein) of fraction A was chromatographed on a Sephadex G-200 column (65 by 2.6 cm). Fractions with HA activity were pooled (fraction B) and concentrated. (iii) Step 3. A 20-ml amount (120 mg of protein) of fraction B was subjected to hydrophobic interaction chromatography on a column of phenyl-Sepharose CL-4B (36 by 1.6 cm) previously equilibrated with 10 mM Tris hydrochloride buffer, pH 7.4 (buffer A). The column was washed with 100 ml of buffer A, and the bound protein was eluted by a linear gradient of ethylene glycol from 0 to 100% in buffer A. Active fractions were pooled (fraction C), dialyzed against water, and concentrated. (iv) Step 4. A 5-ml amount (15 mg of protein) of fraction C was subjected to gel filtration on a column (38 by 1.6 cm) of Sepharose CL-4B previously equilibrated with buffer A containing 6 M urea and 1% sodium deoxycholate. Urea and deoxycholate were removed by ultrafiltration through a PM-10 membrane (Amicon). The HA activity was monitored in detergent-free fractions. Active fractions were pooled (fraction D). Preparation of envelope and outer membrane. Crude envelope preparations were obtained from bacteria grown in BHI broth at 37°C for 18 h. Cells were washed with phosphatebuffered saline and disrupted in an ultrasonic disintegrator (MSE). Unbroken cells were removed by centrifugation at 2,000 x g for 10 min, and the supernatant was centrifuged at 100,000 x g for 1 h to collect the envelope fraction. The outer membrane components were obtained from the crude
3699
envelope preparation by using sodium lauroyl sarcosinate (11). SDS-PAGE. Protein samples were analyzed for molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by the method of Laemmli (20). Samples were run on a 12.5% polyacrylamide gel (2 mm thick) with a 3% stacking gel at a constant voltage of 100 V for 4 h. Proteins were denatured by incubating samples with an equal volume of 10 mM Tris hydrochloride buffer, pH 7.0, containing 2% SDS and 5% 2-mercaptoethanol for 5 min in a boiling-water bath. Protein standards of known molecular weights (Pharmacia Fine Chemicals, Uppsala, Sweden) were also run with the sample for the determination of subunit molecular weight. Gels were fixed and stained with Coomassie blue. Amino acid analysis. The purified HA was hydrolyzed in 6 M HCl at 110°C for 20 h, and amino acids were separated on an amino acid analyzer (Durrum) by using postcolumn derivatization with o-phthalaldehyde (1). Tryptophan was estimated spectrophotometrically (14). Electron microscopy. Carbon-coated, 300-mesh grids were used for specimen support. Samples were stained with 2% (wt/vol) uranyl acetate in glass distilled water. Grids were examined in an electron microscope (model 420T; Philips, Holland), using appropriate magnification. Analytical isoelectric focusing. Isoelectric focusing was carried out under nondenaturing conditions with 1% agarose as the support matrix. The pH gradient was formed by 2% Pharmalyte, pH 3 to 10 range (Pharmacia). The gel was fixed and stained with Coomassie blue. The pl of the purified HA was determined by comparing its mobility with those of a set of standard proteins of known pIs (Pharmacia Fine Chemicals). Immunochemical methods. Antisera to the HA were raised by injecting rabbits intramuscularly with 100 ,ug of purified HA emulsified with Freund complete adjuvant (Difco Laboratories). Three booster injections with HA alone were administered at 15-day intervals. Animals were bled 7 days after the last injection and antisera were collected. The reactivity of the anti-HA antisera to the purified HA as well as to the envelope components of homologous and heterologous strains of V. cholerae was examined by an immunoblotting technique (29). After separation by SDS-PAGE, the proteins were transferred to nitrocellulose (pore size, 0.45 ,um; Millipore, Bedford, Mass.) at 4°C at a constant current of 250 mA for 16 h. Blots were incubated for 2 h at 25°C with the anti-HA antiserum at a dilution of 1:100 and then incubated with peroxidase-labeled anti-rabbit immunoglobulin G (Sigma Chemical Co., St. Louis, Mo.) diluted 1:1,000. Color was developed by treatment with a solution of 3,3'diaminobenzidine hydrochloride (1 mg/ml; Sigma) in buffer A containing H202 (30%) at a concentration of 0.3 Ixl/ml. RESULTS Purification of HA. The HA activity released in the culture supernatant of V2 reached a maximum after growth of the bacteria for 16 to 20 h in BHI broth at 37°C with shaking. The activity could be quantitatively precipitated by 50% (NH4)2SO4 saturation of the culture supernatant. Following gel filtration of fraction A on Sephadex G-100, 90% of the HA activity eluted in the void volume (Table 1). Most of the HA activity in fraction B remained adsorbed to the matrix during hydrophobic interaction chromatography on phenylSepharose CL-4B and could be eluted when the concentration of ethylene glycol in the gradient mixture exceeded 80%
3700
BANERJEE ET AL.
INFECT. IMMUN.
TABLE 1. Purification of the HA
Sp act
Prepn
Total protein
% LPS
by wt
(HAU/mg of protein)
Fraction A [50% (NH4)2SO4Ia Fraction B from Sephadex G-100 chromatography Fraction C from phenyl-Sepharose CL-4B chromatography Fraction D (purified HA) from Sepharose CL-4B chromatography in the presence of urea and deoxycholate
1,330 140 18 10
NDb
1.2 1.0 5.5 1.0
(mg)
75 80 75% (by weight) of fraction C (Table 1), could, however, be removed by gel filtration on Sepharose CL-4B in the presence of 6M urea-1% sodium deoxycholate (Fig. 2). The HA activity was associated with the higher-molecular-weight fraction. The purification procedure resulted in approximately 80-fold enhancement of specific activity, with the final preparation agglutinating rabbit erythrocytes at a concentration as low as 10 ng/ml (Table 1). Recovery of HA activity was approximately 60-fold (Table 1). The HA failed to show any protease activity towards azoalbumin or azocasein. Biochemical characterization of HA. The polypeptide composition of the purified HA was analyzed by SDS-PAGE. The HA was composed of a single polypeptide chain of Mr 62,000 (Fig. 3). The mobility remained unaffected by the omission of treatment with 2-mercaptoethanol (data not shown), indicating the absence of any interchain disulfide linkage. Notably, the crude HA preparation apparently shared several polypeptide components with the outer membrane protein (OMP) preparation. During gel filtration on Sepharose CL-4B under nondenaturing conditions, the purified HA was largely excluded from the matrix (data not shown), suggesting that the HA existed as high-molecular-weight aggregates. However, the HA could not be sedimented by ultracentrifugation at 100,000 x g for 1 h. In contrast, >75% of the HA activity of fraction C could be pelleted under the same conditions. During isoelectric focusing in agarose gel (Fig. 4), the purified HA focused as a sharp band, corresponding to a pl of 6.0.
The amino acid composition of the purified HA revealed a relatively high percentage (24%) of aspartic and glutamic acid residues (Table 2). The nonpolar amino acids constituted about 46% of the total amino acid residues, a value close to the average (6). Electron microscopy. An electron micrograph of the purified HA showed it to be a homogeneous population of disk-shaped particles of 10-nm diameter (Fig. SA). While the morphology was typical of globular proteins, the tendency to form aggregates of varying stoichiometry is clearly discernible. Electron micrographs of fraction C revealed that the material existed as vesicles of varying sizes and shapes (Fig. SB). Effect of protease digestion and heat treatment. The HA activity was sensitive to digestion (2 h at 37°C) with proteases such as trypsin, chymotrypsin, and thermolysin, indicating the involvement of protein in HA activity. The purified HA remained stable at room temperature for at least 24 h and for months during storage at 4°C. The thermal stabilities of the purified HA and fraction C were compared by incubating them at different temperatures for 10 min. While the purified HA became inactivated above 50°C, fraction C was affected only above 70°C, suggesting that association of LPS with protein considerably enhanced its thermal stability. Erythrocyte specificity and sugar inhibition of purified HA. The purified HA was found to be active on a relatively broad spectrum of erythrocytes, including those of rabbits, guinea pigs, mice, sheep, chickens, and humans. Rabbit erythrocytes were the most sensitive, being agglutinated at a con-
80
2-0
1-2 00
.1000
z -J
so50XFI. LU
0.
FIG. 1. Hydrophobic interaction chromatography of fraction B on phenyl-Sepharose CL-4B (See text). Fractions pooled for HA activity are marked by the bar.
VOL. 58, 1990
a 49
0
40
20
~. 60
NOVEL HEMAGGLUTININ FROM V. CHOLERAE a
3AML
as
low
as
pi
6.55
5.85 mIlow
so
VOLUME() FIG. 2. Gel filtration of fraction C on Sephacryl S-300 in the presence of 6 M urea and 1% sodium deoxycholate (see text). Void volume (VO) was determined by blue dextran (Pharmacia). Fractions pooled for HA activity are marked by the bar.
centration
b
3701
10 ng/ml. Human and chicken erythro-
cytes were the least sensitive, requiring 1 ,ug of the purified
HA per ml for visible clumping. Erythrocytes of other animals tested were of intermediate sensitivity. Simple sugars and sialic acid failed to inhibit agglutination of rabbit erythrocytes by the purified HA (Table 3). However, the HA activity was strongly inhibited by fetuin and asialofetuin and weakly by mucin, thereby indicating the requirement of complex carbohydrate structure for binding. The cell-associated HA activity of V2 cells was, however, not inhibited by these glycoproteins, suggesting a difference in receptor specificity. Inhibition of cell-free and cell-associated HA activities of homologous and heterologous strains of V. cholerae. The anti-HA antiserum inhibited agglutination of rabbit erythro-
KD 94 6'7 43
30
20
14
b
c
d
FIG. 3. SDS-PAGE profiles of OMP fraction B and the purified HA of V2: OMP preparation, 30 ,g (lane b); fraction B, 30 p.g (lane c); and purified HA 20 ,g (lane d). Protein standards of known molecular weights are shown in lane a. KD, Kilodaltons.
FIG. 4. Isoelectric focusing of the purified HA in an agarose gel. Purified HA, 5 ,ug (lane a). Protein standards of known pl are shown in lane b.
cytes by the purified HA at a dilution of 1:1,024 (Table 4). The antiserum to cholera toxin failed to inhibit the HA at a dilution as low as 1:10, indicating the specificity of the inhibition reaction. The anti-HA antiserum also neutralized the activities of crude HA preparations isolated from 18-h culture supernatants of several other 01 and non-O1 V. cholerae strains (Table 4), implying antigenic relatedness of HAs excreted by different V. cholerae strains. Notably, the antiserum failed to inhibit, even partially, the cell-associated HA activities of the parent strain (V2) or of other strains examined, suggesting that the cell-free and cell-associated HA activities were mediated by immunochemically distinct TABLE 2. Amino acid composition of V. cholerae HA
mol% in:
Amino acid
HA
Avg protein (6)
Aspartic acid Glutamic acid Serine Glycine Histidine Arginine Threonine Alanine Proline Tyrosine Valine Methionine Cystine Isoleucine Leucine Phenylalanine Lysine Tryptophan
13.6 10.3 8.1 9.0 1.6 5.5 7.0 9.5 3.5 3.2 5.9 0.8 0.5 4.2 8.3 4.3 4.6 1.1
4.3 3.9 7.0 8.4 2.0 4.9 6.1 8.6 5.2 3.4 6.6 1.7
4.5 7.4 3.6 6.6
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BANERJEE ET AL.
INFECT. IMMUN.
FIG. 5. Electron micrographs of (A) purified HA (bar, 100 nm) and (B) fraction C (bar, 200 nm).
components. The antiserum also failed to agglutinate parent or other V. cholerae cells. Cellular localization of the HA. No significant HA activity could be detected in the supernatant of the cell lysate of V2 cells, suggesting that the HA was unlikely to be a component of the cytosol.
The envelope preparations of V2 and two V. cholerae 01 strains, A17 and AD29, were next examined for the presence of HA. Figure 6 shows that, while the protein profiles of the envelope preparations of the three strains were strikingly similar, they also contained a minor polypeptide component with electrophoretic mobility corresponding to that of the
'S,-.';,m
v . ':, . . ,
,a
't_
3703
NOVEL HEMAGGLUTININ FROM V. CHOLERAE
VOL. 58, 1990
TABLE 4. Inhibition of HA-mediated agglutination of rabbit erythrocytes by anti-HA antiserum Strain
Specific HA activity of 50%0 (NH4)2SO4 fraction of culture supernatant (HAU/ mg of protein)a
Dilution of anti-HA antiserum causing
160 20 18 24
1:1,024 1:512 1:512 1:512
inhibitionb
.'
Non-O1 V2 66N S18 10259
01; serotype Ogawa, biotype El Tor 8 10 10
A 17 739 AD 29
1:512 1:512 1:512
a Bacteria were grown in BHI broth at 37°C for 18 h. b Crude cell-free HA solutions containing protein minimum hemagglutinating doses were used.
corresponding
to 8
B:.'....' 'F2 FIG. 5-Continued
HA. This component was also recognized by the anti-HA antiserum during immunoblotting (Fig. 7), suggesting that the HA was of envelope origin. DISCUSSION
Clarification of the relevance of V. cholerae HAs in the pathogenesis of choler presupposes the availability of a purified and biochemically defined preparation of HA. The present communication describes a convenient method for the purification of an HA, hitherto unrecognized, from the culture supernatant of a V. cholerae non-O1 strain. The purified HA was essentially free of biochemically detectable amounts of impurities, including LPS. As shown by SDSPAGE (Fig. 3) and the chemical composition (Table 1) of fraction B, the HA is released, along with many OMP components and LPS, presumably in the form of vesicles from outer membrane blebs during bacterial degeneration (16). Most of the contaminating proteins, but not LPS, could be separated by hydrophobic interaction chromatography on phenyl-Sepharose CL-4B (Fig. 1). Subsequent removal of LPS by gel filtration in the presence of urea and deoxycho-
late (Fig. 2) yielded a product essentially composed of a single polypeptide chain of Mr 62,000 (Fig. 3), with very high specific hemagglutinating activity. The inhibition of the HA activity by fetuin, asialofetuin, and mucin, but not by simple sugars (Table 3), indicated that the HA represented a true lectin. The HA, unlike cholera toxin (7), has no affinity for gangliosides as indicated by its inability to agglutinate phospholipid vesicles in which a crude mixture of gangliosides was incorporated (Banejee et al., unpublished observation). However, the sugar-binding specificity of the HA remains to be defined in detail.
b
a
d
c
e
KD
p~-
.~
-
-#
::. .....m. .....
94 67 43
-lsO"
30
TABLE 3. Inhibition of HA-mediated agglutination of rabbit erythrocytes by glycoconjugates Sugars and
Sugaycoprstea
glycoproteins
Minimu Minimum
inhibitory (,ug/ml)concn
Not inhibitableb 62.5 Fetuin ....................................... 62.5 Asialofetuin ....................................... 500 Mucin ........................................ ...................... >1,000 Ovalbumin ................. >100 Bovine serum albumin .....................................
Simple sugarsa .......................................
a Sugars used:
D-glucose, methyl a-D-glucopyranoside, methyl P-D-glyco-
pyranoside, D-galactose, L-fucose, methyl a-D-galactoside, methyl J3-D-galactoside, D-mannose, N-acetyl-D-glucosamine, N-acetyl-D-mannosamine, maltose, malibiose, rhamnose, raffinose, and N-acetylneuraminic acid. b Not inhibited up to 0.2 M concentration of sugars.
..
W
;.
-
20
.;~
_goo
14
FIG. 6. SDS-PAGE profiles of fraction B and envelope preparations of homologous and heterologous strains V. cholerae: Envelope preparations of V. cholerae serovar 01 strain A17, Ogawa, El Tor (lane a); serovar 01 strain AD 29, Inaba, El Tor (lane b); non-O1 V. cholerae V2 (lane c). Fraction B (lane d) and standard proteins (lane e) of known molecular weight are shown. KD, Kilodaltons.
3704
BANERJEE ET AL.
a
b
INFECT. IMMUN.
c
d
e
KD 94
67 43
>k
30
20
14
FIG. 7. Immunoblot analysis of anti-HA anti- serum against fraction B and envelope preparations of homologous an(s heterologous strains
of V. cholerae. Envelope preparations of V. chol erae serovar 01 strain A17, Ogawa, El Tor (lane a); serovar 01 strain AD 29, Inaba, El Tor (lane b); non-01 strain V2 (lane c); fraction IB (lane d). Standard proteins of known molecular weight were transferred to nitrocellulose paper and stained by amido black (lane e). KD, Kilodaltons.
Electron micrographs of the purified HA revealed a morphology typical of globular proteins (Fig. 5), with subunits apparently being arranged in a ringlike configuration. The subunit composition as well as the morphology of this HA are in sharp contrast to those of the fimbrial HAs, which are usually composed of subunits in the Mr range of 15,000 to 25,000 and characterized by a filamentous structure (22). It may be mentioned, in this connection, that although the presence of nonfimbrial HAs of surface origin was described as early as 1955 by Duguid et al. (9) in Escherichia coli strains, recent studies (26) have failed to confirm their nonfimbrial nature. This HA, thus, provides an interesting example of a true nonfimbrial lectin. A notable feature of this HA is its strong tendency to undergo self-aggregation as indicated by its virtual exclusion from the Sepharose CL-4B matrix during gel filtration as well as by the presence of aggregated particles in electron micrographs (Fig. 5). The aggregation was not reversed by the addition of common protein-dissociating agents such as urea, guanidine hydrochloride, Triton X-100, and deoxycholate (Baneree et al., unpublished observation). However, since the HA was not sedimented at 100,000 x g for 1 h, it did not appear to be particulate in nature. The HA also formed a strong, noncovalent association with LPS as revealed by the vesiclelike morphology as well as the increased effective molecular mass of fraction C compared with the purified HA. The LPS also increased considerably the resistance of the HA to heat inactivation, suggesting stabilization of the biologically active conformation. These features probably reflected a strong hydrophobic nature of the HA molecule, which was also suggested by the relatively high content of nonpolar amino acids (Table 2), and might help in the anchorage of the HA in the lipid-rich milieu of the cell envelope. Complete inhibition of the crude HAs released by the parent as well as several 01 and non-O1 strains of V.
cholerae by anti-HA antiserum (Table 4) suggested that it might represent the only HA released during growth in BHI broth. However, it did not contribute to the cell-associated HA activities of these strains, an observation not unexpected in view of the expression of multiple HAs by V. cholerae (15). This HA is different from the soluble HA with protease activity described earlier (12, 17) in subunit composition, morphology, and biochemical properties. Since only limited information is available about other cholera HAs (5, 28), it is difficult to conclude whether our preparation is related to any of them. Immunochemical detection of the HA as a component of the envelopes of several V. cholerae 01 and non-O1 strains (Fig. 7) confirmed the somatic origin of the HA. Since the HA was not sedimented at 100,000 x g, it could not be expected to be present in the OMP (Fig. 3) prepared from envelope on the basis of their insolubility in sarcosyl solution (11). Several V. cholerae OMPs are, indeed, solubilized by detergents milder than SDS (23). However, since the anti-HA antiserum failed to agglutinate parent cells, the HA was apparently not sufficiently exposed on the bacterial surface during growth in vitro. Further studies are needed to locate the HA in the cell envelope. V. cholerae is also known to express novel surface proteins during in vivo growth (19, 27). It remains to be determined whether this HA is expressed in sufficient quantity or is present in a proper orientation on the surface of in vivo grown cells to enable them to adhere to host cells during intestinal infection. ACKNOWLEDGMENT We are indebted to Ranjit Roy, University of Alabama, Birmingham, for amino acid analysis. LITERATURE CITED 1. Anantharamaiah, G. M., T. A. Hughes, M. Iqbal, A. Gawish, P. J. Neame, and J. P. Segrest. 1983. Effect of oxidation on the properties of apolipoproteins A-I and A-II. J. Lipid Res. 29:309318. 2. Banerjee, K. K., and A. Sen. 1981. Purification and properties of a lectin from the seeds of Croton tiglium with hemolytic activity toward rabbit red cells. Arch. Biochem. Biophys. 212:740-753. 3. Bennett, V., and P. Cuatresasas. 1977. Cholera toxin. Membrane gangliosides and activation of adenylate cyclase, p. 1-66. In P. Cuatresasas (ed.), The specificity and action of animal, bacterial and plant toxins. Chapman and Hall, London. 4. Booth, B. A., C. V. Sciortino, and R. A. Finkelstein. 1986. Adhesins of Vibrio cholerae, p. 169-182. In D. Mirelman (ed.), Bacterial lectins and agglutinins. John Wiley & Sons, New York. 5. Chaicumpa, W., U. Peungjesda, B. Martinez, and N. Atthasishtha. 1982. Soluble hemagglutinin of classical vibrios: isolation and protection against cholera by its antibodies. Southeast Asian J. Trop. Med. Public Health 13:637-645. 6. Dayhoff, M. 0. 1978. Atlas of protein sequence and structure, vol. 5, Suppl., p. 363. National Biomedical, Silver Spring, Md. 7. Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28:350-356. 8. Duguid, J. P., and D. Old. 1980. Adhesive properties of Enterobacteriaceae, p. 185-217. In E. Beachey (ed.), Bacterial adherence. Receptor and recognition, Ser. B, vol. 6. Chapman and Hall, London. 9. Duguid, J. P., I. W. Smith, G. Dempster, and P. N. Edmunds. 1955. Nonflagellar filamentous appendages (fimbriae) and hemagglutinating activity in Bacterium coli. J. Pathol. Bacteriol. 70:335-348.
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10. Dutta-Roy, K., K. Banerjee, S. P. De, and A. C. Ghosh. 1986. Comparative study of expression of hemagglutinins, hemolysins, and enterotoxins by clinical and environmental isolates of non-O1 Vibrio cholerae in relation to their enteropathogenicity. Appl. Environ. Microbiol. 52:875-879. 11. Filip, C., G. Fletcher, J. L. Wulf, and C. F. Earhart. 1973. Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium lauroyl sarcosinate. J. Bacteriol.
NOVEL HEMAGGLUTININ FROM V. CHOLERAE
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115:717-722.
12. Finkelstein, R. A., and L. F. Hanne. 1982. Purification and characterization of the soluble hemagglutinin (cholera lectin) produced by Vibrio cholerae. Infect. Immun. 36:1199-1208. 13. Ghosh, A. K., R. Ganguli, and D. L. Srivastava. 1965. Studies in immunochemistry of Vibrio cholerae. VI. Hemagglutination. Ind. J. Med. Res. 50:1-7. 14. Goodwin, T. W., and R. A. Morton. 1946. The spectrophotometric determination of tyrosine and tryptophan in proteins. Biochem. J. 40:628-632. 15. Hanne, L. F., and R. A. Finkelstein. 1982. Characterization and distribution of the hemagglutinins produced by Vibrio cholerae. Infect. Immun. 36:209-214. 16. Hoekstra, D., J. W. van der Laar, L. de Leij, and B. Wilholt. 1976. Release of outer membrane fragments from normally growing Escherichia coli. Biochim. Biophys. Acta 455:889-899. 17. Honda, T., K. Lertpocasombat, A. Hata, T. Miwatani, and R. A. Finkelstein. 1989. Purification and characterization of a protease produced by Vibrio cholerae non-O1 and comparison with a protease of Vibrio cholerae 01. Infect. Immun. 57:2799-2803. 18. Jones, G. W., and R. Freter. 1976. Adhesive properties of Vibrio cholerae: nature of interaction with isolated rabbit brush border membrane and human erythrocytes. Infect. Immun. 39:10481058.
19. Jonson, G., A. M. Svennerholm, and J. Holmgren. 1989. Vibrio cholerae expresses cell surface antigens during intestinal infection which are not expressed during in vitro culture. Infect. Immun. 57:1809-1815. 20. Laemmli, U. K. 1970. Cleavage of structural proteins during the
24.
25.
26. 27. 28.
29.
30. 31.
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assembly of the head of bacteriophage T4. Nature (London) 227:680-685. Lankford, C. E. 1960. Factors of virulence of Vibrio cholerae. Ann. N.Y. Acad. Sci. 88:1203-1212. Lis, H., and N. Sharon. 1986. Lectins as molecules and as tools. Annu. Rev. Biochem. 55:35-67. Manning, P. A., F. Imbesi, and D. R. Haynes. 1982. Cell envelope proteins in Vibrio cholerae. FEMS Microbiol. Lett. 14:159-166. Markwell, M. A. K., S. M. Haas, L. L. Beilur, and N. E. Tolbert. 1978. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem. 87:206-210. Mirelman, D., and I. Ofek. 1986. Introduction to microbial lectins and agglutinins, p. 1-19. In D. Mirelman (ed.), Microbial lectins and agglutinins: properties and biological activity. John Wiley & Sons, New York. Orskov, I., A. Birch-Anderson, J. P. Duguid, J. Stenderup, and F. Orskov. 1985. An adhesive protein capsule of Escherichia coli. Infect. Immun. 47:191-200. Sciortino, C. V., and R. A. Finkelstein. 1983. Vibrio cholerae expresses iron-regulated outer membrane proteins in vivo. Infect. Immun. 42:990-996. Svennerholm, A. M., G. J. Stromberg, and J. Holmgren. 1983. Purification of Vibrio cholerae soluble hemagglutinin and development of enzyme-linked immunosorbent assays for antigen and antibody quantitations. Infect. Immun. 41:237-243. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354. Westphal, O., and K. Jann. 1965. Bacterial lipopolysaccharides. Extraction with phenol-water and further applications of the procedure. Methods Carbohydr. Chemi. 5:83-91. Wright, B. E., and R. A. Rebers. 1972. Procedure for determining heptose and hexose in lipopolysaccharides. Modifications of the cysteine-sulfuric acid method. Anal. Biochem. 49:307-319.
AND IMMUNITY,
Nov. 1990,
p.
Vol. 58, No. 11
3698-3705
0019-9567/90/113698-08$02.00/0 Copyright C) 1990, American Society for Microbiology
Purification and Characterization of a Novel Hemagglutinin from Vibrio cholerae K. K.
BANERJEE,1* A. N. GHOSH,2
K. DUTTA-ROY,' S. C. PAL,' AND A. C. GHOSE't
Division of Immunology' and Division of Electron Microscopy,2 National Institute of Cholera and Enteric Diseases, Calcutta-700 010, India Received 3 April 1990/Accepted 8 August 1990
A lectin with strong hemagglutinating activity toward erythrocytes of several animal species was isolated from an 18-h culture supernatant of a diarrheagenic strain, V2, of non-O1 Vibrio cholerae. The hemagglutinin (HA) was purified free of lipopolysaccharide by salt fractionation followed by gel filtration, hydrophobic interaction chromatography, and, finally, gel filtration in the presence of urea and deoxycholate. The purification procedure resulted in an HA preparation with 80-fold enhancement of specific activity. The HA consisted of noncovalently bound subunits of Mr 62,000 and behaved essentially as a single component with pI 6.0. Nonpolar and acidic amino acids contributed 46 and 24%, respectively, to the total amino acid residues. Electron micrographs of the HA showed it to consist of large, nonstoichiometric aggregates of disklike molecules of 10-nm diameter. Inhibition of the HA by the glycoproteins fetuin, asialofetuin, and mucin, but not by ovalbumin and simple sugars, suggested the specific requirement of complex carbohydrates for binding. Rabbit antisera to the purified HA inhibited the hemagglutinating activities of the crude cell-free HA preparations, but not cell-associated HA activities of the parent (V2) or of other 01 and non-Ol V. cholerae strains. This suggested that the released and cell-associated HA activities were mediated by antigenically distinct components. Immunoblotting experiments showed that the antisera recognized a polypeptide component of Mr 62,000 in the cell envelope preparations of the parent and several other V. cholerae 01 and non-Ol strains. These data suggested that the HA was a nonfimbrial lectin of somatic origin with no protease activity and was apparently distinct from V. cholerae HAs described so far.
Bacterial lectins are known to mediate attachment of microorganisms to host tissue by specific, noncovalent binding to carbohydrate receptors and are, thus, designed to play a potentially significant role in microbial pathogenicity (8, 25). However, in contrast to the extensive study of the structure and function of plant lectins (22), the bacterial lectins have been subjected to systematic study only recently. Hemagglutinating or lectinlike activities are associated with fimbrial as well as nonfimbrial surface structures of gram-negative bacteria and also with apparently soluble substances released in the culture medium (25). While a number of fimbrial hemagglutinins (HA) have been purified to homogeneity, characterized, and recognized as critical virulence determinants (22, 25), the nonfimbrial HAs have been much less explored. Vibrio cholerae, a gram-negative bacterium associated with cholera, expresses more than one cell-associated HA with distinct carbohydrate-binding specificities during in vitro growth (15, 21). The organism is also known to elaborate apparently soluble or cell-free HA in the culture medium (13). Recently, several groups of investigators (5, 12, 28) have attempted to isolate and characterize cholera HAs. However, no clear picture has emerged, presumably due to the multiplicity of HAs and to the lack of availability of purified materials sufficiently free of lipopolysaccharide (LPS). In particular, Finkelstein and Hanne (12) reported the purification and characterization of a soluble HA with protease activity from the culture supernatant of V. cholerae. This HA was initially described as a lectin (12), but Booth et
*
al. (4) later concluded that its hemagglutination activity could be due to protease modification of erythrocyte surface rather than carbohydrate receptor-mediated interaction. Despite the possible involvement of cholera HAs in the attachment of the organisms to the intestinal epithelium (4, 18), the molecular identity of the true cholera lectin remained elusive. The present communication describes the purification and properties of a novel HA from the culture supernatant of a diarrheagenic strain of V. cholerae non-O1. The HA was of envelope origin and shared by several 01 and non-O1 V. cholerae strains.
MATERIALS AND METHODS Bacterial strains and media. Non-O1 V. cholerae strain V2 used in this study was a clinical isolate kindly made available to us by R. Sakazaki, Tokyo, Japan. This strain evoked cholera toxin-mediated fluid accumulation in the rabbit ligated ileal loop assay, as described previously (10). Brain heart infusion (BHI) broth and nutrient agar were obtained as dehydrated media from Difco Laboratories, Detroit, Mich. HA assay. Twofold serial dilutions of 100-,ul samples of HA preparations in 10 mM sodium phosphate buffer, pH 7.2, containing 150 mM NaCl, (phosphate-buffered saline), were mixed with a 100-pul suspension of erythrocytes of rabbit or other animal species in the same buffer in glass tubes (10 by 1 cm). The mixture was incubated for 1 h at 25°C and agglutination was monitored visually. The HA titer was defined as the reciprocal of the highest dilution of the sample causing visible agglutination of erythrocytes. The specific activity was expressed as the number of minimum HA doses per milligram of the sample. The cell-associated HA activity of the whole organism was determined by the same procedure, using a bacterial suspension adjusted to a turbidity
Corresponding author.
t Present address: Department of Microbiology, Bose Institute, Calcutta-700 009, India. 3698
HEMAGGLUTININ FROM V. CHOLERAE VNOVEL VOL. 58, 1990
corresponding to 200 Klett units against a green filter (KlettSummerson photoelectric colorimeter). Inhibition of hemagglutination by simple sugars and glycoconjugates was performed as described elsewhere (2). Protease assay. The protease activity of the purified HA preparation was examined by using azoalbumin or azocasein as substrate (12). Protein estimation. Protein was estimated by a modified Folin-Ciocalteu method (24), with bovine serum albumin as the standard. Preparation and estimation of LPS. The LPS was extracted, by the phenol-water extraction method (30), from acetone-dried cells of V. cholerae V2 grown overnight in BHI broth at 37°C with shaking. Contaminating protein was removed by digestion with pronase and subsequent dialysis, and the LPS was further purified by ultracentrifugation at 100,000 x g for 1 h. Total carbohydrate content of the LPS was estimated by the phenol-sulfuric acid method (7). Heptose, the LPS-specific sugar, was independently determined by the cysteine-sulfuric acid method (31). Quantitative determinations of LPS in different HA preparations were carried out by estimation of total carbohydrate as well as heptose and comparison of these values with those determined from a weighed sample of lyophilized LPS. Purification of HA. All chromatographic procedures were performed at 25°C. Protein concentrations of the column eluents were monitored by A280. (i) Step 1. Bacteria were grown in BHI broth with shaking at 37°C for 18 h. Cells were removed by centrifugation at 12,000 x g for 10 min. The supernatant was made 50% saturated with solid ammonium sulfate. The precipitate, collected by centrifugation, was dissolved in and dialyzed against 50 mM Tris hydrochloride buffer, pH 7.4, containing 50 mM NaCl and 1 mM EDTA. Any precipitate formed during dialysis was discarded, and the supernatant (fraction A) was retained. (ii) Step 2. A 20-ml amount (360 mg of protein) of fraction A was chromatographed on a Sephadex G-200 column (65 by 2.6 cm). Fractions with HA activity were pooled (fraction B) and concentrated. (iii) Step 3. A 20-ml amount (120 mg of protein) of fraction B was subjected to hydrophobic interaction chromatography on a column of phenyl-Sepharose CL-4B (36 by 1.6 cm) previously equilibrated with 10 mM Tris hydrochloride buffer, pH 7.4 (buffer A). The column was washed with 100 ml of buffer A, and the bound protein was eluted by a linear gradient of ethylene glycol from 0 to 100% in buffer A. Active fractions were pooled (fraction C), dialyzed against water, and concentrated. (iv) Step 4. A 5-ml amount (15 mg of protein) of fraction C was subjected to gel filtration on a column (38 by 1.6 cm) of Sepharose CL-4B previously equilibrated with buffer A containing 6 M urea and 1% sodium deoxycholate. Urea and deoxycholate were removed by ultrafiltration through a PM-10 membrane (Amicon). The HA activity was monitored in detergent-free fractions. Active fractions were pooled (fraction D). Preparation of envelope and outer membrane. Crude envelope preparations were obtained from bacteria grown in BHI broth at 37°C for 18 h. Cells were washed with phosphatebuffered saline and disrupted in an ultrasonic disintegrator (MSE). Unbroken cells were removed by centrifugation at 2,000 x g for 10 min, and the supernatant was centrifuged at 100,000 x g for 1 h to collect the envelope fraction. The outer membrane components were obtained from the crude
3699
envelope preparation by using sodium lauroyl sarcosinate (11). SDS-PAGE. Protein samples were analyzed for molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by the method of Laemmli (20). Samples were run on a 12.5% polyacrylamide gel (2 mm thick) with a 3% stacking gel at a constant voltage of 100 V for 4 h. Proteins were denatured by incubating samples with an equal volume of 10 mM Tris hydrochloride buffer, pH 7.0, containing 2% SDS and 5% 2-mercaptoethanol for 5 min in a boiling-water bath. Protein standards of known molecular weights (Pharmacia Fine Chemicals, Uppsala, Sweden) were also run with the sample for the determination of subunit molecular weight. Gels were fixed and stained with Coomassie blue. Amino acid analysis. The purified HA was hydrolyzed in 6 M HCl at 110°C for 20 h, and amino acids were separated on an amino acid analyzer (Durrum) by using postcolumn derivatization with o-phthalaldehyde (1). Tryptophan was estimated spectrophotometrically (14). Electron microscopy. Carbon-coated, 300-mesh grids were used for specimen support. Samples were stained with 2% (wt/vol) uranyl acetate in glass distilled water. Grids were examined in an electron microscope (model 420T; Philips, Holland), using appropriate magnification. Analytical isoelectric focusing. Isoelectric focusing was carried out under nondenaturing conditions with 1% agarose as the support matrix. The pH gradient was formed by 2% Pharmalyte, pH 3 to 10 range (Pharmacia). The gel was fixed and stained with Coomassie blue. The pl of the purified HA was determined by comparing its mobility with those of a set of standard proteins of known pIs (Pharmacia Fine Chemicals). Immunochemical methods. Antisera to the HA were raised by injecting rabbits intramuscularly with 100 ,ug of purified HA emulsified with Freund complete adjuvant (Difco Laboratories). Three booster injections with HA alone were administered at 15-day intervals. Animals were bled 7 days after the last injection and antisera were collected. The reactivity of the anti-HA antisera to the purified HA as well as to the envelope components of homologous and heterologous strains of V. cholerae was examined by an immunoblotting technique (29). After separation by SDS-PAGE, the proteins were transferred to nitrocellulose (pore size, 0.45 ,um; Millipore, Bedford, Mass.) at 4°C at a constant current of 250 mA for 16 h. Blots were incubated for 2 h at 25°C with the anti-HA antiserum at a dilution of 1:100 and then incubated with peroxidase-labeled anti-rabbit immunoglobulin G (Sigma Chemical Co., St. Louis, Mo.) diluted 1:1,000. Color was developed by treatment with a solution of 3,3'diaminobenzidine hydrochloride (1 mg/ml; Sigma) in buffer A containing H202 (30%) at a concentration of 0.3 Ixl/ml. RESULTS Purification of HA. The HA activity released in the culture supernatant of V2 reached a maximum after growth of the bacteria for 16 to 20 h in BHI broth at 37°C with shaking. The activity could be quantitatively precipitated by 50% (NH4)2SO4 saturation of the culture supernatant. Following gel filtration of fraction A on Sephadex G-100, 90% of the HA activity eluted in the void volume (Table 1). Most of the HA activity in fraction B remained adsorbed to the matrix during hydrophobic interaction chromatography on phenylSepharose CL-4B and could be eluted when the concentration of ethylene glycol in the gradient mixture exceeded 80%
3700
BANERJEE ET AL.
INFECT. IMMUN.
TABLE 1. Purification of the HA
Sp act
Prepn
Total protein
% LPS
by wt
(HAU/mg of protein)
Fraction A [50% (NH4)2SO4Ia Fraction B from Sephadex G-100 chromatography Fraction C from phenyl-Sepharose CL-4B chromatography Fraction D (purified HA) from Sepharose CL-4B chromatography in the presence of urea and deoxycholate
1,330 140 18 10
NDb
1.2 1.0 5.5 1.0
(mg)
75 80 75% (by weight) of fraction C (Table 1), could, however, be removed by gel filtration on Sepharose CL-4B in the presence of 6M urea-1% sodium deoxycholate (Fig. 2). The HA activity was associated with the higher-molecular-weight fraction. The purification procedure resulted in approximately 80-fold enhancement of specific activity, with the final preparation agglutinating rabbit erythrocytes at a concentration as low as 10 ng/ml (Table 1). Recovery of HA activity was approximately 60-fold (Table 1). The HA failed to show any protease activity towards azoalbumin or azocasein. Biochemical characterization of HA. The polypeptide composition of the purified HA was analyzed by SDS-PAGE. The HA was composed of a single polypeptide chain of Mr 62,000 (Fig. 3). The mobility remained unaffected by the omission of treatment with 2-mercaptoethanol (data not shown), indicating the absence of any interchain disulfide linkage. Notably, the crude HA preparation apparently shared several polypeptide components with the outer membrane protein (OMP) preparation. During gel filtration on Sepharose CL-4B under nondenaturing conditions, the purified HA was largely excluded from the matrix (data not shown), suggesting that the HA existed as high-molecular-weight aggregates. However, the HA could not be sedimented by ultracentrifugation at 100,000 x g for 1 h. In contrast, >75% of the HA activity of fraction C could be pelleted under the same conditions. During isoelectric focusing in agarose gel (Fig. 4), the purified HA focused as a sharp band, corresponding to a pl of 6.0.
The amino acid composition of the purified HA revealed a relatively high percentage (24%) of aspartic and glutamic acid residues (Table 2). The nonpolar amino acids constituted about 46% of the total amino acid residues, a value close to the average (6). Electron microscopy. An electron micrograph of the purified HA showed it to be a homogeneous population of disk-shaped particles of 10-nm diameter (Fig. SA). While the morphology was typical of globular proteins, the tendency to form aggregates of varying stoichiometry is clearly discernible. Electron micrographs of fraction C revealed that the material existed as vesicles of varying sizes and shapes (Fig. SB). Effect of protease digestion and heat treatment. The HA activity was sensitive to digestion (2 h at 37°C) with proteases such as trypsin, chymotrypsin, and thermolysin, indicating the involvement of protein in HA activity. The purified HA remained stable at room temperature for at least 24 h and for months during storage at 4°C. The thermal stabilities of the purified HA and fraction C were compared by incubating them at different temperatures for 10 min. While the purified HA became inactivated above 50°C, fraction C was affected only above 70°C, suggesting that association of LPS with protein considerably enhanced its thermal stability. Erythrocyte specificity and sugar inhibition of purified HA. The purified HA was found to be active on a relatively broad spectrum of erythrocytes, including those of rabbits, guinea pigs, mice, sheep, chickens, and humans. Rabbit erythrocytes were the most sensitive, being agglutinated at a con-
80
2-0
1-2 00
.1000
z -J
so50XFI. LU
0.
FIG. 1. Hydrophobic interaction chromatography of fraction B on phenyl-Sepharose CL-4B (See text). Fractions pooled for HA activity are marked by the bar.
VOL. 58, 1990
a 49
0
40
20
~. 60
NOVEL HEMAGGLUTININ FROM V. CHOLERAE a
3AML
as
low
as
pi
6.55
5.85 mIlow
so
VOLUME() FIG. 2. Gel filtration of fraction C on Sephacryl S-300 in the presence of 6 M urea and 1% sodium deoxycholate (see text). Void volume (VO) was determined by blue dextran (Pharmacia). Fractions pooled for HA activity are marked by the bar.
centration
b
3701
10 ng/ml. Human and chicken erythro-
cytes were the least sensitive, requiring 1 ,ug of the purified
HA per ml for visible clumping. Erythrocytes of other animals tested were of intermediate sensitivity. Simple sugars and sialic acid failed to inhibit agglutination of rabbit erythrocytes by the purified HA (Table 3). However, the HA activity was strongly inhibited by fetuin and asialofetuin and weakly by mucin, thereby indicating the requirement of complex carbohydrate structure for binding. The cell-associated HA activity of V2 cells was, however, not inhibited by these glycoproteins, suggesting a difference in receptor specificity. Inhibition of cell-free and cell-associated HA activities of homologous and heterologous strains of V. cholerae. The anti-HA antiserum inhibited agglutination of rabbit erythro-
KD 94 6'7 43
30
20
14
b
c
d
FIG. 3. SDS-PAGE profiles of OMP fraction B and the purified HA of V2: OMP preparation, 30 ,g (lane b); fraction B, 30 p.g (lane c); and purified HA 20 ,g (lane d). Protein standards of known molecular weights are shown in lane a. KD, Kilodaltons.
FIG. 4. Isoelectric focusing of the purified HA in an agarose gel. Purified HA, 5 ,ug (lane a). Protein standards of known pl are shown in lane b.
cytes by the purified HA at a dilution of 1:1,024 (Table 4). The antiserum to cholera toxin failed to inhibit the HA at a dilution as low as 1:10, indicating the specificity of the inhibition reaction. The anti-HA antiserum also neutralized the activities of crude HA preparations isolated from 18-h culture supernatants of several other 01 and non-O1 V. cholerae strains (Table 4), implying antigenic relatedness of HAs excreted by different V. cholerae strains. Notably, the antiserum failed to inhibit, even partially, the cell-associated HA activities of the parent strain (V2) or of other strains examined, suggesting that the cell-free and cell-associated HA activities were mediated by immunochemically distinct TABLE 2. Amino acid composition of V. cholerae HA
mol% in:
Amino acid
HA
Avg protein (6)
Aspartic acid Glutamic acid Serine Glycine Histidine Arginine Threonine Alanine Proline Tyrosine Valine Methionine Cystine Isoleucine Leucine Phenylalanine Lysine Tryptophan
13.6 10.3 8.1 9.0 1.6 5.5 7.0 9.5 3.5 3.2 5.9 0.8 0.5 4.2 8.3 4.3 4.6 1.1
4.3 3.9 7.0 8.4 2.0 4.9 6.1 8.6 5.2 3.4 6.6 1.7
4.5 7.4 3.6 6.6
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BANERJEE ET AL.
INFECT. IMMUN.
FIG. 5. Electron micrographs of (A) purified HA (bar, 100 nm) and (B) fraction C (bar, 200 nm).
components. The antiserum also failed to agglutinate parent or other V. cholerae cells. Cellular localization of the HA. No significant HA activity could be detected in the supernatant of the cell lysate of V2 cells, suggesting that the HA was unlikely to be a component of the cytosol.
The envelope preparations of V2 and two V. cholerae 01 strains, A17 and AD29, were next examined for the presence of HA. Figure 6 shows that, while the protein profiles of the envelope preparations of the three strains were strikingly similar, they also contained a minor polypeptide component with electrophoretic mobility corresponding to that of the
'S,-.';,m
v . ':, . . ,
,a
't_
3703
NOVEL HEMAGGLUTININ FROM V. CHOLERAE
VOL. 58, 1990
TABLE 4. Inhibition of HA-mediated agglutination of rabbit erythrocytes by anti-HA antiserum Strain
Specific HA activity of 50%0 (NH4)2SO4 fraction of culture supernatant (HAU/ mg of protein)a
Dilution of anti-HA antiserum causing
160 20 18 24
1:1,024 1:512 1:512 1:512
inhibitionb
.'
Non-O1 V2 66N S18 10259
01; serotype Ogawa, biotype El Tor 8 10 10
A 17 739 AD 29
1:512 1:512 1:512
a Bacteria were grown in BHI broth at 37°C for 18 h. b Crude cell-free HA solutions containing protein minimum hemagglutinating doses were used.
corresponding
to 8
B:.'....' 'F2 FIG. 5-Continued
HA. This component was also recognized by the anti-HA antiserum during immunoblotting (Fig. 7), suggesting that the HA was of envelope origin. DISCUSSION
Clarification of the relevance of V. cholerae HAs in the pathogenesis of choler presupposes the availability of a purified and biochemically defined preparation of HA. The present communication describes a convenient method for the purification of an HA, hitherto unrecognized, from the culture supernatant of a V. cholerae non-O1 strain. The purified HA was essentially free of biochemically detectable amounts of impurities, including LPS. As shown by SDSPAGE (Fig. 3) and the chemical composition (Table 1) of fraction B, the HA is released, along with many OMP components and LPS, presumably in the form of vesicles from outer membrane blebs during bacterial degeneration (16). Most of the contaminating proteins, but not LPS, could be separated by hydrophobic interaction chromatography on phenyl-Sepharose CL-4B (Fig. 1). Subsequent removal of LPS by gel filtration in the presence of urea and deoxycho-
late (Fig. 2) yielded a product essentially composed of a single polypeptide chain of Mr 62,000 (Fig. 3), with very high specific hemagglutinating activity. The inhibition of the HA activity by fetuin, asialofetuin, and mucin, but not by simple sugars (Table 3), indicated that the HA represented a true lectin. The HA, unlike cholera toxin (7), has no affinity for gangliosides as indicated by its inability to agglutinate phospholipid vesicles in which a crude mixture of gangliosides was incorporated (Banejee et al., unpublished observation). However, the sugar-binding specificity of the HA remains to be defined in detail.
b
a
d
c
e
KD
p~-
.~
-
-#
::. .....m. .....
94 67 43
-lsO"
30
TABLE 3. Inhibition of HA-mediated agglutination of rabbit erythrocytes by glycoconjugates Sugars and
Sugaycoprstea
glycoproteins
Minimu Minimum
inhibitory (,ug/ml)concn
Not inhibitableb 62.5 Fetuin ....................................... 62.5 Asialofetuin ....................................... 500 Mucin ........................................ ...................... >1,000 Ovalbumin ................. >100 Bovine serum albumin .....................................
Simple sugarsa .......................................
a Sugars used:
D-glucose, methyl a-D-glucopyranoside, methyl P-D-glyco-
pyranoside, D-galactose, L-fucose, methyl a-D-galactoside, methyl J3-D-galactoside, D-mannose, N-acetyl-D-glucosamine, N-acetyl-D-mannosamine, maltose, malibiose, rhamnose, raffinose, and N-acetylneuraminic acid. b Not inhibited up to 0.2 M concentration of sugars.
..
W
;.
-
20
.;~
_goo
14
FIG. 6. SDS-PAGE profiles of fraction B and envelope preparations of homologous and heterologous strains V. cholerae: Envelope preparations of V. cholerae serovar 01 strain A17, Ogawa, El Tor (lane a); serovar 01 strain AD 29, Inaba, El Tor (lane b); non-O1 V. cholerae V2 (lane c). Fraction B (lane d) and standard proteins (lane e) of known molecular weight are shown. KD, Kilodaltons.
3704
BANERJEE ET AL.
a
b
INFECT. IMMUN.
c
d
e
KD 94
67 43
>k
30
20
14
FIG. 7. Immunoblot analysis of anti-HA anti- serum against fraction B and envelope preparations of homologous an(s heterologous strains
of V. cholerae. Envelope preparations of V. chol erae serovar 01 strain A17, Ogawa, El Tor (lane a); serovar 01 strain AD 29, Inaba, El Tor (lane b); non-01 strain V2 (lane c); fraction IB (lane d). Standard proteins of known molecular weight were transferred to nitrocellulose paper and stained by amido black (lane e). KD, Kilodaltons.
Electron micrographs of the purified HA revealed a morphology typical of globular proteins (Fig. 5), with subunits apparently being arranged in a ringlike configuration. The subunit composition as well as the morphology of this HA are in sharp contrast to those of the fimbrial HAs, which are usually composed of subunits in the Mr range of 15,000 to 25,000 and characterized by a filamentous structure (22). It may be mentioned, in this connection, that although the presence of nonfimbrial HAs of surface origin was described as early as 1955 by Duguid et al. (9) in Escherichia coli strains, recent studies (26) have failed to confirm their nonfimbrial nature. This HA, thus, provides an interesting example of a true nonfimbrial lectin. A notable feature of this HA is its strong tendency to undergo self-aggregation as indicated by its virtual exclusion from the Sepharose CL-4B matrix during gel filtration as well as by the presence of aggregated particles in electron micrographs (Fig. 5). The aggregation was not reversed by the addition of common protein-dissociating agents such as urea, guanidine hydrochloride, Triton X-100, and deoxycholate (Baneree et al., unpublished observation). However, since the HA was not sedimented at 100,000 x g for 1 h, it did not appear to be particulate in nature. The HA also formed a strong, noncovalent association with LPS as revealed by the vesiclelike morphology as well as the increased effective molecular mass of fraction C compared with the purified HA. The LPS also increased considerably the resistance of the HA to heat inactivation, suggesting stabilization of the biologically active conformation. These features probably reflected a strong hydrophobic nature of the HA molecule, which was also suggested by the relatively high content of nonpolar amino acids (Table 2), and might help in the anchorage of the HA in the lipid-rich milieu of the cell envelope. Complete inhibition of the crude HAs released by the parent as well as several 01 and non-O1 strains of V.
cholerae by anti-HA antiserum (Table 4) suggested that it might represent the only HA released during growth in BHI broth. However, it did not contribute to the cell-associated HA activities of these strains, an observation not unexpected in view of the expression of multiple HAs by V. cholerae (15). This HA is different from the soluble HA with protease activity described earlier (12, 17) in subunit composition, morphology, and biochemical properties. Since only limited information is available about other cholera HAs (5, 28), it is difficult to conclude whether our preparation is related to any of them. Immunochemical detection of the HA as a component of the envelopes of several V. cholerae 01 and non-O1 strains (Fig. 7) confirmed the somatic origin of the HA. Since the HA was not sedimented at 100,000 x g, it could not be expected to be present in the OMP (Fig. 3) prepared from envelope on the basis of their insolubility in sarcosyl solution (11). Several V. cholerae OMPs are, indeed, solubilized by detergents milder than SDS (23). However, since the anti-HA antiserum failed to agglutinate parent cells, the HA was apparently not sufficiently exposed on the bacterial surface during growth in vitro. Further studies are needed to locate the HA in the cell envelope. V. cholerae is also known to express novel surface proteins during in vivo growth (19, 27). It remains to be determined whether this HA is expressed in sufficient quantity or is present in a proper orientation on the surface of in vivo grown cells to enable them to adhere to host cells during intestinal infection. ACKNOWLEDGMENT We are indebted to Ranjit Roy, University of Alabama, Birmingham, for amino acid analysis. LITERATURE CITED 1. Anantharamaiah, G. M., T. A. Hughes, M. Iqbal, A. Gawish, P. J. Neame, and J. P. Segrest. 1983. Effect of oxidation on the properties of apolipoproteins A-I and A-II. J. Lipid Res. 29:309318. 2. Banerjee, K. K., and A. Sen. 1981. Purification and properties of a lectin from the seeds of Croton tiglium with hemolytic activity toward rabbit red cells. Arch. Biochem. Biophys. 212:740-753. 3. Bennett, V., and P. Cuatresasas. 1977. Cholera toxin. Membrane gangliosides and activation of adenylate cyclase, p. 1-66. In P. Cuatresasas (ed.), The specificity and action of animal, bacterial and plant toxins. Chapman and Hall, London. 4. Booth, B. A., C. V. Sciortino, and R. A. Finkelstein. 1986. Adhesins of Vibrio cholerae, p. 169-182. In D. Mirelman (ed.), Bacterial lectins and agglutinins. John Wiley & Sons, New York. 5. Chaicumpa, W., U. Peungjesda, B. Martinez, and N. Atthasishtha. 1982. Soluble hemagglutinin of classical vibrios: isolation and protection against cholera by its antibodies. Southeast Asian J. Trop. Med. Public Health 13:637-645. 6. Dayhoff, M. 0. 1978. Atlas of protein sequence and structure, vol. 5, Suppl., p. 363. National Biomedical, Silver Spring, Md. 7. Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28:350-356. 8. Duguid, J. P., and D. Old. 1980. Adhesive properties of Enterobacteriaceae, p. 185-217. In E. Beachey (ed.), Bacterial adherence. Receptor and recognition, Ser. B, vol. 6. Chapman and Hall, London. 9. Duguid, J. P., I. W. Smith, G. Dempster, and P. N. Edmunds. 1955. Nonflagellar filamentous appendages (fimbriae) and hemagglutinating activity in Bacterium coli. J. Pathol. Bacteriol. 70:335-348.
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