Pili of an Aeromonas hydrophila strain as a possible colonization factor

Aeromonas hydrophila Ae6 had 2 morphologically distinctive kinds of pili. One appeared rigid and straight with a diameter of 9 nm (R-pili). The other appeared wavy and flexible with a diameter of 7 nm (W-pili). W-pili were very few on the cell as compared with R-pili. In this study, W-pili were purified and characterized. The pili consisted of a subunit protein with a molecular weight of 21 kDa as estimated by SDS-PAGE. There was no immunological cross-reaction between W-pili and other cellular components. The strain Ae6 and its purified W-pili adhered to human and rabbit intestine and agglutinated human and rabbit erythrocytes. Organisms pretreated with the Fab fraction of anti-pilus antibody failed to adhere to the intestine. Pretreatment of intestine with purified W-pili blocked adherence of the organisms to the intestine. These results suggest that the W-pili are the colonization factor of A. hydrophila Ae6.     

Characterization of Aeromonas sobria TAP13 pili: a possible new colonization factor.

Pili of Aeromonas sobria TAP13 were purified and characterized. The molecular mass of the pilin was estimated to be about 23 kDa by SDS-PAGE. The TAP13 pili were immunologically different from A. sobria Ae1 pili and A. hydrophila Ae6 W pili as previously reported, nevertheless all three had indistinguishable morphology and shared a high degree of homology in their N-terminal amino acid sequences. Strain TAP13 and its purified pili did not agglutinate human, rabbit or sheep erythrocytes. However, they adhered to rabbit intestine. Organisms pretreated with the Fab fraction of an antipilus antibody failed to adhere to rabbit intestine, and organisms did not adhere to intestine pretreated with purified pili. These results suggest that the pili are a colonization factor of A. sobria TAP13 for the rabbit intestine.     

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.     

Properties of pili from Escherichia coli SS142 that mediate mannose-resistant adhesion to mammalian cells

We isolated pili from Escherichia coli SS142. These pili had a diameter of 6 nm and an average length of 400 nm. They were composed of subunits with a molecular weight of 18,000. Their amino acid composition was determined; methionine and proline were not detected. The isolated pili retained mannose-resistant hemagglutinating activity. Proteolytic digestion and glutaraldehyde fixation led to partial or complete loss of the hemagglutinating activity of the pili without causing any detectable damage to their supramolecular structure, which was only disintegrated by treatment with hot sodium dodecyl sulfate. The hemagglutinating activity of E. coli SS142 was inhibited by the glycoproteins fetuin and Tamm-Horsfall protein, as well as by the glycolipids phytyl lactoside, dansyl-sphingosine lactoside, and digalactosyl diglyceride. Isolated pili inhibited the adhesion of the homologous strain E. coli SS142 to Intestine 407 cell monolayers, but did not inhibit the adhesion of E. coli strain B-413, B-506, or 2699. This indicates that E. coli SS142 binds to a receptor different from those recognized by the other strains and that mannose-resistant adhesion to tissue culture cells can be classified into different subtypes.     

Characterization of type 1 pili of Salmonella typhimurium LT2.

Type 1 pili from Salmonella typhimurium LT2 were purified and characterized. The pilus filaments were 6 nm in diameter and over 1 microns long. Estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of the pilin was 21,000. The isoelectric point of the filament was 4.1. Hydrophobic amino acids comprised 40.3% of the total amino acids of the pilin, which contained more proline, serine, and lysine than reported for the type 1 pilin of Escherichia coli. Purified pili agglutinated both horse and chicken erythrocytes and yeast cells but not bovine, sheep, or human erythrocytes. Horse erythrocyte agglutination was inhibited at lower concentrations by alpha-methyl-D-mannoside than by yeast mannane and D-fructose. Agglutination was not affected by D-galactose or sucrose. Results of the present study confirm the role of type 1 pili as Salmonella hemagglutinins and show chemical differences between the type 1 pili of S. typhimurium and E. coli.     

Purification and characterization of a pore-forming protein from the marine sponge Tethya lyncurium.

A pore-forming protein was detected and purified for the first time from a marine sponge (Tethya lyncurium). The purified protein has a polypeptide molecular mass of 21 kDa and a pI of 6.4. Tethya pore-forming protein (also called Tethya hemolysin) rapidly lysed erythrocytes from a variety of organisms. After binding to target membranes, the hemolysin resisted elution with EDTA, salt or solutions of low ionic strength and hence resembled an integral membrane protein. Erythrocytes could be protected from hemolysis induced by Tethya hemolysin by addition of 30 mM dextran 4 (4-6 kDa; equivalent hydrodynamic diffusion radius, 1.75-2.3 nm) to the extracellular medium, but not by addition of uncharged molecules of smaller size [sucrose, raffinose and poly(ethylene glycol) 1550; equivalent hydrodynamic diffusion radii, 0.46, 0.57 and 1.2 nm, respectively]. This result indicates that hemolysin is able to form stable transmembrane pores with an effective diameter of about 2-3 nm. Treatment of osmotically protected erythrocytes with Tethya hemolysin caused a rapid efflux of intracellular K+ and ATP, and a rapid influx of extracellularly added Ca2+ and sucrose. In negative-staining electron microscopy, target erythrocyte membranes exposed to purified Tethya hemolysin displayed ultrastructural lesions but without visible pores.     

Purification,characterization and molecular cloning of Vibrio fluvialis hemolysin

Hemolysin of Vibrio fluvialis (VFH) was purified from culture supernatants by ammonium sulfate precipitation and successive column chromatographies on DEAE-cellulose and Mono-Q. N-terminal amino acid sequences of the purified VFH were determined. The purified protein exhibited hemolytic activity on many mammalian erythrocytes with rabbit erythrocytes being the most sensitive to VFH. Activity of the native VFH was inhibited by the addition of Zn2+, Ni2+, Cd2+ and Cu2+ ions at low concentrations. Pores formed on rabbit erythrocytes were approximately 2.8-3.7 nm in diameter, as demonstrated by osmotic protection assay. Nucleotide sequence analysis of the vfh gene revealed an open reading frame (ORF) consisting of 2200 bp which encodes a protein of 740 amino acids with a molecular weight of 82 kDa. Molecular weight of the purified VFH was estimated to be 79 kDa by SDS-PAGE and N-terminal amino acid sequence revealed that the 82 kDa prehemolysin is synthesized in the cytoplasm and is then secreted into the extracellular environment as the 79 kDa mature hemolysin after cleavage of 25 N-terminal amino acids. Deletion of 70 amino acids from the C-terminus exhibited a smaller hemolytic activity, while deletion of 148 C-terminal amino acids prevented hemolytic activity.     

The role of pili in colonization of the rabbit intestine by Vibrio parahaemolyticus Na2.

Vibrio parahaemolyticus Na2 and its pili were studied in relation to intestinal colonization. The isolated pili were adhesive to the intestinal epithelium. The adhesion of the organisms was blocked by masking the epithelial receptor with the purified pili, or by treating the organisms with anti-pilus antibody (Fab fraction). These results suggest that the pili of V. parahaemolyticus Na2 play an important role in the adhesion of the organisms to the rabbit intestine.     

Isolation and characterization of pili (fimbriae) from Synechocystis CB3

Abstract Synechocystis CB3, isolated from the Gulf of Finland, was covered by innumerable flexible pili (fimbriae) with an approximate diameter of 6 nm. The Synechocystis CB3 pili had a tendency to attach side by side thus forming characteristic bundles consisting of several dozens of individual pilus filaments. The Synechocystis CB3 pili were isolated and purified using deoxycholate and urea, and found to be very similar to other bacterial pili in their subunit M _r (21 kDa) and amino acid composition (46% hydrophobic amino acids). The amino acid analysis revealed also small amounts of glucosamine in the pilus preparation.     

Biochemical studies on pili isolated from Pseudomonas aeruginosa strain PAO.

Pseudomonas aeruginosa strains PAO and PAK bear polar pili which are flexible filaments having a diameter of 6 nm and an average length of 2500 nm. Both types of pili are retractile and promote infection by a number of bacteriophages. The present communication describes the partial biochemical characterization of PAO pili isolated from a multipiliated nonretractile mutant of PAO. The observed properties are compared to those of PAK pili which were characterized previously. PAO pili were found to contain a single polypeptide subunit of 18 700 daltons. This is similar to PAK pili which contain a single polypeptide of 18 100 daltons. The amino acid composition of PAO pilin was also similar to that of PAK pilin. Neither protein contained phosphate or carbohydrate residues and both were found to contain N-methylphenylalanine at the amino terminus. Sequencing of 20 amino acid residues at the amino terminal end of PAO pilin revealed the sequence to be identical with that of PAK pilin, while tryptic peptide analyses of PAO and PAK pilin indicated that the two proteins probably contain a number of homologous regions within the polypeptide. It was concluded that PAO and PAK pili were closely related structures.     

Phage Folac: an Folac plasmid-dependent bacteriophage

By enriching sewage with Escherichia coli or Salmonella typhimurium strains harbouring the plasmid EDP208, a constitutive pilus-producing derivative of plasmid F olac, a phage was isolated which plated on these two organisms but not on isogenic strains without the plasmid. The phage was named F olac; it has a hexagonal outline with a diameter of 28 nm, contained RNA, was resistant to chloroform, and probably adsorbed preferentially to the sides of EDP208 pili very near the tip. Phage multiplication could be demonstrated on E. coli or S. typhimurium strains carrying the plasmid F olac, but an increase in titre did not occur on E. coli strains carrying plasmids of the F complex. Results of phage multiplication experiments on strains carrying the depressed pilus-producing plasmids R71 or TP224-Tc, which determine pili serologically related to those of EDP208, were inconclusive. Phage F olac was found to be serologically related to phage UA-6, another isolate specific for EDP208.     

Nanoscale characterization and determination of adhesion forces of Pseudomonas aeruginosa pili by using atomic force microscopy

Type IV pili play an important role in bacterial adhesion, motility, and biofilm formation. Here we present high-resolution atomic force microscopy (AFM) images of type IV pili from Pseudomonas aeruginosa bacteria. An individual pilus ranges in length from 0.5 to 7 microm and has a diameter from 4 to 6 nm, although often, pili bundles in which the individual filaments differed in both length and diameter were seen. By attaching bacteria to AFM tips, it was possible to fasten the bacteria to mica surfaces by pili tethers. Force spectra of tethered pili gave rupture forces of 95 pN. The slopes of force curves close to the rupture force were nearly linear but showed little variation with pilus length. Furthermore, force curves could not be fitted with wormlike-chain polymer stretch models when using realistic persistence lengths for pili. The observation that the slopes near rupture did not depend on the pili length suggests that they do not represent elastic properties of the pili. It is possible that this region of the force curves is determined by an elastic element that is part of the bacterial wall, although further experiments are needed to confirm this.     

Studies on novel pili from Shigella flexneri. I. Detection of pili and hemagglutination activity.

Pili were detected using electron microscopy in clinical isolates of Shigella flexneri which had been continuously subcultivated in liquid media. Morphologically, the pili appeared as thin, flexible, cylindrical structures of up to 2-5 microns in length and about 3-5 nm in diameter. Two strains showed mannose-resistant (MR) hemagglutination to fresh fowl erythrocytes (type 4), and one to tannic acid-treated horse erythrocyte (type 3) pili. These pili are novel and different from the mannose-sensitive (MS) type 1 pili described by Duguid and Gillies.     

Biochemical characteristics of Eiseniapore, a pore-forming protein in the coelomic fluid of earthworms.

The cytolytic protein Eiseniapore (38 kDa) from coelomic fluid of the earthworm Eisenia fetida functionally requires sphingomyelin as revealed by using mammalian erythrocytes and phospholipid vesicles. The effects of ions, glycoproteins and phospholipids were investigated for the two-step Eiseniapore action mode, binding and pore formation in different assays. Eiseniapore lysis is activated by thiol groups but inhibited by metal ions. Eiseniapore binding to target membranes is inhibited by Eiseniapore-regulating factor, vitronectin, heparin and lysophosphatidylcholine. Ca2+ and Mg2+ were found to be not necessary for membrane binding or lytic activity. Sphingomyelin was essential for Eiseniapore-induced leakage of liposomes. We describe a cytolytic protein/toxin in Eiseniapore which differs from the established classification; it can be activated by thiol groups and is inhibited by sphingomyelin. Electron microscopy of erythrocyte membranes confirmed ring-shaped structures (pores) with a central channel with outer (10 nm) and inner (3 nm) diameters as shown previously [Lange, S., Nüssler, F., Kauschke, E., Lutsch, G., Cooper, E.L. & Herrmann, A. (1997) J. Biol. Chem. 272, 20 884-20 892] using artificial membranes. Functional evidence of pore formation by Eiseniapore was revealed as protection of lysis by carbohydrates occurred at an effective diameter above 3 nm. From these results, we suggest a plausible explanation for the mechanism by which components of the earthworm's immune system destroy non-self components.     

Purification and characterization of Vibrio cholerae O139 fimbriae

Abstract A Vibrio cholerae O139 (strain Al-1841) isolated from a patient with a cholera-like disease in Bangladesh predominantly produced new curved, wavy fimbriae (Al-1841 fimbriae) and small numbers of previously reported V. cholerae non-O1 S7-like pili. The former was purified and characterized. The molecular mass of the Al-1841 fimbrial subunit was less than 2.5 kDa, and it was immunologically different from that of V. cholerae non-O1 S7 pili. This novel fimbrial antigen was detected in all 182 Gram-negative strains from five genera tested but was absent from the Gram-positive bacteria tested. The purified Al-1841 fimbriae did not agglutinate human or rabbit erythrocytes.     

Assembly of Staphylococcus aureus leukocidin into a pore-forming ring-shaped oligomer on human polymorphonuclear leukocytes and rabbit erythrocytes.

Staphylococcal leukocidin consists of two separate proteins, LukS and LukF, which cooperatively lyse human and rabbit polymorphonuclear leukocytes and rabbit erythrocytes. Here we studied the pore-forming properties of leukocidin and the molecular architecture of the leukocidin pore. (1) Leukocidin caused an efflux of potassium ions from rabbit erythrocytes and swelling of the cells before hemolysis. However, ultimate lysis of the toxin-treated swollen erythrocytes did not occur when polyethylene glycols with hydrodynamic diameters of > or = 2.1 nm were present in the extracellular space. (2) Electron microscopy showed the presence of a ring-shaped structure with outer and inner diameters of 9 and 3 nm, respectively, on leukocidin-treated human polymorphonuclear leukocytes and rabbit erythrocytes. (3) Ring-shaped structures of the same dimensions were isolated from the target cells, and they contained LukS and LukF in a molar ratio of 1:1. (4) A single ring-shaped toxin complex had a molecular size of 205 kDa. These results indicated that LukS and LukF assemble into a ring-shaped oligomer of approximately 200 kDa on the target cells, forming a membrane pore with a functional diameter of approximately 2 nm.     

Helical structure of P pili from Escherichia coli. Evidence from X-ray fiber diffraction and scanning transmission electron microscopy.

The structure of the P pili from Escherichia coli has been studied using X-ray fiber diffraction and scanning transmission electron microscopy (STEM). Analysis of the fiber diffraction data indicates that the pili are constituted largely of structural subunits arranged helically with approximately 33 subunits in 10 turns in an axial repeat of 244.5 +/- 1.8 A. Radial electron density distributions calculated from equatorial diffraction data and STEM data indicate that the pili are about 65 A in diameter with a small central cavity roughly 15 A across. The principal protein component of the pili is PapA, which has a molecular weight of 16.5 kDa. Assuming that each subunit consists of a single PapA molecule, the mass-per-unit-length of the pili predicted from the X-ray data is 2.23 kDa/A. Measurements of mass-per-unit-length were also made through the analysis of STEM images. These measurements indicate a value of 2.13 +/- 0.14 kDa/A. STEM images demonstrated the presence of thin, thread-like structures emerging from the ends of pili and spanning breaks in the pili structure. These structures, which have been observed under other conditions, have been termed fibrillae. In the STEM images the fibrillae appear about 20 A in diameter. The mass-per-unit-length of the fibrillae was estimated using the STEM data to be 0.4 kDa/A. These data are consistent with the fibrillae representing an unwound or unraveled form of the pili proteins overstretched to about five times the length they would have in the intact pili.     

Pleurotolysin, a novel sphingomyelin-specific two-component cytolysin from the edible mushroom Pleurotus ostreatus, assembles into a transmembrane pore complex

Self-assembling, pore-forming cytolysins are illustrative molecules for the study of the assembly and membrane insertion of transmembrane pores. Here we purified pleurotolysin, a novel sphingomyelin-specific two-component cytolysin from the basidiocarps of Pleurotus ostreatus and studied the pore-forming properties of the cytolysin. Pleurotolysin consisted of non-associated A (17 kDa) and B (59 kDa) components, which cooperatively caused leakage of potassium ions from human erythrocytes and swelling of the cells at nanomolar concentrations, leading to colloid-osmotic hemolysis. Hemolytic assays in the presence of poly(ethylene glycol)s with different hydrodynamic diameters suggested that pleurotolysin formed membrane pores with a functional diameter of 3.8-5 nm. Pleurotolysin-induced lysis of human erythrocytes was specifically inhibited by the addition of sphingomyelin-cholesterol liposomes to the extracellular space. Pleurotolysin A specifically bound to sphingomyelin-cholesterol liposomes and caused leakage of the internal carboxyfluorescein in concert with pleurotolysin B. Experiments including solubilization of pleurotolysin-treated erythrocytes with 2% (w/v) SDS at 25 degrees C and SDS-polyacrylamide gel electrophoresis/Western immunoblotting showed that pleurotolysin A and B bound to human erythrocytes in this sequence and assembled into an SDS-stable, 700-kDa complex. Ring-shaped structures with outer and inner diameters of 14 and 7 nm, respectively, were isolated from the solubilized erythrocyte membranes by a sucrose gradient centrifugation. Pleurotolysin A and B formed an SDS-stable, ring-shaped complex of the same dimensions on sphingomyelin-cholesterol liposomes as well.     

Pili of Vibrio cholerae Widely Distributed in Serogroup O1 Strains

The distribution of Vibrio cholerae O1 pili consisting of 16 kDa subunit protein (16K-pili) was examined by Western blotting, using 211 strains from various origins and specific anti-16K-pili sera. The 16 kDa protein was detected in all 211 strains. The pili were purified from 3 El Tor and 3 classical strains, and characterized by hemagglutination and inhibition tests. All purified pili were hemagglutinative. However, the hemagglutinating activity of classical pili disappeared after exposure to 5 M urea and the agglutination induced by the classical pili was inhibited by D -mannose, alpha-methylmannoside, D -glucose and N-acetylglucosamine. On the contrary, El Tor pili were resistant to these sugars and urea.     

Monoclonal antibody to human IgG Fc receptors. Cross-linking of receptors induces lysosomal enzyme release and superoxide generation by neutrophils

The monoclonal antibody KuFc79 binds to a determinant on the Fc receptors (Fc gamma R) of human leukocytes. We examined the biologic effects of the interaction of this antibody with Fc gamma R on human neutrophils (PMNL). The univalent Fab fragment of KuFc79 inhibits the formation of rosettes with IgG-sensitized sheep erythrocytes by as much as 91.7%. In other experiments in which PMNL were washed after exposure to Fab of KuFc79, phagocytosis of IgG-sensitized sheep erythrocytes was inhibited by 36%. Fab fragments of other mouse IgG2b monoclonal proteins did not have these effects. When PMNL are exposed to coverslips coated with univalent Fab fragments of this antibody, the Fc gamma R are removed from the surface of the PMNL. Under these conditions, rosetting could be inhibited by 85.4%. We examined cross-linking of receptor bound monoclonal antibody or its Fab fragment by either Protein A or F(ab')2 of an anti-mouse Ig. As much as 31.7% of beta-glucuronidase, a marker for lysosomal enzymes, is specifically released by cross-linking the Fc gamma R on PMNL. The generation of O2- is also induced by specifically cross-linking Fc gamma R with Fab and anti-Fab. The data constitute the first formal demonstration that cross-linking of Fc gamma R on PMNL leads to enzyme release and superoxide generation.