Pili of Aeromonas hydrophila: purification, characterization, and biological role

Aeromonas hydrophila (Ae6) has 2 morphologically distinctive kinds of pili. One appeared rigid, channeled, and straight with a diameter of 9 nm (Ae6-R pili). The other looked flexible, wavy, and having helical structure with a diameter of 7 nm (Ae6-W pili). Ae6-R pili were purified and characterized. The pili consisted of a subunit protein with a molecular weight of 18 kDa as estimated by SDS-PAGE, and contained 42.3% hydrophobic amino acids and one cysteine residue. The pilus was solubilized to 18 kDa subunit protein by 2-mercaptoethanol, dithiothreitol, hydrochloric acid, or heating at 120 C for 5 min. The organism Ae6 was strongly adhesive to rabbit intestines as well as human intestines, and agglutinated erythrocytes. Anti-pili antibody (Fab fraction) did not block the adhesion. Purified Ae6-R pili did not adhere to the intestine or to the erythrocytes. However, the anti-pili Fab inhibited pellicle formation of the organisms cultured in broth, and also inhibited salt agglutination with ammonium sulfate. From these results, Ae6-R pili are not likely a colonization factor but probably play a role in the autoaggregation of the organisms.     

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.     

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.     

A new method for the extraction and purification of K99 pili from enterotoxigenic Escherichia coli and their characterization.

It was found that K99 pili from enterotoxigenic Escherichia coli (of bovine origin) could be extracted by treatment with 3M-KSCN solution. The K99 pili were purified by preparative isoelectric focusing to apparent homogeneity as judged by the presence of a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; the molecular weight of this component was calculated to be 12 600 +/- 300. This indicated that the K99 pili were composed of a single subunit. On analytical ultracentrifugation, a single boundary with an s20,w of 12.2 S at a concentration of 0.42 mg/ml was observed. The average length of purified pili at zero concentration was approx. 160 nm and the diameter was 7.4 +/- 0.6 nm. Amino acid analysis of the purified K99 pili revealed that sulphur-containing amino acids, cysteine and methionine, were absent. Aromatic amino acids, phenylalanine and tyrosine, previously reported to be absent [Isaacson (1977) Infect. Immun. 15. 272-279], constituted 7.14% of the total amino acid residues present. On immunoelectrophoresis, purified K99 pili migrated towards the cathode and caused mannose-resistant haemagglutination of horse, but not of sheep or guinea-pig, red blood cells. Pili from enterotoxigenic E. coli of porcine and human origin and from another bacterial species, namely Fusiformis nodosus, could also be extracted by the treatment of respective micro-organisms with 3 M-KSCN.     

Characterization of a pilus produced by Aeromonas hydrophila

A pilus produced by Aeromonas hydrophila was purified and partially characterized. The pilin monomers had an apparent molecular weight of 17,000. Agglutination studies indicated serological cross-reactivity in the pili of A. hydrophila strains. Presence of pili did not correlate with hydrophobicity or haemagglutinating ability of the bacteria.     

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.     

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.     

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.     

Purification and Characterization of Thin Pili of IncI1 Plasmids ColIb-P9 and R64: Formation of PilV-Specific Cell Aggregates by Type IV Pili

Thin pili of the closely related IncI1 plasmids ColIb-P9 and R64 are required only for liquid mating and belong to the type IV family of pili. They were sedimented by ultracentrifugation from culture medium in which Escherichia coli cells harboring ColIb-P9- or R64-derived plasmids had been grown, and then the pili were purified by CsCl density gradient centrifugation. In negatively stained thin pilus samples, long rods with a diameter of 6 nm, characteristic of type IV pili, were observed under an electron microscope. Gel electrophoretic analysis of purified ColIb-P9 thin pili indicated that thin pili consist of two kinds of proteins, pilin and the PilV protein. Pilin was demonstrated to be the product of the pilS gene. Pilin was first synthesized as a 22-kDa prepilin from the pilS gene and subsequently processed to a 19-kDa protein by the function of the pilU product. The N-terminal amino group of the processed protein was shown to be modified. The C-terminal segments of the pilV products vary among six or seven different types, as a result of shufflon DNA rearrangements of the pilV gene. These PilV proteins were revealed to comprise a minor component of thin pili. Formation of PilV-specific cell aggregates by ColIb-P9 and R64 thin pili was demonstrated and may play an important role in liquid mating.     

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.     

Hemagglutination by Neisseria meningitidis

The direct agglutination of erythrocytes by Neisseria meningitidis was studied as a marker for adherence. Hemagglutination (HA) was studied by slide test (5-min incubation) and by dilutions in microtitre plates (20-h incubation). Meningococci that were freshly isolated from subjects agglutinated only human cells by slide test but human, dog, rabbit, guinea pig, and rat cells were agglutinated in the microtitre system. Newly isolated strains were piliated and HA positive but pili were lost after 10 passages on agar, and bacteria became HA negative. HA could be maintained by "affinity culturing," which selected markedly adhesive bacteria: erythrocytes with adherent meningococci were isolated and cultured on agar. This procedure was repeated daily. HA titres were unaffected by mannose but were reduced by sonic disruption, trypsinization, ultraviolet irradiation, heating (65 degrees C), and formaldehyde. Encapsulated (serogroupable) bacteria had low HA titres compared with nongroupable strains, and purified capsular polysaccharides A and C inhibited HA. Meningococcal HA is probably mediated by pili and modified by other factors such as encapsulation. Colonial variation was not a reliable indicator of piliation, and HA is best used for this purpose.     

Purification and characterization of the CS2 pili of colonization factor antigen II produced by human enterotoxigenic Escherichia coli

A subtype (CS2) of the colonization factor antigen II (CFA/II) of human enterotoxigenic Escherichia coli (ETEC) was studied. Analysis revealed that CS2-possessing ETEC was predominant among isolates from traveller's diarrhea at Osaka, Japan. TH61 pili produced by a clinical strain (TH61) were purified as a native form to homogeneity by zone electrophoresis and successive column chromatographies on Sepharose 4B and Phenyl-Sepharose CL-4B. It was demonstrated by immunogold staining technique and bacterial agglutination test that antisera against the purified pili of strain TH61 recognized pili of both strain TH61 and strain #C91f, a control strain possessing only CS2 pili. This suggests that TH61 pili purified in this study are CS2 pili. Subunit (pilin) of the purified pili has a molecular weight of about 16,000. Strains bearing CS2 could attach to human jejunal epithelial cells, and this attachment was inhibited by pretreating the enterocytes with purified pili. These indicate that CS2 pili are a factor responsible for attachment of ETEC bearing CS2 to human intestinal cells.     

Pili mediated agglutination of Serratia marcescens in human urine

Of 51 strains of Serratia marcescens isolated from patients with urinary or respiratory tract infections, 35 agglutinated in human urine. The agglutinating strains possessed numerous pili which were morphologically distinct from common pili or type I pili. The diameter of the pili was 3 nm and the average length was 0.3 micrometer. Electron microscopic examination showed that 80% or more of the cells of the agglutinating strains and 0 to 8% of the cells of the nonagglutinating strains were piliated. When an agglutinating strain was heated at 55 C for 10 min, it lost its agglutinating capacity and concomitantly its pili. These results suggest that the agglutination might occur because of interactions between the pili and some factors in human urine. The urinary slime appears to contain these agglutinating factors.     

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.     

A prostaglandin omega-hydroxylase cytochrome P-450 (P-450PG-omega) purified from lungs of pregnant rabbits

Cytochrome P-450-dependent prostaglandin omega-hydroxylation is induced over 100-fold during late gestation in rabbit pulmonary microsomes (Powell, W.S. (1978) J. Biol. Chem. 253, 6711-6716). Purification of cytochromes P-450 from lung microsomes of pregnant rabbits yielded three fractions. Two of these fractions correspond to rabbit lung P-450I (LM2) and P-450II (LM5), which together constitute 70-97% of total cytochrome P-450 in lung microsomes from nonpregnant rabbits. The third form, which we designate rabbit cytochrome P-450PG-omega, regioselectively hydroxylates prostaglandins at the omega-position in reconstituted systems with a turnover of 1-5 min-1. Titration with purified pig liver cytochrome b5, demonstrated a 4-fold maximum stimulation at a cytochrome b5 to a P-450 molar ratio of 1-2. Rabbit lung P-450PG-omega formed a typical type I binding spectrum upon the addition of prostaglandin E1 with a calculated K8 of 1 microM, which agreed reasonably well with the kinetically calculated Km of 3 microM. Cytochrome P-450PG-omega was isolated as a low-spin isozyme with a lambda max (450 nm) in the CO-difference spectrum distinguishable from P-450I (451 nm) and P-450II (449 nm). Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis demonstrated that although purified P-450PG-omega had a relatively low specific content (12.1 nmol mg-1), it appeared homogeneous with a calculated minimum Mr of 56,000, intermediate between rabbit LM4 and LM6. When lung microsomes from pregnant and nonpregnant rabbit were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a protein band, with a Mr identical to P-450PG-omega, was observed in the pregnant rabbit, whereas this band appeared to be very faint or absent in microsomes from the nonpregnant rabbit. Purification of cytochromes P-450 from nonpregnant rabbit lung yielded only P-450I and P-450II. P-450PG-omega appears to be a novel rabbit P-450, possessing high activity towards omega-hydroxylation of prostaglandins, and is greatly induced during pregnancy in rabbit lung.     

Fast uncoiling kinetics of F1C pili expressed by uropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> are revealed on a single pilus level using force-measuring optical tweezers

Uropathogenic Escherichia coli (UPEC) express various kinds of organelles, so-called pili or fimbriae, that mediate adhesion to host tissue in the urinary tract through specific receptor-adhesin interactions. The biomechanical properties of these pili have been considered important for the ability of bacteria to withstand shear forces from rinsing urine flows. Force-measuring optical tweezers have been used to characterize individual organelles of F1C type expressed by UPEC bacteria with respect to such properties. Qualitatively, the force-versus-elongation response was found to be similar to that of other types of helix-like pili expressed by UPEC, i.e., type 1, P, and S, with force-induced elongation in three regions, one of which represents the important uncoiling mechanism of the helix-like quaternary structure. Quantitatively, the steady-state uncoiling force was assessed as 26.4 ±1.4 pN, which is similar to those of other pili (which range from 21 pN for S_I to 30 pN for type 1). The corner velocity for dynamic response (1,400 nm/s) was found to be larger than those of the other pili (400–700 nm/s for S and P pili, and 6 nm/s for type 1). The kinetics were found to be faster, with a thermal opening rate of 17 Hz, a few times higher than S and P pili, and three orders of magnitude higher than type 1. These data suggest that F1C pili are, like P and S pili, evolutionarily selected to primarily withstand the conditions expressed in the upper urinary tract.     

Structural characterization of calcitonin gene-related peptide purified from rabbit intestine

Calcitonin gene-related peptide (CGRP) immunoreactive material has been found in extracts of the intestine, however, the structure of intestinal CGRP is not known. Analytical reverse phase HPLC and ion-exchange FPLC revealed one predominant immunoreactive CGRP peak in rabbit intestinal extracts. This material was purified from rabbit intestine by sequential steps of reverse phase HPLC and ion-exchange FPLC. Microsequence and mass spectral analysis of the purified peptide and its chymotryptic fragments were consistent with the structure: GCNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSEAF-amide. Rabbit intestinal CGRP is identical to human CGRP-II in 35 of 37 amino acid residues. Two amino acid differences were detected at position 1, with Gly in rabbit CGRP instead of Ala in human CGRP-II, and at position 35, with Glu instead of Lys, respectively. Rabbit CGRP differed from human CGRP-I by three additional amino acids at positions 3, 22, and 25. This report shows that a CGRP form which closely resembles human CGRP-II, by means of chemical characterization, is the predominant form in rabbit intestine. Rabbit CGRP is the only CGRP form which has Gly as the amino terminal amino acid. Since the amino terminus of CGRP seems to be important for expression of bioactivity, the biological activity of rabbit CGRP may differ from human, rat and porcine CGRP.     

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.