Agglutination Of Human Erythrocytes Research Articles

The DraC usher in Dr fimbriae biogenesis of uropathogenic E. coli Dr+ strains

Biogenesis of Dr fimbriae encoded by the dra gene cluster of uropathogenic Escherichia coli strains requires the chaperone-usher pathway. This secretion system is based on two non-structural assembly components, the DraB periplasmic chaperone and DraC outer-membrane usher. The DraB controls the folding of DraE subunits, and DraC forms the assembly and secretion platform for polymerization of subunits in linear fibers. In this study, mutagenesis of the DraC N-terminus was undertaken to select residues critical for Dr fimbriae bioassembly. The DraC-F4A, DraC-C64, DraC-C100A and DraC-W142A significantly reduced the adhesive ability of E. coli strains. The biological activity of the DraC mutants as a assembly platform for Dr fimbriae polymerization was verified by agglutination of human erythrocytes and adhesion to DAF localized at the surface of CHO-DAF(+) and HeLa cells. The residue F4 of the DraC usher conserved among FGL and FGS chaperone-assembled adhesive organelles can be used to design pillicides blocking the biogenesis of Dr fimbriae. Because the draC and afaC-III genes share 100% identity the range of the virulence determinant inhibitors could also be extended to E. coli strains encoding afa-3 gene cluster. The investigations performed showed that the usher N-terminus plays an important role in biogenesis of complete fiber.

English

  The studies were carried out with plantlets of two indigenous cultivars of mulberry species Morus alba and Morus nigra grown in soil less cultures, where extracts from roots, shoots, leaves, and reproductive organs were determined for protein content, agglutination of human erythrocytes and the activity of the ammonium assimilatory enzyme glutamine synthetase. M. nigra and M. alba extracts contained potent phytoagglutinins in various tissues with highest contents in M.  nigra. The leaves and roots of both species of mulberry were used to determine the glutamine synthetase activity and high level of activity was found in both tissues. The glutamine synthetase enzymatic activity was higher in roots (1.37 µmol.gfw-1.min-1). The extract from all tissues of both species were used to determine the total protein contents and proteins found higher in the leaves (2.129 µg/gfw in M.  nigra and 0.973 µg/gfw in M.  alba).   Key words: Lectins, GS Assay, ammonium assimilation, mulberry species, phytoagglutanins.

Adhesion of Escherichia coli in urinary tract infection.

In individuals prone to urinary tract infections the intestine is colonized by E. Coli strains that possess a combination of properties determining virulence. Such an E. coli strain may colonize the vaginal and periurethral area and ascend the urinary tract. The ability to attach to the mucosal surface is thought to be essential for E. coli to colonize and to remain in the urinary tract. Most E. coli from patients with urinary tract infection show one or both of two adherence properties. One may depend on the recognition by type 1 fimbriae of mannose-containing residues in the urinary slime. It is measured as mannose-sensitive haemagglutination and is found on most E. coli strains. The second adherence property is detected as attachment to human urinary tract epithelial cells and as mannose-resistant agglutination of human erythrocytes. This may depend on the recognition of globo-series glycolipids in the epithelial cell surface. Possession of this adherence factor is strongly related to virulence. Most strains from patients with acute pyelonephritis and cystitis have this property but it is rare in strains from patients with asymptomatic bacteriuria and strains from normal faeces. Local antibodies may interfere with bacterial attachment, thus possibly preventing the colonization that precedes urinary tract infection or modifying an established infection. Vaginal antibodies are known to coat E. coli from the stools. Antibodies in the urine of patients with acute pyelonephritis inhibit attachment of the infecting strain to uroepithelial cells. Antibodies directed against several bacterial surface structures, for example O antigen and fimbriae, are likely to inhibit attachment by steric hindrance or agglutination. The role of antibodies in adhesion-mediating structure such as fimbriae in susceptibility to and the outcome of human urinary tract infection remains to be investigated.

Purification of a PHA-Like Chitin-binding Protein from Acacia farnesiana Seeds: A Time-dependent Oligomerization Protein

A lectin-like protein from the seeds of Acacia farnesiana was isolated from the albumin fraction, characterized, and sequenced by tandem mass spectrometry. The albumin fraction was extracted with 0.5 M NaCl, and the lectin-like protein of A. farnesiana (AFAL) was purified by ion-exchange chromatography (Mono-Q) followed by chromatofocusing. AFAL agglutinated rabbit erythrocytes and did not agglutinate human ABO erythrocytes either native or treated with proteolytic enzymes. In sodium dodecyl sulfate gel electrophoresis under reducing and nonreducing conditions, AFAL separated into two bands with a subunit molecular mass of 35 and 50 kDa. The homogeneity of purified protein was confirmed by chromatofocusing with a pI = 4.0 +/- 0.5. Molecular exclusion chromatography confirmed time-dependent oligomerization in AFAL, in accordance with mass spectrometry analysis, which confers an alteration in AFAL affinity for chitin. The protein sequence was obtained by a liquid chromatography quadrupole time-of-flight experiment and showed that AFAL has 68% and 63% sequence similarity with lectins of Phaseolus vulgaris and Dolichos biflorus, respectively.

Algae of Peter the Great Bay of the Sea of Japan as a source of lectins

Algae of Peter the Great Bay (Sea of Japan) were for the first time bioassayed as a source of lectins. From 28 algal species of three orders, only some extracts from brown (Phaeophyta) and red (Rhodophyta) seaweeds were found to cause agglutination of human erythrocytes. The hemagglutinating activity of extracts from three species of brown algae and the red alga Tichocarpus crinitus was caused by lectins; for a majority of extracts from the investigated algae, this activity was due to the presence of substances of non-lectin nature.

Immunopotentiating effect of a Fomitella fraxinea-derived lectin on chicken immunity and resistance to coccidiosis

This study reports a novel immunopotentiating effect of a lectin (FFrL) extracted from the mushroom Fomitella fraxinea on poultry cell-mediated immunity and poultry coccidiosis. We describe the extraction of FFrL, its in vitro mitogenic activity and in vivo protection against an oral challenge infection with Eimeria acervulina. When tested on several cell types, crude FFrL agglutinated mouse erythrocytes and thymocytes and various other cells including murine and human cell lines. However, crude FFrL did not agglutinate human erythrocytes. Crude FFrL showed a potent mitogenic activity on chicken splenic lymphocytes, and at lower concentrations it exerted stronger mitogenic activity than Concanavalin A, a well-known potent mitogen for lymphocytes. Further, FFrL significantly induced (P < 0.05) nitric oxide secretion in HD11 cells and suppressed (P < 0.05) RP9 tumor cell growth in a dose-dependent fashion. When injected into 18-d-old chicken embryos followed by a posthatch oral E. acervulina challenge infection, FFrL treatment significantly protected chickens against weight loss associated with coccidiosis (P < 0.05). Injecting embryos with FFrL also resulted in significant reduction in oocyst shedding as compared with the control saline-injected birds (P < 0.05). The results of this study demonstrate that FFrL can be an effective growth promoting and immunostimulating agent in poultry during coccidiosis.

Anti-human erythrocyte antibodies in horse-derived antivenoms used in the treatment of snakebite envenomations

This work examined the presence of antibodies reacting with human erythrocytes in horse-derived antivenoms used in the treatment of snakebite envenomations, and assessed the efficacy of various fractionation protocols in the elimination of agglutinating antibodies. A number of antivenoms produced by various fractionation protocols were tested for direct agglutination of human erythrocytes. Reactions were observed visually and microscopically, and an indirect anti-equine globulin test was also used. In addition, rabbits and mice were injected intravenously with antivenoms to observe possible intravascular hemolysis and erythrocyte sequestration. All tested antivenoms agglutinated human erythrocytes, albeit to different extent, and also gave a positive anti-globulin test. Agglutination was due to IgG(T) subclass of antibodies. Pepsin digestion of horse IgG, to obtain F(ab′) 2 fragments, reduced the direct agglutination, but not the indirect anti-globulin test. Ion-exchange chromatography of IgG in a strongly basic quaternary ammonium cellulose membrane abrogated direct agglutination and reduced the indirect anti-globulin test. Binding of antivenom antibodies to erythrocytes in vivo was demonstrated in rabbits, although there was no evidence of intravascular hemolysis or erythrocyte sequestration in rabbits and mice. It is concluded that anti-human erythrocyte antibodies are present in horse-derived antivenoms, and that fractionation of horse plasma by pepsin digestion, and especially by anion-exchange chromatography, reduces the titer of these antibodies. Our in vivo experimental results do not support a role for these antibodies in early adverse reactions occurring after antivenom administration.

Ribosome-inactivating proteins and other lectins from Adenia (Passifloraceae)

The caudices of 10 Adenia species contain galactose-binding lectins that were purified by affinity chromatography. All lectins but three agglutinate human erythrocytes. Six lectins consist of two unequal chains, which can be separated by reduction, and inhibit protein synthesis both by a rabbit reticulocyte lysate and by HeLa and Raji cells. The lectins from A. goetzii, A. lanceolata and A. stenodactyla had the highest cytotoxicity, inhibiting cell protein synthesis with IC 50s (concentration inhibiting by 50%) below 0.1 ng/ml, and deadenylate DNA, thus being type 2 ribosome-inactivating proteins.

Crystallization and preliminary X-ray characterization of a lectin from Cicer arietinum (chickpea).

The lectin isolated from mature seeds of Cicer arietinum (CAL) agglutinates pronase-treated rabbit and human erythrocytes and its haemagglutination activity is inhibited by fetuin and desialated fetuin but not by simple monosaccharides or oligosaccharides. The purified lectin is a dimer of molecular weight 43,000 Da composed of two identical subunits (MW 21,500), as confirmed by SDS-PAGE. The lectin has been crystallized using the hanging-drop vapour-diffusion method at 295 K over a well solution containing 0.2 M sodium acetate, 0.1 M sodium phosphate buffer pH 6.5 and 14%(w/v) polyethylene glycol 8000. The triangular prism-shaped crystals belong to space group R3 and have unit-cell parameters a = b = 81.2, c = 69.4 A. The diffraction data are 93.8% complete to 2.3 A Bragg spacing with an Rmerge of 0.103.

Mannose-specific plant lectins from the Amaryllidaceae family qualify as efficient microbicides for prevention of human immunodeficiency virus infection.

The plant lectins derived from Galanthus nivalis (Snowdrop) (GNA) and Hippeastrum hybrid (Amaryllis) (HHA) selectively inhibited a wide variety of human immunodeficiency virus type 1 (HIV-1) and HIV-2 strains and clinical (CXCR4- and CCR5-using) isolates in different cell types. They also efficiently inhibited infection of T lymphocytes by a variety of mutant virus strains. GNA and HHA markedly prevented syncytium formation between persistently infected HUT-78/HIV cells and uninfected T lymphocytes. The plant lectins did not measurably affect the antiviral activity of other clinically approved anti-HIV drugs used in the clinic when combined with these drugs. Short exposure of the lectins to cell-free virus particles or persistently HIV-infected HUT-78 cells markedly decreased HIV infectivity and increased the protective (microbicidal) activity of the plant lectins. Flow cytometric analysis and monoclonal antibody binding studies and a PCR-based assay revealed that GNA and HHA do not interfere with CD4, CXCR4, CCR5, and DC-SIGN and do not specifically bind with the membrane of uninfected cells. Instead, GNA and HHA likely interrupt the virus entry process by interfering with the virus envelope glycoprotein. HHA and GNA are odorless, colorless, and tasteless, and they are not cytotoxic, antimetabolically active, or mitogenic to human primary T lymphocytes at concentrations that exceed their antivirally active concentrations by 2 to 3 orders of magnitude. GNA and HHA proved stable at high temperature (50 degrees C) and low pH (5.0) for prolonged time periods and can be easily formulated in gel preparations for microbicidal use; they did not agglutinate human erythrocytes and were not toxic to mice when administered intravenously.

Specificity of human anti-NOR antibodies, a distinct species of "natural" anti-alpha-galactosyl antibodies.

Natural anti-NOR antibodies are common in human sera and agglutinate human erythrocytes of a rare NOR phenotype. The NOR phenotype-related antigens are unique neutral glycosphingolipids recognized by these antibodies and Griffonia simplicifolia IB4 isolectin (GSL-IB4). The oligosaccharide chains of NOR glycolipids are terminated by Galalpha1-4GalNAcbeta1-3Galalpha units. To characterize the specificity of anti-NOR antibodies and compare it with specificities of GSL-IB4 and known anti-Galalpha1,3Gal antibodies, alpha-galactosylated saccharides and saccharide-polyacrylamide conjugates were used. New synthetic oligosaccharides, corresponding to the terminal di- and trisaccharide sequence of NOR glycolipids and the conjugate of the NOR-tri with HSA were included. These compounds were tested by microtiter plate ELISA and hemagglutination inhibition. Anti-NOR antibodies reacted most strongly with Galalpha1-4GalNAcbeta1-3Gal (NOR-tri), and over 100 times less strongly with Galalpha1-4GalNAc (NOR-di). The antibodies reacted also with Galalpha1-4Gal and Galalpha1-4Galbeta1-4GlcNAc, similarly as with NOR-di but not with other tested compounds. In turn, anti-Galalpha1,3Gal antibodies reacted most strongly with Galalpha1-3Gal and were very weakly inhibited by the NOR-related oligosaccharides (weaker than by galactose), and NOR-tri was less active than NOR-di. GSL-IB4 reacted with all tested alpha-galactosylated saccharides and conjugates, including the similarly active NOR-tri and NOR-di. These results showed that anti-NOR represent a new species of anti-alpha-galactosyl antibodies with high affinity for the Galalpha1-4GalNAcbeta1-3Gal sequence present in rare NOR erythrocytes.

Brucella sp. bind to sialic acid residues on human and animal red blood cells.

We report that Brucella abortus and Brucella melitensis agglutinate human (A+ and B+), hamster and rabbit erythrocytes, a heretofore undescribed feature in this genus. This activity was associated with a 29-kDa surface protein (SP29) that bound selectively to these erythrocytes and this binding was inhibited by rabbit anti-SP29 antibodies. Hemagglutination was inhibited by pretreatment of erythrocytes with neuraminidase and by preincubation of B. abortus with chondroitin sulfate, N-acetylneuraminic acid and N-acetylneuramin-lactose.

Brucella sp. bind to sialic acid residues on human and animal red blood cells

We report that Brucella abortus and Brucella melitensis agglutinate human (A+ and B+), hamster and rabbit erythrocytes, a heretofore undescribed feature in this genus. This activity was associated with a 29-kDa surface protein (SP29) that bound selectively to these erythrocytes and this binding was inhibited by rabbit anti-SP29 antibodies. Hemagglutination was inhibited by pretreatment of erythrocytes with neuraminidase and by preincubation of B. abortus with chondroitin sulfate, N-acetylneuraminic acid and N-acetylneuramin-lactose.

Inhibition of Helicobacter pylori adherence by a peptide derived from neuraminyl lactose binding adhesin.

Helicobacterpylori, like many other gut colonizing bacteria, binds to sialic acid rich macromolecules present on the gastric epithelium. NLBH (neuraminyl lactose binding haemagglutinin) a 32 kDa adhesin located on the surface of H. pylori has been shown to have specific affinity towards NeuAcalpha2,3Galbeta1,4Gluc(3'SL). This sialic acid moiety is over-expressed in an atrophic stomach undergoing parietal cell depletion. Antibodies against a lysine rich peptide fragment of NLBH inhibit agglutination of human erythrocytes. This lysine rich sequence from NLBH was proposed to be the receptor-binding site. In order to elucidate the binding of NLBH to gastric epithelium, a peptide (D-P-K-R-T-I-Q-K-K-S) was synthesized. A series of experiments were performed involving adherence inhibition assays, 2D-NMR, molecular modelling and measurement of modulation in acid secretion. Results indicated that the peptide fragment could be involved in receptor recognition, which is important for the binding of H. pylori to gastric epithelium. The binding is possibly through hydrogen bonding. Two lysines and a threonine residue seem to be within the hydrogen bonding distance of NeuAcalpha2,3Galbeta1,4Gluc. Further, in vitro assays were performed to evaluate the role of the peptide on acid secretion by parietal cells isolated from human fundal biopsies. Interestingly, the peptide increases acid secretion only in H. pylori negative and in treated patients but not in H. pylori positive patients. This highlights the role of NLBH in acid secretion and could be of some consequence in the prognosis of the disease.

A conserved residue in the tip proteins of CS1 and CFA/I pili of enterotoxigenic Escherichia coli that is essential for adherence.

Enterotoxigenic Escherichia coli associated with human diarrheal disease utilize any of a limited group of serologically distinguishable pili for attachment to intestinal cells. These include CS1 and CFA/I pili. We show here that chemical modification of arginyl residues in CS1 pili abolishes CS1-mediated agglutination of bovine erythrocytes, which serves as a model system for attachment. Alanine substitution of the single arginyl residue in CooA, the major pilin, had no effect on the assembly of pili or on hemagglutination. In contrast, substitution of alanine for R181 in CooD, the minor pilin associated with the pilus tip, abolished hemagglutination, and substitution of R20 reduced hemagglutination. Neither of these substitutions affected CS1 pilus assembly. This shows that CooD is essential for CS1-mediated attachment and identifies specific residues that are involved in receptor binding but not in pilus assembly. In addition to mediating agglutination of bovine erythrocytes, CFA/I also mediates agglutination of human erythrocytes. Substitution of R181 by alanine in the CooD homolog, CfaE, abolished both of these reactions. We conclude that the same region of the pilus tip protein is involved in adherence of CS1 and CFA/I pili, although their receptor specificities differ. This suggests that the region of the pilus tip adhesin protein that includes R181 might be an appropriate target for therapeutic intervention or for a vaccine to protect against human diarrhea caused by enterotoxigenic E. coli strains that have serologically different pili.

Purification and characterization of a cell-surface lectin (Lectin II) from Agrobacterium radiobacter NCIM 2443.

A lectin was isolated from Agrobacterium radiobacter cell surface and purified. It is a monomer of 40 kDa as shown by SDS-PAGE. The lectin has a pI of 9.15 and amino acid composition of the lectin shows that 44% of the amino acids are hydrophobic. The lectin agglutinates rabbit erythrocytes but does not agglutinate human erythrocytes. It does not show specificity for monosaccharides except for D-glucosamine. Fetuin and its N-linked glycopeptide also inhibit the activity of the lectin but greater inhibition is shown by locust bean gum and Nicotiana tobaccum (tobacco) tissue extracts.

Purification and characterization of a lectin from seeds and cotyledonary callus of Zizyphus mauritiana

A lectin from Zizyphus mauritiana lamk. has been purified from the 25-50% (NH4)2SO4 fraction of crude seed and cotyledonary leaf callus extracts. The lectins purified from the two sources had the same structure and properties. Upon specific adsorption on Sephadex G-100, the lectin (ZML) could be displaced with 0.1 M D-glucose. ZML yielded a single band corresponding to a Mr of 66 kDa both in the absence and presence of β-mercaptoethanol on native- as well as in SDS-PAGE. It is thermostable but pH sensitive and agglutinates human erythrocytes only. Lectin activity could also be detected in the cotyledons, leaf, and stem, and in their cultures, as well as in in vitro regenerants. Cotyledons, cotyledonary leaf and its callus showed much higher lectin activity than seeds.

Molecular characterization of hemagglutination domains on the fibers of subgenus D adenoviruses.

The adenovirus fiber mediates the agglutination of erythrocytes. Based on differential hemagglutinating properties, subgenus D adenoviruses can be subdivided into clusters DI, DII, and DIII. While subgenus DI adenoviruses agglutinate rat and human erythrocytes, DII adenoviruses simply agglutinate rat erythrocytes and DIII adenoviruses display no or only weak rat erythrocyte agglutination. Amino acid sequence comparisons revealed distinct domains on the fiber knob which could be involved in hemagglutination. In order to localize and characterize the domains responsible for the interaction with rat and human erythrocytes, potential hemagglutination domains of the adenovirus type 9 (Ad9) (subgenus DI) fiber knob were introduced into Ad17 (subgenus DII) and Ad28 (subgenus DIII) fiber knobs by primer-directed mutagenesis. Furthermore, rat erythrocyte hemagglutination domains were also introduced into the Ad3 (subgenus B) fiber knob, which only agglutinated monkey erythrocytes. Altogether, 27 chimeric and mutated fiber proteins were expressed in Escherichia coli and subsequently tested for hemagglutination activity. The hemagglutination tests revealed that at least two domains can mediate the agglutination of rat erythrocytes. While one domain is located on the GH loop, the other domain extends from the C beta strand to the CD loop. The domain on the GH loop was partially conserved in all adenoviruses showing an incomplete hemagglutination pattern with rat erythrocytes. The domains involved in the agglutination of human erythrocytes are located on the CD and HI loops of the subgenus DI fiber knob.

Adhesive property of toxin-coregulated pilus of Vibrio cholerae O1.

The adhesive property of toxin-coregulated pilus (TCP) to the human intestine jejunum), and whether or not TCP mediates the adhesion of Vibrio cholerae 395 organisms to the intestinal epithelium were investigated using visually proving methods. The purified TCP did not agglutinate human erythrocytes nor adhere to the surface of human intestinal epithelium. V. cholerae 395 adhered to the epithelium, but the adhesion was not inhibited by blocking the pili with the Fab fraction of anti-TCP IgG. The organisms adhered to the intestine treated with purified TCP in advance, as well as to the intact intestine. These findings suggest that TCP is not involved in the adhesion of these organisms to the intestinal epithelium.

Antirotaviral activity of milk proteins: lactoferrin prevents rotavirus infection in the enterocyte-like cell line HT-29.

Different milk proteins were analyzed for their inhibitory effect on either rotavirus-mediated agglutination of human erythrocytes or rotavirus infection of the human enterocyte-like cell line HT-29. Proteins investigated were alpha-lactalbumin, beta-lactoglobulin, apo-lactoferrin, and Fe(3+)-lactoferrin, and their antiviral action was compared with the activity of mucin, a milk glycoprotein known to affect rotavirus infection. Results obtained demonstrated that beta-lactoglobulin, apo- and Fe(3+)-lactoferrin are able to inhibit the replication of rotavirus in a dose-dependent manner, apo-lactoferrin being the most active. It was shown that apo-lactoferrin hinders virus attachment to cell receptors since it is able to bind the viral particles and to prevent both rotavirus haemagglutination and viral binding to susceptible cells. Moreover, this protein markedly inhibited rotavirus antigen synthesis and yield in HT-29 cells when added during the viral adsorption step or when it was present in the first hours of infection, suggesting that this protein interferes with the early phases of rotavirus infection.