The present report describes the identification, purification, and characterization of a hemolysin produced by Streptococcus suis type 2. The hemolysin was purified from the culture supernatant by using different filtration steps, Superose-12 column chromatography, and selective (NH4)2SO4 precipitation. The purified hemolysin, designated suilysin, had an apparent molecular mass of 54,000 Da and exhibited a specific activity of 0.7 x 106 hemolytic units per mg. Suilysin appeared to belong to a family of toxins known as the thiol-activated toxins, with which it had several characteristics in common: loss of activity upon oxidation, reactivation upon reduction, and inhibition of activity by small amounts of cholesterol. The N-terminal amino acid sequence of suilysin showed many similarities with parts of the deduced N-terminal amino acid sequences of perfringolysin 0, streptolysin 0, listeriolysin 0, alveolysin, and pneumolysin. Mice immunized with a vaccine containing purified suilysin appeared to be completely protected against a lethal S. suis type 2 challenge, indicating that suilysin is an important factor and that the neutralization of this single factor is sufficient to protect mice against the detrimental effects of an S. suis type 2 infection. Most of the different (serotype) strains appeared to secrete hemolytic activity which was biochemically and immunologically indistinguishable from suilysin into the culture supernatant in vitro, indicating that suilysin might be a cross-protection factor.

Three groups of three pigs were vaccinated either with vaccine VAC-SLY, containing purified suilysin derived from Streptococcus suis strain P1/7 (serotype 2), or with vaccine VAC-SCF, containing most of the other extracellular antigens produced by strain P1/7 (but essentially free from suilysin), or with a placebo vaccine. The pigs were vaccinated twice at four weeks and six weeks of age and were challenged intravenously with S suis strain P1/7 at eight weeks of age. On the day of challenge, only the VAC-SLY vaccinated pigs showed an increase in haemolysin neutralisation titre. After challenge the placebo vaccinated pigs developed severe clinical signs characterised by lameness involving several joints, a depressed appearance, high temperatures and/or neurological signs. The VAC-SCF vaccinated pigs showed the same clinical signs but less severely. The VAC-SLY vaccinated pigs were the least affected and showed only mild signs which subsided more quickly than those of the other groups. A post mortem investigation and histology of brain tissue samples confirmed the clinical findings; fibrinous arthritis was less severe and less frequently observed in the VAC-SLY vaccinated pigs than in the VAC-SCF or placebo vaccinated pigs, and none of the VAC-SLY vaccinated pigs had meningitis whereas two of the VAC-SCF and two of the placebo vaccinated pigs did so. All the samples of brain, lung and tarsus taken from the VAC-SLY vaccinated pigs were sterile whereas S suis was reisolated from most of these tissues from the other groups.

Thiol-activated cytolysins share a conserved hydrophobic, Trp-rich undecapeptide that is suggested to be involved in membrane binding and intercalation. The neutralizing monoclonal antibody PLY-5 recognizes all members of this toxin family and peptide mapping assigned its epitope to the undecapeptide motif. This antibody inhibited binding of the toxins to host cell membranes and the epitope was no longer available for binding when a preformed toxin/membrane complex was tested. These results confirm the model of cytolysin binding suggested by structural data.z 1999 Federation of European Biochemical Societies.

The gene encoding the K99 fibrillar adhesin of Escherichia coli has been modified by oligonucleotide-directed, sitespecific, mutagenesis. The tryptophan-67, lysine-132, lysine-133 or arginine-136 were replaced by leucine, threonine, threonine and serine, respectively. The threonine-133 mutant fibrillae were indistinguishable from wild-type fibriliae. In contrast, replacement of lysine-132 or arginine-136 by threonine or serine, respectively, resulted in mutant fibrillae which had completely lost adhesive capacity, suggesting that the positive charges of these residues are essential for the interaction with the negatively charged sialic acid residue of the receptor molecules. After the replacement of tryptophan-67 with leucine neither fibrillae nor subunits were detectable, indicating that the mutant product is unstable and that tryptophan-67 has an essential structural role in the K99 subunit.

A cyanogen bromide fragment derived from the K88ab adhesin inhibited the hemagglutinating activity of K88 fibrillae. Smaller fragments which inhibited the adherence of K88 fibrillae to erythrocytes or to intestinal epithelial cells were obtained by digestion of K88ab fibrillae with oa-chymotrypsin. Active peptides were isolated from the digestion mixture and identified as Ser-Leu-Phe and Ala-Ile-Phe. Both tripeptides correspond to the peptide stretches Ser-148-Leu-Phe-150 and Ala-156-Ile-Phe-158, respectively, which are part of conserved regions in the primary structure of the K88 variants ab, ac, and ad. The isolated tripeptides inhibited the hemagglutinating activity of purified K88 fibrillae in the 1 to 5 ,uM range, while adherence of the fibrillae to intestinal epithelial cell brush borders was inhibited in the 10 to 50 ,uM range. Furthermore, the tripeptides were capable of eluting attached bacteria from agglutinated erythrocytes. The inhibitory activity of the isolated peptides was confirmed by testing various synthetic peptides for their ability to inhibit the interaction of the different K88 variants with various species of erythrocytes. The significance of these findings for the localization of the receptor-binding domain is discussed.K88 fibrillae are nonflagellar, filamentous adhesins found on many enterotoxigenic Escherichia coli strains that cause neonatal diarrhea in pigs (4). They enable the bacteria to colonize the small intestinal epithelium, which is considered to be a prerequisite for the establishment of diarrheal disease. The K88 fibrillae consist of multimers of the K88 adhesin subunit with a molecular weight of 27,500 (4). Three serological variants of K88 fibrillae have been described and designated K88ab, K88ac, and K88ad (7). The primary structure of all three proteins has been determined, showing both conserved and variable regions (5,6,12 Purification of fibrillae. K88ab, K88ac, K88ad, F41, and K99 fibrillae were isolated and purified as described previously (10).Isolation of brush borders. Brush borders were prepared from the pig intestine as described by Middeldorp and Witholt (15) and stored in 50% glycerol at -20°C.Hemagglutination inhibition test. Suspensions of washed erythrocytes (2%) in PBSM (50 mM sodium phosphate [pH 7.3] containing 0.9% NaCl and 0.5% mannose) were mixed 1:1 with various amounts of peptide fractions in the same buffer. Subsequently, 50-pl portions of these suspensions were added to serial twofold dilutions (50 ,ul) of purified fibrillae in PBSM, using polystyrene microtiter trays with V-shaped cups. After 2 h of incubation at 4°C, the trays were examined. The initial concentrations of K88ab, -ac, and -ad fibrillae used were 50, 500, and 25 [Lg/ml, respectively