Surface-exposed antibody-accessible outer membrane proteins of Bordetella pertussis Department of Microbiology, U n i v e r s i ~of Alberta, Edntonton, Alta., Ccrnadcr T6G 2E9
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Received November 2. 1990 Revision accepted February 22, 199 1 ANWAR,H. 199 1. Surface-exposed antibody-accessible outer membrane proteins of Borderella pertussis. Can. J . Microbiol. 37: 590-593. The convalescent sera from a patient recovered form whooping cough were used to identify the surface-exposed antibodyaccessible outer membrane proteins (OMPs) of Bordetellapertu.ssis. The results indicated that the 69 000 OMP, the 40 000 porin, agglutinogens 2 and 3, and a number of presently unknown OMPs were exposed on the surface. The importance of these surfaceexposed antigens in the protection against whooping cough is discussed. Key words: Borderella pertussis, whooping cough, immune response, surface antigens, outer membrane protein. ANWAR,H. 1991. Surface-exposed antibody-accessible outer membrane proteins of Borderella pertussis. Can. J . Microbiol. 37 : 590-593. . Le sCrum d'un patient convalescent atteint de coqueluche a CtC utilisC pour identifier les protCines de surface de la membrane externe qui sont accessibles a l'anticorps chez Borderella pertussis. Les rCsultats indiquent que la membrane externe 69 000, la porine 40 000, les agglutinogknes 2 et 3 et un certain nombre de membrares externes inconnues sont accessibles sur la surface. On discute l'importance de ces antigknes dans les facteurs de protection contre la coqueluche. Mots elks: Bordetella pertussis, coqueluche, rCponse immunitaire, antigknes de surface, protCines de la membrane externe. [Traduit par la rCdaction]
Introduction Whooping cough is an inflammation of the lining of the respiratory &act of young children and is often chara~t~rized by a typical paroxysmal cough. The aetiologial agent is Bordetella pertussis and, less frequently, Bordetella parapertussis. Killed whole-cell vaccines are immensely successful in that they have virtually eliminated what was once regarded as a widely prevalent, frequently fatal, disease. Despite the tremendous success of whole-cell vaccines in the prevention of whooping cough, pertussis vaccination has always been a major concern to parents and clinicians because of the rare problematical association of the vaccines with brain damage. The toxic side effects of vaccination are a major concern in any vaccination program and form the other side of the benefit-risk equation (Miller et al. 1982). In the United Kingdom, diminished public confidence in the safety of killed whole-cell pertussis vaccine led to a reduction in vaccine acceptance, which correlated with an increase in the incidence of pertussis (Miller et al. 1982; Robinson et al. 1985).The precise identity of the component that causes the side effects in the whole-cell vaccine is still unknown. However, pertussis toxin (PT) has been suggested to be the component that causes the toxic side effect (Robinson et al. 1985; Weiss and Hewlett 1986). The presence of large amounts of lipopolysaccharide in the whole-cell vaccine is also of concern since this component is pyrogenic (Robinson et al. 1985). Whole-cell pertussis vaccines are crude preparations containing thousands of antigens. It is believed that those antigens that are exposed on the cell surface are probably important since they are likely to play a role in the prevention of whooping cough. It is, therefore, of interest to identify the outer membrane proteins (OMPs) of B. pertussis, which have conformational epitopes that are exposed on the cell surface and accessible to antibody so that the importance of these surface antigens in the prevention of the infection can be thoroughly investigated. To explore the accessibility to the surface antigens of B. pertussis to antibody, the method described by Swanson (198 1) was adopted and modified accordingly. The recognition of the OMPs of B. pertussis by
antibody present in the convalescent sera of a patient recovered from the infection was investigated by immunoblotting.
Materials and methods Strain and cultirral conditions Borderella pertussis Wellcome 28 was used throughout the study. It was maintained as freeze-dried cultures and recovered by growth on charcoal agar plates containing 10% (v/v) defibrinated horse blood (Gibmar Laboratories, Edmonton, Canada). After 48 h at 35"C, the growth from plates was inoculated into 100 mL Stainer and Scholte's medium (Stainer and Scholte 1970) containing I g/L of 2,6-0-dimethylP-cyclodextrin (Imaizumi et al. 1983) and incubated at 30°C for 24 h on an orbital shaker (1 80 rpm). Medium (300 mL) in 2-L conical flasks was inoculated with 10 mL of primary culture and incubated on an orbital shaker at 35OC. After 4 0 4 6 h, the cells were harvested by centrifugation (5000 X g for 30 min) and the cell pellet was retained. Preparation of outer membrane ( O M ) and SDS-PAGE The outer membrane (OM) was prepared using sodium lauryl sarcosinate (Sarkosyl; Sigma Chemicals Co., St. Louis, MO) as described by Filip et al. (1973). The bacterial pellet was suspended in 20 mL of distilled water and broken by two 60-s pulses of sonification in an ice bath, with one 60-s interval for cooling. Unbroken cells were removed by centrifugation at 3000 X g for 5 min. Sarkosyl was added to the supernatant to give a final concentration of 2%. The mixture was incubated at room temperature for 30 min and then centrifuged at 38 000 X g for 1 h. The membrane pellets were washed twice with distilled water, resuspended in a small volume of distilled water, and stored at - 20°C. Membrane preparations were subjected to sodium dodecyl sulphate - polyacrylamide gel electrophoresis (PAGE) by the system described by Anwar et al. (1987) with 12% acrylamide separating gel and ultrapure SDS. Immunoblotting The OMPs separated by SDS-PAGE were transferred onto nitrocellulose (NC) paper and visualized immunologically by the method of Towbin et al. ( 1979) as modified by Anwar et al. (1984). The NC paper was incubated first with 10 rnM Tris-hydrochloride - 0.85% saline (pH 7.4) (TBS) containing 0.3% Tween-20 for 1 h to saturate nonspecific protein-binding sites and then with patient serum diluted 1/50 in TBS - 0.3% Tween-20 for 4 h at 37OC. The paper was then washed
ANWAR
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thoroughly with TBS and incubated for an additional 2 h at 37°C with horseradish peroxidase goat anti-human immunoglobulin G (Sigma Chemicals Co., St. Louis, MO) diluted 112000 in TBS-0.3% Tween-20. After incubation, the NC paper was again washed thoroughly, and antigenic sites were visualized with a 25 p,glmL solution of 4-chloro- 1naphthol (Sigma Chemicals Co., St. Louis, MO) in TBS containing 0.0 1% H202.To show complete qualitative transfer of protein bands from polyacrylamide gels to the NC paper, I directly visualized blotted protein bands on duplicate NC paper by staining the paper with 1% amido black (data not shown). Surface-exposed antibody-uccessible antigen preparation The surface-exposed antigens were investigated by modifications of the method described by Swanson ( I98 l), in which the iodine labelling step was omitted and the antigenic sites were visualized by immunoblotting (Kadurugamuwa et ul. 1985). The bacterial culture (2 L) was harvested by centrifugation (5000 X g for 30 min) and washed once with 10 mM phosphate buffer saline (PBS), pH 7.4. The cells were treated with 5 mL convalescent sera and the sample was incubated at 35°C in an orbital shaker for 20 min. The cell suspension was then centrifuged at 5000 X g for 30 min and washed three times with PBS. The cells were disrupted by suspension of the pellet in 10 mL 1% N-tetradecyl-N,N-dimethyl-3-ammonio- I -propane sulfonate (Zwittergent 3-14; Calbiochem-Behring Corp., La Jolla, CA) in 10 mM PBS, pH 7.4. The mixture was well mixed, incubated at 37°C for 30 min, and centrifuged at 5000 rpm for 30 min. The supernatant was carefully removed and transferred to a cylindrical 10-mL column containing 5 mL previously swollen protein A - Sepharose 4B CL (Sigma Chemical Co., St Louis, MO). The mixture was incubated at room temperature for 20 min with regular end-to-end rotation, and the excess fluid was drawn through the column, which was then washed with 120 mL PBS. The antigen-antibody complexes bound to protein A Sepharose were eluted wit 100 rnL of 0.1 M glycine (pH 2.5) with regular end-to-end rotation (Kadurugamuwa et al. 1985). The eluate was dialyzed against distilled water at 4°C overnight, lyophilized and suspended in I mL of denaturing buffer containing 4% ultrapure SDS (British Drug House, Poole, U.K.), 8% 2-mercaptoethanol, 20% glycerol, and 0.0 1 % bromophenol blue in 0.125 M Tris-hydrochloride buffer, pH 6.8. The sample was stored at - 20°C.
Results and discussion The accessibility of the OMPs of B. pertussis to antibody was investigated by identification of the OMPs of intact cells of B. pertussis that are involved in the binding of IgG present in the convalescent sera. This method was successfully used in the investigation of the exposure of the OMPs of encapsulated strains of Klebsiella pneumoniae following growth of the cells in the presence of subinhibitory concentrations of cephalosporins (Kadurugamuwa et al. 1985). Figure 1 shows the OM (lane 2) and the surface-exposed antibody-accessible OMPs (lane 3) of B. pertussis. A number of protein bands that were found in the OMP profile (lane 2) could also be detected in the surfaceexposed antibody-accessible OMP preparation (lane 3). These are proteins with M, of 69 000, 40 000, 22 000, 20 000 and 15 000. The 69 000 OMP of B. pertussis has recently been identified as an agglutinogen and its usefulness as a vaccine candidate has been studied (Brennan et al. 1988; Charles et al. 1989).Molecular studies revealed that the biosynthesis of the 69 000 OMP of B. pertussis is under genetic control of the vir locus (Charles et al. 1989). The 40 000 protein has previously been identified to be a pore-forming protein (porin) that forms anionic-selective channels in lipid bilayer membrane (Armstrong et al. 1986). Agglutinogens were prepared by the method described by Rutter et al. (1988) for comparison. Lane 1 shows the electrophoretic positions of agglutinogen 2 (Agg 2; 22 000) and agglutinogen 3
FIG.1. SDS-PAGE of OMPs of Bordetella pertussis. Lane 1 , Agg 2 and Agg 3 preparation; lane 2, outer membrane preparation; and lane 3, the surface-exposed antibody accessible OMP preparation.
(Agg3; 20 000) on the acrylamide system used in this study. Agg 2 (Gorringe et al. 1985; Robinson et al. 1985) is identical with Agg 6 described by other workers (Steven et al. 1986; Zhang et al. 1985). The intensity of the Agg 2 band observed in the surface-exposed antibody-accessible preparation (lane 3) was closely similar to that of the Agg 3 band (lane 3), indicating that the same amounts of Agg 2 and Agg 3 subunits were produced by this strain of B. pertussis. Agg 2 was barely detectable in the OM preparation (lane 2), indicating that this protein is probably not firmly anchored to the OM and was released during the preparation of the OM. The protein band above the 15 000 protein in the surface-exposed antibody-accessible OMP preparation (lane 3) was not found in the OM preparation (lane 2). This protein was possibly loosely bound to the outer leaflet of the OM and was released during the preparation of the OM. Similarly, the 54 000 protein was found to be a major band in the surface-exposed antibody-accessible OMP preparation (lane 3) but not associated with the OM (lane 2). It was, therefore, of interest to carry out some further biochemical analysis to verify the origin of the 54 000 protein. The protein was purified by electroelution as described by Hunkapiller et al. (1983) and the purified 54 000 protein was subjected to N-terminal amino acid sequencing by S. Kielland (University of Victoria, Victoria,
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CAN. J. MICROBIOL. VOL. 37, 1991
FIG. 2. Immunoblot of the OMPs described in Fig. 1 electrophoretically transferred onto nitrocellulose paper and reacted with IgG present in the convalescent sera. Lane 1, Agg 2 and Agg 3 preparation; lane 2, outer membrane preparation; and lane 3, the surface-exposed antibody-accessible OMP preparation.
B.C.). Based on a 14 amino acid sequence, the 54 000 protein was found to have 100% homology to the heavy chain (V-I11 region) of human IgG (data not shown).Therefore, the 54 000 protein observed in the surface-exposed antibody-accessible preparation had not originated from B. pertussis, but was from the heavy chain of the IgG that was copurified during the preparation of surface-exposed antibody-accessible antigens (Kadurugamuwa et al. 1985). Figure 2 illustrates the antigenic profiles of the surface OMPs of B. pertussis after the proteins from Fig. 1 were blotted onto nitrocellulose paper and probed with the convalescent serum. IgG present in the convalescent sera reacted strongly with the agglutinogens (Agg 2 and Agg 3) of B. pertussis (lane 1). The Agg 2 and Agg 3 bands were seen as a diffused band in the immunoblot. The importance of Agg 2 and Agg 3 as potential vaccine candidates has been discussed (Robinson et al. 1985) and these antigens have been included in the development of an acellular pertussis vaccine in the United Kingdom (Rutter et al. 1988). The phase I clinical trial of the vaccine indicated that Agg 2 and Agg 3 were immunogenic and strong antibody responses to these antigens were observed following administration of the vaccine to healthy adult volunteers (Rutter et al. 1988).
Monoclonal antibodies to Agg 2 and Agg 3 have been shown to inhibit the adherence of B. pertussis to Vero cells (Gorringe et al. 1985). The results of this study indicated that strong IgG responses to Agg 2 and Agg 3 were observed following the natural infection with B. pertussis (Fig. 2, lanes 1 and 3). This observation strengthens the importance of adding Agg 2 an Agg 3 in the development of acellular pertussis vaccine in order to give full protection against the disease (Robinson et al. 1985; Rutter et al. 1988). The sera from the patient also contained IgG, which reacted with a number of antigens in the OM preparation of B. pertussis (Fig. 2, lane 2). The porin (40 000) was highly immunogenic and a strong antibody response was launched by the immune system against this anl.igen as the results of host-parasite interaction. The usefulness of the 40 000 porin as a potential vaccine candidate in the prevention of whooping cough remains to be investigated. In this study, a strong antibody response to 69 000 OMP was also observed (Fig. 2, lane 2), confirming the importance of this antigen as a potential vaccine candidate in the prevention of whooping cough (Brennan et al. 1988; Charles et ul. 1989). Five protein bands above the 69 000 protein were immunogenic and reacted strongly with IgG (Fig. 2, lane 2). These protein bands are likely to be the subunits of the filamentous haemaglutinin as observed by other workers (Robinson et ul. 1985; Rutter et ul. 1988; Weiss and Hewlett 1986). These protein bands are currently being investigated in the laboratory. The exposed outer surface of a bacterial cell plays a crucial role in its survival in vivo (Parker and Armstrong 1989; Smith 1977, 1990; Swanson 198 1). The surface components are in constant interaction with the immune system. Humoral responses are launched against the surface components to provide specific recognition of the infecting organism so that the pathogen can be dealt with efficiently and effectively in vivo. The surfaceexposed antibody-accessible antigens are, therefore, of interest to those who are involved in vaccine research. The lactoperoxidase-catalysed iodination technique has previously been used to investigate the exposure of the surface proteins of B. pertussis (Parker and Armstrong 1989; Redhead 1983). The drawback of this technique is that the outer membrane proteins must have residues such as tyrosine exposed on the surface to be readily accessible for labelling by lactoperoxidase. In this study, the OMPs of B. pertussis that are exposed on the surface and accessible to IgG have been identified by using convalescent sera obtained from natural infections. Pertussis toxin and filamentous haemaglutinin of B. pertussis have been extensively studied and these components have been included in the development of acellular pertussis vaccines (Robinson et al. 1985; Rutter et al. 1988; Sato et al. 1984). Agglutinogens produced by B. pertussis have also been investigated and included in the development of acellular petussis vaccines (Brennan et al. 1988; Charles et al. 1989; Rutter et nl. 1988). In this study, a simple method was used to investigate the surface exposure of the outer membrane antigens of B. pertussis. The method can also be used in the study of the surface antigens of other clinically important Gram-negative pathogens. More convalescent sera (10-20 human immune serum specimens) should be investigated before a general conclusion can be made regarding the immune responses to the surface antigens of B. pertussis. Stimulation of the immune system to launch specific antibody responses to pertussis toxin, filamentous haemaglutinin, agglutinogens, and other important surfaceexposed antibody-accessible antigens should assist the swift eradication of the infecting pathogen that is trying to colonize the respiratory tract. Given the present high levels of interest in the
ANWAR
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development of acellular pertussis vaccines, I am confident that an effective acellular pertussis vaccine consisting of these protective antigens of B. pertussis will be introduced into the immunization program to prevent infants and children from contracting whooping cough.
593
IMAIZLIMI, A. SUZUKI, Y., ONO, S., SATO, H., and SATO, Y. 1983. Effect of heptakis (2,6-0-dimethyl) P-cyclodextrin on the production of pertussis toxin by Bordetella pertu.s.si.s. Infect. Immun. 41: 1138-1 143. H., BROWN,M. R. W., and ZAK,0 . KADURUGAMUWA, J. L., ANWAR, 1985. Protein antigens of encapsulated Kleb,siellu pneumoniue surface exposed after growth in the presence of subinhibitory Acknowledgements concentrations of cephalosporins. Antimicrob. Agents Chemother. The author thanks William J. Page (Department of Microbi28: 1 95- 199. ology, University of Alberta) for helpful discussion and for MILLER,D. L., ALDERSLADE, R., and Ross, E. M. 1982. Whooping critically reviewing the manuscript. This work was supported by cough and whooping cough vaccine: the risks and benefits debate. Epidemiol. Rev. 4: 1-24. operating grants from the Central Research Fund of the UniverPARKER,C. D., and ARMSTRONG, S. K. 1989. Surface proteins of sity of Alberta and the Natural Sciences and Engineering Bordetella pertu.s.si.s. Rev. Infect. Dis. 10: S327-S330. Research Council of Canada. REDHEAD, K. 1983. Variability of the surface exposure of the outer membrane proteins of Bordetella pertus.si,s. FEMS Microbiol. Lett. ANWAR,H. ASHWORTH, L. A. E., FUNNELL, S., ROBINSON, A., and 17: 35-39. IRONS,L. I. 1987. Neutralisation of biological activities of pertussis ROB~NSON, A., IRONS, L. I., and ASHWORTH,L. A. E. 1985. Pertussis toxin with a monoclonal antibody. FEMS Microbiol. Lett. 44: vaccine: present status and future prospects. Vaccine, 3: 1 1-22. 141-145. RU~ER D., A., ASHWORTH, L. A. E., DAY,A., FUNNELL, S., LOVELL, ANWAR,H., BROWN,M. R. W., DAY,and A., WELLER,P. H. 1984. F., and ROBINSON, A. 1988. Trial of a new acellular pertussis vaccine Outer membrane antigens of mucoid Pseudornonas r~erugirzoscr in healthy adult volunteers. Vaccine, 6: 29-32. isolated directly from the sputum of a cystic fibrosis patient. FEMS SATO, Y., KIMURA, M., and FUKUMI,H. 1984. Development of a Microbiol. Lett. 24: 235-239. pertussis component vaccine in Japan. Lancet, 1: 122-1 26. ARMSTRONG, S. K., PARR.T. R., PARKER,C. D. and HANCOCK, SMITH,H. 1977. Microbial surface in relation to pathogenicity. R. E. W. 1986. Bordetella pertussis major outer membrane porin Bacteriol. Rev. 41: 475-500. protein forms small, anionic-selective channels in lipid bilayer 1990. Pathogenicity and the microbe in vivo. J. Gen. Microbiol. membranes. J. Bacteriol. 166: 2 12-2 16. 136: 377-383. BRENNAN, M. J., LIM,Z. M. COWELL,J. L., BISHER,M. E., STEVEN, STAINER, D. W., and SCHOLTE,M. J. 1970. A simple chemically A. C., NOVOTNY, P., and MANCLARK, C. R. 1988. Identification of defined medium for the production of phase I Bordetellu pertussis. a 69 kilodalton nonfimbrial protein as an agglutinogen of Bordetella J. Gen. Microbiol. 63: 2 11-220. pertussis. Infect. Immun. 56: 3 189-3 195. STEVEN, A. C., BISHER, M. E., TRUS,B. L., THOMAS, D., ZHANG,J. M., I. G., DOUGAN, G., PICKARD, D., CHATFIELD, S., SMITH,M., CHARLES, and COWELI.,J. L., 1986. Helical structure of Bordetella pertu.ssi.s NOVOTNY,P., MORRISSEY, P., and FAIRWEATHER, N. F. 1989. timbriae. J. Bacteriol. 167: 968-974. Molecular cloning and characterization of protective outer membrane SWANSON, J. 1981. Surface-exposed protein antigen so gonococcal protein P. 69 from Bordetellapertus.sis. Proc. Natl. Acad. Sci. U.S.A outer membrane. Infect. Immun. 34: 804-8 16. 86: 3554-3558. TOWBIN,H., STACHELIN, T., and GORDON,J. 1979. Electrophoretic FILIP,C., FLETCHER, G., WULFF,J. L., and EARHART, C. F. 1973. transfer of proteins from acrylamide gels to nitrocellulose sheets. Solubilisation of the cytoplasmic membrane of Escherichia coli. J. Procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76: Bacteriol. 115: 7 17-722. 43504354. GORRINGE, A., ASHWORTH, L. A. E., IRONS,L. I., and ROBINSON, A. WEISS,A. A., and HEWLETT,E. L. 1986. Virulence factors of Borde1985. Adhesion of B. pertussis to Vero cells. FEMS Microbiol. Lett. tellu pertussi.~.Annu. Rev. Microbiol. 40: 661 -686. 26: 5-9. ZHANG, J. M., COWELL,J. L., STEVEN,A. C., CARTER,P. H., HANKAPILLER, M. W., LUJAN,E., OSTRANDER. F., and HOOD,L. E. MCGRATH,P. P. and MANCLARK, C. R., 1985. Purification and 1983. Isolation of microgram quantities of protein from acrylamide characterization of timbriae isolated from Bordetella pertu.s.si.s. gels for amino acid sequence analysis. In Methods in enzymology Infect. Immun. 48: 4 2 2 4 2 7 . Vol. I. Edited by C. H. W. Hirs and S. N. Timasheff. Academic Press, New York. pp. 227-236.
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Received November 2. 1990 Revision accepted February 22, 199 1 ANWAR,H. 199 1. Surface-exposed antibody-accessible outer membrane proteins of Borderella pertussis. Can. J . Microbiol. 37: 590-593. The convalescent sera from a patient recovered form whooping cough were used to identify the surface-exposed antibodyaccessible outer membrane proteins (OMPs) of Bordetellapertu.ssis. The results indicated that the 69 000 OMP, the 40 000 porin, agglutinogens 2 and 3, and a number of presently unknown OMPs were exposed on the surface. The importance of these surfaceexposed antigens in the protection against whooping cough is discussed. Key words: Borderella pertussis, whooping cough, immune response, surface antigens, outer membrane protein. ANWAR,H. 1991. Surface-exposed antibody-accessible outer membrane proteins of Borderella pertussis. Can. J . Microbiol. 37 : 590-593. . Le sCrum d'un patient convalescent atteint de coqueluche a CtC utilisC pour identifier les protCines de surface de la membrane externe qui sont accessibles a l'anticorps chez Borderella pertussis. Les rCsultats indiquent que la membrane externe 69 000, la porine 40 000, les agglutinogknes 2 et 3 et un certain nombre de membrares externes inconnues sont accessibles sur la surface. On discute l'importance de ces antigknes dans les facteurs de protection contre la coqueluche. Mots elks: Bordetella pertussis, coqueluche, rCponse immunitaire, antigknes de surface, protCines de la membrane externe. [Traduit par la rCdaction]
Introduction Whooping cough is an inflammation of the lining of the respiratory &act of young children and is often chara~t~rized by a typical paroxysmal cough. The aetiologial agent is Bordetella pertussis and, less frequently, Bordetella parapertussis. Killed whole-cell vaccines are immensely successful in that they have virtually eliminated what was once regarded as a widely prevalent, frequently fatal, disease. Despite the tremendous success of whole-cell vaccines in the prevention of whooping cough, pertussis vaccination has always been a major concern to parents and clinicians because of the rare problematical association of the vaccines with brain damage. The toxic side effects of vaccination are a major concern in any vaccination program and form the other side of the benefit-risk equation (Miller et al. 1982). In the United Kingdom, diminished public confidence in the safety of killed whole-cell pertussis vaccine led to a reduction in vaccine acceptance, which correlated with an increase in the incidence of pertussis (Miller et al. 1982; Robinson et al. 1985).The precise identity of the component that causes the side effects in the whole-cell vaccine is still unknown. However, pertussis toxin (PT) has been suggested to be the component that causes the toxic side effect (Robinson et al. 1985; Weiss and Hewlett 1986). The presence of large amounts of lipopolysaccharide in the whole-cell vaccine is also of concern since this component is pyrogenic (Robinson et al. 1985). Whole-cell pertussis vaccines are crude preparations containing thousands of antigens. It is believed that those antigens that are exposed on the cell surface are probably important since they are likely to play a role in the prevention of whooping cough. It is, therefore, of interest to identify the outer membrane proteins (OMPs) of B. pertussis, which have conformational epitopes that are exposed on the cell surface and accessible to antibody so that the importance of these surface antigens in the prevention of the infection can be thoroughly investigated. To explore the accessibility to the surface antigens of B. pertussis to antibody, the method described by Swanson (198 1) was adopted and modified accordingly. The recognition of the OMPs of B. pertussis by
antibody present in the convalescent sera of a patient recovered from the infection was investigated by immunoblotting.
Materials and methods Strain and cultirral conditions Borderella pertussis Wellcome 28 was used throughout the study. It was maintained as freeze-dried cultures and recovered by growth on charcoal agar plates containing 10% (v/v) defibrinated horse blood (Gibmar Laboratories, Edmonton, Canada). After 48 h at 35"C, the growth from plates was inoculated into 100 mL Stainer and Scholte's medium (Stainer and Scholte 1970) containing I g/L of 2,6-0-dimethylP-cyclodextrin (Imaizumi et al. 1983) and incubated at 30°C for 24 h on an orbital shaker (1 80 rpm). Medium (300 mL) in 2-L conical flasks was inoculated with 10 mL of primary culture and incubated on an orbital shaker at 35OC. After 4 0 4 6 h, the cells were harvested by centrifugation (5000 X g for 30 min) and the cell pellet was retained. Preparation of outer membrane ( O M ) and SDS-PAGE The outer membrane (OM) was prepared using sodium lauryl sarcosinate (Sarkosyl; Sigma Chemicals Co., St. Louis, MO) as described by Filip et al. (1973). The bacterial pellet was suspended in 20 mL of distilled water and broken by two 60-s pulses of sonification in an ice bath, with one 60-s interval for cooling. Unbroken cells were removed by centrifugation at 3000 X g for 5 min. Sarkosyl was added to the supernatant to give a final concentration of 2%. The mixture was incubated at room temperature for 30 min and then centrifuged at 38 000 X g for 1 h. The membrane pellets were washed twice with distilled water, resuspended in a small volume of distilled water, and stored at - 20°C. Membrane preparations were subjected to sodium dodecyl sulphate - polyacrylamide gel electrophoresis (PAGE) by the system described by Anwar et al. (1987) with 12% acrylamide separating gel and ultrapure SDS. Immunoblotting The OMPs separated by SDS-PAGE were transferred onto nitrocellulose (NC) paper and visualized immunologically by the method of Towbin et al. ( 1979) as modified by Anwar et al. (1984). The NC paper was incubated first with 10 rnM Tris-hydrochloride - 0.85% saline (pH 7.4) (TBS) containing 0.3% Tween-20 for 1 h to saturate nonspecific protein-binding sites and then with patient serum diluted 1/50 in TBS - 0.3% Tween-20 for 4 h at 37OC. The paper was then washed
ANWAR
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thoroughly with TBS and incubated for an additional 2 h at 37°C with horseradish peroxidase goat anti-human immunoglobulin G (Sigma Chemicals Co., St. Louis, MO) diluted 112000 in TBS-0.3% Tween-20. After incubation, the NC paper was again washed thoroughly, and antigenic sites were visualized with a 25 p,glmL solution of 4-chloro- 1naphthol (Sigma Chemicals Co., St. Louis, MO) in TBS containing 0.0 1% H202.To show complete qualitative transfer of protein bands from polyacrylamide gels to the NC paper, I directly visualized blotted protein bands on duplicate NC paper by staining the paper with 1% amido black (data not shown). Surface-exposed antibody-uccessible antigen preparation The surface-exposed antigens were investigated by modifications of the method described by Swanson ( I98 l), in which the iodine labelling step was omitted and the antigenic sites were visualized by immunoblotting (Kadurugamuwa et ul. 1985). The bacterial culture (2 L) was harvested by centrifugation (5000 X g for 30 min) and washed once with 10 mM phosphate buffer saline (PBS), pH 7.4. The cells were treated with 5 mL convalescent sera and the sample was incubated at 35°C in an orbital shaker for 20 min. The cell suspension was then centrifuged at 5000 X g for 30 min and washed three times with PBS. The cells were disrupted by suspension of the pellet in 10 mL 1% N-tetradecyl-N,N-dimethyl-3-ammonio- I -propane sulfonate (Zwittergent 3-14; Calbiochem-Behring Corp., La Jolla, CA) in 10 mM PBS, pH 7.4. The mixture was well mixed, incubated at 37°C for 30 min, and centrifuged at 5000 rpm for 30 min. The supernatant was carefully removed and transferred to a cylindrical 10-mL column containing 5 mL previously swollen protein A - Sepharose 4B CL (Sigma Chemical Co., St Louis, MO). The mixture was incubated at room temperature for 20 min with regular end-to-end rotation, and the excess fluid was drawn through the column, which was then washed with 120 mL PBS. The antigen-antibody complexes bound to protein A Sepharose were eluted wit 100 rnL of 0.1 M glycine (pH 2.5) with regular end-to-end rotation (Kadurugamuwa et al. 1985). The eluate was dialyzed against distilled water at 4°C overnight, lyophilized and suspended in I mL of denaturing buffer containing 4% ultrapure SDS (British Drug House, Poole, U.K.), 8% 2-mercaptoethanol, 20% glycerol, and 0.0 1 % bromophenol blue in 0.125 M Tris-hydrochloride buffer, pH 6.8. The sample was stored at - 20°C.
Results and discussion The accessibility of the OMPs of B. pertussis to antibody was investigated by identification of the OMPs of intact cells of B. pertussis that are involved in the binding of IgG present in the convalescent sera. This method was successfully used in the investigation of the exposure of the OMPs of encapsulated strains of Klebsiella pneumoniae following growth of the cells in the presence of subinhibitory concentrations of cephalosporins (Kadurugamuwa et al. 1985). Figure 1 shows the OM (lane 2) and the surface-exposed antibody-accessible OMPs (lane 3) of B. pertussis. A number of protein bands that were found in the OMP profile (lane 2) could also be detected in the surfaceexposed antibody-accessible OMP preparation (lane 3). These are proteins with M, of 69 000, 40 000, 22 000, 20 000 and 15 000. The 69 000 OMP of B. pertussis has recently been identified as an agglutinogen and its usefulness as a vaccine candidate has been studied (Brennan et al. 1988; Charles et al. 1989).Molecular studies revealed that the biosynthesis of the 69 000 OMP of B. pertussis is under genetic control of the vir locus (Charles et al. 1989). The 40 000 protein has previously been identified to be a pore-forming protein (porin) that forms anionic-selective channels in lipid bilayer membrane (Armstrong et al. 1986). Agglutinogens were prepared by the method described by Rutter et al. (1988) for comparison. Lane 1 shows the electrophoretic positions of agglutinogen 2 (Agg 2; 22 000) and agglutinogen 3
FIG.1. SDS-PAGE of OMPs of Bordetella pertussis. Lane 1 , Agg 2 and Agg 3 preparation; lane 2, outer membrane preparation; and lane 3, the surface-exposed antibody accessible OMP preparation.
(Agg3; 20 000) on the acrylamide system used in this study. Agg 2 (Gorringe et al. 1985; Robinson et al. 1985) is identical with Agg 6 described by other workers (Steven et al. 1986; Zhang et al. 1985). The intensity of the Agg 2 band observed in the surface-exposed antibody-accessible preparation (lane 3) was closely similar to that of the Agg 3 band (lane 3), indicating that the same amounts of Agg 2 and Agg 3 subunits were produced by this strain of B. pertussis. Agg 2 was barely detectable in the OM preparation (lane 2), indicating that this protein is probably not firmly anchored to the OM and was released during the preparation of the OM. The protein band above the 15 000 protein in the surface-exposed antibody-accessible OMP preparation (lane 3) was not found in the OM preparation (lane 2). This protein was possibly loosely bound to the outer leaflet of the OM and was released during the preparation of the OM. Similarly, the 54 000 protein was found to be a major band in the surface-exposed antibody-accessible OMP preparation (lane 3) but not associated with the OM (lane 2). It was, therefore, of interest to carry out some further biochemical analysis to verify the origin of the 54 000 protein. The protein was purified by electroelution as described by Hunkapiller et al. (1983) and the purified 54 000 protein was subjected to N-terminal amino acid sequencing by S. Kielland (University of Victoria, Victoria,
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CAN. J. MICROBIOL. VOL. 37, 1991
FIG. 2. Immunoblot of the OMPs described in Fig. 1 electrophoretically transferred onto nitrocellulose paper and reacted with IgG present in the convalescent sera. Lane 1, Agg 2 and Agg 3 preparation; lane 2, outer membrane preparation; and lane 3, the surface-exposed antibody-accessible OMP preparation.
B.C.). Based on a 14 amino acid sequence, the 54 000 protein was found to have 100% homology to the heavy chain (V-I11 region) of human IgG (data not shown).Therefore, the 54 000 protein observed in the surface-exposed antibody-accessible preparation had not originated from B. pertussis, but was from the heavy chain of the IgG that was copurified during the preparation of surface-exposed antibody-accessible antigens (Kadurugamuwa et al. 1985). Figure 2 illustrates the antigenic profiles of the surface OMPs of B. pertussis after the proteins from Fig. 1 were blotted onto nitrocellulose paper and probed with the convalescent serum. IgG present in the convalescent sera reacted strongly with the agglutinogens (Agg 2 and Agg 3) of B. pertussis (lane 1). The Agg 2 and Agg 3 bands were seen as a diffused band in the immunoblot. The importance of Agg 2 and Agg 3 as potential vaccine candidates has been discussed (Robinson et al. 1985) and these antigens have been included in the development of an acellular pertussis vaccine in the United Kingdom (Rutter et al. 1988). The phase I clinical trial of the vaccine indicated that Agg 2 and Agg 3 were immunogenic and strong antibody responses to these antigens were observed following administration of the vaccine to healthy adult volunteers (Rutter et al. 1988).
Monoclonal antibodies to Agg 2 and Agg 3 have been shown to inhibit the adherence of B. pertussis to Vero cells (Gorringe et al. 1985). The results of this study indicated that strong IgG responses to Agg 2 and Agg 3 were observed following the natural infection with B. pertussis (Fig. 2, lanes 1 and 3). This observation strengthens the importance of adding Agg 2 an Agg 3 in the development of acellular pertussis vaccine in order to give full protection against the disease (Robinson et al. 1985; Rutter et al. 1988). The sera from the patient also contained IgG, which reacted with a number of antigens in the OM preparation of B. pertussis (Fig. 2, lane 2). The porin (40 000) was highly immunogenic and a strong antibody response was launched by the immune system against this anl.igen as the results of host-parasite interaction. The usefulness of the 40 000 porin as a potential vaccine candidate in the prevention of whooping cough remains to be investigated. In this study, a strong antibody response to 69 000 OMP was also observed (Fig. 2, lane 2), confirming the importance of this antigen as a potential vaccine candidate in the prevention of whooping cough (Brennan et al. 1988; Charles et ul. 1989). Five protein bands above the 69 000 protein were immunogenic and reacted strongly with IgG (Fig. 2, lane 2). These protein bands are likely to be the subunits of the filamentous haemaglutinin as observed by other workers (Robinson et ul. 1985; Rutter et ul. 1988; Weiss and Hewlett 1986). These protein bands are currently being investigated in the laboratory. The exposed outer surface of a bacterial cell plays a crucial role in its survival in vivo (Parker and Armstrong 1989; Smith 1977, 1990; Swanson 198 1). The surface components are in constant interaction with the immune system. Humoral responses are launched against the surface components to provide specific recognition of the infecting organism so that the pathogen can be dealt with efficiently and effectively in vivo. The surfaceexposed antibody-accessible antigens are, therefore, of interest to those who are involved in vaccine research. The lactoperoxidase-catalysed iodination technique has previously been used to investigate the exposure of the surface proteins of B. pertussis (Parker and Armstrong 1989; Redhead 1983). The drawback of this technique is that the outer membrane proteins must have residues such as tyrosine exposed on the surface to be readily accessible for labelling by lactoperoxidase. In this study, the OMPs of B. pertussis that are exposed on the surface and accessible to IgG have been identified by using convalescent sera obtained from natural infections. Pertussis toxin and filamentous haemaglutinin of B. pertussis have been extensively studied and these components have been included in the development of acellular pertussis vaccines (Robinson et al. 1985; Rutter et al. 1988; Sato et al. 1984). Agglutinogens produced by B. pertussis have also been investigated and included in the development of acellular petussis vaccines (Brennan et al. 1988; Charles et al. 1989; Rutter et nl. 1988). In this study, a simple method was used to investigate the surface exposure of the outer membrane antigens of B. pertussis. The method can also be used in the study of the surface antigens of other clinically important Gram-negative pathogens. More convalescent sera (10-20 human immune serum specimens) should be investigated before a general conclusion can be made regarding the immune responses to the surface antigens of B. pertussis. Stimulation of the immune system to launch specific antibody responses to pertussis toxin, filamentous haemaglutinin, agglutinogens, and other important surfaceexposed antibody-accessible antigens should assist the swift eradication of the infecting pathogen that is trying to colonize the respiratory tract. Given the present high levels of interest in the
ANWAR
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development of acellular pertussis vaccines, I am confident that an effective acellular pertussis vaccine consisting of these protective antigens of B. pertussis will be introduced into the immunization program to prevent infants and children from contracting whooping cough.
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IMAIZLIMI, A. SUZUKI, Y., ONO, S., SATO, H., and SATO, Y. 1983. Effect of heptakis (2,6-0-dimethyl) P-cyclodextrin on the production of pertussis toxin by Bordetella pertu.s.si.s. Infect. Immun. 41: 1138-1 143. H., BROWN,M. R. W., and ZAK,0 . KADURUGAMUWA, J. L., ANWAR, 1985. Protein antigens of encapsulated Kleb,siellu pneumoniue surface exposed after growth in the presence of subinhibitory Acknowledgements concentrations of cephalosporins. Antimicrob. Agents Chemother. The author thanks William J. Page (Department of Microbi28: 1 95- 199. ology, University of Alberta) for helpful discussion and for MILLER,D. L., ALDERSLADE, R., and Ross, E. M. 1982. Whooping critically reviewing the manuscript. This work was supported by cough and whooping cough vaccine: the risks and benefits debate. Epidemiol. Rev. 4: 1-24. operating grants from the Central Research Fund of the UniverPARKER,C. D., and ARMSTRONG, S. K. 1989. Surface proteins of sity of Alberta and the Natural Sciences and Engineering Bordetella pertu.s.si.s. Rev. Infect. Dis. 10: S327-S330. Research Council of Canada. REDHEAD, K. 1983. Variability of the surface exposure of the outer membrane proteins of Bordetella pertus.si,s. FEMS Microbiol. Lett. ANWAR,H. ASHWORTH, L. A. E., FUNNELL, S., ROBINSON, A., and 17: 35-39. IRONS,L. I. 1987. Neutralisation of biological activities of pertussis ROB~NSON, A., IRONS, L. I., and ASHWORTH,L. A. E. 1985. Pertussis toxin with a monoclonal antibody. FEMS Microbiol. Lett. 44: vaccine: present status and future prospects. Vaccine, 3: 1 1-22. 141-145. RU~ER D., A., ASHWORTH, L. A. E., DAY,A., FUNNELL, S., LOVELL, ANWAR,H., BROWN,M. R. W., DAY,and A., WELLER,P. H. 1984. F., and ROBINSON, A. 1988. Trial of a new acellular pertussis vaccine Outer membrane antigens of mucoid Pseudornonas r~erugirzoscr in healthy adult volunteers. Vaccine, 6: 29-32. isolated directly from the sputum of a cystic fibrosis patient. FEMS SATO, Y., KIMURA, M., and FUKUMI,H. 1984. Development of a Microbiol. Lett. 24: 235-239. pertussis component vaccine in Japan. Lancet, 1: 122-1 26. ARMSTRONG, S. K., PARR.T. R., PARKER,C. D. and HANCOCK, SMITH,H. 1977. Microbial surface in relation to pathogenicity. R. E. W. 1986. Bordetella pertussis major outer membrane porin Bacteriol. Rev. 41: 475-500. protein forms small, anionic-selective channels in lipid bilayer 1990. Pathogenicity and the microbe in vivo. J. Gen. Microbiol. membranes. J. Bacteriol. 166: 2 12-2 16. 136: 377-383. BRENNAN, M. J., LIM,Z. M. COWELL,J. L., BISHER,M. E., STEVEN, STAINER, D. W., and SCHOLTE,M. J. 1970. A simple chemically A. C., NOVOTNY, P., and MANCLARK, C. R. 1988. Identification of defined medium for the production of phase I Bordetellu pertussis. a 69 kilodalton nonfimbrial protein as an agglutinogen of Bordetella J. Gen. Microbiol. 63: 2 11-220. pertussis. Infect. Immun. 56: 3 189-3 195. STEVEN, A. C., BISHER, M. E., TRUS,B. L., THOMAS, D., ZHANG,J. M., I. G., DOUGAN, G., PICKARD, D., CHATFIELD, S., SMITH,M., CHARLES, and COWELI.,J. L., 1986. Helical structure of Bordetella pertu.ssi.s NOVOTNY,P., MORRISSEY, P., and FAIRWEATHER, N. F. 1989. timbriae. J. Bacteriol. 167: 968-974. Molecular cloning and characterization of protective outer membrane SWANSON, J. 1981. Surface-exposed protein antigen so gonococcal protein P. 69 from Bordetellapertus.sis. Proc. Natl. Acad. Sci. U.S.A outer membrane. Infect. Immun. 34: 804-8 16. 86: 3554-3558. TOWBIN,H., STACHELIN, T., and GORDON,J. 1979. Electrophoretic FILIP,C., FLETCHER, G., WULFF,J. L., and EARHART, C. F. 1973. transfer of proteins from acrylamide gels to nitrocellulose sheets. Solubilisation of the cytoplasmic membrane of Escherichia coli. J. Procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76: Bacteriol. 115: 7 17-722. 43504354. GORRINGE, A., ASHWORTH, L. A. E., IRONS,L. I., and ROBINSON, A. WEISS,A. A., and HEWLETT,E. L. 1986. Virulence factors of Borde1985. Adhesion of B. pertussis to Vero cells. FEMS Microbiol. Lett. tellu pertussi.~.Annu. Rev. Microbiol. 40: 661 -686. 26: 5-9. ZHANG, J. M., COWELL,J. L., STEVEN,A. C., CARTER,P. H., HANKAPILLER, M. W., LUJAN,E., OSTRANDER. F., and HOOD,L. E. MCGRATH,P. P. and MANCLARK, C. R., 1985. Purification and 1983. Isolation of microgram quantities of protein from acrylamide characterization of timbriae isolated from Bordetella pertu.s.si.s. gels for amino acid sequence analysis. In Methods in enzymology Infect. Immun. 48: 4 2 2 4 2 7 . Vol. I. Edited by C. H. W. Hirs and S. N. Timasheff. Academic Press, New York. pp. 227-236.
