(Received for publication, August 14, 1995)
From the
Streptococcus suis causes meningitis, sepsis, and other
serious infections in newborn and young pigs and in adult humans. The
Gal1-4Gal-binding adhesin of S. suis was purified
to homogeneity by ultrasonic treatment, fractional ammonium sulfate
precipitation, and preparative polyacrylamide gel electrophoresis.
Pigeon ovomucoid, a glycoprotein with Gal
1-4Gal terminals,
was used to detect the adhesin by blotting. The purified adhesin
appeared as single band of an apparent size of 18 kDa and of a pI of
6.4; no disulfide bridges were present. The amount of adhesin as
revealed by pigeon ovomucoid binding correlated with the
hemagglutination activity of different S. suis strains. The
purified adhesin bound to latex particles induced hemagglutination
which was specifically inhibited with the same inhibitors as
hemagglutination by the intact bacteria, thus demonstrating that the
purified protein was the Gal
1-4Gal-recognizing adhesin of S. suis. Two adhesin variants (P
and
P
) with differing Gal
1-4Gal binding specificity
had the similar electrophoretic mobilities and the same N-terminal
peptide sequences, indicating that they were closely related. This
represents the first isolation of an adhesin with well-defined cell
surface carbohydrate binding activity from Gram-positive bacteria
associated with meningitis.
The first event in the establishment of infectious diseases is the adhesion of bacteria to the surface of host cells(1) . Adhesins mediating this interaction are thus essential factors in bacterial pathogenesis and important virulence factors(2, 3) . Many adhesins act as lectins, recognizing specific carbohydrate moieties on host cell surface glycoconjugates. Most of the knowledge on bacterial carbohydrate-binding adhesins is derived from studies on Gram-negative bacteria (reviewed in (4) ), whereas only little information exists on the molecular identity and detailed binding properties of such adhesins in Gram-positive bacteria.
Streptococcus suis is an important Gram-positive pathogen which causes meningitis, sepsis, and other serious infections in piglets (5, 6, 7) and meningitis in humans who have been in contact with pigs(8, 9) . The identification and characterization of the molecules responsible for the interaction of the bacteria with host cells would give valuable information for the understanding of the pathogenesis of the infection and is a key factor in the development of new antibacterial agents and vaccines.
In
previous studies, the binding of S. suis bacteria to host
cells was found to be mediated by an adhesion activity which recognizes
the disaccharide
galactosyl-1-4-galactose(10, 11) . In the
present study, the adhesin was identified, purified to homogeneity, and
characterized and was found to retain the hemagglutination activity and
specificity of the intact bacteria.
Todd-Hewitt broth and a Gas-Pak anaerobic system were purchased from Becton Dickinson and Co., Cockeysville, MD. Microwell Microtiter Plates were from Dako, Roskilde, Denmark, and polystyrene flat bottom Microstrips from Labsystems, Helsinki, Finland. IODOBEADS were purchased from Pierce. Bio-Gel P-6DG Desalting Gel was from Bio-Rad. Nitrocellulose sheets were from Schleicher & Schuell, Dassel, Germany and PVDF-P membrane from Millipore.
Nonspecific binding sites were saturated by incubation of the
membranes for 1.5 h in phosphate buffer C (0.1 M sodium
phosphate buffer, 0.5% Tween 20, 150 mM NaCl, pH 5.3). The
membranes were incubated with the I-pigeon ovomucoid (6
10
cpm/15 cm
, specific activity about
2.5
10
cpm/µg) in phosphate buffer C for 1 h at
+8 °C. The membranes were washed three times for 10 min in
phosphate buffer C, dried between filter papers, and exposed to an
x-ray film with an intensifying screen at -80 °C for
5-48 h.
Latex hemagglutination and hemagglutination
inhibition assays were performed as described before(14) .
Adhesin-covered latex particles (5, 10, or 25 µl) were mixed with a
4% suspension of sialidase-treated human erythrocytes (5, 10, or 25
µl, respectively) on a ceramic slide and incubated on ice for 10
min. In agglutination inhibition assays, 10 µl of adhesin-covered
latex particles were mixed with 10 µl of the inhibitory compounds
on a slide and incubated on ice. After 15 min, 20 µl of
sialidase-treated 4% human erythrocytes were added, and the mixture was
incubated for 15 min on ice. Similar agglutination reactions were
obtained in volumes of 5 to 25 µl of latex with 5 to 25 µl of
sialidase-treated erythrocytes, respectively. Bovine serum
albumin-coated latex particles (0-0.1 mg/1.25 10
particles) used as controls gave no hemagglutination reactions.
Figure 1:
Binding of pigeon ovomucoid to S. suis bacterial cells in a dot assay. Two strains of S.
suis cells having different hemagglutinating activity (628, 1:64
and 836, 1:8) and a strain with no hemagglutinating activity (598) were
pipetted onto nitrocellulose membrane at the dilutions of 1:1, 1:10,
and 1:50 in a volume of 1 µl. After blocking nonspecific binding
sites, the membranes were incubated for 1 h with I-pigeon
ovomucoid and processed for
autoradiography.
In fractional ammonium sulfate precipitation of the sonicate (50 mg of protein/16 ml of sonicate), the adhesin activity was precipitated with ammonium sulfate with a saturation degree of 70% (Fig. 2). Ammonium sulfate having a saturation degree of 60% precipitated the contaminating proteins running near the adhesin, as analyzed by polyacrylamide gel electrophoresis in the absence of SDS and Western blotting with radiolabeled pigeon ovomucoid.
Figure 2:
Purification of the adhesin protein by
fractional ammonium sulfate precipitation. The sonication supernatant
of S. suis strain 628 was subjected to fractional ammonium
sulfate precipitation, and the precipitates of the 60, 70, and 80%
ammonium sulfate saturation precipitates and the starting sonication
supernatant (S) were subjected to electrophoresis in 6%
polyacrylamide gels in the absence of SDS. The gel was stained for
protein (A), or the proteins were transferred to PVDF-P
membrane which was probed for adhesin activity with I-labeled pigeon ovomucoid (B).
The adhesin was finally purified to homogeneity from the 70% ammonium sulfate precipitate (1 mg of protein/16 ml of sonicate) by preparative gel electrophoresis. Fractions from the preparative gel were collected and analyzed by gel electrophoresis. The adhesin was eluted as a single band (Fig. 3). Usually about 150 µg of pure adhesin was isolated from 16 ml of a bacterial suspension of S. suis strain 628 with a hemagglutination titer of 1:32.
Figure 3: Purification of the adhesin by preparative gel electrophoresis. The 70% ammonium sulfate precipitate was subjected to preparative electrophoresis in a 6% polyacrylamide gel in the absence of SDS. Fractions of 4 ml were collected and analyzed by analytical gel electrophoresis in a 6% gel stained for protein. The numbers of the fractions are given. The pure adhesin was eluted in fractions 78-83.
Figure 4: Polyacrylamide gel electrophoresis and isoelectric focusing of purified S. suis adhesin. A, the purified adhesin was run in 15% polyacrylamide gel electrophoresis in the presence of SDS which was stained with Serva Blue. The molecular masses of the standard proteins (St1 and St2) are indicated in kDa. B, isoelectric focusing of the adhesin by the Phast Gel system. The gel was stained with silver. The isoelectric points of the standard proteins (St) and the sample application point (S) are indicated.
The P
adhesin activity of S. suis was previously shown to occur as
two variant activities P and P
differing in
their Gal
1-4Gal binding specificity(11) . The N
termini of adhesins determined from electrophoretic blots of four other S. suis strains, two of type P
(TEW/2, R75/L1) and
two of type P
(752, 825), were identical with the purified
adhesin.
Figure 5:
Binding of pigeon ovomucoid to adhesin in S. suis strains with different hemagglutinating activities.
Sonicated extracts of the S. suis strains indicated (top) with differing hemagglutinating activity were separated
by electrophoresis in 6% polyacrylamide gels in the absence of SDS and
transferred to PVDF-P membrane which was probed for adhesin activity
with I-labeled pigeon ovomucoid. The hemagglutinating
activities are given as the reciprocal of the titer (bottom).
Bacteria of strain 628 extracted in high or low hemagglutination titer
phases were included in the analysis.
The intensity of the band correlated with the hemagglutination titers of the corresponding strain. Like in many other bacteria(24) , the P hemagglutination of S. suis undergoes spontaneous phase variation. Also in bacteria extracted in the highly agglutinating or low agglutinating phases, the intensity of the adhesin bands correlated with the agglutinating titer (Fig. 5, strain 628).
In previous studies, we found that pigeon ovomucoid, due to
the presence of the Gal1-4Gal-containing blood group P
active glycans, is an effective inhibitor of the P adhesins of S. suis(10, 11) . This glycoprotein was
therefore used as an indicator for the presence of the adhesin in S. suis cells by dot binding assay and for the identification
of the adhesin protein during its purification. Different lines of
observations suggest that the protein isolated was indeed the adhesin
responsible for the hemagglutinating activity of the bacteria. The
hemagglutination activity correlated with pigeon ovomucoid binding both
in whole cells and in the sonication extracts of the cells. In the
latter, only one pigeon ovomucoid-binding component was present and
corresponded in mobility to the purified protein. Furthermore, the
purified protein adsorbed to latex particles induced hemagglutination,
which was inhibited by the same inhibitory compounds as agglutination
induced by the intact bacteria.
Two variant P adhesion specificities
are present in Gal1-Gal-binding S. suis, the adhesion
activity inhibitable by galactose and N-acetylgalactosamine
(type P
) or by galactose only (type
P
)(11) . In the present study, the adhesin was
isolated from strain 628 which is of type P
. However,
pigeon ovomucoid also bound to whole bacteria and the adhesin band of
type P
bacteria. Furthermore, the adhesin bands of
P
and P
had similar mobilities in gel
electrophoresis, and their N-terminal amino acid sequences were
identical. These findings indicate that the two variant P adhesins are
closely related. No homology to previously known adhesins was apparent.
Recently, a 110-kDa protein of S. suis serotype 2 has been identified as a virulence marker(25) . Also, the capsular polysaccharides, occurring as at least 29 serotypes, have been suggested to play a role in the pathogenesis of infections caused by S. suis(26, 27) . The contribution of these factors to the pathogenic mechanisms of S. suis are at present not known.
Only few adhesins have been identified and isolated from Gram-positive bacteria. Some of these are oral strains and are related to the binding of the bacteria to hydroxyapatite(28) , saliva(29) , or other bacteria (30) , or bind to extracellular components such as fibronectin (31) or fibrinogen(32) . These adhesins differ in molecular properties from the P adhesin of S. suis, and their possible lectin and cell-surface binding activities are in most cases not known or have not been characterized in detail. In Gram-negative bacteria, adhesins from bacteria associated with meningitis include the S-fimbrial adhesin from Escherichia coli(33, 34) and an adhesin from Neisseria meningitidis(35) . Interestingly, an N-acetylglucosamine-specific adhesin of 17 kDa has been reported from type III group B streptococci associated with newborn meningitis (36) but further details of the interaction and the molecules involved are missing.
The present report represents the
first isolation of Gal1-4Gal-binding adhesins from
Gram-positive bacteria and from bacteria associated with meningitis.
Among Gram-negative bacteria, extraintestinal E. coli strains
that cause urinary tract infections frequently produce P-fimbrial
adhesins as virulence factors (37, 38) . These
adhesins interact with glycolipids containing the Gal
1-4Gal
structure. On the other hand, E. coli isolates associated with
newborn meningitis contain S-fimbrial adhesins recognizing
Sia
2-3Gal(33, 39) . Sia
2-3Gal binding
has also been described in S. suis, but the adhesion activity
differs from that of E. coli in the recognition of the inner
sugars in the receptor oligosaccharide(40) . In pigs, many
tissues express Gal
1-4Gal-containing
glycolipids(41) , and these are able to serve as binding
ligands for S. suis containing P adhesin to frozen sections of
pig pharyngeal tissue(10) . However, the in vivo roles
of the different adhesion specificities remain so far unresolved.
Identification and purification of the P adhesin of S. suis will now make possible the cloning of the corresponding gene and
thus facilitate studies designed to elucidate the molecular mechanism
of the binding interaction, the basis of the differential binding of
the variant adhesins, and the pathogenic role of the adhesin.