Veterinary Microbiology, 29 ( 199 i ) 85-93 Elsevier Science Publishers B.V., Amsterdam

85

Immunological characterization of breakdown peptides of the 104 kilodalton hemolysin of Actinobacillus pleuropneumoniae serotype l J. Devenish and S. Rosendal Departmen! of I'eterinary Microbiology and hnmunology. University of Guelph. Guelph. Ontario NIG 2Wl, Canada (Accepted 28 February 1991 )

ABSTRACT Devenish, J. and RosendaL S., 199 l. Immunological characterization of breakdown peptides of the 104 kilodalton hemolysin ofActinobacil/us pleurcpneumoniae serotype !. [i,I. Microbiol. 28: 8593. The 104 kilodalton (kDa) hemolysin ofActinobacillus pleuropneumoniae serotype i, strain CM-5 was precipitated from RPMI-1640 culture supernatant usi_ng ammonium sulfate to 80% saturation. In immunoblots, a rabbit polyclonal antiserum against the 104 kDa hemolysin protein, recognized not only the original 104 kDa monomeric form of the hemolysin but other proteins in the crude antigen mixture ranging in molecular mass from 43 to > 125 kDa. The antiserum was able to crosslink these proteins to active hemolysin in RPMI-1640 culture supernatant resulting in bands of hemolysis in blood agar used in a contact assay. Corresponding to these bands of hemolysis, denatured peptides with molecular masses of 5 I, 85° i04 and > 125 kDa were excised and injecled into rabbits. in immunoblots, the r~ultant antibodies recognized the injected peptide and lhe monomeric 104 kDa protein. However, only the rabbit antisera produced against the 104 and 125 kDa proteins contained antibodies which neutralized the active 104 kDa hemolysin in cul!ure supernatant. These results indicate that (i) the 104 kDa protein hemolysin can exist in a higher molecular weight aggregate ~greater than 125 kDa) but can also break down to peptides which have molecular masses smaller than the. 104 kDa parent molecule and (ii) while several epitopes are present in the hemolysin molecule, there seems to be a restricted number of antigenic determinants responsible for inducing neutralizing antibodies and these seem *.o reside only in the 104 kDa p~,ent molecule. This may have consequences. in terms of vaccine development, for :he control of pleuropneumonia in swine herds.

INTRODUCTION

Actinobacillus pleuropneumoniae is the etiological agent of fibrinous pleuropneumonia of swine, a disease which can cause high morbidity and mortality in infected herds (Rosendal et al., 1985; Sebunya and Saunders, 1983). Whole cell bacterins, which decrease mortality but not morbidity and can cause adverse reactions at the injection site, have been used to contro~ this 0378-1135/91/$03.50

© 1991 - - Elsevier Science Publishers B,V.

86

J. DEVEN,:SHAND S. ROSENDAL

disease (Rosendal et al., 1981; Sebunya and Saunders, 1983 ). A pleuropneumoniae, serotype I produces a heat-labile hemolysin protein of 104 to 110 kilodaltons (kDa) (Devenish and Rosendal, 1989; Fedorka-Cray et al, 1990; Frey and Nicolet, 1988; Laionde et al., 1989), requires Ca e+ for biological activity (Devenish and Rosendal, 1991 ) and has been shown to be cytotoxic for neutrophils, macrophages and erythmeytes (Rosendal et al., ! 988 ). This toxin is immunologically and genetically related to the RTX group of bacterial cytolysins (Chang et al., 1989; Devenish et al., 1989; Gygi et al., 1990; Lo, 1990), and it has also been demonstrated that a common but not identical protein of approximately the same molecular mass is produced by the other 11 serotypes ofA. pleuropneumoniae (Devenish et al., 1989; Frey and Nicolet, 1990). Pigs recovering |'rom pleuropneumonia l'esist reinfection and have high titers of neutralizing antibody to the hemolysin in serum. However, available bacterins induce low levels of antibodies which will neutralize the 104 kDa hemolysin of A. pleuropneumoniae (Rosendal et al., 1988). This may explain the inability of whole cell bacterins to confer effective protection against challenge with virulent bacteria (Ro~endal et al. 1981; Rosendal et al., 1988; Sebunya and Saunders, 1983 ). Thus, the hemolysin of A. pleuropneumoniae may h~ve a potential virulence function and more efficacious vaccination procedures may have to include this hemolysin in order to protect swine herds against disease. However, both biological activity and the presence of the 104 kDa hemolysin molecule in crude culture supernatants decreases rapidly with time (Devenish and Rosendal, 1989; Frey and Nicolet, 1988; Lalonde et al., 1990). What effect this instability may have on the protective immunogenicity in animals needs to be clarified. In this paper we describe the breakdown and immunological characterization of the 104 kDa hemolysin of,4. pleuropneumoniae serotype 1, strain CM-5. MATERIALS AND M E T I | O D S

Producli,n. c~mc;'ntralion, and l¢',¥llrtg ¢~.fcrude hemolysin ,4 pleur~pneumemi~aeserotype I, strain CM-5 was grown in Roux flasks containing 150 ml oftryptic soy agar (Difco Laboratories, Detroit, Mi. ), supplemented with l0 mM CaCl_, and 0.01% NAD: for 18 h at 37°C. The bacteria were suspended in 75 ml of RPMi-1640, incubated for I h at 37°C, centrifuged ( 16 000 g for 20 rain ), and the supernatant was filtered using polycarbonate filters (Nucleopore Corp., Pleasanton, CA) with an exclusion of 0.~t l~m. The filtered supernatant was placed in an ice bath and ammonium sulfate (Sigma Chemical Co., St. Louis, MI) was added slowly to 80% saturation with continuous agitation, centrifuged (21 000 g for 45 rain ), and the precipitated pellet resuspended in 1.5 ml phosphate buffered saline pH 7.4 (PBS). This fraction was the crude hemolysin and had a hemolytic activity of 10 4 hemolytic units/ml and a protein concentration of ]00 pg/ml. The hemolytic

CHARACTERIZATION OF PEPTIDESOF HEMOLYSIN

87

assay was conducted as described elsewhere (Devenish and Rosendal, 1989; Rosendal et al., 1988) and the protein determination was done using the BioRad protein dye reagent (Bio-Rad Laboratories, Richmond, CA).

Electrophoresis, immunoblotting, and hemolysis testing of proteins in the crude hemolysin The crude hemolysin preparation was denatured by heating for 5 rain at 100°C in Laemmli's sample buffer. The denatured proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 4% stacking gel and an 11% separating gel. Electrophoresed proteins in the gel were electroblotted onto Biotrans nylon membranes (Pall Biosupport, East Hills, NY). Proteins on the filters were stained with 0.0C 1% India ink in PBS-tween 89. For immunoblotting, the filters were blocked as described by Johnson et al. ( ! 984) and probed with a I : 200 dilution of a rabbit polyclonal antiserum specifically raised against the 104 kDa hemolysin of CM-5 (Devenish and Rosendai, 1989). Staining of bound antibodies to proteins on the nylon membranes was observed by the hydrolysis of H20., in the presence of 4-chioro-l-naphthol (Sigma Chemical Co., St. Louis, MI) after the membranes were incubated with horseradish peroxidase-protein A conjugate (Boehringer Mannheim, Dorval, Quebec). The ability to cross-link the active hemolysin was studied as follows. After the electroblot was probed with the specific rabbit antiserum and washed, it was placed into 75 mi of filtered CM5 RPMI-1640 culture supernatant with 0.055 sodium azide (Sigma Chemical Co., St. Louis, MI ) for 4 h at 22 °C with gentle shaking. After washing, the membrane was inverted onto agar containing 5% washed bovine e_,ryth___roc~es in 10 mM Tris, 0.14 M NaCI, and 0.02% sodium azide pH 7.4. After incubation at 37°C for 18 h, the membranes were removed and the agar observed for bands of hemolysis. As controls, the same procedure was followed except that the electroblotted proteins were not exposed to serum or were exposed to normal rabbit serum prior to incubation in the CM-5 RPMi-1640 culture supernatant containing active hemolysin.

Prodl,wtion and testing of rabbit antisera to denatured proteins in crude hemolysin. The crude hemolysin was denatured and electrophoresed by SDS-PAGE as described above. The gel was stained with Coomassie Blue and protein bands with molecular masses of 51, 85, 104, > 125 kDa, corresponding to stained bands in immunoblots and bands of hemolysis ~n blood agar, were excised from the gel, minced with a glass rod and added to an equivalent volume of PBS. The gel bands were mixed with an equal volume of Freund's complete adjuvant and injected subcu~.aneously into New Zealand white rabbits. The subcutaneous injection was repeated two weeks later. After one month, the rabbits were given a final intravenous injection of the same protein electroe-

88

J. DEVENISH AND S. ROSENDAL

luted from the SDS-PAGE gel. Briefly, the gel band was minced in twice the volume of elec~.roelution buffer (9 mM Tris-borate buffer with 0.005% SDS pH 8.0), placed into separate sample concentration cups (Isco Inc., Lincoln, NE) and electroeluted for 90 min at 150 volts. Protein concentrations were determined in these aqueous eluates prior to injection and found to be 18, 16, 300, and 8pmg/ml for the 5 l, 85, 104, and > 125 kDa proteins, respectively. The rabbits wei-e exsanguinated one week later and the serum was heat inactivated (56 °C for 30 min) and tested (i) for neutralizing antibody activity to CM-5 hemolysin in culture supernatant as described previously (Devenish and Rosendal, 1989; Devenish et al., 1989; Rosendal et al., 1988), and (ii) diluted l: 100 in 20 mM Tris with 0.14 M NaC! pH 7.4 and used to immunoblot electroblotted proteins, from CM-5 crude hemolysin, on nylon membranes as described above.

I

2

3

4

200 ~,~ ~-125 116 ~

104

97 ,-~

85

~

51

67

43

Fig. I..4. pleuropneumoniaeCM-5 crude extracellular hemolysin antigens electrophoresed using SDS-PAGE and electroblotted to nylon membranes. Lane l, membrane stained by lndia lnk; Lane 2, membrane immunoblotted with a rabbit polyclon.ai antiserum specifically produced to the 104 kDa hemolysin protein of CM-5; Lane 3, immu,~oblotted membrane exposed to CM-5 RPMI-1640 culture supernatant and placed onto 5% bovine blood agar for 18 h at 37°C; arid Lane 4. electroblotted membrane exposed to CM-5 RPMl-1640 culture supernatant and placed onto 5% bovine blood agar for 18 h at 37°C. Bio-Rad high molecular weight protein standards are indicated to the left. The arrows to the cight indicate the molecular mass proteins selected for injection into rabbits.

89

CHARACTERIZATION OF PEPTIDES OF HEMOLYSIN

1

200

2

3

4

-

-,,~>!25 116-~11 97-~

~

104

~

~5

67 " ~

51

4:3-

Fig. 2. Electroblotted CM-5 crude extracellular hemolysin antigens on nylon membranes probed with antiserum from rabbits immunized with the > 125 kDa (Lane 1 ), 104 kDa (Lane 2), 85 kDa ( Lane 3 ), and 51 kDa ( Lane 4) proteins. Bio-Rad high molecular weight protein standards are indicated to the left. The arrows to the right indicate the position of the injected proteins and their molecular masses.

TABLE 1 Anti-CM-5 hemolysin neutralizing titers of sera from rabbits immunized with peptides associated with the 104 kDa hemolysin produced by A. pleuropneumoniae serotype 1, strain CM-5. Protein inoculated ~

Anti-CM-5 hemolysin titer-"

> 125 kDa 104 kDa 85 kDa 51 kDa None (NRS) ~

1:650 1 : 6200 l : 30 1:30 1:30

' ~ll injected proteins were recognized in immunoblots by a rabbit polyclonal antiserum specifically produced to the 104 kDa hemolysin of CM-5, and were able to cross-link active hemolysin in CM-5 culture supernatant and produce zones of hemolysis in blood agar. -"The dilution of serum that neutralized 50% hemolysis of a 1% bovine erythrocyte suspension exposed to 10 hemolytic units ofCM-5 RPMI- 1640 culture supernatant. -~Normal rabbit serum.

90

J. DEVENISH AND S. ROSENDAL

RESULTS

The proteins in the crude hemolysin which were stained by India ink on electroblots are shown in Fig. 1 (lane 1 ). The corresponding proteins which were stained in immunoblots using the rabbit polyclonal antiserum specific to the 104 kDa hemolysin ranged in molecular mass from 43 to > 125 kDa (Fig. i, lane 2). Using the same antiserum, these immunostained proteins were able to cross-link active hemolysin in culture supernatant and produce bands of hemolysis in blood agar (Fig. l, lane 3). These bands of hemolysis were not observed in the negative controls (Fig. 1, lane 4). Proteins with molecular masses of 5 l, 85, 104, and > 125 kDa were selected for rabbit injection. All four proteins were observed readily in the India ink stained blots facilitating their excision from the SDS-PAGE gels. The 51 kDa protein stained very. weakly in the immunoblot yet produced a pronounced band of hemolysis when cross-linked to active hemolysin. The 85 kDa protein stained moderately in the immunoblot and produced a strong band of hemolysis in blood agar. The 104 kDa monomeric form of the hemolysin stained darkest in the immunoblot and had the most prominent band of hemolysis and the > 125 kDa protein while prominently displayed in the immunoblot produced a weak band of hemolysis in the blood agar. The immunoblots, of the CM-5 crude hemolysin, using the four rabbit hyperimmune antisera, produced against the 5 l, 85, 104, and greater than 125 kDa proteins, are presented in Fig. 2. The antisera against the 51 and 85 kDa proteins recognized the homologous protein band and the 104 kDa protein monomer. The antisera against the 104 kDa and > 125 kDa proteins recognized the homologous proteins, the i04 kDa parent protein, and other peptides in the crude hemolysin mixture. Unlike the antisera produced to the 51 and 85 kDa peptides, the antisera produced to the 104 and > 125 kDa proteins were able to neutralize the active hemolysin in RPMI-1640 culture supernatant (Table l ). DISCUSSION

Extraceilular peptides on electroblots, ranging in size from 43 to > 125 kDa, produced by CM-5 were able to cross-link active hemolysin in culture supernatant when probed with a specific rabbit antiserum to the 104 kDa hemolysin of CM-5. This shows that these peptides are probably associated with the 104 kDa hemolysin of CM-5 and indicates that the hemolysin occurs as higher molecular mass and smaller peptides; which are likely aggregates and breakdown products, respectively, of the parent 104 kDa molecule. Although the results shown were obtained with a specific rabbit polyclonal antiserum to the 104 kDa hemolysin (Devenish and Rosendal, 1989), we have also used both a convalescent swine serum with a high neutralizing antibody titer to the hemolysin (Devenish and Rosendal, 1989) and a monoclonal antibody produced from this molecule (Devenish et al., 1989) and obtained similar results (data not shown).

CHARACTERIZATION OF PEPTIDES OF HEMOLYSIN

91

In addition, antibodies were induced in rabbits to peptides which were either aggregated or breakdown forms of the parent 104 kDa hemolysin. All four injected peptides induced antibodies which recognized the 104 kDa monomeric form of the hemolysin supporting their close association with this molecule. However, the antisera against the 51 and 85 kDa peptides recognized only the homologous peptide and the 104 kDa monomer, and these antisera did not neutralize hemolytic activity. In contrast, the antisera from rabbits receiving the 104 kDa monomer and the > 125 kDa aggregate recognized the full range of potential breakdown and aggregate forms of the hemolysin and were also able to neutralize the active hemolysin in culture supernatant. The term > 125 kDa is used in this paper because it was impossible to determine with any precision the actual molecular mass of this protein. We cannot discount the possibility of individual animal differences in immune response being responsible for the results but all four rabbits responded to the injected peptides as observed in immunoblots and the differences in neutralizing response were not likely due to the differences in the amcunt of protein injected since the rabbit receiving the > 125 kDa peptide was given the lowest amount of protein and yet produced neutralizing antibodies to the hemolysin. From these data, it can be suggested that while all peptides associated with the parent 104 kDa molecule contain epitopes which will induce an immune response to the hemolysin, epitopes necessary for generating neutralizing antibodies reside in the 104 kDa monomer, but not in breakdown peptides. It is because the > 125 kDa peptide induced a neutralizing antibody response to the hemolysin that we feel it is an aggregated complex that contains the complete 104 kDa parent molecule. This conclusion remains tentative, however, .until . . . :' this com pl ex has been "~--"~uny cna~acte!-' ht.,a proteln r,~,uu,.,~u . . . . . . .- :--"~Lcu.A ~'zu"'-',.,-,n • _.~.~ . . . . .~ by A. pleuropneumoniae has been reported to be cytolytic for macrophages (Kamp et al., 1990; Rycraft and Cullen, 1990). However, this protein was found only in serotype 2 strains and did not cross-react immunologically with the 104 kDa hemolysin. Thus it is probably not the same molecule as the greater than 125 kDa aggregate reported here. The 104 kDa hemolysin of CM-5 has been shown to be related to the alpha hemolysin of Escherichia coli and the leukotoxin of Pasteurella haemolytica (Devenish et al., 1989). Both of these cytolysins have been shown to break down to smaller peptides in culture supernatants but also to exist in some form of higher molecular weight complex which has not been well defined (Bohach and Snyder, 1986; Mosier et al,. 1989). The breakdown of these cytotoxins has been speculated, but not proven, to be caused by proteolytic cleavage following release from the bacterium (Bohach and Snyder, 1986; Mosier et al., 1989). This may also be the case for the 104 kDa hemolysin of CM-5 but, this has been difficult to prove. However, this breakdown may explain the lack of biological and structural stability reported by researchers during attempts to purify this cytolysin from A pleuropneumoniae (Devenish and Rosendal, I989; Frey and Nicolet, 1988; Lalonde et al,. 1989). -

-

-

92

J. DEVENISH AND S. ROSENDAL

Injection of the purified 104 kDa protein will offer protection in swine against challenge with virulent A. pleuropneumoniae if a high neutralizing antibody response can be induced (Devenish et al., 1990). However the breakdown of this protein in culture supernatant, as reported here, may be significant in terms of vaccine development. Attempts to use crude concentrated extracellular extracts ofA pleuropneumoniae to vaccinate pigs resulted in partial protection, only (Fedorka-Cray et al., 1990). One reason may be the instability of the hemolysin. While breakdown products may still generate an immune response, when measured by tests such as ELISA, they may induce an antibody response that is insufficient to neutralize the cytolytic effects produced by the 104 kDa hemolysin in vivo. It would seem that for use in a vaccine, the 104 kDa hemolysin must be present in a stable form in sufficient concentration. ACKNOWLEDGEMENTS

The technical assistance provided by Mr. Paul Huber was greatly appreciated. This study was supported by the Ontario Pork Producers Marketing Board and the Ontario Ministry of Agriculture and Food. REFERENCES

Bohach, G.A. and Snyder, I.S., 1986. Composition of affinity-purified alpha-hemolysin of Escherichia coil Infect. Immun., 53: 435-437. Chang, Y.. Young, R. and Struck, D.K., 1989. CIoeing and characterization of a hemolysin gene from .4ctinobacilh~s pleuropneumo~dae. DNA, 8: 635-647. Devenish, J. and Rosendal, S., 1989. Identification of the heat-labile hemolysin of Actinobacilh~s pleuropneumoniae serotype 1. Can. J. Vet. Res., 53:251-254. Devenish, J., Rosendal, S. Johnson, R and Hubler, S., 1989. Immunoserological comparison of 104-kilodalton proleins associated with hemolysis and cytolysis in Actinobacilhts pleuropneumoniae. Actinobacillus suis. Pasteurella haemolytica, and Escherichia coil. Infect. Immun., 57: 3210-3213. Devenish, J., Rosendal, S. and BossY, J.T., 1990. Humoral antibody response and protective immunity in swine following immunization with the 104-kilodalton hemolysin of,4ctinobacillus pleuropneumoma. Infect. lmmun., 58: 3829-3832. Dcvenish, J. and Rosendal, S., 1991. Calcium binds to and is required for biological activity of the 104 kilo dalton hemolysin produced by Actinobacilhtspleuropnetononiae serotype I. Can. J. Microbiol.. 37: 317-321. Fedorka-Cray, P.J., Huether, M.J., Stine, D.L. and Anderson, G.A., 1990. Efficacy of a cell extract from Actinobacillus ( tlaemophilus ) pleuropneun',oniae serotype ! against disease in swine. Infect. lmmun., 58: 358-365. Frcy, J. and Nicolet, J., 1988. Regulation of hemolysin expression in .4ctinobacilluspleuropneumomae serotye I by Ca -'+. Infect. lmmun., 56: 2570-2575. F:cy. J. and Nicolet, J., 1990. Hemolysin patterns ofActinobacillus pleuropneumoniae. J. Clin. Microbiol., 28: 232-236. Gygi, D., Nicolet, J., Frey, J., Cross, M., Koronakis, V. and Hughes, C., 1990. Isolation and

CHARACTERIZATION OF PEPTIDES OF HEMOLYSIN

93

expression of the Actinobacillus pleuronpneumoniae hemolysin gene; activation and secretion of the prohemolysin by the HlyC, HlyB, and HIyD proteins ofE. coli. Mol. Microbiol.. 4: 123-128. Johnson, D.A., Gautsch, J.W., Sportsman, J.R. and Elder, J.H., 1984. Improved technique utilizing non-fat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose. Gene Anal. Tech., 1: 3-8. Kamp, E.M., Popma, J.K. and Smits, M.A. Abstr. Proc. 1 lth Congr. Int. Pig Vet. Soc. Lausanne. 1990, p. 20. Lalonde, G., McDonald, T.V. Gardner, P. and O'Hanley, P.D., 1989. Identification of the hemolysin from Actinobacillus pleuropneurnoniae and characterization of its channel properties in planar phospholipid bilayers. J. Biol. Chem., 264:13559-13564. Lo, R.Y.C., 1990. Molecular characterization of cytotoxins produced by Haemophilus, Actinobacillus, and Pasteurella. Can. J. Vet. Res., 54: $33-$35. Mosier, D.A., Simons, K.R., Confer, A.W., Panciera, R.J. and Clinkenbeard, K.D., 1989. Pasteurella haemolytica antigens associated with resistance to pneumonic pasteurellosis. Infect. lmmun., 57:711-716. Rosendal, S., Carpenter, D.S., Mitchell, W.R. and Wilson, M.R., 1981. Vaccination against pleuropneumonia of pigs caused by Haemophilus pleuropneumoniae. Can. Vet. J., 22: 3435. Rosendal, S., Boyd, D.A. and Gilbride, K.A., 1985. Comparative virulence of porcine Haemophilus bacteria. Can. J. Comp. Med., 49: 68-74. Rosendal, S., Devenish, J., Maclnnes, J.l., Lumsden, J.H., Watson, S. and Xun, H., 1988. Evaluation of heat-sensitive, neutrophil-toxic, and hemolytic activity of Haemophilus (Actinobacillus) pleuropneumoniae. Am. J. Vet. Res., 49:1053-1058. Rycroft, A.N. and Cullen, J.M. Abstr. Proc. 1 lth Congr. Int. Pig Vet. Soc., Lausanne, 1990, p. 22. Sebunya, T.N.K. and Saunders, J.R, 1983. Haemophilus pleuropneuoloniae infection in swine: a review. J. Am. Vet. Med. Assoc., 182: 1331-1337.

Immunological characterization of breakdown peptides of the 104 kilodalton hemolysin of Actinobacillus pleuropneumoniae serotype 1.

The 104 kilodalton (kDa) hemolysin of Actinobacillus pleuropneumoniae serotype 1, strain CM-5 was precipitated from RPMI-1640 culture supernatant usin...
597KB Sizes 0 Downloads 0 Views