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Campylobacter jejuni major outer membrane protein and a 59-kDa protein are involved in binding to fibronectin and INT 407 cell membranes

Irmgard Moser, W. Schroeder, Johann Salnikow
DOI: http://dx.doi.org/10.1111/j.1574-6968.1997.tb12778.x 233-238 First published online: 1 December 1997

Abstract

Campylobacter jejuni is one of the major causes of human diarrhea throughout the world. Attachment to host cells and extracellular matrix proteins is considered to be an essential primary event in the pathogenesis of enteritis. Outer membrane proteins of three C. jejuni strains, one of which was aflagellate, were investigated for their contribution to the process of adhesion to INT 407 cell membranes and the extracellular matrix protein fibronectin. Using a ligand-binding immunoblotting assay the flagellin, the major outer membrane protein and a 59-kDa protein were detected to be involved in adhesion to both substrates. The MOMP was able to inhibit the attachment of the bacteria to INT 407 cell membranes partly, when the protein was isolated under native conditions. However, it was totally lost when the protein was isolated in the presence of SDS. The 59-kDa protein of one strain was identified by N-terminal sequencing, and regarding the first 14 amino acids it was found to be identical to the 37-kDa CadF protein just recently described as fibronectin-binding protein of C. jejuni. Especially for the aflagellate strain this protein may be of special importance for adhesion of the bacteria to different substrates.

Keywords
  • Campylobacter
  • Major outer membrane protein
  • 59-kDa outer membrane protein
  • Adhesion
  • INT 407 cell
  • Fibronectin

1 Introduction

Campylobacter (C.) jejuni is one of the major causative agents of human diarrhea worldwide [1, 2]. Additionally it may cause the onset of polyneuropathy of the Guillain-Barré type, since in many cases a C. jejuni infection is detected preceding this disease [3]. The pathogenic mechanisms of the microorganism are still not well understood [46]. However in analogy to other pathogenic microorganisms adhesion to host tissue, cells and extracellular matrix proteins, is considered to be of special importance in establishing the infectious process. Several outer membrane proteins (OMPs) with apparent molecular masses from 26 to 32 kDa, the flagella and lipopolysaccharide have been described to be involved in adhesion of C. jejuni to HeLa cells and to Hep-2 cells by several authors [7, 8]. Previously published adhesion experiments performed in our laboratory indicated that other outer membrane proteins (OMPs), especially the major outer membrane protein (MOMP) of C. jejuni, may also play a role in adhesion to INT 407 cells [9]. Binding to extracellular matrix proteins, including fibronectin, laminin, collagen has been described for several pathogenic microorganisms including Staphylococcus aureus, enterotoxigenic Escherichia coli, and C. jejuni[1013]. In the present study we present data that the MOMP is able to inhibit adhesion to INT 407 cell membranes partially and another membrane protein with the apparent molecular mass of 59-kDa is involved in adhesion to both INT 407 cell membranes and the extracellular matrix protein fibronectin. The 59-kDa protein was identified by N-terminal amino acid sequencing of the first 15 amino acid residues.

The capacity to bind to these substances may help to establish the infection in the initial stage. In the present study we have shown that the same proteins, which mediate adhesion to INT 407 cell membranes are also involved in fibronectin-binding.

2 Materials and methods

2.1 Bacteria and growth conditions

C. jejuni 10945, the reference strain of serotype O 13, was received from the Culture Collection of the University of Göteborg, the wild-type strains were isolated from a healthy cat (K 22) and a cow (1767), identified as source of outbreak of human enteritis in Germany. The bacteria were cultivated on Mueller-Hinton agar containing 5% sheep erythrocytes under microaerophilic conditions as described before [9].

2.2 Outer membrane (OM) preparation

The OM preparation was performed according to the method of Blaser et al. as described previously [14, 15].

2.3 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE was performed according to Laemmli [16] at constant current (stacking gel 5% acrylamide, separating gel 12% acrylamide, 18 mA).

2.4 Isolation of the MOMP using preparative SDS-PAGE

The MOMP was isolated from OM preparations by preparative SDS-PAGE (stacking gel 5%, separating gel 12%, 50 mA) using a PrepCell apparatus (Model 491, Biorad) followed by electroelution as described previously [9].

2.5 Native preparation of the MOMP

The OM preparation was subjected to native gel electrophoresis according to Schägger and Jagow [17] with slight modifications as described previously [9].

2.6 Preparation of INT 407 cell membranes

Membrane fractions of INT 407 cells were prepared as described previously [15, 18].

2.7 Microadhesion assay and adhesion inhibition

Adhesion was measured as described previously using an enzyme-linked immunosorbent assay (ELISA; [15]) with slight modifications. Briefly, microtiter plates (Greiner) were coated with membrane fractions of INT 407 cells (20 μg/ml) or fibronectin (20 μg/ml; from bovine plasma; Sigma, Germany) in 50 mM carbonate buffer (pH 9.6) at 37°C overnight. Uncoated sites were blocked by incubation with horse serum diluted 1:50 in 10 mM phosphate-buffered saline (PBS; pH 7.4). Bacteria harvested from plates were suspended in PBS to OD600nm= 1. Subsequently bacteria (OD600nm= 1) or crude OM preparations (100 μg/ml) labelled with Sulfo-NHS-LC-Biotin (Pierce Rockford, IL, USA) according to Overath and Ziegelbauer [19] were incubated in the coated microtiter plates at 37°C. Adherent bacteria or OM preparations were detected with HRPO-conjugated streptavidin (Amersham Buchler, Braunschweig, Germany). Extinction was measured at 405 nm after 30 min and 60 min of incubation at 37°C. Incubations were performed for 1 h at 37°C. All washing steps between the incubations and the dilutions before the incubation of plates with viable bacteria were performed with PBS, all washings and dilutions following incubation with bacteria were performed with PBS-Tween (0.05%). For adhesion inhibition different concentrations (10–100 μg/ml) of the purified MOMP, isolated under native and denaturating conditions and horse serum (diluted 1:50 in PBS), respectively, were allowed to bind to INT 407 cell membranes immobilized in microtiter plates (see above) for 60 min at 37°C, followed by an adhesion assay as described above.

2.8 Ligand immunoblot assay

Unlabelled intact bacteria and OM preparations, respectively, were allowed to adhere to INT 407 cell membranes or fibronectin immobilized in microtiter plates. After 90 min of incubation the non-adhering bacteria or membrane components were removed by washing the plates 5 times with PBS-Tween, and the adhering components were solubilized by incubation of the plates with SDS-PAGE sample buffer for 90 min at 60°C. The material was subjected to SDS-PAGE and blotted onto nitrocellulose. The blotted components were detected by incubation of the blot membrane with rabbit antiserum raised against the homologous C. jejuni strains and horseradish peroxidase-conjugated goat anti-rabbit IgG (Boehringer, Mannheim, Germany) as described previously [15].

2.9 Antisera

Antibodies against whole bacteria were raised in adult white New Zealand rabbits as described previously [20]. Antiserum against OM preparations were raised by immunizing rabbits intravenously with bacterial OM preparations suspended in PBS (1 mg/ml).

2.10 Western blot

Western blots were performed as described previously [15, 21].

2.11 Protein determination

The protein content of membrane samples was determined according to Lowry [22].

2.12 N-terminal amino acid sequencing

N-terminal amino acid sequences were determined by automatic Edman degradation of proteins blotted onto polyvinylidene difluoride membranes as described previously [9].

2.13 Calculations

The adhesion tests were set six-fold. Adhesion values were calculated as means and standard deviations, if the tests were performed more than three times. The values presented without standard deviations are values of single representative tests. Statistical significance was not determined.

3 Results and discussion

From a large collection of C. jejuni strains adhering to INT 407 cell membranes and fibronectin three strains were investigated in more detail as representatives using a microadhesion ELISA procedure (Fig. 1, black bars). The C. jejuni strains 1767 and K 22, both flagellate, exhibited high and relatively low adhesion values, respectively, whereas strain C. jejuni 10945, aflagellate as confirmed by SDS-PAGE and Western blot analysis with flagellin-specific antiserum (data not shown), exhibited again intermediate adhesion values.

Figure 1

Adhesion and adhesion inhibition assay with the C. jejuni strains K 22, 1767 and 10945. Adhesion to INT 407 cell membranes was performed with biotin-labelled bacteria and OM preparations and measured using ELISA with HRPO-conjugated streptavidin. The bacteria and OM preparations were suspended in PBS/glucose and allowed to adhere for 60 min at 37°C. For adhesion inhibition the incubation of bacteria or OM preparation with INT 407 cell membranes was preceded by incubation of the coated microtiter plates with different concentrations of purified MOMP. Extinction was measured after 30 min of incubation with the substrate at 37°C.

Ligand immunoblot assays performed with both INT 407 cell membranes and fibronectin, immobilized in microtiter plates, revealed that in binding to both substrates the same bacterial membrane proteins were involved. When viable bacteria were allowed to adhere to INT 407 cell membranes, numerous adhesive membrane components were detected on the blot membrane (Fig. 2A). However, when preformed OM preparations were used in the adhesion test, the number of bacterial components binding to cell membranes or fibronectin was reduced to three proteins: the flagellin (63 kDa), the MOMP (43 kDa) and a 59-kDa protein (Fig. 2B). In case of binding to fibronectin lipopolysaccharide was also detected (Fig. 2C). When the aflagellate strain was used in the binding assay the flagellin band was missing on the binding blot. None of these proteins was detected by the C. jejuni-specific antiserum in negative control samples, which contained only INT 407 cell membranes or fibronectin as solid phase (Fig. 2B, C). The flagella have been extensively characterized on their molecular level and regarding their contribution to adhesion to cells [23]. The MOMP has been purified and sequenced on protein level recently and was also shown to bind to INT 407 cell membranes in its purified state [9]. For further characterization of its binding properties the MOMP of C. jejuni K 22 was tested for its capacity to inhibit adhesion of intact bacteria and OM preparations to INT 407 cell membranes using the microadhesion ELISA procedure. The adhesion values of intact bacteria of the C. jejuni strains K 22, 1767, 10945 and the OM preparation of C. jejuni K 22 were reduced partially in a dose-dependent fashion by the native MOMP (Fig. 1). The adhesion values were reduced by up to 40% for the OM preparation and up to 15% to 20% for bacteria. Significant differences in the capacity of the MOMP to inhibit adhesion of the homologous or the heterologous bacterial strains were not detected. When denatured MOMP at the same concentrations or horse serum (50 μg of protein/ml) were used for inhibition, no inhibitory effect was detected (data not shown).

Figure 2

A: Western blot analysis of OM components of intact bacteria adherent to INT 407 cell membranes. The C. jejuni strains 10945 (A; aflagellate) and 1767 (B; flagellate) are presented in this figure. B: Western blot analysis of OM components of the strains K 22 (A), 1767 (B), 10945 © adherent to INT 407 cell membranes. C: Western blot analysis of OM preparations of the strains K 22 (A), 1767 (B), 10945 © adherent to fibronectin. a, OM preparation; b, adhering OM components; c, negative control; MW, molecular mass (given in kDa); *> flagellin band; > MOMP; LPS, lipopolysaccharide; *, 59-kDa protein band.

While the primary structure of the MOMP has been analyzed, the 59-kDa protein was still unidentified. Therefore we determined the amino acid sequence of this protein, taken from the aflagellate strain (10945), by automatic Edman degradation of the protein band blotted onto PVDF membrane. The first 15 amino acid residues were identified and the following sequence was found: A D N N V K F E I T P T L N I.

This sequence is for the first 14 amino acid residues identical with the 37-kDa CadF protein, which was identified just recently by Konkel et al. [24]. In position 15, however, an isoleucine residue was found in our strain instead of an histidine residue. No indication for the flagellin sequence was found in the OMP preparation of this strain on the molecular mass level of 59–62 kDa.

As shown in Fig. 2, on the ligand-binding blot of strain C. jejuni K 22, exhibiting weak binding affinity to INT 407 cell membranes or fibronectin, both the 45-kDa protein and the 59-kDa protein could not be detected. N-terminal amino acid sequencing of two different proteins appearing on the molecular mass level around 59 kDa after separating OMPs by SDS-PAGE revealed that one of these proteins was the flagellin subunit exhibiting the amino acid sequence: G F R I N T N V A A L N A K A N A D L. The second protein did also not possess a CadF-like sequence.

These findings indicate that in addition to flagella the MOMP and the 59-kDa protein may contribute to adhesion of C. jejuni to both cellular and extracellular substrate. The reason, why we found the 59-kDa protein with the N-terminal amino acid sequence identical to the CadF 37-kDa protein of C. jejuni, is not known. It may be a partially dimeric formation of this protein. It is however interesting that on the C. jejuni strain with weak affinity to both substrates we detected a protein with the flagellar amino acid sequence instead of a CadF-like protein with the apparent molecular mass of 59 kDa.

The relevance of these findings for the situation in vivo has to be investigated further, however, since antibodies against C. jejuni-LPS are suspected to be involved in post-infectious complications, like the Guillain-Barré syndrome or the Miller-Fisher syndrome, it seems to be desirable to develop a vaccine, which is devoid of carbohydrate components. In this context the MOMP and the 59-kDa CadF-like protein may be a useful component of a vaccine.

Acknowledgements

The authors wish to thank Mrs. Haeselbarth and Mr. P. Schwerk for their excellent technical assistance.

References

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