Characterization of the Renibacterium salmoninarum haemagglutinin

Water-extracted proteins from nine geographically diverse strains of Renibacterium salmoninarum, all of which agglutinated rabbit erythrocytes and rainbow trout spermatozoa, were compared by SDS-PAGE. Extracts from eight strains, including the type strain, ATCC 33209, were similar, containing a major protein of 57 kDa and a minor protein of 58 kDa. The SDS-PAGE protein profile of the Char strain did not contain the 58 kDa protein. A non-agglutinating strain, MT-239, which was also non-hydrophobic, did not produce any water-extractable protein. Immunoblot reactions with rabbit antiserum prepared against whole cells of the type strain demonstrated that the water-extracted haemagglutinins from the various strains were antigenically related. When purified by polyacrylamide gel zone electrophoresis, the haemagglutinin from R. salmoninarum ATCC 33209 formed a doublet band with molecular masses of 57 and 58 kDa, similar to the previously described F antigen. The water-extracted haemagglutinin agglutinated salmonid spermatozoa, was degraded by protease K and trypsin, and was shown to self-assemble onto the cell surface.     

Identification of erythrocyte-binding antigens in Helicobacter pylori.

The surface antigens of Helicobacter pylori conferring erythrocyte-binding activity were obtained by adsorption onto formaldehyde-treated dog and goat erythrocytes from supernatant fractions of sonicated bacteria and elution using a high concentration of NaCl. The desorbed material was analysed by SDS-PAGE and immunoblotting with anti-whole-cell serum to agar-grown bacteria which had been absorbed with broth-grown, non-haemagglutinating cells (haemagglutination-associated antiserum). Two polypeptides with molecular masses of 25 and 59 kDa were revealed as erythrocyte-binding antigens. Strains which agglutinated both dog and goat erythrocytes possessed both these erythrocyte-binding antigens, whereas an antigenically cross-reactive 24 kDa polypeptide was present in a strain which only agglutinated goat erythrocytes. Haemagglutinin material was extracted from H. pylori using n-octylglucopyranoside and purified by Sepharose chromatography and sucrose density gradient ultracentrifugation. The purified extract directly agglutinated erythrocytes in a neuraminyl-lactose-sensitive and neuraminidase-sensitive manner. The 59 kDa polypeptide was not present in the purified haemagglutinin preparation. The haemagglutination-associated antiserum reacted strongly with the 25 kDa polypeptide band which was the most prominent polypeptide band on analysis of the purified haemagglutinin preparation by SDS-PAGE and silver staining. Thus, H. pylori possesses at least two adhesins, one of which recognises a N-acetylneuraminic acid (alpha 2-3) moiety of receptors, the other being of unknown receptor specificity. Differences in the antigenicity and molecular masses of these adhesins in individual strains may underlie differences in receptor-binding specificities and haemagglutination profiles.     

A new haemagglutinin from the amoebocytes of the horseshoe crab Carcinoscorpius rotundicauda. Purification and role in cellular aggregation.

The present paper describes the purification and function of a haemagglutinin from the amoebocyte lysate of the horseshoe crab Carcinoscorpius rotundicauda. The purified protein consisted of a single subunit of Mr 24 000 and agglutinated human blood-group-A+ erythrocytes. Its haemagglutinin activity was inhibited by purified lysate, coagulogen, but not by sugars. The haemagglutinin differed immunologically and in activity from the sialic-acid-binding lectin carcinoscorpin present in the haemolymph. It caused aggregation of forma-fixed amoebocytes, and on the basis of this observation its role in cell-cell adhesion is proposed. This new haemagglutinin promotes cell-cell aggregation in amoebocytes in a manner that shares some similarities with thrombospondin-mediated platelet aggregation in vertebrates [Jaffe, Leuang, Nachman, Levin & Moseher (1981) Nature (London) 295, 246-248].     

Surface components of shigellae involved in adhesion and haemagglutination

Strains of Shigella species were studied for their ability to adhere and agglutinate mammalian erythrocytes. Shigella dysenteriae and Sh. flexneri exhibited haemagglutinating (HA) properties when cultured in Casamino Acids-Yeast Extract (CYE) broth in the presence of 1 mmol 1^-1 calcium chloride, but other shigellae did not show this property under the same culture conditions. Repeated subcultivation of Sh. boydii, Sh. sonnei and HA negative strains of Sh. dysenteriae and Sh. flexneri in CYE broth medium induced adhesive and haemagglutinating properties that were inhibited by sodium periodate. HA activities of Shigella spp. were also inhibited by N -acetylneuraminic acid, α₁-glycoprotein and fetuin, but not by protease. Electron microscopy of Sh. dysenteriae 1, Sh. flexneri 2a, Sh. boydii 12 and Sh. sonnei 1 grown in CYE broth showed the presence of an extracellular slime layer that promoted agglutination of erythrocytes. The slime layer extracted from the cell surface of Shigella spp. showed HA properties, whereas lipopolysaccharide (LPS) obtained from the same strains, except Sh. dysenteriae 1, did not agglutinate erythrocytes. This evidence suggests that the cell surface haemagglutinin is a loosely bound slime layer which is expressed in CYE broth medium.     

Hemagglutinating properties of Proteus mirabilis strains isolated from clinical specimens

Haemagglutinating properties of 345 P. mirabilis strains isolated from various clinical samples were determined. Red blood cells of different origin as human group 0, bovine, horse, sheep and rat were used for the study. For the detection of MS and MR/P haemagglutinins the haemagglutination reaction was run with and without D-mannose. On the other hand, for the detection of type MR/K haemagglutinins tanned human and bovine erythrocytes were used. The majority of tested strains (90.14%) was polyhaemagglutinating i.e. showed simultaneously the presence of two or three haemagglutinins. Only three strains of P. mirabilis (0.87%) did not agglutinate any of the erythrocytes used for the study. The majority of strains (95.83-100%) in specific groups of clinical materials showed the presence of MR/K+ while MR/P+ 45.45-93.75% of strains and MS+ 45.83-73.1% of tested strains. Out of P. mirabilis strains isolated from urine, faeces and blood the highest percentage possessed at the same time all three haemagglutinin types (MS+, MR/K+, MR/P+) or pattern MR/K+, MR/P+. Bronchial isolates had usually pattern MR/K+ (31.82%) and strains isolated from skin possessed haemagglutinins of pattern MR/K+, MR/P+ (50%) and MS+, MR/K+, MR/P+ (43.75%). Among strains expressing MR/P+ at 37 degrees C a great differentiation of spectrum activity against tested erythrocytes was seen. Undoubtedly, the majority of MR/P+ strains from specific groups of clinical materials (with the exception of urine) agglutinated sheep and horse erythrocytes with and without D-mannose. The majority of strains isolated from urine agglutinated sheep and bovine erythrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

A method for studies of an El Tor-associated antigen of Vibrio cholerae O1

A method for studying the biotype El Tor associated mannose-sensitive haemagglutinin (MSHA) of V. cholerae O1 has been developed. By using crude MSHA adsorbed to chicken erythrocytes as solid phase antigen in an enzyme-linked immunosorbent assay (ELISA), antisera against V. cholerae of the El Tor biotype reacted in high titre with the MSHA-coated cells, whereas antisera against vibrios of the classical biotype did not bind significantly, i.e. in higher titre than pre-immune sera. The binding of anti-MSHA serum, or a monoclonal antibody against MSHA, to the MSHA-coated erythrocytes could be efficiently inhibited by crude MSHA as well as by El Tor vibrios whereas neither V. cholerae lipopolysaccharide nor different strains of classical vibrios had any inhibitory effect. These results support the existence of an El Tor-associated immunogen. They also suggest a possibility of determining antibodies against different haemagglutinins in ELISA without having access to purified antigens.     

Hemagglutinin specificity of Cl. botulinum types A, B, and F in reaction with erythrocytes of various animals]

The following differences were revealed in the haemagglutination reaction with the erythrocytes of man, sheep, rabbit, chicks and mice between the haemagglutinins of Cl. botulinum, types A, B and F, having a close affinity with one another: haemagglutinin of type A actively reacted with the erythrocytes of man, sheep, rabbit, rats and chicks; haemagglutinin of type B reacted only with the erythrocytes of man and rabbits; haemagglutinin of type F failed to react with any of the types of the erythrocytes tested; only with the use of erythrocytes treated with neuraminidase was it possible to establish the presence of haemagglutinin fraction in Cl. botulinum, type F. Treatment of human erythrocytes with neuraminidase and proteolytic enzymes caused a marked increase in the sensitivity of the haemagglutination reaction in haemagglutinins of Cl. botulinum, types A, B and F.     

Hemagglutinating and binding properties of botulinum C2 toxin

To characterize the binding substance(s) for botulinum C2 toxin, the hemagglutinating activity of component II of botulinum C2 toxin (C2II) was studied by hemagglutination and hemagglutination inhibition. Human and animal erythrocytes were agglutinated by trypsinized C2II much more strongly than by untreated C2II. Trypsinized C2II agglutinated neuraminidase-treated erythrocytes more strongly than intact, trypsin- and pronase-treated ones. On the other hand, trypsin- and pronase-treated erythrocytes were more weakly hemolyzed by trypsinized C2II than intact and neuraminidase-treated ones, and trypsinized C2II showed both hemagglutinating and hemolytic activities to these erythrocytes. Hemagglutination of trypsin-treated human type B erythrocytes was inhibited by galactose, N-acetylgalactosamine, N-acetylglucosamine, L-fucose and mannose. Thyroglobulin and bovine salivary mucin were much stronger inhibitors. From these findings, the binding substance(s) for botulinum C2 toxin on erythrocytes is(are) suggested to be glycoprotein(s).     

Proteins with haemagglutinin activity in larvae of the Colorado beetle, Leptinotarsa decemlineata

The haemagglutinating activity of larval haemolymph of Leptinotarsa decemlineata against red blood cells of various origins has been examined. This activity appeared to be unspecific, since all the different types of erythrocytes were agglutinated by a haemolymph dilution of $\text{1}\text{128}$ to $\text{1}\text{512}$. Only horse erythrocytes were agglutinated to a greater degree ($\text{1}\text{3200}$. Red blood cells became much more sensitive after treatment with trypsin, while formol fixation also resulted in a better agglutinability. Sulphated polysacchrides (heparin, mucin, dextran sulphate) were good inhibitors of the haemagglutination reaction. A weaker inhibition was obtained with hexosamines. As demonstrated by immunoelectrophoresis, two haemagglutinins occur in larval haemolymph. One is specific for larvae and pupae, and is therefore called the larval-pupal haemagglutinin. It is absent in adults. The second haemagglutinin is the well-known chromoprotein 2, which is present in all developmental stages, including the egg, where it constitutes an important element of yolk proteins. The affinity of chromoprotein 2 toward dextran sulphate was confirmed by precipitation tests in agarose.     

Haemagglutinin of the antarctic seaweed Georgiella confluens (Reinsch) Kylin: isolation and partial characterization

The marine red alga Georgiella confluens collected from Mackellar Inlet, King George Island, South Shetland Islands, Antarctic, was used in the isolation of a protein with agglutinating activity. The Georgiella confluens haemagglutinin (GCH) was extracted with 20 mM phosphate buffer, pH 7.0, and purified through ion exchange chromatography, followed by affinity chromatography on immobilized porcine stomach mucin. Among the erythrocytes analysed (human A, B and O groups, rabbit and chicken), GCH agglutinated specifically chicken erythrocytes. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of the haemagglutinin revealed a single band of 21.5 kDa, while by gel filtration on Sephadex G-100 its native molecular mass was 25.5 kDa, suggesting that GCH is a monomeric protein. Haemagglutination studies showed that the GCH activity was stable through temperature variations and did not exhibit divalent cation dependence. Furthermore, the haemagglutinin was inhibited by the complex glycoproteins of porcine stomach mucin and fetuin, whereas the mono-, di-, and trisaccharides tested showed no effect.     

Biochemical Characterization and Agglutinating Properties of Xenorhabdus nematophilus F1 Fimbriae

Xenorhabdus spp., entomopathogenic bacteria symbiotically associated with nematodes of the family Steinernematidae, occur spontaneously in two phases. Only the phase I variants of Xenorhabdus nematophilus F1 expressed fimbriae when the bacteria were grown on a solid medium (nutrient agar; 24 and 48 h of growth). These appendages were purified and characterized. They were rigid, with a diameter of 6.4 (plusmn) 0.3 nm, and were composed of 16-kDa pilin subunits. The latter were synthesized and assembled during the first 24 h of growth. Phase II variants of X. nematophilus did not possess fimbriae and apparently did not synthesize pilin. Phase I variants of X. nematophilus have an agglutinating activity with sheep, rabbit, and human erythrocytes and with hemocytes of the insect Galleria mellonella. The purified fimbriae agglutinated sheep and rabbit erythrocytes. The hemagglutination by bacteria and purified fimbriae was mannose resistant and was inhibited by porcine gastric mucin and N-acetyl-lactosamine. The last sugar seems to be a specific inhibitor of hemagglutination by X. nematophilus.     

Different polysaccharides in the external layers (capsule and slime) of the cell envelope of Rhodopseudomonas capsulata Sp11

Two different acidic polysaccharides (I and II) were detected in the external cell envelope layers (slime and capsule) of Rhodopseudomonas capsulata Sp11. Polysaccharide I contains rhamnose, fucose, glucosamine and an unknown acidic sugar, it represents the slime material of the strain. Polysaccharide II contains rhamnose, galactose, 3-amino-3,6-dideoxygalactose, an unknown amino sugar and galacturonic acid, it represents very likely the capsule of R. capsulata Sp11. Polysaccharide I has a serological specificity different from that of polysaccharide II as shown by immunoprecipitation using antisera against living cells. Polysaccharide II, but not polysaccharide I, reacts in antiserum against heat-treated cells (100 degrees C, 2.5 h). Whole cells are agglutinated in the antisera against living but not in those against heat-treated cells.     

The marine red alga Eucheuma serra J. Agardh, a high yielding source of two isolectins

Aqueous ethanolic extracts from five species of the genus Eucheuma (Rhodophyta) i.e. E. serra, E. amakusaensis, E. cottonii, E. gelatinae and E. denticulatum, were examined for hemagglutinating activity with vertebrate erythrocytes. All the extracts tested agglutinated trypsin-treated sheep and rabbit erythrocytes as well as untreated sheep erythrocytes. From the extract of E. serra, which exhibited the highest activity, a lectin was purified by precipitation with cold ethanol followed by gel filtration to exhibit a single band on SDS-PAGE. The yield was surprisingly as high as 1000 mg from 100 g powdered alga. The purified lectin was further separated into two isoforms, designated ESA-1(90 mg) and ESA-2 (890 mg), by ion exchange chromatography. Both lectins showed a single protein band with the same molecular weight of 29 000 on SDS-PAGE and differed from each other only in isoelectric point (pI 4.75 for ESA-1 and pI 4.95 for ESA-2). Biochemical studies revealed that both are monomeric proteins without a carbohydrate moiety. The hemagglutinating activities were stable over a wide pH range and at a relatively high temperature. The activities were inhibited by a number of glycoproteins, but not by any of the monosaccharides and disaccharides tested. The lectins showed strong mitogenic activities for mouse lymphocytes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Interaction of cells of Helicobacter pylori with human polymorphonuclear leucocytes: Possible role of haemagglutinins

Abstract The interaction of fluorescein isothiocynate (FITC)-labelled cells of Helicobacter pylori with human polymorphonuclear leucocytes (PMNs) was studied. Two strains with surface haemagglutinins expressing different receptor specificity were used in order to decide if cell surface haemagglutinins of H. pylori may play a role in lectin-mediated binding to/uptake by phagocytes: (1) strain 17874 (NCTC 11637) which expresses sialic acid-specific haemagglutin; and (2) strain 17875 (NCTC 11638) which expresses a sialic acid-independent haemagglutinin. Cells of strain 17874 were poorly attached to/ingested by PMNs compared to cells of strain 17875. Pre-treatment of bacteria with fetuin or rabbit antibodies against partly purified sialic acid-specific haemagglutinin enhanced interaction of cells of strain 17874 with PMNs. The enhancement did not occur in the case of strain 17875. Phagocytosis of H. pylori 17874 bacteria was slightly increased by fresh human sera positive for anti- H. pylori antibodies. The results suggest that the sialic-acid-specific haemagglutinin complex of 17874 bacteria might disturb their uptake by human PMNs.     

Analysis of the specificity of five murine anti-blood group A monoclonal antibodies, including one that identifies type 3 and type 4 A determinants

The specificity of five mouse monoclonal anti-A blood group antibodies (Ab), four of which were produced by immunization with cultured human cancer cells and one with a synthetic antigen, has been determined by examining their reactivity with purified A glycolipids, erythrocyte glycolipids, oligosaccharides, ovarian cyst glycoproteins, and salivary glycoproteins. Two of the antibodies (HT29-36 and CB) reacted with all A variant structures tested and have a broad anti-A reactivity. Ab CLH6 did not agglutinate A erythrocytes and reacted preferentially with the type 1A structure. Ab S12 agglutinated all A1 erythrocytes and reacted best with simple, monofucosyl type 2 A structures, such as Aa-2, Ab-2, and A tetrasaccharide. Ab M2 has a novel, but complex, spectrum of reactivity. It reacts with type 3 and type 4 A chains and not with type 1 and type 2 A chains. It appears to recognize both an external A structure (formula; see text) (I) (found) in type 3 and type 4 chains) and also an internal structure (II) found in type 3 chains. Ab M2 agglutinates all A and AB erythrocytes but does not react with salivary glycoproteins.     

A surface polysaccharide of Escherichia coli O111 contains O-antigen and inhibits agglutination of cells by O-antiserum

The repeating pentasaccharide of O-antigen from Escherichia coli O111 contains galactose, glucose, N-acetylglucosamine, and colitose, the latter representing the major antigenic determinant. Phenol extraction of this strain was previously shown to release two fractions (I and II) containing O-antigen carbohydrate, and both fractions were believed to be lipopolysaccharide. We have now characterized fractions I and II and conclude that only fraction II represents lipopolysaccharide. Fraction II contains phosphate, 2-keto-3-deoxyoctonate, beta-hydroxymyristic acid, and potent endotoxin activity, whereas fraction I was deficient in all of these properties of the lipid A and core oligosaccharide regions of lipopolysaccharide. Fractions I and II each represented 50% of the total cellular O-antigen, and both were present on the cell surface. Both fractions were metabolically stable, and no precursor-product relationship existed between them. Fraction II had a number-average molecular weight of 15,800, corresponding to an average of 12 O-antigen repeats per molecule. In contrast, fraction I had a number-average molecular weight of 354,000, corresponding to an average of 404 O-antigen repeats per molecule. Before heat treatment, cells of E. coli O111 are poorly agglutinated by O-serum; although this indicates the presence of a capsule, the corresponding K-antigen was never detected. We conclude that fraction I, when present on the cell surface, inhibits agglutination of unheated cultures of E. coli O111 by O-serum because: (i) a variant strain which lacks fraction I was agglutinated by O-serum without prior heating; (ii) erythrocytes coated with purified fraction I behaved like bacteria containing fraction I in showing inhibition of O-serum agglutination; and (iii) heat treatment released fraction I and rendered bacterial cells agglutinable in O-serum.     

A protein with lectin activity in penetration glands of Diplostomum pseudospathaceum cercariae

Homogenates of Diplostomum pseudospathaceum cercariae agglutinated mouse erythrocytes. The haemagglutination could be inhibited by certain glycoconjugates containing beta-1,3- and beta-1,4-glycan chains and also by some simple saccharides. The most potent inhibitors were heparin and some other glycosaminoglycans, bacterial lipopolysaccharides, laminarin (a beta-1,3-glucan) and lactulose. After electrophoresis of cercarial proteins, a dominant double band appeared in the 22-24 kDa region of gels. On blots, this protein bound labelled laminarin and it was also one of the few proteins recognised by mouse antibodies raised against cercarial haemagglutinins. In addition, mouse polyclonal antibodies against the beta-1,3-glucan-binding protein bound exclusively to the 22-24 kDa region on Western blots. Histochemistry revealed strong binding of labelled laminarin to cercarial penetration glands; this reaction was fully blocked by unlabelled laminarin. Other labelled glycoconjugates such as heparin, hyaluronic acid and a bacterial lipopolysaccharide also bound to the glands. Immunohistochemistry confirmed the localisation of the beta-1,3-glucan-binding protein in penetration glands. Reaction of the cercarial protein with immunoglobulins from non-immunised mice was observed on both nitrocellulose membranes and histological sections; this could be blocked by laminarin in incubation buffers. We consider the cercarial haemagglutinin to be a lectin which is identical with the 22-24 kDa beta-1,3-glucan-binding protein. According to the binding specificity and localisation we speculate on a role of this lectin in cercarial penetration into the host, probably as a tissue recognition or antibody rendering factor.     

A genetic and monoclonal analysis of high-yielding reassortants of influenza A virus used for human vaccines

This paper describes two methods of analysis using monoclonal antibodies and RNA hybridization to characterize variation in the haemagglutinins of seven high-yielding influenza virus reassortants used for inactivated vaccine production. The results show that variants' were selected in producing these genetic reassortants. The haemagglutinins of two reassortants showed both antigenic and structural differences from their wild-type (wt) parents as detected by the two methods of analysis. These variants were more closely related to other subtype strains which had previously been differentiated from the wt parent by post-infection ferret sera and which also had amino acid sequence differences in antigenically significant sites on the HA 1 polypeptide chain of the haemagglutinin molecule. The haemagglutinins of four of the seven reassortants showed antigenic differences but no apparent structural differences from their wt parents. The haemagglutinin of only on reassortant was antigenically and structurally identical to its wt parent. The variants could not be reliably distinguished with hyperimmune rabbit serum or immune ferret serum to the wt parent virus. It is therefore important to use more discriminatory tests to identify influenza strains correctly. It is also essential for vaccine strains to be as completely characterized as possible. It is considered desirable that both methods of analysis be used to characterize influenza virus reassortant strains.     

Characterization of Acid-Cyclodextrin Glycosyltransferase of an Alkalophilic Bacillus sp.

Three kinds of lectins (LOL-I, II and III) were isolated from seeds of Lathyrus odoratus (sweet pea) in a homogenous form. The three fractions agglutinated the erythrocytes of laying hens, and the agglutination was strongly inhibited by α-methyl d-mannoside and d-mannose. However, they did not agglutinate those of the males and nonlyaing hens, differing from concanavalin A which showed a similar binding specificity for monosaccharide to LOL and agglutinated all types of erythrocytes derived from chicken in this study. LOL–I and II had a molecular weight of 52,000 and both consisted of two large (20,000 daltons) and two small subunits (6000 daltons). LOL–III had a molecular weight of 55,000, and its subunit structure was different from those of LOL–I and II. The amino acid compositions of the three fractions were very similar. They contained large amounts of aspartic acid, threonine, serine and valine, but no cysteine or methionine. Circular dichroism measurements indicated that β-structure was a major secondary structure of these lectins. The addition of α-methyl d-mannoside or d-mannose had significant effects on the CD spectra in the near-ultraviolet region, but no detectable change was observed in the 200~250 nm region. LOL–I had two binding sites for d-mannose, and the association constant was about 1000 liters per mol.     

Purification and characterization of CS2, a sialic acid-specific haemagglutinin of enterotoxigenic Escherichia coli.

CS2 fimbriae of enterotoxigenic Escherichia coli were purified and characterized. The surface haemagglutinins (fimbriae) were detached by sonication from a strain producing only the CS2 fimbriae. Isolation was carried out by gel filtration on a Sepharose 4B column. After depolymerization, the fimbriae subunits were purified on a Sephacryl S-300 column in 8.0 M-guanidinium chloride. From 1 litre of medium, 4-6 mg of purified fimbriae was obtained. We found that CS2 fimbriae were completely dissociated by saturated guanidinium chloride into subunits with a molecular mass of 16.5 kDa. CS2 fimbriae was sialic acid-specific, since sialic acids were the most potent inhibitors, and neuraminidase treatment of erythrocytes abolished haemagglutination. Both fimbriae and fimbrial subunits were found to bind to bovine erythrocytes. The binding of subunits to erythrocytes could be inhibited with low concentrations of sialyl-lactose.