Immunochemical characterization of a haemagglutinating antigen of Arcobacter spp.

Transcription

1 ELSEVIER FEMS Microbiology Letters 136 (1996) Immunochemical characterization of a haemagglutinating antigen of Arcobacter spp. Abstract Raymond S.W. Tsang aq *, John M.C. Luk b, David L. Woodward a, Wendy M. Johnson a a National Laboratory for Enteric Pathogens, Bureau of Microbiology, Laboratov Centre for Disease Control. HPB Building, Room 188, TunneJ s Pasture, Ottawa, Ontario KIA OL2, Canada b Department of Pediatrics, Case Western Reserve Unir,ersity, Cleveland, Ohio, USA Received 12 October 1995; revised 1 December 1995; accepted 17 December 1995 The Arcobacter haemagglutinin has been identified by Western immunoblot to be an immunogenic protein of about 20 kda. The haemagglutinating activity is sensitive to proteolytic enzyme digestion and heat treatment of 80 C and above. The Arcobacter haemagglutinin is possibly a lectin-like molecule binding to erythrocytes via a glycan receptor containing D-galactose as part of its structure. Keywords: Arcobacter; Lectin-like haemagglutinating antigen 1. Introduction 2. Materials and methods Arcobacter spp. are potential emerging enteropathogens which can cause severe diarrhea and occasionally systemic infections such as bacteremia and peritonitis in human [ 1,2]. To understand the pathogenesis of this emerging pathogen, putative adhesive factor(s) in this organism was studied by examining their ability to agglutinate erythrocytes from human and other animal species. Corresponding author. Tel: + 1 (613) ; Fax: + I (613) ; rtsang@hpb.hwc.ca This work was presented as a poster in the First International Rushmore Conference on Mechanisms in the Pathogenesis of Enteric Diseases, South Dakota, USA, September 27-30, I. Bacterial strains and cultivation condition Two strains of Arcobacter, LCDC isolated from a human blood specimen, and LCDC 15792, isolated from poultry were grown aerobically on Muller-Hinton blood agar (with 10% sheep blood) at 30 C. They were identified by biochemical reactions according to phenotypic tests described by Vandamme et al., [3] and serologically by slide agglutination method according to Lior and Woodward [4]. Proteinase K treatment of bacteria was done by mixing equal volumes of Arcobacter (at about 10 cells/ml) and serial dilutions of proteinase K (Sigma Chem. Co., St. Louis, Missouri, USA) from 2 mg or 28 units per ml to 2 ng/ml in ph 7.4 PBS at 37 C /96/$ Federation of European Microbiological Societies. All rights reserved SSDl (79)

2 for 1 h. Control was done by exposing bacteria to an identical treatment with PBS substituting for the proteinase K solution ~uem~gglut~nation and huemagglut~nuti~)n inhibition Haemagglutination was performed by mixing 25 ~1 of a suspension containing about 10 cells/ml of Arcobacter in PBS with an equal volume of a 3% erythrocyte suspension in PBS on a clean microscope slide. Macroscopic haemagglutination was scored as either positive or negative after 10 min of mixing at room temperature on a platform rotator. Haemagglutination inhibition was carried out by adding 5 ~1 of either PBS as control or 5 ~1 of inhibitor (5% w/v solution of different sugars in PBS) to of bacteria and 25 ~1 of erythrocytes with mixing and observation of the reaction as described above. Those sugars giving positive inhibition at 5% concentration were serially diluted twofold and the haemagglutination inhibition reaction was repeated as described above to find out the minimum amount required to give inhibition Sonic lysate preparation Muller-Hinton blood agar plate grown Arcobacter (40 h at 37 C) were resuspended in PBS to a cell density of about 10 cells/ml for sonication in a Braun-sonic 1510 sonicator (B. Braun Biotech., USA). Sonication was done with the sample cooled in an ice-bath and the power output of the instrument set at 350 watt. Five cycles of 1 min each were performed with 1 min of cooling between each cycle of sonication. After sonication, unbroken cells were removed by centrifugation at 8000 X g for 20 min in a Beckman model refrigerated centrifuge (Beckman Instruments Inc., USA). Supematant from the centrifuged lysate was filtered through a 0.45 pm cellulose acetate membrane filter (Pro-X filter, Lida Manufacturing Corp., USA) and stored at 4 C before use SDS-PAGE and Western immunoblot SDS-PAGE was carried out on a 12.5% polyacrylamide gel using dissociating buffer condition as described by Laemmli [5]. Western immunoblot was performed after SDS-PAGE according to procedures described by Towbin et al. [6]. Excess binding sites on the nitrocellulose membrane were blocked with a 2% solution of bovine serum albumin (BSA) in PBS at 37 C for 1 h. Immune reaction was carried out with a rabbit antiserum (1: 100 dilution) to whole cells of A. butzleri serotype 4 [4] followed by horseradish peroxidase enzyme conjugated goat anti-rabbit serum (1:3000 dilution) and 4 chloro-lnaphthol. Incubation of the membrane with antibodies was done at room temperature for 1 h, and the diluent for the antibodies was 2% BSA-PBS. Membrane was washed after blocking and reactions with antibodies using the following buffers: once for 10 min in 10 mm Tris-HCl buffer, ph 7.4 with 0.9% NaCl (Tris-saline); twice for 10 min each in Trissaline with 0.05% Tween-20. and once for 10 min in Tris-saline. Samples of erythrocytes used for Western immunoblot were prepared by mixing 1 ml of 3% human group 0 red cells with an equal volume of Arcobacter sonic lysate or with PBS as negative control at room temperature for 2 h to allow the haemagglutinin to absorb onto the red cells. After three washes with large volumes of sterile PBS, the washed erythrocytes with their bound Arcobacter haemagglutinin were redissolved in 150 ~1 of SDS- PAGE sample buffer, boiled for 10 min and analysed using 30 ~1 per lane. Prestained protein molecular mass standards (ISS mid range kit with proteins in the molecular mass range of 95 kda to 14 kda, Integrated Separation Systems, Enprotech Corporation, USA) were used for monitoring the electrotransfer process and for estimating the molecular mass of the HA band in the immunoblot. 3. Results and discussion 3.1. Strains and characteristics qf their haemagglutination reaction Two strains of aerotolerant Gram-negative motile curve bacilli were identified by biochemical reactions to belong to the genus Arcobacter. Using a serotyping scheme based on heat labile antigens [4], the two strains of Arcobacter were identified as serotype 4 (strain 13198) and serotype 28 (strain 15792).

3 R.S. W. Tsang et al./ FEMS Microbiologr Letters 136 ( Fresh cultures of both strains agglutinated erythrocytes from human (blood groups A, and 01, sheep, and rabbit. Twenty five microliters of a suspension containing 2.1 X IO8 cells/ml (determined by standard spread plate colony count technique) for strain or 2.05 X IO9 cells/ml for strain 13198, gave strong macroscopic haemagglutination within 10 min after mixing with an equal volume of a 3% erythrocyte suspension on a clean microscope slide. The ability of the bacteria to haemagglutinate was destroyed by heat and proteolytic enzyme treatments of the bacteria (Table l), suggesting that the Arcobacfer haemagglutinin is a protein. Electron microscopic examination of the two Arcobacter strains revealed neither pili nor fimbriae (data not shown), therefore the Arcobacter haemagglutinin does not appear to be similar surface appendages known to function as haemagglutinin and adhesin in other bacteria such as Escherichia coli or Salmonella. Nonfimbrial haemagglutinins have also been described in other bacteria such as Escherichia coli [7], Vibrio cholerue [8], and Aeromonas hydrophilu [9]. To understand the nature of the red cell receptor for the haemagglutinin, various sugars were used to inhibit the haemagglutination reaction (Table 2). D- Table 1 Effect of heat and protease K treatment of Arcobacterbacteria their ability to agglutinate erythrocytes Treatment Proteinase K (concentration) digestion: (2 mg/ml) - ve (200/Lg/rnl) - ve (20 kg/ml) - ve (2 pg/ml) - ve (200 ng/ml) - ve (20 ng/ml) - ve (2 ng/ml) + ve (0 ng/ml; PBS control) + ve Haemagglutination Result Heat treatments: 100 C for 10 mitt - ve * 80 C for 60 min - ve * 50 C for 60 min - ve * 37 C for 60 min + ve * Although no haemagglutination was observed with bacteria treated at 50 C. 80 C and loo C, soluble haemagglutinin could be demonstrated by Western immunoblot in the supematant of the heated bacterial extract at 50 C but not at 80 C or 100 C. on Table 2 Inhibition of the Arcobacfer haemagglutination (HA) reaction by monosaccharides, and amino sugars Inhibitors Minimum amount (mm) required for inhibition of the HA D-Glucose + BMannose L-Fucose Methyl-o-DMannopyranoside Methyl-/.I-tSlucopyranoside N-Acetyl-DGlucosamine N-Acetylneuraminic Acid t&alactose D-Fucose N-Acetyl-DGalactosamine z 5.56 D-Galactosamine z No inhibition when tested at 5% (w/v) concentration. Galactose was found to be the most potent inhibitor followed by D-fucose (about 4-fold less effective compared with D-galactose), N-Acetyl-Dgalactosamine (about 25fold less effective) and o-galactosamine (about looo-fold less effective). This indicated that o-galactose containing glycolipid or glycoprotein on the surface of the red cells serves as a receptor for the binding and cross-linking by Arcobacter Immunological characterization of the Arcobacter haemagglutinin Haemagglutinin extracted from the Arcobacter, by sonication and millipore filtration to remove intact bacteria, did not cause agglutination of erythrocytes. This observation that extracted haemagglutinin from the bacteria is unable to agglutinate erythrocytes is not unique to our study. Similar findings have also been reported for the outer membrane haemagglutinin of Aeromonas hydrophila [9], and the haemagglutinating adhesin of group B streptococci [lo]. However, the haemagglutinin could be demonstrated to bind to erythrocytes by analysing erythrocytes exposed to a sonic lysate of a haemagglutinating strain of Arcobacter by Western immunoblot with an antiserum raised against whole cells of Arcobacter. Fig. 1 compared the reactions of human group 0 erythrocytes, either exposed to a sonic lysate of Arcobacter or exposed to PBS as

4 212 R.S. W. T.sang et (11./ FEMS Mkrobiology Letters 1.36 (IY96I 20%213 Fig. I. Western immunoblot to detect Arcobacter haemagglutinin in sonic lysates and erythrocytes exposed to sonic lysates ot Arcobacter. Lanes: I, sonic iysate of Arcobtrctrr 13198, serotype 4; 2, sonic lysate of Arcobactrr serotype 28: 3, erythrocytes exposed to sonic lysate of 13198: 4, erythrocytes exposed to sonic lysate of 15792: 5, erythrocytes not exposed to Arcobtrcter. Arrow indicates position of the 20 kda haemagglutinin. negative control. with an immune serum developed against whole cells of Arcobacter [4]. Erythrocytes exposed to Arcobacter reacted with the anti- Arcobacter serum to give an additional band with an apparent molecular mass of 20 kda not found in the reaction with erythrocytes not exposed to Arcobacter. This unique 20 kda band was also found in the fraction eluted from erythrocytes that had been exposed to sonic lysate of a haemagglutinating Arcobacter strain, using a solution of either 3% D- galactose or 3% D-fucose. Similar results were obtained when rabbit red cells were used for detecting the soluble Arcobacter haemagglutinin. The finding of several bands of reaction between erythrocytes not exposed to Arcobacter and the immune serum against Arcobacter was not expected but might be heterophile antigen-antibody reactions between rabbit serum and human red cell antigens. However using normal rabbit serum, the reactions observed with erythrocytes were much diminished suggesting that immunizing rabbits with Arcobacter might induce antibody formation against normal tissue components. This finding may warrant futher investigation. Control immunoblots with only the first antibody (rabbit anti-arcobacter serum) or with only the second antibody (enzyme conjugated goat anti-rabbit serum) showed no immuno-staining of the membrane. The ability of erythrocytes to adsorb the Arcobacter haemagglutinin provides a basis for future development of an affinity chromatography method for isolating and purifying the haemagglutinin antigen. Based on knowledge and information available on the red cells carbohydrate antigens, synthetic glycoconjugates on solid matrix medium may be prepared for fractionating the haemagglutinin antigen from sonic lysates of Arcobacter strains. The ability of red cells from rabbits and other animal species to adsorb the haemagglutinin antigen can also provide a unique approach to prepare an immunogen (red cells with bound Arcobacter haemagglutinin) for immunizing rabbits to raise antiserum. With the availability of specific antiserum, the distribution of the haemagglutinin antigen and the function of this potential adhesin can also be studied in more detail, not only in Arcobacter but also in related species of organisms. Acknowledgements The authors wish to thank Dr. J. Austin for performing the electron microscopic examination of the Arcobacter strains. One of us (R.S.W.T.) also wishes to thank Dr. H.J. Jennings for helpful discussion. References [I] Kiehlbauch. J.A.. Tauxe, R.V. and Wachsmuth, I.K. (1991) Clinical features of Cump~lobncter butzleri associated diarrhea1 illness. Microb. Ecol. Health Dis. 4, S92. [2] Lerner. J., Brumberger, V. and Preac-Mursic. V. (1994) Severe diarrhea associated with Arcobucter baleri. Eur. J. Clin. Microbial. Infect. Dis. 13, [3] Lior, H. and Woodward. D. (1991) A serotyping scheme for Cmnp~lobacter burden. Microb. Ecol. Health Dis. 4, S93.

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