Wodd Journal
of Microbiology
& Siotechnology
10, 438438
Recognition of immunologically important antigens in Bacter0ides hagi/& infection -an experimental study in mice T. Khanna, D. Panigrahi,* N.K. Ganguly and S. Majumdar When the outer membrane proteins (OMP) of Bacteroides fragilis ATCC 23745 grown under seven different culture conditions were analysed by SDS-PAGE, five of the seven preparations showed similar protein profiles. When the three different OMP preparations were subsequently analysed by Western blotting, that from B. fragilis grown in brain heart infusion broth supplemented with haemin and menadione alone showed more immunodominant bands than those grown with additional cholesterol and methionine or cholesterol and cysteine. Only the former showed immunodominant proteins around 45.6 kDa and 66.2 kDa throughout the course of infection in mice. A protein corresponding to 97.4 kDa was found under all culture conditions. The results emphasise the importance of culture conditions in the expression of immunologically reactive antigens and also the possible use of such antigens in the serodiagnosis of B. fragilis infection. Key words: Antigens,
Bacteroides fragilis, immunodominant.
Bacteroides fragilis is the most common Bacteroides species, accounting for about 70% of human anaerobic infections (Lindberg et al. 1979). Diagnosis of Bacteroides infections poses a problem because of lack of laboratory expertise. Several workers have used a serological approach to investigate Bacteroides infections. However, poor immunogenicity, presence of specific antibodies in the sera of healthy individuals and antigenic heterogenicity complicate the interpretation of the results (Danielsson et al. 1974). Culture conditions are known to influence the composition of the outer membrane proteins (OMP) of Bacteroides spp. OMP of B. fragilis grown under different culture conditions were therefore analysed in the present study. Immunodominant OMP expressed during the course of infection in an experimental mouse model have been identified, with the aim of developing a good antibody-detection system.
T. Khanna is and D. Panigrahi was with the Department of Medical Microbiology and N.K. Ganguly and S. Majumdar are with the Department of Experimental Medicine, at the Postgraduate Institute of Medical Education and Research, Chandigarh, India. D. Panigrahi is now with Kuwait University, Faculty of Allied Health Science, P.O. Box 31470 Sulaibikhat, 90805, Kuwait; fax: 965 4830937. ‘Corresponding author. 0
7994 Rapid Communications
436
of Oxford Ltd
World Journal ofMicrobiology
b Biotechnology, Vol IO, 1994
Materials and Methods Bacteria and Growth Conditions Bacteroides fragilis ATCC 23745 was grown in seven different media: (1) brain heart infusion (BHI) broth (HI-Media) supplemented with 0.05% haemin and 0.005% menadione (= medium 1); (2) medium 1 plus 100 mg% cholesterol; (3) medium 1 plus 20 mg% cysteine, 20 mg% methionine and 100 mg% cholesterol; (4) medium 1 plus 20 mg% cysteine; (5) medium 1 plus 20 mg% methionine; (6) medium 1 plus 20 mg% methionine and 100 mg% cholesterol; (7) medium 1 plus 20 mg% cysteine and 100 mg% cholesterol. OMP Preparation Three isolated colonies of B.fragiZis grown overnight on BHI agar plates were inoculated into 400 ml of each of the seven media and incubated anaerobically for 24 h at 37°C. The cultures were centrifuged at 1200 xg for 10 min at 4°C and the bacterial pellets processed for OMP preparation according to Kasper & Seiler (1975). Protein in the OMP preparations was estimated by the Lowry method. Production of Infection in Mice Twenty mice were each inoculated subcutaneously with 1.5 x 108 organisms. Abscesses developed on day 4 post-infection. Blood
Immunodominant was collected from all the mice on days 3, 9, 15 and 22 postinfection by the retro-orbital route and the sera collected on the same day were pooled and stored at -70°C. SDS-PAGE Seven different OMP were studied by SDS-PAGE by the methods of Laemmli (1970). Protein concentration of each OMP preparation was adjusted to 3 mg/ml and 5 pl was loaded into each well. High and low molecular weight standards were used for comparison. Western Blotting Western blots were made by the methods of Towbin et al. (1979). After SIX-PAGE of the OMP, the gels were subjected to electroblot transfer for 1 h at 100 V and 0.2 mA. Immunoblotting was then carried out using 4% BSA as blocking solution, pooled infected mice sera (1:5001 as antibody source, horse-radish peroxidase anti-mouse IgG (1:2000) as conjugate and diaminobenzidine as substrate.
Results and Discussion Between 17 and 20 polypeptide bands from 14 to 116 kDa were observed in all OMP preparations. OMP prepared from cells grown under culture conditions 1 to 5 shared a similar polypeptide pattern while those from culture conditions 6 and 7 each showed unique profiles. Protein bands with a M, of 21.5 kDa were only present in cells grown under culture conditions 6 and 7; proteins of 31.4 and 58 kDa were present in those grown under culture conditions 1 to 5 and 26 and 28 kDa proteins in those grown under culture conditions 1 to 5 and 7 (Figure 1). Hence, for further studies, OMP prepared
antigens of Bacteroides
fragilis
from cells grown under culture conditions 1, 6 and 7 were investigated. Western blotting showed a protein band at 97.4 kDa in all three of these OMP preparations with all four antisera. Two groups of immunodominant proteins were observed, one, around 45.6 kDa, expressed only under culture condition 1 and another, around 66.2 kDa, under culture conditions 1 and 6. These two immunodominant proteins were labelled by all four antisera, whereas another protein, of about 66.2 kDa, was only labelled by sera collected on days 15 and 22 (Figure 2). Culture conditions therefore play an important role in the production of OMP. In B. thetaiotaomicron, OMP vary with the sugars used in the culture medium (Kotarski & Salyer’s 1984). In Escherichia coli, OMP composition changes if glycerol is used instead of glucose in the medium (Schnaitman 1974). In the present study, the abundance of OMP of 21.5, 31.4 and 58 kDa varied with the culture conditions. OMP prepared from B. fragilis grown in BHI supplemented with only haemin and manadione expressed the largest number of protein bands. Elhag & Alkarmi (1991) demonstrated 21 immunodominant proteins in crude antigens of the fragilis group of Bacteroides, ranging from 10.8 to 102.3 kDa. In the present study, the crude Oh@ preparations from supplemented BHI broth showed two groups of immunodominant proteins, at about 45.6 and 66.2 kDa. Loeb & Smith (19821 have shown elevated antibody titres and the presence of new immunogenic bands in the convalescent sera of a patient infected with Haemophilus influenzae. In the present study, antibodies against 45.6 and 66.2 kDa proteins were present throughout the post-infection period. Antibodies against a different, 66.2 kDa protein appeared only on day 15 postinfection as the abscess began to heal.
kDa
kDa
-
974 --------1'g.f~ ------65.2-==x==-- =I---__-45.6 _ ? s 2 -TLIZ z: --zzz=_:= -_- _..21.5 ..~.. ___.. --.-_ -- _~ 14.4 1 -. -
200 a.2
-_ g; _;: * _ ----- =;::. .-.;;-- --~ - -.--
45.6
kDa 1 6 ----I---.-_-'-.__. __-- -.. -__. .. . . _. _. ____ .~~..-- --_ _- .. . . ---- _ _
7 --
---..---
97.4 66.2 45.6 21.5
L1234je7~ 1. SDS-PAGE of outer membrane proteins of Bacteroides fragi/isATCC 23745 grown under seven different culture conditions. Figure drawn from original gels after Coomassie Blue staining. I-BHI broth supplemented with haemin and menadione (= medium 1); 2-medium 1 plus cholesterol; 3-l plus cysteine, methionine and cholesterol; 4-l plus cysteine; 5-l plus methionine; 6-1 plus methionine and cholesterol; 7-l plus cysteine and cholesterol; L-low molecular weight standards; H-high molecular weight markers.
14.4
Figure
3
g
15
22
3
9 15
22 3 9 15 22
Days post-infection Figure
lmmunoblotting of outer membrane proteins of grown under culture conditions 1,6 and 7, using sera from 6. fragiis-infected mice collected on days 3, 9, 15 and 22 post-infection. (Figure drawn from original blots.) Becteroides
2.
fagilis
World pm~al
ofMicrobiology
& Biotechnology, Vd IO.1994
437
T. Khnna
et al.
We are now in the process of evaluating the role of these immunodominant proteins as protective antigens and their potential value in the serodiagnosis of B. frugilis infection.
References Danielsson, D., Lambe, D.W. & Persson, S. 1974 Immune response to anaerobic infections. In Anaerobic Bacteria: Role in Disease, ed Barlows, A. pp. 173-191. Springfield, IL: Charles C. Thomas Press. Elhag, K.M. & Alkarmi, T.O. 1991 A study of the antigenic composition of the fragilis group of Bacferoides. journal of Medical Microbiology 35,1X3-122. Kasper, D.L. & Seiler, M.W. 1975 Immunological characterization of outermembrane complex of Bacferoides fragilis ssp fragilis. Journal of Infectious Diseases 132,440-450. Kotarski, S.F. & Salyer’s, A.A. 1984 Isolation and characterization of outer membrane of Bacferoides frasiZis grown on different carbohydrates. Journal of Bacteriology 1!58,102-109.
438
World /ownal
of Microbiology
b Biotechnology, Vol lo,1994
Laemmli, U.K. 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Lindberg, A.A., Berthold, I’., Nord,C.E. & Weintraub, A. 1979 Encapsulated strains of Bacferoides fragilis in clinical specimens. Medical Microbiology and Immunology 167,29-36. Loeb, M.R. & Smith, D.H. 1982 Human antibody response to individual outer membrane proteins of Haemophilus influenzae type-b. Infection and Immunity 37,1032-1036. Schnaitman, C.A. 1974 Outer membrane protein of Escherichiu coli. IV. Differences in outer membrane proteins due to strain and cultural differences. Journal of Bacteriology 118, 454-464. Towbin, H.J., Staehelin, J. & Gordon, J. 1979 Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America 76,4350-4354.
(Received in revised form 23 February 1994; accepted 25 February 2994)
of Microbiology
& Siotechnology
10, 438438
Recognition of immunologically important antigens in Bacter0ides hagi/& infection -an experimental study in mice T. Khanna, D. Panigrahi,* N.K. Ganguly and S. Majumdar When the outer membrane proteins (OMP) of Bacteroides fragilis ATCC 23745 grown under seven different culture conditions were analysed by SDS-PAGE, five of the seven preparations showed similar protein profiles. When the three different OMP preparations were subsequently analysed by Western blotting, that from B. fragilis grown in brain heart infusion broth supplemented with haemin and menadione alone showed more immunodominant bands than those grown with additional cholesterol and methionine or cholesterol and cysteine. Only the former showed immunodominant proteins around 45.6 kDa and 66.2 kDa throughout the course of infection in mice. A protein corresponding to 97.4 kDa was found under all culture conditions. The results emphasise the importance of culture conditions in the expression of immunologically reactive antigens and also the possible use of such antigens in the serodiagnosis of B. fragilis infection. Key words: Antigens,
Bacteroides fragilis, immunodominant.
Bacteroides fragilis is the most common Bacteroides species, accounting for about 70% of human anaerobic infections (Lindberg et al. 1979). Diagnosis of Bacteroides infections poses a problem because of lack of laboratory expertise. Several workers have used a serological approach to investigate Bacteroides infections. However, poor immunogenicity, presence of specific antibodies in the sera of healthy individuals and antigenic heterogenicity complicate the interpretation of the results (Danielsson et al. 1974). Culture conditions are known to influence the composition of the outer membrane proteins (OMP) of Bacteroides spp. OMP of B. fragilis grown under different culture conditions were therefore analysed in the present study. Immunodominant OMP expressed during the course of infection in an experimental mouse model have been identified, with the aim of developing a good antibody-detection system.
T. Khanna is and D. Panigrahi was with the Department of Medical Microbiology and N.K. Ganguly and S. Majumdar are with the Department of Experimental Medicine, at the Postgraduate Institute of Medical Education and Research, Chandigarh, India. D. Panigrahi is now with Kuwait University, Faculty of Allied Health Science, P.O. Box 31470 Sulaibikhat, 90805, Kuwait; fax: 965 4830937. ‘Corresponding author. 0
7994 Rapid Communications
436
of Oxford Ltd
World Journal ofMicrobiology
b Biotechnology, Vol IO, 1994
Materials and Methods Bacteria and Growth Conditions Bacteroides fragilis ATCC 23745 was grown in seven different media: (1) brain heart infusion (BHI) broth (HI-Media) supplemented with 0.05% haemin and 0.005% menadione (= medium 1); (2) medium 1 plus 100 mg% cholesterol; (3) medium 1 plus 20 mg% cysteine, 20 mg% methionine and 100 mg% cholesterol; (4) medium 1 plus 20 mg% cysteine; (5) medium 1 plus 20 mg% methionine; (6) medium 1 plus 20 mg% methionine and 100 mg% cholesterol; (7) medium 1 plus 20 mg% cysteine and 100 mg% cholesterol. OMP Preparation Three isolated colonies of B.fragiZis grown overnight on BHI agar plates were inoculated into 400 ml of each of the seven media and incubated anaerobically for 24 h at 37°C. The cultures were centrifuged at 1200 xg for 10 min at 4°C and the bacterial pellets processed for OMP preparation according to Kasper & Seiler (1975). Protein in the OMP preparations was estimated by the Lowry method. Production of Infection in Mice Twenty mice were each inoculated subcutaneously with 1.5 x 108 organisms. Abscesses developed on day 4 post-infection. Blood
Immunodominant was collected from all the mice on days 3, 9, 15 and 22 postinfection by the retro-orbital route and the sera collected on the same day were pooled and stored at -70°C. SDS-PAGE Seven different OMP were studied by SDS-PAGE by the methods of Laemmli (1970). Protein concentration of each OMP preparation was adjusted to 3 mg/ml and 5 pl was loaded into each well. High and low molecular weight standards were used for comparison. Western Blotting Western blots were made by the methods of Towbin et al. (1979). After SIX-PAGE of the OMP, the gels were subjected to electroblot transfer for 1 h at 100 V and 0.2 mA. Immunoblotting was then carried out using 4% BSA as blocking solution, pooled infected mice sera (1:5001 as antibody source, horse-radish peroxidase anti-mouse IgG (1:2000) as conjugate and diaminobenzidine as substrate.
Results and Discussion Between 17 and 20 polypeptide bands from 14 to 116 kDa were observed in all OMP preparations. OMP prepared from cells grown under culture conditions 1 to 5 shared a similar polypeptide pattern while those from culture conditions 6 and 7 each showed unique profiles. Protein bands with a M, of 21.5 kDa were only present in cells grown under culture conditions 6 and 7; proteins of 31.4 and 58 kDa were present in those grown under culture conditions 1 to 5 and 26 and 28 kDa proteins in those grown under culture conditions 1 to 5 and 7 (Figure 1). Hence, for further studies, OMP prepared
antigens of Bacteroides
fragilis
from cells grown under culture conditions 1, 6 and 7 were investigated. Western blotting showed a protein band at 97.4 kDa in all three of these OMP preparations with all four antisera. Two groups of immunodominant proteins were observed, one, around 45.6 kDa, expressed only under culture condition 1 and another, around 66.2 kDa, under culture conditions 1 and 6. These two immunodominant proteins were labelled by all four antisera, whereas another protein, of about 66.2 kDa, was only labelled by sera collected on days 15 and 22 (Figure 2). Culture conditions therefore play an important role in the production of OMP. In B. thetaiotaomicron, OMP vary with the sugars used in the culture medium (Kotarski & Salyer’s 1984). In Escherichia coli, OMP composition changes if glycerol is used instead of glucose in the medium (Schnaitman 1974). In the present study, the abundance of OMP of 21.5, 31.4 and 58 kDa varied with the culture conditions. OMP prepared from B. fragilis grown in BHI supplemented with only haemin and manadione expressed the largest number of protein bands. Elhag & Alkarmi (1991) demonstrated 21 immunodominant proteins in crude antigens of the fragilis group of Bacteroides, ranging from 10.8 to 102.3 kDa. In the present study, the crude Oh@ preparations from supplemented BHI broth showed two groups of immunodominant proteins, at about 45.6 and 66.2 kDa. Loeb & Smith (19821 have shown elevated antibody titres and the presence of new immunogenic bands in the convalescent sera of a patient infected with Haemophilus influenzae. In the present study, antibodies against 45.6 and 66.2 kDa proteins were present throughout the post-infection period. Antibodies against a different, 66.2 kDa protein appeared only on day 15 postinfection as the abscess began to heal.
kDa
kDa
-
974 --------1'g.f~ ------65.2-==x==-- =I---__-45.6 _ ? s 2 -TLIZ z: --zzz=_:= -_- _..21.5 ..~.. ___.. --.-_ -- _~ 14.4 1 -. -
200 a.2
-_ g; _;: * _ ----- =;::. .-.;;-- --~ - -.--
45.6
kDa 1 6 ----I---.-_-'-.__. __-- -.. -__. .. . . _. _. ____ .~~..-- --_ _- .. . . ---- _ _
7 --
---..---
97.4 66.2 45.6 21.5
L1234je7~ 1. SDS-PAGE of outer membrane proteins of Bacteroides fragi/isATCC 23745 grown under seven different culture conditions. Figure drawn from original gels after Coomassie Blue staining. I-BHI broth supplemented with haemin and menadione (= medium 1); 2-medium 1 plus cholesterol; 3-l plus cysteine, methionine and cholesterol; 4-l plus cysteine; 5-l plus methionine; 6-1 plus methionine and cholesterol; 7-l plus cysteine and cholesterol; L-low molecular weight standards; H-high molecular weight markers.
14.4
Figure
3
g
15
22
3
9 15
22 3 9 15 22
Days post-infection Figure
lmmunoblotting of outer membrane proteins of grown under culture conditions 1,6 and 7, using sera from 6. fragiis-infected mice collected on days 3, 9, 15 and 22 post-infection. (Figure drawn from original blots.) Becteroides
2.
fagilis
World pm~al
ofMicrobiology
& Biotechnology, Vd IO.1994
437
T. Khnna
et al.
We are now in the process of evaluating the role of these immunodominant proteins as protective antigens and their potential value in the serodiagnosis of B. frugilis infection.
References Danielsson, D., Lambe, D.W. & Persson, S. 1974 Immune response to anaerobic infections. In Anaerobic Bacteria: Role in Disease, ed Barlows, A. pp. 173-191. Springfield, IL: Charles C. Thomas Press. Elhag, K.M. & Alkarmi, T.O. 1991 A study of the antigenic composition of the fragilis group of Bacferoides. journal of Medical Microbiology 35,1X3-122. Kasper, D.L. & Seiler, M.W. 1975 Immunological characterization of outermembrane complex of Bacferoides fragilis ssp fragilis. Journal of Infectious Diseases 132,440-450. Kotarski, S.F. & Salyer’s, A.A. 1984 Isolation and characterization of outer membrane of Bacferoides frasiZis grown on different carbohydrates. Journal of Bacteriology 1!58,102-109.
438
World /ownal
of Microbiology
b Biotechnology, Vol lo,1994
Laemmli, U.K. 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Lindberg, A.A., Berthold, I’., Nord,C.E. & Weintraub, A. 1979 Encapsulated strains of Bacferoides fragilis in clinical specimens. Medical Microbiology and Immunology 167,29-36. Loeb, M.R. & Smith, D.H. 1982 Human antibody response to individual outer membrane proteins of Haemophilus influenzae type-b. Infection and Immunity 37,1032-1036. Schnaitman, C.A. 1974 Outer membrane protein of Escherichiu coli. IV. Differences in outer membrane proteins due to strain and cultural differences. Journal of Bacteriology 118, 454-464. Towbin, H.J., Staehelin, J. & Gordon, J. 1979 Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America 76,4350-4354.
(Received in revised form 23 February 1994; accepted 25 February 2994)
