Glycobiology, 2000, Vol. 10, No. 1 31-37
© 2000 Oxford University Press
Purification and characterization of an adhesin from Pasteurella haemolytica
CENID-Microbiología, Instituto Nacional de Investigaciones Forestales y Agropecuarias, SAGAR. Mexico, 2Departamento de Bioquímica, Instituto Nacional de Enfermedades Respiratorias, Secretaría de Salud, México, 3Laboratoire de Chimie Biologique, UMR 8576 du CNRS, Université des Sciences et Technologies de Lille, 59655 Villeneuve d’Ascq, France, 4Departamento de Patología, Facultad de Medicina Veterinaria y Zootecnia, UNAM, México, and 5Departamento de Bioquímica, Facultad de Medicina UNAM, PO Box 70159, 04510 Mexico DF
Received on March 22, 1999; revised on June 15, 1999; accepted on July 2, 1999.
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We purified an adhesin from Pasteurella. haemolytica by affinity chromatography using glutaraldehyde treated rabbit erythrocytes stroma. The adhesin is a protein of 68 kDa, as determined by SDS–PAGE, and the most abundant amino acids constituting this protein were Gly, Ser, Glx, and Ala, and low concentrations of Cys, Met, and Tyr residues were also found. The N-terminal sequence of the adhesin is ANEVNVYIYKQPYLI. No carbohydrate residues were detected. The adhesin agglutinated rabbit erythrocytes but when the latter were desialylated or pronase treated the agglutinating activity was abolished. The agglutinating activity of the adhesin was inhibited with N-acetyl-D-glucosamine (GlcNAc), and in a lesser degree with N-acetyl-neuraminic acid (NeuAc). GalNAc, N-glycolyl-neuraminic acid, N-deacetylated GlcNAc, or neutral sugars do not modify the activity of the adhesin. The equatorial -OH on C4 and the NH-acetylated group on C2 from GlcNAc, as well as the 4-OH and NH-acetylated group on C5 from NeuAc seem to be responsible for the interaction with the adhesin. The protein is divalent cation-dependent and thermolabile. As for the agglutinating activity, the adhesion of P.haemolytica to tracheal cell-cultures was inhibited by GlcNAc, NeuAc or the purified adhesin, strongly suggesting that the P.haemolytica adhesin plays an important role in infection.
Key words: Pasteurella haemolytica/glycoproteins/bacterial adhesin/N-acetyl-glucosamine/sialic acid
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Introduction |
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Pasteurella haemolytica, the main causative agent of enzootic septicemia in sheep, has been classified into two biotypes, A and T, and 16 serotypes (Biberstein, 1978
; Fraser et al., 1982). The A1 serotype is associated with outbreaks of shipping fever, a respiratory disease that affects feedlot livestock (Yates, 1982; Whiteley et al., 1992). Although the bacterial capsular substance of A1 plays a role in the pathogenicity of the organism, it has recently been shown that the supernatants of P.haemolytica A1 cultures possess O-sialoglycoprotein endopeptidase and neuraminidase activity (Frank and Tabatabal, 1981; Abdullah et al., 1990; Straus et al., 1993); nevertheless, the role of these proteins in the pathogenesis and induction of immune responses remains unclear. One key step in bacterial pathogenesis and infection is adherence (reviewed in Jaques and Paradis, 1998). In Pasteurellaceae members, such as Haemophilus, Actinobacillus and Pasteurella, a wide variety of adhesin–host receptor interactions have been reported in this group of bacteria. Some of these interactions have been demonstrated to be due to the participation of specific organelles such as fimbria or outer membrane proteins; in some cases the adhesion mechanisms are mediated through specific recognition of carbohydrates on the host cell surfaces (Yates, 1982; Jaques and Paradis, 1998). Capsular polysaccharides and lipopolysaccharides has been proposed to participate in the adhesion mechanisms for P.haemolytica (Whiteley et al., 1992); however, to date the specific adhesion mechanisms of this bacteria remain unknown (Confer et al., 1995; Jaques and Paradis, 1998). In this work we describe the purification, the partial characterization, and the sugar specificity of a P.haemolytica adhesin, which explains in part the lung tropism properties exerted by this bacteria.
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Purification of the adhesin
Pasteurella haemolytica was obtained from pneumonic processes in bovines and was biochemically and serologically characterized as an A1 serotype. The adhesin was purified from bacterial extracts, by incubating the bacterial suspension at 41°C and then precipitating the solubilized material with ammonium sulfate at 66% saturation; this concentration provided almost 90% of the hemagglutinating activity of the solubilized material. The molecule responsible for the agglutinating activity was eluted with 200 mM GlcNAc or NeuAc from an affinity chromatography column containing glutaraldehyde fixed stroma from rabbit erythrocytes physically entrapped in Sephadex G-25 (Figure 1). No other monosaccharide or N-deacetylated glucosamine, used at the same concentration, nor heparan or chondroitin sulfate at 10 mg/ml, was able to elute the protein. Quantitative elution of the protein from the affinity chromatography column was also obtained by adding 0.1 M glycine/HCl, pH 2.8, instead of the specific sugar. The purification of the adhesin from P.haemolytica, could be performed also by affinity chromatography on GlcNAc coupled on epoxy-activated Sepharose 4B, and the molecular characteristics of the purified adhesin were identical to the fraction purified by affinity chromatography on glutaraldehyde fixed stroma; however, with the immobilized GlcNAc matrix, the yield of protein was 50% lower. As summarized in Table I, the amount of purified protein represented >0.02% of the total protein obtained from the bacterial extract.
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Chemical characterization of the protein
The eluted molecule is a protein of 68 kDa, as determined by SDS–PAGE electrophoresis (Figure 2). Preliminary studies performed with the purified adhesin by gel filtration chromatography on a Sephadex G-150 column, reported fractions with hemagglutinating activity in the exclusion volume as well as in fractions corresponding to 68 kDa, suggesting that the purified protein possesses high tendency to form molecular aggregates (not shown). The most abundant amino acids identified were Gly, Ser, Glx, and Ala, and in minor proportion Cys, Met, and Tyr. The protein does not contain carbohydrates, as determined by gas-liquid-chromatography (Table II), and its N-terminal sequence is ANEVNVYIYK QPYLI.
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Hemagglutinating activity of the purified protein
Bacterial extracts as well as the purified protein showed hemagglutinating activity toward rabbit erythrocytes; the minimal concentration of purified protein that agglutinated a 2% rabbit erythrocyte suspension was 0.1 µg/ml. Treatment of rabbit erythrocytes with pronase or C.perfringens neuraminidase diminished 16- and 32-fold, respectively, the capacity of the purified protein to agglutinate them. Erythrocytes from other animal species such as cow, sheep, rat, mice, and human ABO, were not agglutinated even at concentrations of 12 µg/ml.
Sugar specificity
The sugar specificity of the purified P.haemolytica adhesin was determined by comparing the inhibitory activity of various sugars, glycans, glycoproteins and polysaccharides on the agglutination induced by the agglutinin against rabbit erythrocytes. Table III shows that from all the sugars tested, only GlcNAc and NeuAc inhibited the hemagglutinating activity of the agglutinin; polymers of GlcNAc, such as N,N'-acetylchitobiose and N,N',N''-acetylchitotriose, were also inhibitors. NeuAc 
-methylglycoside was 2-fold better inhibitor than NeuAc ß-methylglycoside, but both methylglycosides were better inhibitors than NeuAc. We found that (2,3) or (2,6) sialyllactosamine, and colominic acid (NeuAc2–8n) showed capacity to inhibit the hemagglutinating activity. From the glycoproteins tested, fetuin was the most powerful inhibitor of the hemagglutinating activity but 1-acid glycoprotein and human transferrin were also inhibitors, although to a lesser extent, of the agglutinin activity. The asialo-forms of these glycoproteins lost their capacity to inhibit the agglutinating activity of the purified protein. Anionic glycosaminoglycans such as heparan and chondroitin sulfate had a lesser inhibitory activity than the glycoproteins. Protamine, a cationic compound, did not inhibit the hemagglutinating activity.
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Bacterial adhesion-specificity
P.haemolytica has the capacity to adhere to the microvilli and the cell body of the epithelial tracheal cells from healthy calf (Figure 3a). As shown on Table IV, adhesion of P.haemolytica to calf epithelial cells was inhibited specifically with GlcNAc and NeuAc (p < 0.005 and 0.025, respectively). Neither N-glycolyl-neuraminic acid nor other monosaccharides inhibited adhesion of bacteria, and no significant inhibitory effect was also found using polymers from GlcNAc (as chitobiose and chitotriose) or from NeuAc (as colominic acid). Similarly to the effect observed upon the hemagglutinating activity by the Pasteurella extract and its purified protein, fetuin was the most powerful inhibitor (p < 0.0005) of bacterial tracheal adhesion (Table IV, Figure 3b). Addition of the purified protein to the epithelial tracheal cells before incubating them with the bacteria indicated that at concentrations of 10 and 25 µg the adhesion of the bacteria was inhibited 50 and 75%, respectively, in comparison with nontreated epithelial cells (p < 0.025) (Table IV).
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Physicochemical properties
Treatment of the P.haemolytica adhesin with 0.1 M EDTA practically eliminated its agglutinating activity; however, addition of divalent cations such as Ca2+, Mg2+, or Mn2+ restored the activity, indicating that metals are essential for the hemagglutinating activity. The hemagglutinating activity of the purified adhesin remained practically unaltered when the protein was incubated at 56°C or above for 30 min; however, at 65°C and over, the activity was drastically diminished after a 15 min incubation and was completely inhibited after 30 min incubation at 65°C or 5 min at 100°C.
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Discussion |
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Members of the Pasteurellaceae are small Gram negative rods that colonize the mucosal surface of the respiratory and genital tracts by specific recognition of saccharidic structures on the host (Whiteley et al., 1992
). Pasteurella haemolytica is the main bacterial pathogen in the bovine respiratory complex, although the mechanisms involved in the colonization of host mucosal surfaces by this bacterium are still unknown (Confer et al., 1995; Jaques and Paradis, 1998). In this work we report the purification of a 68 kDa adhesin from P.haemolytica bacterial extracts, by affinity chromatography on fixed rabbit erythrocyte stroma. The N-terminal sequence of the adhesin is ANEVNVYIYK QPYLI. The presence of adhesins has been described in other members of the Pasteurellaceae group, such as Haemophilus influenzae (Reddy et al., 1996) and Actinobacillus actinomicetemcomitans (Groenink et al., 1996; however, no homology with the amino terminal sequence of these adhesins nor other bacterial adhesins was identified (Swiss Protein Data Bank, version 1998).
The adhesin is dependent of divalent cations and agglutinates rabbit erythrocytes specifically; erythrocytes from other animal species were not agglutinated. Elimination of sialic acid residues by neuraminidase and protease treatment inhibit the interaction of the adhesin with the erythrocytes. These results indicate that the adhesin membrane receptor is a sialoglycoprotein, since it has been described that pronase treatment cleaves glycoproteic components on the erythrocyte surface (Bratosin et al., 1995).
Our results show that P.haemolytica adhesin is specific for GlcNAc and NeuAc. GalNAc did not inhibit the adhesin activity, thus indicating that the OH group on the C4, in axial position, from the GlcNAc, is essential for the interaction. Elimination of the acetylated group on the C2 from GlcNAc, by hydrazinolysis, is detrimental to the adhesin’s binding affinity, indicating that the adhesin prefers the presence of an acetamido group at C-2 rather than the amino group alone, such as in GlcNH2. It is interesting to note that the GlcNAc oligomers chitobiose and chitotriose inhibit also the hemagglutinating activity of the adhesin; however, the GlcNAc is a more powerful inhibitor of the bacterial adhesion than its oligomers, very probably by the presence of a more specific ligand for the adhesin on the epithelial cells than in the rabbit erythrocyte membrane. P.haemolytica shows great affinity for glycoproteins which characteristically possess sialylated bi- and tri-antennary N-glycosidically linked glycans of the N-acetyl-lactosaminic type, such as fetuin (Spiro and Bhoyroo, 1974; Takasaki and Kobata, 1986), 1-acid glycoprotein (Fournet et al., 1978), and transferrin (Montreuil, 1975). O-Glycosylated proteins such as ovine submaxillary mucin, which contained high amounts of NeuAc, and bovine submaxillary mucin, which contained O-acetyl-sialic acid, are less powerful inhibitors than fetuin. Hen ovalbumin, which possesses oligomannosidic and hybrid-type structures and lacks NeuAc residues (Montreuil, 1984), does not inhibit the hemagglutinating activity of the adhesin.
The interaction of the adhesin with rabbit erythrocytes or tracheal cells is inhibited by GlcNAc and to a lesser degree by NeuAc. There are other examples of GlcNAc-binding lectins that bind NeuAc (e.g., wheat germ agglutinin) similarly to P.haemolytica adhesin. This is due to the fact that when the 4-OH and 5-NHAc on the N-acetyl-neuraminic acid ring are lined up with the same group of N-acetyl-D-glucosamine (3-OH and 2-NHAc), they are very similar in conformation (Ichikawa et al., 1990). The fact that NeuAc-methyl-glycosides, preferentially in the -anomeric form inhibited the adhesin hemagglutinating activity, suggests that the position of the carboxyl group of NeuAc plays a relevant stabilization role for the interaction of the carbohydrate-recognizing domain of the adhesin with complex oligosaccharides (Peters et al., 1979). For this reason we identify that anionic compounds, such as chondroitin and heparan sulfate, inhibited the hemagglutinating activity of the adhesin; however, protamine, a cationic protein does not modify the biological activity of the adhesin, thus confirming that negative charged groups are also important for an adequate adhesin-receptor interaction.
The functional relevance of the adhesin as a virulence factor is strongly suggested by the fact that the purified adhesin inhibits the adhesion of the bacterium to tracheal cells. P.haemolytica adhesin shares the GlcNAc sugar specificity with adhesins from some strains of Escherichia coli, which have enterotoxigenic and septicemic activity toward bovines (Bertin et al., 1996). But in contrast to E.coli adhesins, the NeuAc residues seem to play a major role in the interaction of P.haemolytica adhesin with its cellular receptor. The capacity of adhesins to interact with sialic acid–containing oligosaccharides has been identified in H.influenzae (Reddy et al., 1996) and A.actinomycetemcomitans (Groenink et al., 1996. It would be interesting to assess whether this specificity for N-acetylated sugars is a common characteristic of the members of the Pasteurellaceae group. The functional role of P.haemolytica adhesin role in pathogenesis remains to be determined, nevertheless it is highly possible that this adhesin participates, in a sequential process of host-cell recognition preceding the release of exoproteins with catalytic activity, such as endopeptidase (Fraser et al., 1982) and/or neuraminidase (Straus et al., 1993). Our findings indicate the relevance of the P.haemolytica adhesin as a potential target for vaccine development against pasteurelosis (Wizemann et al., 1999).
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Materials and methods |
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Materials
Pronase (from Streptomyces griseus fraction XXV), neuraminidase (from Clostridium perfringens, fraction V EC 3.2.1.18), glycoproteins and all sugars were purchased from Sigma Fine Chemical (St. Louis, MO). Human transferrin as well as its asialo-form were gifts from Prof. Genevieve Spik;
(2–3) and (2–6) sialyl-lactose from human milk and hen ovomucoid were provided by Dr. Gerard Strecker, Université des Sciences et Technologies de Lille, France.
Bacteria
Pasteurella haemolytica classified as A1 at the Instituto Nacional de Investigaciones Agricolas y Pecuarias, Mexico, was isolated from bronchial lavages of sheep with pneumonic pasteurelosis and was grown to a late logarithmic phase in brain heart infusion (Bioxon, Mexico) (Carter, 1986). The stock culture was stored in 10% skim milk at –70°C. Cells were harvested by centrifugation (10,000 x g, 10 min at 4°C), washed with PBS (50 mM sodium phosphate, NaCl 0.15 M, 5 mM CaCl2, pH 7.4) and suspended in 200 ml of PBS. Bacterial extracts were obtained by incubation of the bacteria at 41°C, 1 h, under constant shaking followed by centrifugation at 10,000 x g, 30 min. The clear supernatant representing the crude extract was stored at –70°C until used.
Epithelial cells
These cells were obtained from the trachea of healthy 2-day-old calf, killed at the slaughterhouse. The cells were obtained within the first 2 h after the animals’ death. The trachea was transported to the laboratory in polystyrene boxes containing freezing bags; after extensive washing with sterile PBS, the trachea was cut in 2 cm rings. Epithelial cells were obtained by scraping the inner part of the rings with a surgical knife. Cells were collected in 50 ml Falcon tubes containing 35 ml of sterile PBS, washed thrice by centrifugation at 2500 x g for 10 min, counted, and adjusted to 1 x 106 cells/ml in PBS. Cell viability (>90%) was determined by trypan blue-dye exclusion.
Protein purification
The crude extract was precipitated with ammonium sulfate at a final saturation of 66%. The precipitate was solubilized in PBS, exhaustively dialyzed against PBS, and its protein concentration determined by the method of Lowry et al. (1951) using bovine serum albumin as standard; 750 µg of the precipitate were poured onto a 1 x 5 cm column containing glutaraldehyzed rabbit erythrocytes stroma, physically entrapped in Sephadex G-25 prepared according to Vázquez et al. (1993). The column has been equilibrated previously with PBS at room temperature with a flow rate of 10 ml/h. Unbound material was eluted from the column with PBS until the optical density of the eluent at 280 nm was <0.01. Bound material was eluted with 200 mM GlcNAc or NeuAc in PBS and the 1 ml fractions were dialyzed against PBS before testing their hemagglutinating activity against rabbit red blood cells. Active fractions were pooled, dialyzed against distilled water, lyophilized and kept at –70°C until used. Control experiments were performed using different monosaccharides as well as N-deacetylated GlcNAc or GalNAc as eluent of the affinity chromatography column. The homogeneity of the purified protein was analyzed by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) according to Laemmli (1970), and its molecular weight was determined by comparison with protein standards (Pharmacia, Uppsala, Sweden).
Analytical methods
The amino acid analysis was performed on an automatic amino acid analyzer Durrum 500, according to Bidlingmeyer et al. (1984) using 100 µg of the sample hydrolyzed under vacuum with 2 ml 6 M HCl at 110°C in sealed tubes for 24, 48, and 72 h. Nor-Leucine was used as internal standard. The N-terminal sequence analysis was determined on samples of purified P.haemolytica protein resolved in 15% SDS–polyacrylamide gels and transferred to a PVDF with a Trans-Blot Cell (Bio-Rad). After the protein band was excised from the blot, it was sequenced on a Beckman LF3000 protein sequencer (Beckman Instrument Inc., Fullerton, CA.). Carbohydrate analyses of desialylated glycoproteins and of the purified protein were performed by methanolysis in the presence of meso-inositol as internal standard. The trimethylsilylated methyl-glycosides (after re-N-acetylation) were analyzed by gas-chromatography using a capillary column (25 x 0.32 mm) of 5% Silicone OV 210 (Applied Science Lab., Buffalo, NY), in a Varian 2100 gas chromatograph (Orsay, France) equipped with a flame detector and a glass solid injector. The carrier gas was helium at a pressure of 0.6 bar, and the oven temperature programed from 150°C to 250°C at 3°C per min as described by Montreuil et al. (1986).
Hemagglutinating activity
Human erythrocytes type A, B, O, M, N, Lea, Leb, P, S, and Kell from healthy human donors were obtained from the Central Blood Bank, IMSS, Mexico. Erythrocytes from different animal species were obtained at the School of Veterinary Medicine, UNAM, Mexico. The hemagglutinating activity was assayed in microtiter U plates (NUNC, Denmark) by the twofold serial dilution procedure. The hemagglutinating activity was tested with a 1% (v/v) erythrocyte suspension in PBS. Assays were performed with native erythrocytes, neuraminidase-treated (0.1 U of neuraminidase per 0.5 ml of packed erythrocytes at 37°C for 30 min) or pronase-treated (100 µg of protease per 0.5 ml of packed erythrocytes at 37°C for 30 min) erythrocytes (Vázquez et al., 1993).
Desialylation of glycoproteins and N-deacetylation of sugars
Fetuin, transferrin, and 1-acid glycoprotein were desialylated by incubation at 100°C for 1 h in the presence of 0.02 N sulfuric acid (Spiro and Bhoyroo, 1974) and desalted on a Bio-Gel P-2 column (2 x 60 cm) equilibrated with water. N-Deacetylation of GlcNAc and GalNAc was performed by incubating each carbohydrate in 500 µl of anhydrous hydrazine at 100°C for 8 h, then excess hydrazine was eliminated by repeated evaporations in the presence of toluene under a stream of nitrogen and sugars were freeze-dried until used. N-Reacetylation was performed by an overnight incubation of the N-deacetylated sugar in 500 µl of saturated NaHCO3 containing 50 µl of acetic anhydride (Michalski, 1995).
Sugar specificity
The sugar specificity of the P.haemolytica purified protein was determined by comparing the inhibitory activity of various sugars, glycoproteins, and desialylated glycoproteins on the agglutination induced by the adhesin against rabbit erythrocytes. Results were expressed as the minimal concentration required to completely inhibit four hemagglutinating doses (titer = 4). The molar concentration of asialoglycoproteins was determined on the basis of their monosaccharides content as determined by gas chromatography.
Bacterial adhesion specificity
Tracheal cells (1 x 106cells/1 ml of PBS) were incubated with P.haemolytica (1 x 107 CFU) for 1 h at 37°C. The cells were then centrifuged at 1500 x g for 10 min to remove nonadherent bacteria and washed three times with PBS. The cells were then fixed in a smear and stained with the Gram or the May-Grünwald Giemsa staining method. Assays were performed in triplicate and the number of epithelial cells with at least three bacteria adhered to the microvilli or the body of the cells was determined under light microscopy at 100x. The specificity of the bacterial adherence was determined by inhibition assays using bacteria that had been previously incubated with different concentrations of sugars, glycoproteins, glycosides, or desialylated glycoproteins as well as 1–25 µg of purified adhesin before adding them to the cell suspension.
Physicochemical properties
The effect of ions on the adhesin activity was assessed using purified adhesin dialyzed against 0.1 M EDTA/0.1 M acetic acid, distilled water and finally, PBS (without metals). The adhesin was then dialyzed against PBS supplemented with 5 mM MnCl2, CaCl2, or MgCl2 before testing the hemagglutinating activity of each fraction against a 2% rabbit erythrocyte suspension (Vázquez et al., 1993). This results were compared with those obtained with nontreated and PBS dialyzed protein. Thermal stability of the protein was determined by incubating aliquots of the purified adhesin at different temperatures and time intervals before testing the hemagglutinating activity (Vázquez et al., 1993).
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This work was supported in part by CONACyT (27609 M), DGAPA-UNAM (PAPIIT-IN224598), and Program ECOS Mexico-France (M97B05).
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1 To whom correspondence should be addressed
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