Kinetics of phase variation between S and type-1 fimbriae of Escherichia coli

Abstract The rate of fimbrial phase variation in Escherichia coli strain 3040 was determined. The strain has type-1 and S fimbriae. The bacterial culture was fractionated into homogeneous subpopulations expressing either one of the fimbrial types only; the subpopulations were inoculated into broth and the fimbriation of individual cells was assayed by immunofluorescence as a function of time. The rate of the shift from S- or type-1-fimbriate cells to non-fimbriate ones was of the order of 10⁻² per cell generation and more rapid than a direct shift from one fimbrial phase to another, although both types of phase variations were observed.     

Organization of fimbriate cells in colonies of Escherichia coli strain 3040

Immunofluorescence staining with fimbria-specific antibodies was used to study the organization of fimbriate cells in colonies of Escherichia coli strain 3040. The strain has both type-1 and S fimbriae and shows fast phase variation between the fimbrial types. Colonies stained in sectors whose length and number per colony were dependent on the fimbrial phase of progeny cells. It is proposed that such sectors result from fimbrial phase variation.     

Occurrence of type-1C fimbriae on Escherichia coli strains isolated from human extraintestinal infections

Two monoclonal antibodies specific for type-1C fimbriae of Escherichia coli were produced. In enzyme-linked immunosorbent assay and immunoblotting the antibodies, which were of the IgG1 isotype, reacted with type-1C, but not with P or type-1 fimbriae of E. coli strain KS71. Immunoblotting and immunoprecipitation of crude fimbrial extracts from 25 strains invariably gave an apparent molecular weight of 17 000 for the type-1C fimbrillin. A total of 313 E. coli strains, isolated from patients with extraintestinal infection or from faeces of healthy children, were screened for the presence of type-1C fimbriae using both the monoclonal and polyclonal antibodies. Of these, 45 (14%) strains had type-1C fimbriae, with the highest frequency (27%) on strains isolated from patients with pyelonephritis. No faecal strain had type-1C fimbriae, and the frequency on the other diagnostic groups ranged from 11 to 15%. Thus, no direct correlation between type-1C fimbriae and bacterial virulence in human extraintestinal infections was found. Type-1C fimbriae were detected on only a few E. coli serotypes, notably on all O6:K2:H1 and O22:K13:H1 strains tested.     

Fractionation of a bacterial cell population by adsorption to erythrocytes and yeast cells

Abstract Fluorochrome-labeled antibodies specific for either S or type-1 fimbriae of Escherichia coli were used to show that in broth culture the two fimbrial types of strain 3040 mostly occurred on different cells. 12% of the cells were nonfimbriated. A fractionation procedure that involved adsorption of bacterial cells onto erythrocytes and yeast cells was developed to isolate homogeneous subpopulations (S-fimbriated, type-1-fimbriated, and non-fimbriated) of. E. coli. The level of contamination in each isolated subpopulation was 4% at the highest. The method is useful in obtaining homogeneous bacterial populations for adherence studies and for purification of specific fimbrial antigens.     

Type V collagen as the target for type-3 fimbriae, enterobacterial adherence organelles

Tissue-binding specificity of the type-3 fimbriae of pathogenic enteric bacteria was determined using frozen sections of human kidney. A wild-type Klebsiella sp. strain and the recombinant strain Escherichia coli HB101(pFK12), both expressing type-3 fimbriae, as well as the purified type-3 fimbriae effectively bound to sites at or adjacent to tubular basement membranes, Bowman's capsule, arterial walls, and the interstitial connective tissue. Bacterial adherence to kidney was decreased after collagenase treatment of the tissue sections. Recombinant strains expressing type-3 fimbriae specifically adhered to type V collagen immobilized on glass slides, whereas other collagens, fibronectin or laminin did not support bacterial adherence. In accordance with these findings, specific binding of purified type-3 fimbriae to immobilized type V collagen was demonstrated. Specific adhesion to type V collagen was also seen with the recombinant strain HB101(pFK52/pDC17), which expresses the mrkD gene of the type-3 fimbrial gene cluster in association with the pap-encoded fimbrial filament of E. coli, showing that the observed binding was mediated by the minor lectin (MrkD) protein of the type-3 fimbrial filament. The interaction is highly dependent on the conformation of type V collagen molecules since type V collagen in solution did not react with the fimbriae. Specific binding to type V collagen was also exhibited by type-3 fimbriate strains of Yersinia and Salmonella, showing that the ability to use type V collagen as tissue target is widespread among enteric bacteria.     

Immunological and genetical relatedness of type-1 and type-2 fimbriae in salmonellas of serotypes Gallinarum, Pullorum and Typhimurium

The fimbriae of 50 strains of serotype Gallinarum and 35 strains of serotype Pullorum of the genus Salmonella were compared with the type-1 fimbriae of serotype Typhimurium strains by immune electron microscopy and dot blot hybridization tests with gene probes for type-1 fimbriation in Typhimurium. The fimbriae of Gallinarum and Pullorum strains were coated with Typhimurium type-1 fimbrial antiserum and probes hybridized strongly with DNA of Gallinarum and Pullorum strains under stringent conditions. Furthermore, when Typhimurium type-1 fimbrial antiserum, that had been absorbed with fimbriate Gallinarum or Pullorum bacteria, was used in immune gold labelling experiments, it was shown that residual antibody recognized sites of possible adhesin incorporation at intervals along the length of Typhimurium type-1 fimbriae. These findings suggest that the type-2 fimbriae produced by all Gallinarum and Pullorum strains are non-adhesive forms of adhesive, type-1 fimbriae. This observation is of interest because type-1 fimbriae have never been reported in naturally occurring strains of these two avian-adapted serotypes.     

Complementation and regulatory interaction between two cloned fimbrial gene clusters of Escherichia coli strain KS71

Summary Complementation experiments with cloned DNA fragments encoding either the KS71A, the KS71B or the KS71C fimbriae of the pyelonephritogenic Escherichia coli strain KS71 were used to localise the P-fimbrillin genes and to demonstrate regulatory interactions between the cloned genes. The structural genes of the KS71A and KS71B fimbriae were located within a common 1.1 kilobase pair ClaI-SmaI fragment, and it was shown that the gene clusters for these fimbriae could complement each other in trans. The gene cluster encoding the KS71C fimbriae did not complement for the other KS71 fimbriae. A DNA fragment, located near the KS71A fimbrillin gene, was found to enhance the production of the KS71B fimbriae in trans.     

Immunological and genetical relatedness of type-1 and type-2 fimbriae in salmonellas of serotypes Gallinarum, Pullorum and Typhimurium

The fimbriae of 50 strains of serotype Gallinarum and 35 strains of serotype Pullorum of the genus Salmonella were compared with the type-1 fimbriae of serotype Typhimurium strains by immune electron microscopy and dot blot hybridization tests with gene probes for type-1 fimbriation in Typhimurium. The fimbriae of Gallinarum and Pullorum strains were coated with Typhimurium type-1 fimbrial anti-serum and probes hybridized strongly with DNA of Gallinarum and Pullorum strains under stringent conditions. Furthermore, when Typhimurium type-1 fimbrial antiserum, that had been absorbed with fimbriate Gallinarum or Pullorum bacteria, was used in immune gold labelling experiments, it was shown that residual antibody recognized sites of possible adhesin incorporation at intervals along the length of Typhimurium type-1 fimbriae. These findings suggest that the type-2 fimbriae produced by all Gallinarum and Pullorum strains are non-adhesive forms of adhesive, type-1 fimbriae. This observation is of interest because type-1 fimbriae have never been reported in naturally occurring strains of these two avian-adapted serotypes.     

Surface structures, haemagglutination and cell surface hydrophobicity of Bacteroides fragilis strains.

Nineteen strains of Bacteroides fragilis were examined by negative staining for surface structures. One strain (ATCC 23745) possessed peritrichous fibrils, 16 strains carried peritrichous fimbriae and two strains carried no surface structures. The fimbriae had a diameter of 2.1 +/- 0.25 nm and appeared to be 'curly'. Only a small proportion (4 to 41%, depending on the strain) of cells in a population carried fimbriae or fibrils. Strain A312 Showed phase variation of fimbriae as expression of fimbriae was repressed at 20 degrees C and in early exponential phase at 37 degrees C. The fibrils on strain ATCC 23745 did not exhibit phase variation in response to changes in incubation temperature, growth phase or growth in two different media. Capsules were demonstrated by the Indian ink method on 18 of the 19 strains, varying in size from strain to strain and within the same population. Cultures often contained both capsulate and noncapsulate cells. All strains possessed an electron dense ruthenium red staining layer between 7.9 and 23.9 nm in width attached to the outer membrane. Cell surface hydrophobicity quantified by the hexadecane partition assay gave low values ranging from 6.6 to 52.1%. Only a few strains were able to haemagglutinate and these were only weakly active. There was no correlation between cell surface hydrophobicity, haemagglutinating activity and surface structures.     

Two kinds of P-fimbrial variants of uropathogenic Escherichia coli recognizing forssman glycosphingolipid.

Various types of fimbriae on pathogenic Escherichia coli strains have been classified by their antigenicities and recognition specificities for receptors. However, the antigenicity of fimbrial proteins does not always correlate with the fimbrial recognition specificity. In this communication, the exact carbohydrate structures recognized by the fimbriae of two human uropathogenic E. coli strains, KS71 (O4) and IH11024 (O6), that have P-fimbrial antigen, were examined. Strain KS71 showed mannose-resistant (MR) hemagglutination (HA) of human blood group OP1 phenotype erythrocytes, and its HA was inhibited by blood group Pk antigen, Gal(alpha,1-4)Gal(beta,1-4)Glc-ceramide and P antigen, GalNAc(beta,1-3)Gal (alpha,1-4)Gal(beta,1-4)Glc-ceramide but not by Forssman antigen, GalNAc(alpha,1-3)GalNAc(beta,1-3)Gal(alpha,1-4)Gal (beta,1-4)Glc-ceramide, as previously described in many papers. The cells also showed MR HA of sheep erythrocytes, which was potently inhibited by Forssman, and weakly by P and Pk antigens. These phenomena could not be explained by the above P adhesin specificity. This adhesin was called Forssman-like adhesin. Strain IH11024 also caused MR HA of sheep erythrocytes but not of human erythrocytes. The HA was inhibited specifically by Forssman but neither by Pk nor P antigen. This adhesin was completely different from P adhesin and Forssman-like adhesin in recognition of the carbohydrate epitope. This adhesin, until now called a pseudotype of P fimbriae, was renamed Forssman adhesin.     

Basement membrane carbohydrate as a target for bacterial adhesion: binding of type I fimbriae of Salmonella enterica and Escherichia coli to laminin

Adherence of type-1-fimbriate Salmonella enterica and Escherichia coli to immobilized proteins of the extracellular matrix and reconstituted basement membranes was studied. The type-1-fimbriate strain SH401 of S. enterica serovar Enteritidis showed good adherence to laminin, whereas the adherence to fibronectin, type I, type III, type IV or type V collagens was poor. Only minimal adherence to the matrix proteins was seen with a non-fimbriate strain of S. enterica serovar Typhimurium. A specific and mannoside-inhibitable adhesion to laminin was exhibited by the recombinant E. coli strain HB101(plSF101) possessing fim genes of Typhimurium. Adherence to laminin of strain SH401 was inhibited by Fab fragments against purified SH401 fimbriae, and a specific binding to laminin, of the purified fimbriae, was demonstrated using fimbriae-coated fluorescent microparticles. Periodate treatment of laminin abolished the bacterial adhesion as well as the fimbrial binding. Specific adhesion to immobilized laminin was also shown by the type-1 -fimbriate E. coli strain 2131 and the recombinant strain E. coli HB101(pPKL4) expressing the cloned type-1-fimbriae genes of E. coli. Adhesion to laminin of strain HB101(pPKL4) was inhibited by mannoside, and no adherence was seen with the fimH mutant E. coli HB101(pPKL5/pPKL53) lacking the fimbrial lectin subunit. The type-1 fimbriate strains also adhered to reconstituted basement membranes from mouse sarcoma cells and human placenta. Adhesion of strains HB101(plSF101) and HB101(pPKL4) to both basement membrane preparations was inhibited by mannoside. We conclude that type-1 fimbriae of S. enterica and E. coli bind to oMgomannoside chains of the lamjnjn network in basement membranes.     

Muricauda ruestringensis Has an Asymmetric Cell Cycle

Muricauda ruestringensis B1 is a Gram ‐negative, marine bacterium and a member of the Flavobacteriaceae family. It is characterized by long appendages, which appear at different stages of growth. At the outer end of these appendages there is a bulbous structure. Investigating the cell morphology of strain B1 during batch growth revealed a high diversity of cell types and sizes. Apart from small rod‐shaped cells and rods with appendages, there were large rods and spherical cells of different sizes as well as spherical cells which had fimbriae. To be able to study the cell cycle events, it was essential to monitor the population dynamics of the involved individuals. For this purpose, fluorochromising techniques, multi‐parametric flow cytometry, image analysis and fluorescence microscopy were used. It was demonstrated that all cell types displayed a broad variation in DNA content; the precise number of chromosomes varied depending on the growth phase. The assortment was testified to hold 16S rDNA sequence identity. The cultures consisted of subpopulations whose density within a Percoll gradient varied considerably, ranging from 1.028 to 1.070. Consolidating the results of the morphological data, the chromosome content and the density of the subpopulations at different growth stages enabled us to construct an asymmetric cell cycle for the growth of strain B1 under the specific culture conditions of our experiments.     

Escherichia coli fimbriae recognizing sialyl galactosides

Fimbriae recognizing sialyl galactosides (S fimbriae) were purified from an Escherichia coli strain. The S fimbriae were morphologically identical to type 1 and P fimbriae of E. coli and showed a hemagglutination that was abolished when erythrocytes were treated with neuraminidase. Hemagglutination by the purified fimbriae was inhibited by orosomucoid but not by its desialylated derivative. Of the oligosaccharides tested, sialyl-(alpha 2-3)-lactose and sialyl-(alpha 2-3)-N-acetyllactosamine had the strongest inhibitory activities. It was concluded that S fimbriae have the strongest affinity for (alpha 2-3)-linked sialyl galactosides. In the enzyme-linked immunosorbent assay, the hyperimmune serum to the S fimbriae reacted strongly with the homologous antigen but not with type 1, P, or nonhemagglutinating KS71C fimbriae of E. coli. Analogously, the hyperimmune sera to the other E. coli fimbriae did not react with the purified S fimbriae. The immunoprecipitation assay showed that S fimbriae on different E. coli serotypes shared immunological cross-reactivity.     

Characterization of two genes encoding antigenically distinct type-1 fimbriae of Klebsiella pneumoniae

A uropathogenic isolate of Klebsiella pneumoniae was shown to exhibit a mannose-sensitive hemagglutinating phenotype and to produce type-1 fimbriae consisting of subunits with a different electrophoretic mobility than those previously investigated. The gene cluster encoding expression of fimbriae was cloned and the genetic organization of the encoded polypeptides was determined. The gene encoding the major fimbrial subunit was localized and further examined by nucleotide sequence analysis. Comparison of two K. pneumoniae fimbrial genes revealed a nucleotide sequence agreement of 73%, and amino acid sequence agreement of 84% for the mature fimbrial subunits. Predictions of putative antigenic sites were correlated with regions demonstrating amino acid variability. In agreement with these predictions, no serological cross-reactivity between both fimbrial proteins could be demonstrated using an enzyme-linked immunosorbent assay (ELISA).     

Purification and characterization of thin, aggregative fimbriae from Salmonella enteritidis.

Novel fimbriae were isolated and purified from the human enteropathogen Salmonella enteritidis 27655. These fimbriae were thin (measuring 3 to 4 nm in diameter), were extremely aggregative, and remained cell associated despite attempts to separate them from blended cells by centrifugation. The thin fimbriae were not solubilized in 5 M NaOH or in boiling 0.5% deoxycholate, 8 M urea, or 1 to 2% sodium dodecyl sulfate (SDS) with or without 5% beta-mercaptoethanol. Therefore, an unconventional purification procedure based on the removal of contaminating cell macromolecules in sonicated cell extracts by enzymatic digestion and preparative SDS-polyacrylamide gel electrophoresis (PAGE) was used. The insoluble fimbriae recovered from the well of the gel required depolymerization in formic acid prior to analysis by SDS-PAGE. Acid depolymerization revealed that the fimbriae were composed of fimbrin subunits, each with an apparent molecular mass of 17 kDa. Although their biochemical characteristics and amino acid composition were typical of fimbriae in general, these thin fimbriae were clearly distinct from other previously characterized fimbriae. Moreover, their fimbrin subunits had a unique N-terminal amino acid sequence. Native fimbriae on whole cells were specifically labeled with immune serum raised to the purified fimbriae. This immune serum also reacted with the denatured 17-kDa fimbrin protein in Western blots. The polyclonal immune serum did not cross-react with the other two native fimbrial types produced by this strain or with their respective fimbrins on Western blots (immunoblots). Therefore, these fimbriae represent the third fimbrial type produced by the enteropathogen S. enteritidis.     

Purification and characterization of a novel secondary fimbrial protein from Porphyromonas gingivalis strain 381

We previously reported the existence of two different kinds of fimbriae expressed by Porphyromonas gingivalis ATCC 33277. In this study, we isolated and characterized a secondary fimbrial protein from strain FPG41, a fimA-inactivated mutant of P. gingivalis 381. FPG41 was constructed by a homologous recombination technique using a mobilizable suicide vector, and failed to express the long fimbriae (41-kDa fimbriae) that were produced on the cell surface of P. gingivalis 381. However, short fimbrial structures were observed on the cell surface of FPG41 by electron microscopy. The fimbrial protein was purified from FPG41 by DEAE-Sepharose CL-6B column chromatography. The secondary fimbrial protein was eluted at 0.15 M NaCl, and the molecular mass of this protein was approximately 53 kDa as estimated by SDS-PAGE. An antibody against the 53-kDa fimbrial protein reacted with the short fimbriae of the FPG41 and the wild-type strain. However, the 41-kDa long fimbriae of the wild-type strain and the 67-kDa fimbriae of ATCC 33277 did not react with the same antibody. Moreover, the N-terminal amino acid sequence of the 53-kDa fimbrial protein showed only 2 of 15 residues that were identical to those of the 41-kDa fimbrial protein. These results show that the properties of the 53-kDa fimbriae are different from those of the 67-kDa fimbriae of ATCC 33277 as well as those of the 41-kDa fimbriae.