Investigation of minor components of Escherichia coli Type 1 Fimbriae: protein chemical and immunological aspects

Microbial Pathogenesis 1988 ; 4 : 231-238

Investigation of minor components of Escherichia coli Type 1 Fimbriae: protein chemical and immunological aspects Karen A . Krogfelt and Per Klemm Department of Microbiology, Technical University of Denmark DK-2800 Lyngby, Denmark (Received November 9, 1987 ; accepted January 27, 1988)

Krogfelt K. A . (Dept . of Microbiology, Technical University of Denmark DK-2800 Lyngby, Denmark) and P . Klemm . Investigations of minor components of E. coli Type 1 Fimbriae : protein chemical and immunological aspects . Microbial Pathogenesis 1988 ; 4 : 231-238 . Three minor components of type 1 fimbriae, FimF, FimG and FimH have been characterized . These proteins are integrated in the fimbrial structure ; are responsible for the adhesive properties of the fimbriae but are not necessary for the production of fimbriae' . Fimbriae were purified from different clones harbouring various combinations of the fimF, fimG and fimH genes in addition to the fimA gene . The FimF, FimG and FimH proteins were identified by two dimentional gel electrophoresis . They were found to have molecular weights of 18 .0 kDa, 17 .0 kDa and 30 kDa, respectively . The ratio of FimF, FimG and FimH components to the major subunit was less than 1 :100 . The fimH protein especially was present in very small quantities . Sera raised against fimbriae from two of the clones (HB101/pPKL5 and HB101/pPKL4) were found by immunoblotting to be specific respectively for the major structural protein only (FimA), and for all components . Key words: Bacterial adhesins; minor fimbrial components; organelle structure .

Introduction The majority of Escherichia coli strains are able to produce type 1 fimbriae ; long thread-like, 7 nm wide surface structures . Type 1 fimbriae mediate adhesion to mannosides and thereby enable the fimbriated bacteria to colonize a broad range of epithelial surfaces, due to the ubiquitous presence of mannose in host cell membranes . It has been proposed that the biological role of type 1 fimbriae is to provide bacterial adhesion to mucus in the large intestines, i.e . the natural habitat of E. coil (for reviews see 2 and 3) . The expression of these fimbriae is phase variable, i.e . the bacteria shift periodically between a fimbriated and a non-fimbriated state . The phase variation has been found to result from the stochastic inversion of a 300 base pair DNA segment harbouring the promoter for the fimA gene .` A single type 1 fimbria is made up almost entirely of the product of this gene . The fim gene cluster has been cloned both from an E. coil K-12 strain and from a wild type strains •' The fim genes have been found to include in addition to the fimA gene two genes, fimB and fimE, encoding regulatory proteins directing the phase dependent expression of the fimA gene .' Other genes, namely fimC and fimD, have been identified which are necessary for transport and assembly of the fimbriae .' , ' Recently three new genes, fimF, fimG and fimH were identified .' The corresponding 0882-4010/88/030231 +08 $03 .00/0

( 1988 Academic Press Limited



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- 25K

23K

18K

WIRE 26-5K

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89 K

(1711

175K 16K

I pPKL4(BR) I pPKL66(BR)

~I T M

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I pPKL5 ( BR) pPKL52(AC) pPKL65(AC)

Fig . 1 . Organization of the fim genes on the 10 .5 kilobase Bg//I-SaII fragment . The extent of the various plasmids used in this study is shown . Only passenger DNA is shown .

proteins were found to play a paramount role in the adhesive faculty as well as in the regulation of length of type 1 fimbriae . Thus, in order to produce adhesive fimbriae, it was found that apart from the FimA protein, a combination consisting of either FimF plus FimH, or FimG plus FimH, was necessary . Furthermore, DNA sequence analysis of the fimF, fimG and fimH genes showed approximate 20% homology of the corresponding products to the major subunit protein, suggesting them to be integrated in the fimbriae . However, they were dispensable for production of fimbriae, since a host cell harbouring a plasmid deleted for these 3 genes were able to produce fimbriae, although these did not confer any adhesion .' In this context we have analysed type 1 fimbriae and periplasmic preparations from various clones producing combinations of FimA, FimF, FimG and FimH proteins . Results

Purification of fimbriae The fimF, fimG and FimH genes have been localized, by genetic mapping and DNA sequencing, in the outermost right hand part of the fim gene cluster (Fig . 1) .' Three H13 01 clones were used for purification of fimbriae . These contained plasmids pPKL4 (fimA+, fimF+, fimG+, fimH+), pPKL66 (fimA+, fimF+) and pPKL5 (fimA+), respectively . Host cells containing pPKL4 were shown by electron microscopy to produce many fimbriae (approx . 200 pr . cell) having an average length of 1 Jim, and which were fully adhesive, as measured by adhesion to mannose-covered Sepharose beads and haemagglutination activity . HB101 hosts harbouring plasmid pPKL5 produced considerably fewer (approx . 25 pr . cell), and very long fimbriae (2 .2 µm), which did not show any adhesive properties.' Preparations of purified fimbriae were easily obtained from hosts containing the entire gene cluster (plasmid pPKL4) by usual methods (shearing, salt precipitation, gel filtration) . $ However, preparations from hosts harbouring the pPKL5 plasmid contained large amounts of membrane proteins . It appeared that fimbriae produced by HB1 01 /pPKL5, consisting uniquely of the FimA protein, were very rigid and almost unbreakable structures . Thus, one could imagine that instead of breaking at the cell surface the FimA-only fimbriae rupture the membrane during shearing, and thereby liberate a high background level of membrane proteins . Fimbriae from HB101/pPKL5 were much more resistant, as compared to 'wildtype' fimbriae, to depolymerization . They were however, finally purified by gel chromatography in 6M guanidine chloride, where they eluted in the void volume of the column (data not shown) . The extreme rigidness and high degree of structural integrity of fimbriae from HB101/pPKL5 was



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Fig . 2 . 2D-gel electrophoresis of purified fimbriae (A) Fimbriae from the HB101 /pPKL4 clone encoding FimA, FimF, FimG and FimH proteins; (B) Fimbriae from the HB101 /pPKL5 clone coding for the major subunit, FimA . Point streaking on the silver stained 2D-gels was due to the presence of 2-mercaptoethanol in the SDS equilibration buffer of the immobilized pH gradient gel 21 .

also reflected in the fact that they only depolymerized after boiling in acid . These fimbriae are seen to repolymerize fast at neutral pH ; while applied to polyacrylamide gels the fimbriae precipitate in the stacking gel [Fig . 2(B)] . Purification of fimbriae from hosts containing the plasmid pPKL66 was performed as for HB101/pPKL5, as they showed similar properties, i .e . extreme resistance to shearing and depolymerization . Detection of FimF, FimG and FimH in fimbriae preparations Purified fimbriae from host cells containing plasmids pPKL4, pPKL66 and pPKL5, respectively, were analysed by simple SDS-polyacrylamide gel electrophoresis, as well as by two dimensional gel electrophoresis, employing isoelectric focusing in the first dimension . Fimbriae from hosts harbouring pPKL5 showed only one band in SDSpolyacrylamide electrophoresis, while in 2D-gels, in addition to the band of 17 .0 kDa corresponding to the fimbrial subunit, it was possible to see a 'ladder' effect due to the presence of FimA polymers in various stages of depolymerization . As mentioned before, it was observed that a great deal of the protein remained in the stacking gel . Furthermore, most of the FimA that did enter the separation gel was present as polymer variants, whereas relatively little was present as monomer [Fig 2(B)] . Four proteins could be detected in 2D-gels with fimbriae from HB101/pPKL4 . In addition to the major structural protein, FimA, three minor components were seen having apparent molecular weights of 17 .0 kDa, 18 .0 kDa and 30 kDa, respectively [Fig 2(A)] . The ratio of minor components to FimA was less than approximately 1 :100 as estimated from silver staining intensity, and the FimH protein notably was present in very minute quantities . Two proteins were present in fimbriae from HB101/pPKL66, namely a protein of 18 kDa in addition to FimA . This protein must be FimF, and the proteins of 17 .0 kDa and 30 kDa represent FimG and FimH respectively, according to the data on the extent and genetic information found on plasmids pPKL5, pPKL66 and pPKL4 previously described' (Fig . 1) . In periplasmic preparations from HB101/pPKL52 and HB101/pPKL65 (Fig 1), neither of which produced fimbriae, it was possible to deduce the proteins synthesized from the plasmids, in 2D-gels, by comparison with preparations from the host cell



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Fig . 3 . Western blot of type 1 fimbrial preparations incubated with antiserum raised against : (A) fimbriae from HB101/pPKL4 ; FimA, FimF, FimG, FimH and FimH-dimer was observed ; (B) fimbriae from HB1 01 /pPKL5 . These antibodies showed a faint reaction to FimH .

HB101 and it was found that they produced FimF, FimG and FimH, and only FimH respectively . Analysis of sera produced upon immunization with purified fimbriae Rabbits were immunized with purified fimbriae preparations respectively from HB101/pPKL4 and HB101/pPKL5 clones . The antisera were adsorbed with the host H13 01 in order to remove any background . In Western blotting of 2D-gels, anti-HB101/pPKL4 serum reacted with all four fimbrial constituents. In addition, this serum was able to recognize a dimer of the FimH protein (Fig . 3A), which was not clearly seen by silver staining of the fimbrial proteins (Fig . 2A) Anti-HB101/pPKL4 serum reacted with the FimF, FimG and FimH proteins and with a FimH dimer present in periplasmic preparations from the H13 10 /pPKL52 clone . Antibody binding was also observed to the FimH protein, and its dimer, in periplasmic preparations from the HB101/pPKL65 clone . In the case of Western blots of 2D-gels of fimbriae prepared from the HB101/pPKL5 and HB101/pPKL66 clones anti-HB101/pPKL4 serum was able to detect only the FimA protein as well as its polymer forms and the FimA and FimF proteins, respectively . Antibodies raised against purified fimbriae from HB101/pPKL5 recognized, in Western blots, only the FimA components and very faintly the FimH, but not the FimF and FimG components from fimbrial preparations of the HB101/pPKL4 clone (Fig . 3B) . The above serum reacted only with the FimA protein and its polymers from fimbrial preparations of HB101/pPKL5 and HB101/pPKL66 . In periplasmic preparations of the HB101/pPKL65 clone there was seen binding of the anti-FimA serum to the monomer of the FimH protein but the dimer form was not present . Discussion and conclusions Three minor components of type 1 fimbriae, FimF, FimG and FimH, have been characterized . The proteins are vital for the lectin-like adhesive activity of type 1 fimbriae . The present data strongly indicate these proteins to be integral parts of the fimbrial structure .



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100-200 FimA

0~0+0 0,,0,0 O*O`O 000 Fig . 4 . Model for type 1 fimbriae structure . The adhesin FimH is believed to be integrated as a dimer in the fimbrial structure anchored to either/both of the minor components FimF and FimG (0) . The binding sites are positioned laterally as well as on the tip of the fimbria .

'Wildtype' type 1 fimbriae, consisting of the FimA as well as the three minor components, e .g . fimbriae from a HB101 host containing plasmid pPKL4 (Fig . 1), are much more prone to breakage and depolymerization than pure FimA fimbriae (HB101/pPKL5) . These findings are consistent with a model of type 1 fimbrial structure where the minor components are interspersed at intervals along the fimbriae . We believe, by analogy to the Pap-system 13 .14 , that the FimH protein constitutes the mannose binding lectin, anchored in the fimbrial superstructure by either FimF and/or FimG . Positions occupied by the minor components, FimF, FimG and FimH would then introduce fragile regions where the structure preferentially would break up when exposed to stress . Such a model would have fimbriae with laterally- as well as tippositioned binding sites, the latter resulting from exposure after breakage . Furthermore, since a complex consisting of FimH in concert with either one or both of the FimF and FimG proteins seems to act as initiator of fimbrial synthesis,' the tip of an unbroken fimbria would also be adhesive . This model would explain the extreme rigidness and apparent strength of pure FimA fimbriae since they would have no minor components and thereby no fragile points . From the immunological data it is plausible that FimH appears as a dimer in the fimbrial structure . A model for the structure of type 1 fimbriae is shown in Fig . 4 . The model agrees with reports that suggest a lateral positioning of the adhesive sites on type 1 fimbriae, based on binding studies . 9' 10• 1 1 Very recently Abraham et al." showed by immunogold electron microscopy that FimG and FimH are indeed integral parts of type 1 fimbriae . Our data indicate that FimA and FimH are immunologically cross-reactive . Since they show extended sequence homology' it would not seem surprizing if they had one or a few antigenic determinants in common . It was recently reported that Pap fimbriae from uropathogenic E. coli, contain, in addition to the major subunit PapA the products of the papE, papF and papG genes, as minor components . Furthermore these proteins played a role in the adhesive faculty of these fimbriae, with the PapG protein as lectin ."" , " The minor components from pap fimbriae are very similar to the FimF, FimG and FimH proteins (Fig . 5), being of





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FimH :

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T I I PA NN

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Fig . 5 . Comparison of the structures of the FimH and PapG proteins given in the standard single letter code. Identical amino acids are boxed and gaps have been introduced in order to obtain maximum fit . Sequences were compared by using the program ALIGN ."

the same size and showing approximately 20% direct homology with these proteins . This may be indicative of similar functions, and it is therefore tempting to imply a corresponding function for the FimF, FimG and FimH proteins . The PapE, PapF and PapG proteins have been reported to be located uniquely at the tip of the pap fimbriae . 13 This is however not in accordance with out model lateral and tip positioning of the corresponding minor components in type 1 fimbriae .

Materials and methods Bacterial strains and culture conditions. The Escherichia coli K-12 strain used in this study was HB101 . 75 Plasmids pPKL4, pPKL5, pPKL52, pPKL64, pPKL65 and pPKL66 have previously . 16 been described ." Cells were grown as previously reported Assays for adhesion . The capacity of bacteria to express biologically active type 1 fimbriae was assayed by their ability to agglutinate guinea-pig erythrocytes on glass slides . One drop of a 3% solution of erythrocytes in buffered physiological salt water was gently mixed with one drop of an overnight bacterial culture . In addition the ability of the bacteria to bind to Dmannose coupled to epoxy activated Sepharose CL-6B beads, Pharmacia, was assayed . Bacteria and mannose-coupled beads were incubated for 1 h at 37°C, gently washed and inspected microscopically . Purification of fimbriae. Fimbriae were purified as described by Eshdat et at' Strains containing the pPKL4 plasmid gave rise to a pure fimbriae preparation by this method . Fimbriae preparations from the pPKL5 strains contained a great number of membrane proteins . This crude extract was incubated for 2 h at 37°C in 8 M GndHCI under agitation and then applied on a Sepharose CL6B column . Fimbriae were eluted in the void volume with 6 M GndHCI as eluent . Fimbriae preparations were concentrated with PEG and the fimbriae solution, containing 0 .05% sodium azide, was stored at 4°C . Periplasmic preparations . Periplasmic proteins were released by chloroform treatment ." Immunization and sera . Female albino rabbits were immunized with either of the two fimbriae preparations described above, after emmulsification in Freund's incomplete adjuvant (0 .2 mg protein/injection) . Rabbits were immunized three times at intervals of 2 weeks, and bled after 2 months . The antisera obtained were adsorbed with whole bacteria in order to remove non-



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specific antibodies prior to utilization in Western blotting . Antisera were mixed gently with a bacterial pellet and incubated for 1 h at 37°C . Cells were removed by centrifugation at 5000xg for 10 min at 4°C . The adsorption was repeated three times . Polyacrylamide gel electrophoresis . Two-dimensional gel electrophoresis was performed according to O'Farrell 18 with some modifications . lsoelectricfocusing (IEF) in the first dimension was carried out in cylindrical gels (length 10 cm, diameter 1 .0 mm) ; containing 28 .4% w/v acrylamide, 1 .6% w/v methylenbisacrylamide, 9 M urea, 2% Nonidet P-40 (NP-40) and 2% Ampholines (pH 5 to 7, pH 3 to 10 ; 4:1) . Polymerization was initiated by using 0 .04% ammoniumpersulfate and 0 .02% TEMED (N,N,N',N'-tetramethylendiamine) . Proteins were solubilized in IEF sample buffer containing 9 M urea, 2% NP-40 and 5% 2-mercaptoethanol . IEF was prefocused as recommended 18 and carried out overnight at 400 V, followed by 1 h at 800 V. The electrolytes used were 0 .02 M NaOH in the upper reservoir and 0.01 M H 3 P0 4 in the lower reservoir of a standard electrophoresis apparatus. Gels were transfered to 3 ml equilibration buffer containing 10% glycerol, 3% SIDS, 5% 2-mercaptoethanol and 0 .1 M Tris-HC1 pH 6 .8 and stored frozen at -20°C . After thawing, IEF gels were equilibrated for 30 min in the same buffer and subjected to one-dimensional SDS-PAGE electrophoresis, which was performed in 12% slab gels as described by Laemmli . 19 The proteins were visualized by silver staining .", " Western blotting. The proteins separated by two-dimensional electrophoresis were tested for their affinity to the prepared antisera by Western blotting, which was performed as previously .22 described We wish to thank Flemming Holm for his advice about 2D-gel electrophoresis, and our colleagues for helpful discussions and criticism of this manuscript . This work was supported by the Carlsberg Foundation (86/87 no 238/11) .

References 1 . Klemm P, Christiansen G . Three fim genes required for the regulation of length and mediation of adhesion of Escherichia co/i type 1 fimbriae . Mol Gen Genet 1987 ; 208: 439-445 . 2 . Mooi FR, de Graaf FK . Molecular biology of fimbriae of enterotoxigenic Escherichia coli. Curr Top Microbiol Immunol 1985 ; 118 :119-138 . 3 . Klemm P. Fimbrial adhesins of Escherichia coli . Rev Infect Dis 1985 ; 7 : 321-339 . 4 . Abraham JM, Freitag CS, Clements JR, Eisenstein BI . An invertible element of DNA controls phase variation of type 1 fimbriae of Escherichia coli. Proc Natl Acad Sci USA 1985 ; 82 : 5724-5727 . 5 . Klemm P . Two regulatory fim genes, fimB and fimE, control the phase variation of type 1 fimbriae in Escherichia coli. EMBO J 1986 ; 5:1389-1393 . 6 . Klemm P, Jorgensen BJ, van Die 1, de Ree H, Bergmans H . The fim genes responsible for synthesis of type 1 fimbriae in Escherichia coli. Mol Gen Genet 1985; 199 : 410-414 . 7 . Orndorff PE, Falkow S . Organization and expression of genes responsible for type 1 piliation in Escherichia coli. J Bacteriol 1984; 159 : 736-744 . 8 . Eshdat Y, Sikverblatt FJ, Sharon N . Dissociation and reassembly of Escherichia coli type 1 pili . J Bacteriol 1981 ; 148 : 308-314 . 9 . Sweeney G, Freer J . Location of binding sites on common type 1 fimbriae from Escherichia coli. J Gen Microbiol 1979 ; 112 : 321-328 . 10 . Salit IE, Gotschlich EC . Type 1 Escherichia coli pili : characterization of binding to monkey kidney cells . J Exp Med 1977 ; 146 :1169-1181 . 11 . Goochee CF, Hatch RT, Cadman TW . Some observations on the role of type 1 fimbriae in Escherichia coli auto-flocculation . Biotechn and Bioengineer 1987 ; Vol XXIX : 1024-1034. 12 . Lindberg F, Lund B, Normark S . Gene products specifying adhesion of uropathogenic Escherichia coli are minor components of pili . Proc Natl Acad Sci USA 1986 ; 83 : 1891-1895. 13 . Lindberg F . Lund B, Johansson L, Normark S . Localization of the receptor-binding protein at the tip of the bacterial pilus . Nature 1987 ; 328 : 84-87 . 14 . Lund B, Lindberg F, Marklund BI, Normark S . The PapG protein is the a-D-galactopyranosyl-(1-4)-BD-galactopyranose binding adhesin of uropathogenic Escherichia coli . Proc Natl Acad Sci USA 1987 ; 84 :5898-5902. 15 . Boyer HW, Roulland-Dussoix D . A complementary analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 1969 ; 41 : 459-472. 16 . von Meyenburg K, Hansen FG, Nielsen LD, Rise E . Origin of replication, oriC, of the Escherichia coli chromosome on specialized transducing phages ).asn . Mol Gen Genet 1978; 160 : 287-295 . 17 . Ames GF-L, Prody C, Kustu S . Simple, rapid and quantitative release of periplasmic proteins by chloroform . J Bacteriol 1984 ; 160 : 1181-1183 .



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18. O'Farrell PH . High resolution two-dimensional electrophoresis of proteins . J Biol Chem 1975; 250 : 4007-4021 . 19 . Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature 1970 ; 117 : 680-685 . 20 . Blum H . Beier H, Gross HJ . Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels . Electrophoresis 1987 ; 8 : 93-99 . 21 . Gorg A, Postel W, Weser J, Gunther S, Strahler JR, Hanash SM, Somerlot L . Elimination of point streaking on silver stained two-dimensional gels by addition of iodoacetamide to the equilibration buffer . Electrophoresis 1987; 8: 122-124 . 22 . Krogfelt KA, Meldal M, Klemm P . K88 fimbrial antigens : identification of antigenic determinants by the use of synthetic peptides . Microbial Pathogenesis 1987 ; 2 : 465-472 . 23 . Taylor P. A fast homology program for aligning biological sequences . Nucleic Acids Research 1984 ; 12:447-455 . 24. Abrahan SN, Goguen JD, Sun D, Klemm P, Beachey EH . Identification of two ancillary subunits of Escherichia coli type 1 fimbriae by using antibodies against synthetic oligopeptides of fim gene products . J Bacteriol 1987; 169 : 5530-5536 .