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Glycobiology vol. 10 no. 1 pp. 31–37, 2000

Purification and characterization of an adhesin from Pasteurella haemolytica

Laura Jaramillo, Fernando Díaz, Pedro Hernández2, Henri Debray3, Francisco Trigo4, Guillermo Mendoza5 and Edgar Zenteno1,5 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

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-acetylD-glucosamine (GlcNAc), and in a lesser degree with Nacetyl-neuraminic acid (NeuAc). GalNAc, N-glycolylneuraminic 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

Introduction Pasteurella haemolytica, the main causative agent of enzootic septicemia in sheep, has been classified into two biotypes, A

1To

whom correspondence should be addressed

© 2000 Oxford University Press

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.

Results 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 31

L.Jaramillo et al.

Fig. 1. Purification of Pasteurella haemolytica adhesin by affinity chromatography. P.haemolytica bacterial extract (750 µg) was applied onto a column (1 × 5 cm) containing glutaraldehyde fixed rabbit erythrocytes stroma, physically entrapped in Sephadex G-25 equilibrated in PBS. Unbound material was eluted with PBS, and the adhesin was eluted with 200 mM GlcNAc in PBS. Absorbance at 280 nm and hemagglutinating activity were measured after exhaustive dialysis against PBS.

Table I. Purification process of the Pasteurella haemolytica adhesin by affinity chromatography on glutaraldehyde fixed rabbit erythrocytes stroma physically entrapped in Sephadex G-25* Fraction

Protein (mg)

HAU*

Specific activity (HAU/protein)

Total extract**

15. 2

23,040

1,516

Unretained

12.6

126

10

GlcNAc eluted

0.1

14,200

129,090

*Hemagglutination units determined in the presence of rabbit erythrocytes. **Protein concentration corresponds to the solubilized material from 0.5 g of bacterial mass.

Fig. 2. SDS electrophoresis of the purified Pasteurella haemolytica adhesin. (A) 5 µg of affinity chromatography purified adhesin; (B) 25 µg P.haemolytica bacterial crude extract. The molecular weight markers included are: E.coli βgalactosidase (116 kDa), rabbit muscle phosphorylase-b (97kDa), bovine serum albumin (66 kDa), chicken egg ovalbumin (45 kDa), and bovine carbonic anhydrase (29 kDa). The gel was silver-nitrate stained.

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.

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.

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.

Chemical characterization of the protein

Sugar specificity

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

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.

32

Hemagglutinating activity of the purified protein

Pasteurella haemolytica adhesin

Table II. Amino acid composition of the Pasteurella haemolytica adhesin Residue

Mol %

Res/Mol*

Asx

8

44

Glx

14

78

Ser

16

87

Gly

17

97

Hys

3

15

Arg

2

13

Ala

11

78

Pro

4

22

Tyr

1

30

Val

4

23

Met

1

3

Cys

1

2

Ile

3

17

Leu

5

27

Phe

3

18

Lys

4

23 Total 567

*Considering a molecular weight of 68 kDa.

NeuAc α-methylglycoside was 2-fold better inhibitor than NeuAc β-methylglycoside, but both methylglycosides were better inhibitors than NeuAc. We found that (α2,3) or (α2,6) sialyllactosamine, and colominic acid (NeuAcα2–8n) showed capacity to inhibit the hemagglutinating activity. From the glycoproteins tested, fetuin was the most powerful inhibitor of the hemagglutinating activity but α1-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. 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 Nglycolyl-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

Fig. 3. Microphotograph of adherence of Pasteurella haemolytica to epithelial tracheal cells from healthy calf. (a) the bacteria adhered to tracheal cells, mainly in the microvilli and the cell body. (b) Tracheal cells were incubated previously with 0.01 µM fetuin, we observed the negative adherence of bacteria in this cell group (amplification 100×).

adhesion of the bacteria was inhibited 50 and 75%, respectively, in comparison with nontreated epithelial cells (p < 0.025) (Table IV). 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 33

L.Jaramillo et al.

Table III. Effect of sugars, glycosides and glycoproteins on the hemagglutinating activity of Pasteurella haemolytica adhesin* Compound

Concentration (µM)

Relative inhibitory potency**

GlcNAc

25

4

NeuAc

100

1

NeuAc α-methylglycoside

6

16

NeuAc β-methylglycoside

25

4

(α2,3)Sialyllactosamine

25

4

(α2,6)Sialyllactosamine

25

4

Colominic acid***

0.05



Fetuin

0.01

10,000

Asialofetuin

NI



Transferrin

4

25

Asialotransferrin

NI



α1-Acid-glycoprotein

0.1

1000

Asialo-α1-acid glycoprotein

NI



Bovine submaxillary mucin

2

50

Ovine submaxillary mucin

2

50

N′-Diacetyl-chitobiose

25

4

N,N’,N′′-Triacetyl-chitotriose

25

4

Chondroitin sulfate***

2.5



Heparan sulfate***

1.5



*Minimal concentration needed to inhibit 4 HAU (titer = 4) of P.haemolytica adhesin. The adhesin concentration was 0.2 µg/ml. **Inhibitory potency corresponds to the comparative value of the inhibitory concentration versus that of NeuAc. Other sugars without inhibitory (NI) activity at 200 mM were L- and D-fucose, D-mannose, GalNAc, D-glucose, Dgalactose, D-galactosamine, D-glucosamine, D-mannosamine, melezitose, lactose, raffinose, NeuGc, N-deacetylated GlcNAc, and GalNAc. Neither hen ovalbumin nor protamine was inhibitory at 10 µM and 25 mg/ml concentration, respectively. ***Values are represented in mg/ml since the molecular weight is not known.

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.

Discussion 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 34

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 triantennary N-glycosidically linked glycans of the N-acetyllactosaminic type, such as fetuin (Spiro and Bhoyroo, 1974; Takasaki and Kobata, 1986), α1-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 α-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

Pasteurella haemolytica adhesin

Table IV. Inhibition of the adhesion* of Pasteurella haemolytica to epithelial cells obtained from 2-day-old calf trachea Compound

Concentration (µM)

% Adhered bacteria

Significance

GlcNAc

25

30 ± 6

p < 0.005

76 ± 6

Control

NeuAc

100

34 ± 6

P < 0.025

NeuAc α-methylglycoside

6

28 ± 3

P < 0.005

NeuAc β-methylglycoside

12.5

30 ± 7

P < 0.05

(α2,3)Sialyllactosamine

25

30 ± 8

P < 0.005

(α2,6)Sialyllactosamine

25

28 ± 7

P < 0.005

Colominic acid**

10

68 ± 3

n.s.

Fetuin

0.01

8.0 ± 1

P < 0.0005

Asialofetuin

1

70 ± 6

n.s.

Transferrin

4

68 ± 9

n.s.

Asialotransferrin

5

70 ± 2

n.s.

α1-Acid glycoprotein

0.1

14 ± 3

P < 0.001

Asialo-α1-acid glycoprotein

1

78 ± 3

n.s.

Bovine submaxillary mucin

2

56 ± 5

n.s.

Ovine submaxillary mucin

2

58 ± 4

n.s.

N,N′-Diacetyl-chitobiose

200

64 ± 2

n.s.

N,N′,N′′-Triacetyl-chitotriose

200

62 ± 4

n.s.

Chondroitin sulfate**

10

50 ± 5

P < 0.05

Protamine**

10

72 ± 5

n.s.

Purified adhesin**

0.025

22 ± 4

P < 0.025

*Values represent the percentage of tracheal epithelial cells with at least three adhered bacteria. Results were obtained under microscopic observation (100×), and represent the mean of three different determination ± the SEM. **Values correspond to mg/ml. Statistical analysis was performed using Student’s t test statistics. N.S., Not significant. No inhibitory activity was identified with the sugars and compounds listed in Table III footnote.

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 Nacetylated 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).

Materials and methods 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; α(2–3) and α(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 35

L.Jaramillo et al.

stock culture was stored in 10% skim milk at –70°C. Cells were harvested by centrifugation (10,000 × 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 × 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-dayold 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 × g for 10 min, counted, and adjusted to 1 × 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 × 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
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