Type 3 Fimbriae of Klebsiella sp.: Molecular Characterization and ...

JOURNAL OF BACTERIOLOGY, Aug. 1983, p. 860-865

Vol. 155, No. 2

0021-9193/83/080860-06$02.00/0 Copyright C) 1983, American Society for Microbiology

Type 3 Fimbriae of Klebsiella sp.: Molecular Characterization and Role in Bacterial Adhesion to Plant Roots TIMO K. KORHONEN,' EVELIINA TARKKA,' HELENA RANTA,2 AND KIELO HAAHTELA1*

Department

of General

Microbiology, University of Helsinki,1 and National Public Health Institute,' SF00280, Helsinki 28, Finland Received 14 February 1983/Accepted 15 May 1983

Type 3 fimbriae of Klebsiella were purified and characterized. The fimbriae 4 to 5 nm in diameter and 0.5 to 2 pLm long. In sodium dodecyl sulfatepolyacrylamide gel electrophoresis, the fimbrillin had an apparent molecular weight of 23,500, and it differed from enterobacterial type 1 fimbrillins in its amino acid composition. Hydrophobic amino acids comprised 33.6% of all amino acids in the fimbrillin, which lacked cystine, phenylalanine, and arginine. Serologically, the type 3 fimbriae were also distinct from the type 1 fimbriae. Purified type 3 fimbriae agglutinated tannin-treated human blood group 0 erythrocytes; this confirms the role of type 3 fimbriae as hemagglutinins. Purified 125I-labeled type 3 fimbriae bound to the roots of Poa pratensis, and this binding could be inhibited by Fab fragments to the purified fimbriae. Anti-type 3 fimbriae Fab fragments also inhibited bacterial adhesion to plant roots. These results demonstrate that type 3 fimbriae mediate adhesion of klebsiellas to plant roots. Eight nitrogen-fixing strains of Klebsiella also produced type 3 fimbriae when grown under anaerobic nitrogen fixation conditions. It is proposed that type 3 fimbriae are involved in the establishment of the plant-bacterium association concerning nitrogen-fixing Klebsiella strains. were

Associative nitrogen fixation is carried out by bacteria living on the roots of nonleguminous plants. The bacteria described as associative nitrogen fixers include species of Klebsiella, Enterobacter, Erwinia, Azotobacter, Bacillus, and Pseudomonas (1, 17, 19). These bacteria live attached to the surfaces of plant roots, where they may benefit from the organic material in the root exudates. In some genera, e.g., Azospirillum, this association may involve bacterial invasion into the root (18, 22). Very little is known about the adhesion mechanisms and the specificities involved in associative nitrogen fixation. In contrast, the adhesion of symbiotic nitrogen-fixing bacteria to the roots of leguminous plants is well characterized (8). Adhesion specificity, which in part determines the symbiosis, relies on plant lectins that specifically recognize lipopolysaccharide or capsular antigens or both on bacterial surfaces (8). Duguid (2) noticed that saprophytic Klebsiella strains adhere, apparently by their fimbriae, to the root hairs of red clover and cress seedling plants. Bacterial fimbriae (or pili) are filamentous proteins that protrude from the bacterial surface. They are known to mediate bacterial adhesion to mammalian epithelial cells and erythrocytes (3). Two types of fimbriae, type 1 and type 3, can be found on saprophytic Klebsi-

ella strains (2). The type 1 fimbriae are 5 to 7 nm in diameter and bind specifically to at-D-mannosides, so the adhesion they mediate is mannose sensitive (3, 9, 10). The type 3 fimbriae are thinner and about 4 to 6 nm in diameter (2, 3). The receptor for the type 3 fimbriae is not known, but Duguid (2) noticed that Klebsiella strains carrying type 3 fimbriae agglutinate ox or human 0 erythrocytes treated with tannin. In this work, we purified and characterized the type 3 fimbriae from Klebsiella and demonstrated that they mediate bacterial adhesion to the roots of Poa pratensis. (Part of this work was presented in June 1982 in Helsinki at the Second National Symposium on Biological Nitrogen Fixation.) MATERIALS AND METHODS Bacteria. The strains 69/1 (carrying type 3 fimbriae) and 55/1 (carrying type 1 fimbriae) of K. aerogenes were gifts from J. P. Duguid (Dundee, United Kingdom). Eight strains of nitrogen-fixing klebsiella were isolated from roots of grasses as described previously (7). These strains were typed as K. pneumoniae with an API 20E test kit (API Systems SA, Appareils et Procedds d'Identification, France). For isolation of the fimbriae and for the adherence tests, the bacteria were grown in static malate broth for 48 h at 28°C (7). Under nitrogen fixation conditions, the bacteria were grown in 10 ml of static malate broth without mineral nitrogen 860

VOL. 155, 1983 in 22-ml serum bottles. The bottles were made anaerobic by replacing the gas phase with gaseous nitrogen, as previously described (7). Fimbriae. The type 3 fimbriae of K. aerogenes 69/1 and the type 1 fimbriae of K. aerogenes 55/1 were purified by using deoxycholate and concentrated urea, as described previously (11). Type 1 fimbriae from Escherichia coli 2131 and Salmonella typhimurium SH6749 were available from previous work (12). SDS-PAGE. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) was performed in 1.2-mm-thick slab gels (gel concentration, 17%) by the system of Laemmli (13). Peptide bands were visualized by staining with Coomassie brilliant blue R250. A low-molecular-weight electrophoresis kit (Pharmacia Fine Chemicals, Uppsala, Sweden) was used as a standard. Type 1 fimbrial preparations were denaturated at low pH before treatment with SDS (16). Protein estimation. Protein was estimated by the modified Lowry procedure-as described in reference 15, using bovine serum albumin (BSA) as a standard. Electron microscopy. Purified fimbriae were negatively stained with 2% (wt/vol) phosphotungstic acid in 0.1 M sodium phosphate buffer (pH 6.5). The micrographs were taken at the Department of Electron Microscopy, University of Helsinki, with a JEM-1OOB electron microscope operated at 80 kV. Immunological methods. Antisera against the various purified fimbriae were produced in rabbits as described previously (11), and antibody titers and serological cross-reactivity were estimated by enzyme-linked immunosorbent assay (ELISA [4]) as described recently (12). Purified immunoglobulin G against the type 3 fimbriae was treated with papain (Sigma Chemical Co., St. Louis, Mo.), and the Fab fragments were prepared essentially as described by Porter (20). The Fab fragments were assessed for purity by SDS-PAGE; they were shown to be free from the heavy chain of the immunoglobulin G molecule. Amino acid composition. Hydrolysis was done in 6 M HCl for 24 h at 110°C under nitrogen. Analysis was performed on a Jeol JLC-5AH amino acid analyzer with sodium citrate buffers. No corrections were made for losses of threonine and serine. Radiolabeling of fimbriae and the binding assay. Purified type 3 fimbriae (100 ,ug) was labeled with 1251 (0.5 mCi; Amersham, United Kingdom) by the tetrachlorodiphenylglycouril method described by Fraker and Speck (6) and by Markwell and Fox (14). Unbound iodine was removed by dialyzing the fimbriae first against water and then against phosphate-buffered saline (pH 7.1) containing 0.02% (vol/vol) Triton X-100 (Koch-Light Laboratories Ltd., Buckinghamshire, United Kingdom), until no radioactivity was detected in dialysis buffer. The specific activity obtained was 0.7 ,uCi per ,ig of protein. To test the in vitro binding of labeled fimbriae to plant roots, we surface sterilized seeds of P. pratensis by treating them for 1 min with 94% ethanol and for 8 min with 5% (wt/vol) hypochloric acid, washed them six times with sterile water, and then allowed them to germinate for 7 to 8 days on water agar plates. Roots 1 cm in length only were used. The roots were incubated for 2 h at room temperature with increasing concentrations of labeled fimbriae in phosphate-buffered saline containing 0.1% (wt/vol) BSA and then were washed

TYPE 3 FIMBRIAE OF KLEBSIELLA

861

five times with 40 ml of phosphate-buffered saline. The radioactivity remaining on the roots was determined in a gamma counter (Selectronic, Denmark). To test the inhibition by the Fab fragments, we incubated the labeled fimbriae with the Fab fragments for 30 min at room temperature before the binding assay. The numbers given represent means of five determinations. Bacterial adherence to plant roots. The method used in studying the bacterial adherence to the P. pratensis roots will be described in detail elsehwere (K. Haahtela et al., manuscript in preparation). Briefly, the method was as follows. Bacteria grown in malate broth containing L-[4,5-3H]leucine (5 ,uCi/ml; Amersham) were collected, washed with 17 mM potassium phosphate buffer (pH 6.9), and suspended in buffer to give 109 per ml. Bacteria (5 x 10' per ml of buffer) were incubated with plant roots for 1 h at room temperature with occasional shaking and were washed five times with buffer. The washed roots were incubated with 200 pAl of Lumasolve (Lumac Co., Basel, Switzerland) for 1 h at 37°C, suspended to 2.5 ml of Lipoluma (Lumac Co.), and allowed to stabilize for 5 h at 4°C. Radioactivity was determined in a 1215 Rackbeta scintillation counter (LKB Wallac, Bromma, Sweden). In inhibition tests, the bacteria were incubated with increasing concentrations of Fab fragments or BSA for 30 min at room temperature before adhesion tests. The numbers given represent means of five determinations. Agglutination tests. Hemagglutinations and yeast cell agglutination with whole bacterial cells and purified fimbriae were performed as previously described (9). Human blood group 0 erythrocytes were treated with tannin as described by Duguid (2).

RESULTS Characterization of the 69/1 fimbriae. The type 3 fimbriae from K. aerogenes 69/1 retained their native morphology in the purification process; they were 4 to 5 nm in diameter and 0.5 to 2 ,um long (Fig. 1). No contaminating membrane vesicles were observed in the fimbrial preparation by electron microscopy. In SDS-PAGE, the type 3 fimbrial preparation gave only one peptide band, with an apparent molecular weight of 23,500 (Fig. 2). This was clearly different from those of the type 1 fimbrillins of K. aerogenes 55/1 (18,000) and E. coli 2131 (17,000), which are shown for comparison. The amino acid composition of the 69/1 fimbriae is given in Table 1. Hydrophobic amino acids comprised 33.6% of the amino acids in the

fimbrillin, which lacked cystine, phenylalanine, and arginine. The molecular weight calculated from the amino acid composition was 24,000. Hemagglutination properties of the 69/1 fimbriae. The intact 69/1 cells did not agglutinate yeast cells but did agglutinate human blood group 0 erythrocytes treated with tannin. The purified 69/1 fimbriae retained the hemagglutination properties of the cells; with tannin-treated erythrocytes, the hemagglutination titer (i.e., the smallest concentration of fimbriae showing hemagglutination) was 63 ,ug/ml. No hemagglutina-

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tion was observed with untreated erythrocytes. The type 1 fimbrial preparations agglutinated yeast cells but did not hemagglutinate tannintreated erythrocytes. Serological properties of the 69/1 fimbriae. The serological cross-reactivity of the type 3 fimbriae with the type 1 fimbriae from K. aerogenes, E. coli, and S. typhimurium was tested by

J. BACTERIOL.

ELISA (Table 2). The 69/1 did not show detectable (i.e., less than 3%) immunological crossreaction with the type 1 fimbriae from E. coli 2131 or S. typhimurium SH6749. However, the ELISA titrations revealed a low cross-reaction between the two Klebsiella fimbriae. The type 1 fimbriae from E. coli and S. typhimurium did not show any cross-reaction in ELISA, thus confirming our earlier results (10). The type 1 fimbriae from K. aerogenes 55/1 were antigenically related to the other fimbrial proteins, since the purified 55/1 fimbriae reacted with the four hyperimmune sera and the anti-55/1 serum gave titers with the four fimbrial antigens. Binding of purified 69/1 fimbriae to plant roots. A direct way to demonstrate the role of the type 3 fimbriae in bacterial adherence was to label the fimbriae with 1251 and to test their binding to plant roots in vitro. This was done by incubating roots of P. pratensis with increasing concentrations of labeled fimbriae (Fig. 3). The radioactivity remaining on the roots was dependent on the amount of labeled fimbriae added, and binding could be inhibited by the Fab fragments to the purified 69/1 fimbriae. In the experiment shown in Fig. 3, the concentration of the Fab fragments equaled that of the labeled fimbriae. The inhibition of binding by the Fab fragments was dependent on the dose; maximum inhibition (about 75%) was obtained with a fimbriae/Fab ratio of 5:1 (data not shown). Effect of Fab fragments on bacterial adhesion. The strain 69/1 of Klebsiella adheres to the roots TABLE 1. Amino acid composition of the type 3

fimbriae from K. aerogenes 69/1

a

b

c

d

mm-l

Amino acid

Aspartic acid ................. Threonine ....................

Alanine ......................

39 37 20 21 9 35 34

Cystine (half) .................

NDb

Valine ....................... Methionine .................... Isoleucine .................... Leucine ......................

25 1 5 10 Tr ND 14 ND Tr

Serine ....................... Glutamic acid ................ Proline ......................

Glycine ......................

Tyrosine ..................... Phenylalanine ................ Lysine ....................... FIG. 2. Comparison of type 3 and type 1 fimbriae in SDS-PAGE. Lanes: a, standard proteins, BSA (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (20,100), and alactalbumin (14,400); b, purified type 3 fimbriae from K. aerogenes 69/1 (25 ,ug); c, type 1 fimbriae from K. aerogenes 55/1 (15 ,ug); and d, type 1 fimbriae from E. coli 2131 (12) (15 jig).

Residues per molecule"

Histidine .....................

Arginine

.....................

a The proportion of hydrophobic amino acids (proline, alanine, valine, methionine, isoleucine, leucine, and phenylalanine) was 33.6%. The calculated molecular weight was 24,000. b ND, Not detected. c Recovered as methionine sulfone.

863

TYPE 3 FIMBRIAE OF KLEBSIELLA

VOL. 155, 1983

TABLE 2. Cross-reactions between the purified type 3 fimbriae of K. aerogenes 69/1 and the type 1 fimbriae of K. aerogenes 55/1, E. coli 2131, and S. typhimurium SH6749 Antiserum to fimbriae from:

Antibody titer' to fimbriae from: E. coli

K. aerogenes

K. aerogenes

69/1 4.8 2.1