Characterization of monoclonal antibodies to the outer membrane protein (OmpD) of Salmonella typhimurium SURESHR. P A I , YVONNE ~ UPSHAW,AND SHIVAP. SINGH
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Biomedical Research and Training Programs, Alabama State University, Montgomery, AL 36101, U.S.A. Received July 16, 1991 Revision received April 24, 1992 Accepted April 29, 1992 PAI, S. R., UPSHAW,Y., and SINGH,S. P. 1992. Characterization of monoclonal antibodies to the outer membrane protein (OmpD) of Salmonella typhimurium. Can. J. Microbiol. 38: 1102-1 107. A panel of monoclonal antibodies, seven against the trimeric and seven against the monomeric forms to outer membrane protein D (OmpD) of Salmonella typhimurium were produced. The specificities of these monoclonal antibodies for the porin proteins of S. typhimurium and their cross-reactions with Salmonella porins OmpC and OmpF were determined by Western immunoblotting and enzyme-linked immunosorbent assay. We observed that OmpD shared more epitopes and had greater structural similarity with OmpC than with OmpF. Key words: Salmonella typhimurium, outer membrane protein, monoclonal antibody, trimeric, monomeric. PAI, S. R., UPSHAW,Y., et SINGH,S. P. 1992. Characterization of monoclonal antibodies to the outer membrane protein (OmpD) of Salmonella typhimurium. Can. J. Microbiol. 38 : 1102-1 107. Nous avons produit une serie d'anticorps monoclonaux contre la protkine OmpD de Salmonella typhimurium, soit sept dirigks contre la forme trimkre et sept autres contre la forme monomkre. La specificite de ces anticorps monoclonaux envers les proteines des porines de S. typhimurium et leurs reactions croiskes avec les proteines OmpC et OmpF de Salmonella ont ete evaluees par immunobuvardage de type Western et par immunoessai enzymatique. Nous avons constate que 1'OmpD partageait plus d'epitopes et avait une plus grande parente de structure avec 1'OmpC qu'avec 1'OmpF. Mots clks : Salmonella typhimurium, proteine de la membrane externe, anticorps monoclonal, trimkre, monomkre. [Traduit par la redaction]
Introduction The outer membrane (OM) of Salmonella typhimurium as well as other gram-negative bacteria contains pore-forming proteins called porins (Benz 1988; Garavito and Rosenbusch 1986; Ishii and Nakae 1980; Rosenbusch 1974; Schindler and Rosenbusch 1984). They are present as trimers and form trans outer membrane water-filled channels, which facilitate the transport of small hydrophilic molecules (Hancock 1987). The B-sheet porins span .the .thickness of the outer membrane and produce tight complexes with the underlying peptidoglycan layer and with lipopolysaccharides (LPSs) (Inouye 1979). They are oriented roughly perpendicular to the membrane. They protrude a little on both sides from the plane of the membrane, with three separate openings at the surface, which coalesce into a single channel near the center of the membrane (Rosenbusch 1987). Under normal growth conditions, S. typhimurium LT2 expresses three porins, OmpD (34 000 Da), OmpF (35 000 Da), and OmpC (36 000 Da), whereas Escherichia coli K12 synthesizes two such proteins, OmpC and OmpF (Ishii and Nakae 1980; Lee and Schnaitman 1980; Nurminen et al. 1976; Tokunaga et al. 1979). Other porins such as LamB, PhoE, NmpC, LC, OmpG, and Tx are expressed under certain physiological conditions (Benz 1988; Lugtenberg and Van Alphen 1983; Nakae 1986; Nikaido and Vaara 1985). The OmpF, OmpC, PhoE, NmpC, and LC proteins share significant sequence homologies at both the nucleotide and amino acid levels (Blasband et al. 1986; Mizuno et al. 1983; Overbeeke et al. 1983). The immunological properties of ' ~ u t h o rto whom all correspondence should be sent at the following address: Department of Pathobiology, Auburn University, Auburn, AL 36849-5517, U.S.A. Printed in Canada
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E. coli porin OmpF, OmpC, PhoE, and LamB proteins have been widely studied. The purified porins are immunogenic in mice and rabbits (Hofstra and Dankert 1980, 1981; Overbeeke et al. 1980) as either native trimeric (T) porin or denatured monomeric (M) porin. Porin monomers derived from a number of Enterobacteriaceae, including the OmpF, OmpC, and PhoE porin monomers of E. coli and porin protein P of Pseudomonas aeruginosa, have been shown to cross- react immunologically (Chai and Foulds 1979; Hofstra and Dankert 1980, 1981; Overbeeke et al. 1980; Poole and Hancock 1986). Monoclonal antibodies (MAbs) offer several distinct advantages over polyclonal sera, including defined specificity, reproducibility, and availability. Monoclonal antibodies raised against E. coli LamB (Gabay et al. 1983), PhoE (Van der Ley et al. 1985), and OmpF (Bentley and Klebba 1988; Klebba et al. 1990) have been used to study the structure and topology of porin and their variability among related species. Recently, we tested a library of 43 anti-E. coli B/r OmpF MAbs by Western immunoblots against 12 different enteric and 4 nonenteric gram-negative species, as well as the E. coli porins NmpC, OmpC, and PhoE. The results showed considerable immunological divergence between the pore proteins of these organisms (Klebba et al. 1990). To our knowledge this is the first report of the production of a panel of MAbs against the T and M forms of OmpD protein of S. typhimurium. We describe the isotypes of these MAbs and their interactions with the T and M forms of OmpD, OmpC, and OmpF porins, and the purified OM fragments from S. typhimurium. Our results indicate OmpD to have greater immunological similarity with OmpC than with OmpF porin.
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Materials and methods
A B C
Bacterial strains and growth conditions The bacterial strains used for this study were kindly supplied by Dr. P.H. Makela of the National Public Health Institute, Helsinki, Finland, and were derivatives of S. typhimurium LT2. Strains SH7457 and SH7454 produce OmpC and OmpF, and OmpD and OmpF porins, respectively; SH7455 produces OmpF; SH5014 produces all three porins (Dr. P.H. Makela, personal communication). The production of OmpF by SH7457 and SH7454 was repressed by growing these strains in medium containing 1.5% NaCl (Dr. H. Nikaido, personal communication). Strain SH5014 was grown in nutrient broth. The cultures were grown by aseptically transferring 10 mL of an overnight inoculum into 240 mL prewarmed medium contained in 1-L Erlenmeyer flasks. The flasks were shaken at 220 rpm in a gyrotory water-bath shaker at 37°C. Preparation and purification of proteins Exponentially growing cells were harvested and washed with cold 50 mM Tris-HC1 buffer, p H 7.7, and passed through a chilled French pressure cell (SLM/Amin Co.) twice at 16 000 psi (1 psi = 6.895 kPa) (Nikaido 1983). The extract was centrifuged at 1000 x g for 10 min to pellet the unbroken cells, and the supernatant was centrifuged at 100 000 x g for 1 h at 4°C in a Beckman model L8-70 M ultracentrifuge, using the Ti 70.1 rotor. The nonporin proteins were extracted with 2% sodium dodecyl sulfate (SDS) in 10 mM Tris-HC1, pH 7.7, followed by extraction of the porin proteins with NaCl buffer (1% SDS, 50 mM Tris-HC1, pH 7.7,0.4 M NaC1,5 mM EDTA, 0.05 % 2-mercaptoethanol, and 3 mM sodium azide) (Nikaido 1983). The NaCl extract was applied to a Sephacryl S-200 column (1.6 x 90 cm), eluted with NaCl buffer, precipitated with acetone, and suspended in 0.01 M Tris-HC1, p H 7.5. Protein concentration of the porin suspension was determined on the Gilford Response spectrophotometer equipped with a single cell holder at 280 nm, using the Bio-Rad protein standard. The monomer was prepared by heating the native trimer in boiling water for 10 min at 100°C (Fig. 1) (Bentley and Klebba 1988). Preparation of the outer membrane The cell pellet as obtained above was suspended in 10 mM Hepes buffer, p H 7.4. Each pellet was treated with 0.05 mg of DNAse and RNAse, French pressed at 14 000 psi twice, and centrifuged at 3000 x g for 5 min. The supernatant was centrifuged at 150 000 x g for 1 h at 4°C. The OM, separated and purified by sucrose density gradient centrifugation, was suspended in Dulbecco's phosphate-buffered saline (DPBS) (Gibco) and stored at 4°C until use (Nikaido 1983). Isolation of lipopolysaccharide Lipopolysaccharide of S. typhimurium LT2 was isolated according to the method of Galanos et al. (1969). Polyacrylamide gel electrophoresis The porin and OM preparations were solubilized with SDS mix (0.0625 M Tris, p H 6.8, 2% SDS, 5% 2-mercaptoethanol (BME), and 10% glycerol) and loaded directly for trimeric forms, boiled (5 min) for monomeric forms, and analyzed using separating gels containing 11.5% acrylamide and 0.2% SDS. Electrophoresis was carried out at 25 mA, increasing to 30 mA when the sample reached the separating gel and until the dye front reached the bottom. Coomassie Brilliant Blue was used to detect protein bands (Laemmli 1970). Anti-OmpD MAbs BALB/c mice were immunized with either the T or the M OmpD antigen, depending on the monoclonal antibody to be produced. The 0.05-mL volume of the antigen (100 pg) was inoculated on day 1, in complete Freund's adjuvant, subcutaneously into both foot pads. On days 6, 10, 14, 26, 32, and 42 the antigen was suspended in DPBS, and injections were given in alternating foot pads. Half the concentration of the antigen (50 pg/O.O5 mL) was
FIG. 1. Detection of trimeric (top arrowhead) and monomeric, after boiling the antigen for 5 min (bottom arrow), Omp by SDSPAGE. The proteins were stained with Coomassie Brilliant Blue. Marker proteins had molecular sizes of 92.5, 66.2, 45, 31, 21.5, and 14.4 kDa, as shown on left. used for the last two inoculations. The animal was sacrificed on day 45. Lymph-node cells were fused with P3-x63-Ag8.653, using polyethylene glycol (PEG-4000) (Accurate Chemical and Scientific Corp., New York), and the fusion products were diluted in 96-well plates containing hypoxanthine-aminopterin-thymidine medium (Goding 1983; Kearney et al. 1979). Hybridomas were screened by ELISA either with the T or the M Omp antigen. Those producing anti-porin MAbs were cloned twice by limiting dilution and injected intraperitoneally into pristane-primed BALB/c mice for ascitic tumors. Iso types Heavy- and light-chain isotypes were identified either by double diffusions of hybridoma culture supernatants against class-specific antisera (Miles Laboratories, Inc.) or by ELISA using clonotyping kit system I (Fisher Scientific, Orangeburg). EL ISA ELISA
was performed according t o the method described by Kenneth et al. (1980). The OmpC, OmpD, OmpF, or OM antigen trimer or monomer was suspended in a buffer (0.01 M ammonium acetate, 0.02 M ammonium carbonate, pH 8.2), 0.1-mL aliquots containing 50 pg of the above antigen were dispensed in polystyrene microtiter wells (Immunolon 11; Dynatech), and the plate was incubated overnight at 4°C. The wells were blocked with 0.1% bovine serum albumin (BSA) in borate-buffered saline (BS) (0.1 M boric acid, 0.025 M sodium borate, 0.075 M sodium chloride, 0.015 mM sodium azide, p H 8.2) for 1 h. Supernatant (0.1 mL) from hybridoma-containing wells or ascites was added to each well and incubated overnight in a humidified chamber. After the BS-BSA block, between each stage, the plate was washed with (BS) on a Titertek Microplate washer 120. The substrate, 0.1 mL of
CAN. J. MICROBIOL. VOL. 38, 1992
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A
B
C
D
E
F
G
H
I
TABLE1. Anti-Salmonella OmpD MAbs and their isotypes
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MAb
FIG. 2. Western immunoblots of anti-T MAbs against the denatured whole-cell lysate of S. typhimurium SH7454. After Western transfer, the nitrocellulose paper was cut into strips and incubated with various MAbs as follows: lane A, 3C6; lane B, 5D4; lane C, 8D4; lane D, 10D1; lane E, 14A2; lane F, 15A4; lane G, Fll-2. All the selected monoclonals were positive on Western blots and are indicated by an arrowhead at 34 000 Da. The last two strips were probed with ascites fluid from plasmacytoma cells, P3-x63-Ag8.653, and normal mouse serum, respectively.
Isotype
Trimeric 3C6 5D4 8D4 1OD 1 14A2 15A4 Fll-2 Monomeric C12 91D8 4.1G5 1B4 1A5 2All 3 1B9 NOTE: The light chain of all the MAbs was found to be of the k type.
FIG. 3. Western immunoblots of anti-M MAbs against the denatured whole-cell lysate of S. typhimurium SH7454 as described in Materials and methods. After Western transfer, the nitrocellulose paper was cut into strips and incubated with various MAbs as follows: lane A, C12; lane B, 91D8; lane C, 41G5; lane D, 1B4; lane E, 1A5; lane F, 2A11; and lane G, 3 1B9. Positive reaction is indicated at 34 000 Da (indicated by arrowhead). The last two strips were probed with ascites fluid from plasmacytoma cells, P3-x63Ag8.653, and normal mouse serum, respectively.
p-nitrophenylphosphate diluted 1:1000 in a buffer (0.5 1 mM MgCl,, 9.6% diethanolamine, pH 9.8), was added for an enzymatic color change in 15-30 min. The reaction was stopped by adding 3 M NaOH (50 pL), and the absorbance at 405 nm was read in the BioTek model EL 310 reader. Western blot analysis The antigens separated by PAGE were electrophoretically transferred onto a nitrocellulose (NC) paper (BA 85 0.45 pM; Schleicer and Schuell, Inc.) at 15 V for 21 h in a Hoefer model TE 52 transbolt cell (Bentley and Klebba 1988; Burnette 1981; Towbin et al. 1979). The NC paper was sliced into 5 mm wide strips on an Accutran strip cutter (Schleicher and Schuell) and incubated with MAbs (130 dilution) overnight. The immunodetection of OmpD and its cross-reaction with OmpC, OmpF, and OM was carried out by incubation of the strips in goat antimouse immunoglobin (Fisher Scientific) overnight. The strips were then incubated with alkaline phosphatase conjugate anti-goat IgG in rabbit for 90 min. The blots were developed with 5-bromo-4chloro-3-indolyl phosphate - nitro blue tetrazolium. The reaction was stopped after 15-20 min by washing the strips in distilled water. Anti-porin immunoblot reactions were evaluated by comparing these immunoblots with those using normal mouse serum and ascitic fluid from P3-x63-Ag8.653, the cell fusion partner, which is a nonsecretor of immunoglobulins (Kearney et al. 1979).
Results and discussion Hybridomas were observed in more than 60% of .the wells in all fusion experiments. They were cloned by limiting dilution following the Poisson distribution and until only a single clone in one-third of .the wells was evident. A total of 28 anti-T and 21 anti-M OmpD specific clones were observed and were found to be positive by ELISA with their respective antigen. Eight anti-T and four anti-M ascites that were anti OmpD porin specific also showed reactivity with LPS by ELISA and Western blots at a concentration of 10, 1, and 0.01 pg/mL. There is a possibility that the porins become associated with LPS during electrophoretic separation in Western blots since whole cell lysates were used, which may give a false-positive LPS reaction (Poxton et al. 1985). These antibodies were excluded from the present study. The remaining MAbs did not bind to LPS, as indicated by ELISA and Western blots. Twelve anti-T and 10 anti-M MAbs were positive on the ELISA but negative on Western blots. This may be attributed to a change in configuration of the epitope as a result of the SDS treatment prior to electrophoresis, which causes unfolding and denaturation. Alternatively some epitopes may have been buried too deep in the cell lysate used in the Western Blots, and these were not accessible to its homologous antibody. Two MAbs (T), F11-1 and F11-2, obtained from the same master well from the initial plating indicated the probable identification of a common epitope. Of these two MAbs, F11-2 was used in the present study. Seven anti-T and seven anti-M MAbs were subsequently judged to produce monospecific antibody on the basis of their specific affinity to pure antigen when assayed by ELISA and Western blots (Figs. 2 and 3). The T antibodies were observed to be IgG, except for antibody 15A4, which was an IgM. The monomeric antibodies belonged to isotype IgM, except for 31B9, which was IgG (Table 1). The ELISA on the T and M MAbs with the homologous antigen was highly positive as a result of the initial selection of the dilution for a strong positive titre of r 0.5. The same concentration was used for normalization of the conditions for all cross-reactivity experiments (Tables 2 and 3).
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TABLE2. Anti-Salmonella Omp T MAbs and their cross-reactivity with Salmonella porins in ELISA Cross-reacting porina
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OmpD
OmpC
OmpF
OM
TMAb
Antibody dilution
T
M
T
M
T
M
T
M
3C6 5D4 8D4 lODl 14A2 15A4 Fll-2
1:1000 1 :2000 1:1000 1 : 25 000 1:25000 1:1000 1:300
1.25 1.28 1.07 1.60 1.56 1.44 0.50
0.39 0.28 1.56 0.61 1.43 1.86 0.49
0.32 0.34 1.83 0.24 0.19 1.63 0.59
0.30 0.24 1.96 0.45 0.17 1.86 0.57
0.19 0.16 0.46 0.14 1.85 0.98 0.38
0.17 0.15 0.45 0.17 1.67 1.09 0.33
0.74 0.24 2.51 2.18 2.41 1.90 0.83
0.43 0.26 2.29 2.23 1.18 2.02 0.56
"Antibody reading of 0.5 or higher at A405in homologous reaction at the dilution shown in Table 2. 'b~ntibodiesreading 0.5 or higher were considered strongly cross-reactive, and their numbers are indicated outside the parentheses. The numbers inside the parentheses represent weakly positive antibodies, which produce reading of 0.3 to 0.49 in heterologous reaction. Values below 0.3 were considered negative.
TABLE3. Anti-Salmonella OmpD M MAbs and their cross-reactivity with Salmonella porins in ELISA
OmpD MMAb
Antibody dilution
T
M
OmpC T
M
OmpF
T
M
OM T
M
"See footnote a in Table 2. b ~ e footnote e b in Table 2.
The T MAbs 3C6, 5D4, and lODl showed less binding to the M than to the T form of OmpD antigen. This nonrecognition could be due to .the loss of symmetry or denaturation as a result of boiling the antigen. The trimeric MAbs 8D4 and 15A4 showed greater specificity for the monomer compared with the trimer. The T Omp antigen was initially subjected to SDS-PAGE to determine its molecular weight before inoculation of mice for the T MAbs. It is possible that owing to steric hindrance the large trimeric antibody molecule was unable to recognize the epitope on the trimeric antigen and produce a strong reaction. However, when the antigen was monomeric, the antigenic determinants were exposed to produce a strong reaction. It is also possible that anti-M antibodies were isolated as well as anti-T antibodies when trimers were the immunogens as a result of antigen processing in the mouse. This would require confirmation of specificity of the MAb on a nondenaturing gel with porin trimer as antigen. Cross-reaction of OmpD MAbs to OmpC antigen was greater than to OmpF antigen, with the exception of T MAb 14A2. Both the T and M MAbs 3C6, 8D4, 15A4, and F11-2 reacted with greater binding affinity to the
T and M OmpC antigen compared with the T and M OmpF antigens. The T MAb 5D4 showed greater binding to the T OmpC antigen, and the T MAb lODl showed greater binding to the M OmpC antigen. Only MAb 5D4 crossreacted with OmpC and OmpF antigens, whereas F l l - 2 antibody cross-reacted with OmpC antigen and 15A4 antibody cross-reacted with OmpF antigen on Western blot. Cross-reaction of monoclonals with OmpF and OmpC in ELISA suggests the possibility of some common epitopes in all three OM proteins. An earlier study (Tokunaga et al. 1979) on the OM proteins of S. typhimurium indicated that the three proteins were coded by separate genes. Structural homologies between the porins as well as reproducible differences, showing lack of homology, were expected on the basis of amino acid analysis. Tryptic fingerprints showed that a small fraction of the primary structure of the three porins shared a common sequence. Alanine and phenylalanine are observed to be common by the amino terminal and the carboxyl terminal analysis, respectively. Isoelectric points have suggested some resemblance as well as reproducible differences. Ultraviolet absorption spectra give
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TABLE 4. Western blots of anti-Salmonella OmpD MAbs and their cross-reactions with OmpC, OmpF, and OM
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MAb Trimeric 3C6 5D4 8D4 1OD1 14A2 15A4 Fll-2 Monomeric C12 91D8 4 1G5 1B4 1A5 2All 3IB9
SH7454 SH7457 SH7455 SH5014 ( O ~ P D ) ( O ~ P C ) ( O ~ P F ) (OM)
+ + + + + + + + + + +. + + +
-
+ -
+ + + + -
-
+
+ +
-
-
-
-
+ -
-
-
+
-
-
-
+
-
+ -
essentially similar profiles. The common sequences show signs that the Omp may have evolved by domain shuffling from a single ancestral gene that duplicated in the course of evolution to give rise to other genes to produce a related protein with new functions. Using SDS-PAGE, it is possible that some of the antigens of SH5014 lost their native conformation as a result of denaturation by SDS, BME, or heat and thus were not detected on immunoblots, whereas in the ELISA,the antigens retained their native conformation. Also, owing to its sensitivity, it is possible that ELISA was able to detect even weak antigen presence, except in the case of T MAb 5D4, where both the T and M OM were negative, and M MAb 1A5, where only M OM was positive (Klebba et al. 1990). Monoclonal antibody 5D4 was able to identify a common epitope in all the three Omp porins on Western blots (Table 4). However, all ELISA titres to M Omp were negative, but reaction with T OmpD was strongly positive, with T OmpC was weak, and with T OmpF was negative. It is difficult to determine the causes for a positive immunoblot and negative ELISA with antibodies against 5D4 and when similar results were observed with 1A5 against M OmpF. However, the positive reaction may be due to the difference in the dilution of the antibody for Western blots, 1:50 as compared with the dilutions 1:2000 and 1:500 for MAbs 5D4 and 1A5, respectively, for ELISA. Additional methodologies such as immunoprecipitation and peptide mapping may also be helpful. Cross-reaction of anti-OmpD M antibodies was higher with T and M OmpC antigen than with T and M OmpF antigen. The M OmpD antibodies were observed to bind more strongly with their homologous M OmpD antigen, except for antibody 31B9, indicating the presence of large number of common exposed epitopes. The binding of the M OmpD antibodies was also stronger with OmpC, OmpF, and OM M than T antigens, except for C12, where a lower binding was seen for unknown reasons. Binding of the monomeric antibodies was stronger with OmpC than with OmpF antigen, indicating higher homology and greater structural similarity of OmpD to OmpC than OmpD t o
OmpF. Only C 12,41G5, and 1B4 M antibodies cross-reacted with OmpC antigen on Western blot. M antibody 1A5 crossreacted with OmpF but escaped detection on ELISA. Reactions with OM on ELISA were as expected, as it carries all the three OM proteins. The high probability of strong homologous and heterologous cross-reaction between antiOmpD M MAbs and the respective trimeric and monomeric OM proteins suggests the Omp receptors are covered by reactive epitopes. OmpC shares a larger percentage of common epitopes with OmpD than OmpF with OmpD. A high percentage of common epitopes with structural similarity suggests a similar evolutionary origin. Our conclusion that OmpD is more closely related to OmpC than to OmpF is based solely on the overall performance of the MAbs and the total number of positive cross-reactions. It is unlikely that all the MAbs are similar and react with the same antigenic determinants, as they were produced from different experiments and belong to different antibody types and subtypes. In conclusion, we have raised an extensive panel of MAbs against OmpD T and M forms of S. typhimurium and studied their cross-reactions with ELISA and Western blots. The evidence supports the conclusion that the structural epitopes of OmpD, OmpC, and OmpF porins recognized by these MAbs are broadly overlapping but not identical. The OmpC appears to be more closely related to OmpD than OmpF antigenically. The feasibility of these monoclonals for epidemiological typing of serotype strains of S. typhimurium along with monoclonals against OmpC and OmpF (Pai et al. 1992) also needs to be investigated.
Acknowledgements The authors thank Dr. Richard Curtis Bird, Professor Gerald Wilt, and Dr. Victor Panangala for critical reading of the manuscript. The work was supported by Public Health Service grants GM 08167 and GM 08219. Bentley, A.T., and Klebba, P.E. 1988. Effect of lipopolysaccharide structure on reactivity of antiporin monoclonal antibodies with the bacterial cell surface J . Bacteriol. 170: 1063-1068. Benz, R. 1988. Structure and function of porins from gram-negative bacteria. Annu. Rev. Microbiol. 42: 359-393. Blasband, A.J., Marcotte, W.R., Jr., and Schnaitman, C.A. 1986. Structure of the lc and nmpC outer membrane porin protein genes of lambdoid bacteriophage. J. Biol. Chem. 261: 12 723 - 12 732. Burnette, W.N. 1981. Western blotting: electrophoretic transfer of proteins from sodium dodecyl sulfate - polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112: 195-203. Chai, T. J., and Foulds, J. 1979. Isolation and partial characterization of protein E, a major protein found in certain Escherichia coli K-12 mutant strains: relationship to other membrane proteins. J. Bacteriol. 139: 418-423. Gabay, J., Benson, S., and Schwartz, M. 1983. Genetic mapping of antigenic determinants on a membrane protein. J. Biol. Chem. 258: 2410-2414. Galanos, C., Luderitz, O., and Westphal, 0 . 1969. A new method for the extraction of R lipopolysaccharides. Eur. J . Biochem. 9: 245-249. Garavito, R.M., and Rosenbusch, J .P. 1986. Isolation and crystallization of bacterial porin. Methods Enzymol. 125: 309-328.
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Goding, J . W. 1983. Monoclonal antibodies: principles and practice. Academic Press Inc., London. Hancock, R.E.W. 1987. Role of porins in outer membrane permeability. J . Bacteriol. 169: 929-933. Hofstra, H., and Dankert, J . 1980. Major outer membrane proteins: common antigens in Enterobacteriaceae species. J. Gen. Microbiol. 117: 437-447. Hofstra, H., and Dankert, J . 1981. Porin from the outer membrane of Escherichia coli: immunological characterization of native and heat-dissociated forms. J. Gen. Microbiol. 125:
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/15/14 For personal use only.
285-292.
Inouye, M. 1979. What is the outer membrane? In Bacterial outer membranes. Edited by M. Inouye. John Wiley & Sons, Inc., New York. pp. 1-12. Ishii, J., and Nakae, T. 1980. Subunit constituent of the porin trimers that form the permeability channels in the outer membrane of Salmonella typhimurium. J. Bacteriol. 142: 27-3 1. Kearney, J.F., Radbruch, A., Liesgang, B., and Rajewsky, K. 1979. A new mouse myeloma line that has lost immunoglobulin expression but permits construction of antibody-secreting hybrid cell lines. J. Immunol. 123: 1548-1550. Kenneth, R.H., McKearn, T. J., and Bechtol, K.B. (Editors).1980. Monoclonal antibodies: a new dimension in biological analysis. Plenum Press, New York. p. 423. Klebba, P.E., Benson, S.A., Bala, S., Abdullah, T., Reid, J., Singh, S.P., and Nikaido, H. 1990. Determinants of ompF porin antigenicity and structure. J . Biol. Chem. 265: 6800-6810. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227: 680-685. Lee, D.R., and Schnaitman, C.A. 1980. Comparison of outer membrane porin proteins produced by Escherichia coli and Salmonella typhimurium. J . Bacteriol. 142: 1019- 1022. Lugtenberg, B., and Van Alphen, L. 1983. Molecular architecture and functioning of the outer membrane of Eschericha coli and other gram-negative bacteria. Biochim. Biophys. Acta, 737: 51-1 15.
Mizuno, T., Chou, M.Y., and Inouye, M. 1983. A comparative study on the genes for three porins of the Escherichia coli outer membrane. J. Biol. Chem. 258: 6932-6940. Nakae, T. 1986. Outer-membrane permeability in bacteria. CRC Crit. Rev. Microbiol. 13: 1-62. Nikaido, H. 1983. Proteins forming large channels from bacterial and mitochondria1 outer membranes: porins and phage lambda receptor protein. Methods Enzymol. 97: 85-100. Nikaido, H., and Vaara, M. 1985. Molecular basis of bacterial outer membrane permeability. Microbiol. Rev. 49: 1-32.
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Nurminen, M., Lounatmaa, K., Sarvas, M., Makela, P.H., and Nakae, T. 1976. Bacteriophage-resistant mutants of Salmonella typhimurium deficient in two major outer membrane proteins. J . Bacteriol. 127: 941-955. Overbeeke, N., van Scharrenburg, G., and Lugtenberg, B. 1980. Antigenic relationships between pore proteins of Escherichia coli K12. Eur. J . Biochem. 110: 247-254. Overbeeke, N., Bergmans, F., van Mansfield, F., and Lugtenberg, B. 1983. Complete nucleotide sequence of phoE, the structural gene for the phosphate limitation inducible outer membrane pore protein of Escherichia coli K12. J. Mol. Biol. 163: 513-532. Pai, S.R., Upshaw, Y., and Singh, S.P. 1992. (Alabama State University.) Unpublished data. Poole, K., and Hancock, R.E. W. 1986. Phosphate-starvation induced outer membrane proteins of members of the families Enterobacteriaceae and Pseudomonodaceae: demonstration of immunological cross reactivity with an antiserum specific for porin protein P of Pseudomonas aeruginosa. J . Bacteriol. 165: 987-993.
Poxton, I.R., Bell, G.T., and Barclay, G.R. 1985. The association of SDS-polyacrylamide gels of lipopolysaccharide and outer membrane proteins of Pseudomonas aeruginosa as revealed by monoclonal antibodies and western blotting. FEMS Microbiol. Lett. 27: 247-251. Rosenbusch, J.P. 1974. Characterization of the major envelope protein from Escherichia coli. Regular arrangement on the peptidoglycan and unusual dodecyl sulfate binding. J . Biol. Chem. 249: 8019-8029. Rosenbusch, J.P. 1987. Three-dimensional structure of membrane proteins. In Bacterial outer membranes as model systems. Edited by M. Inouye. John Wiley & Sons, Inc., New York. pp. 141-162. Schindler, M, and Rosenbusch, J.P. 1984. Structural transitions of porin, a transmembrane protein. FEBS Lett. 173: 85-89. Tokunaga, H., Tokunaga, M., and Nakae, T. 1979. Characterization of porins from the outer membrane of Salmonella typhimurium. 1. Chemical analysis. Eur. J. Biochem. 95: 434-439.
Towbin, M., Staehelin, T., and Gordon, J . 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76: 4350-4354. Van der Ley, P., Amesz, H., Tommassen, J., and Lugtenberg, B. 1985. Monoclonal antibodies directed against the cell surface exposed part of PhoE pore protein of the Escherichia coli K-12 outer membrane. Eur. J . Biochem. 147: 401-407.
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Biomedical Research and Training Programs, Alabama State University, Montgomery, AL 36101, U.S.A. Received July 16, 1991 Revision received April 24, 1992 Accepted April 29, 1992 PAI, S. R., UPSHAW,Y., and SINGH,S. P. 1992. Characterization of monoclonal antibodies to the outer membrane protein (OmpD) of Salmonella typhimurium. Can. J. Microbiol. 38: 1102-1 107. A panel of monoclonal antibodies, seven against the trimeric and seven against the monomeric forms to outer membrane protein D (OmpD) of Salmonella typhimurium were produced. The specificities of these monoclonal antibodies for the porin proteins of S. typhimurium and their cross-reactions with Salmonella porins OmpC and OmpF were determined by Western immunoblotting and enzyme-linked immunosorbent assay. We observed that OmpD shared more epitopes and had greater structural similarity with OmpC than with OmpF. Key words: Salmonella typhimurium, outer membrane protein, monoclonal antibody, trimeric, monomeric. PAI, S. R., UPSHAW,Y., et SINGH,S. P. 1992. Characterization of monoclonal antibodies to the outer membrane protein (OmpD) of Salmonella typhimurium. Can. J. Microbiol. 38 : 1102-1 107. Nous avons produit une serie d'anticorps monoclonaux contre la protkine OmpD de Salmonella typhimurium, soit sept dirigks contre la forme trimkre et sept autres contre la forme monomkre. La specificite de ces anticorps monoclonaux envers les proteines des porines de S. typhimurium et leurs reactions croiskes avec les proteines OmpC et OmpF de Salmonella ont ete evaluees par immunobuvardage de type Western et par immunoessai enzymatique. Nous avons constate que 1'OmpD partageait plus d'epitopes et avait une plus grande parente de structure avec 1'OmpC qu'avec 1'OmpF. Mots clks : Salmonella typhimurium, proteine de la membrane externe, anticorps monoclonal, trimkre, monomkre. [Traduit par la redaction]
Introduction The outer membrane (OM) of Salmonella typhimurium as well as other gram-negative bacteria contains pore-forming proteins called porins (Benz 1988; Garavito and Rosenbusch 1986; Ishii and Nakae 1980; Rosenbusch 1974; Schindler and Rosenbusch 1984). They are present as trimers and form trans outer membrane water-filled channels, which facilitate the transport of small hydrophilic molecules (Hancock 1987). The B-sheet porins span .the .thickness of the outer membrane and produce tight complexes with the underlying peptidoglycan layer and with lipopolysaccharides (LPSs) (Inouye 1979). They are oriented roughly perpendicular to the membrane. They protrude a little on both sides from the plane of the membrane, with three separate openings at the surface, which coalesce into a single channel near the center of the membrane (Rosenbusch 1987). Under normal growth conditions, S. typhimurium LT2 expresses three porins, OmpD (34 000 Da), OmpF (35 000 Da), and OmpC (36 000 Da), whereas Escherichia coli K12 synthesizes two such proteins, OmpC and OmpF (Ishii and Nakae 1980; Lee and Schnaitman 1980; Nurminen et al. 1976; Tokunaga et al. 1979). Other porins such as LamB, PhoE, NmpC, LC, OmpG, and Tx are expressed under certain physiological conditions (Benz 1988; Lugtenberg and Van Alphen 1983; Nakae 1986; Nikaido and Vaara 1985). The OmpF, OmpC, PhoE, NmpC, and LC proteins share significant sequence homologies at both the nucleotide and amino acid levels (Blasband et al. 1986; Mizuno et al. 1983; Overbeeke et al. 1983). The immunological properties of ' ~ u t h o rto whom all correspondence should be sent at the following address: Department of Pathobiology, Auburn University, Auburn, AL 36849-5517, U.S.A. Printed in Canada
/
lmprime au Canada
E. coli porin OmpF, OmpC, PhoE, and LamB proteins have been widely studied. The purified porins are immunogenic in mice and rabbits (Hofstra and Dankert 1980, 1981; Overbeeke et al. 1980) as either native trimeric (T) porin or denatured monomeric (M) porin. Porin monomers derived from a number of Enterobacteriaceae, including the OmpF, OmpC, and PhoE porin monomers of E. coli and porin protein P of Pseudomonas aeruginosa, have been shown to cross- react immunologically (Chai and Foulds 1979; Hofstra and Dankert 1980, 1981; Overbeeke et al. 1980; Poole and Hancock 1986). Monoclonal antibodies (MAbs) offer several distinct advantages over polyclonal sera, including defined specificity, reproducibility, and availability. Monoclonal antibodies raised against E. coli LamB (Gabay et al. 1983), PhoE (Van der Ley et al. 1985), and OmpF (Bentley and Klebba 1988; Klebba et al. 1990) have been used to study the structure and topology of porin and their variability among related species. Recently, we tested a library of 43 anti-E. coli B/r OmpF MAbs by Western immunoblots against 12 different enteric and 4 nonenteric gram-negative species, as well as the E. coli porins NmpC, OmpC, and PhoE. The results showed considerable immunological divergence between the pore proteins of these organisms (Klebba et al. 1990). To our knowledge this is the first report of the production of a panel of MAbs against the T and M forms of OmpD protein of S. typhimurium. We describe the isotypes of these MAbs and their interactions with the T and M forms of OmpD, OmpC, and OmpF porins, and the purified OM fragments from S. typhimurium. Our results indicate OmpD to have greater immunological similarity with OmpC than with OmpF porin.
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Materials and methods
A B C
Bacterial strains and growth conditions The bacterial strains used for this study were kindly supplied by Dr. P.H. Makela of the National Public Health Institute, Helsinki, Finland, and were derivatives of S. typhimurium LT2. Strains SH7457 and SH7454 produce OmpC and OmpF, and OmpD and OmpF porins, respectively; SH7455 produces OmpF; SH5014 produces all three porins (Dr. P.H. Makela, personal communication). The production of OmpF by SH7457 and SH7454 was repressed by growing these strains in medium containing 1.5% NaCl (Dr. H. Nikaido, personal communication). Strain SH5014 was grown in nutrient broth. The cultures were grown by aseptically transferring 10 mL of an overnight inoculum into 240 mL prewarmed medium contained in 1-L Erlenmeyer flasks. The flasks were shaken at 220 rpm in a gyrotory water-bath shaker at 37°C. Preparation and purification of proteins Exponentially growing cells were harvested and washed with cold 50 mM Tris-HC1 buffer, p H 7.7, and passed through a chilled French pressure cell (SLM/Amin Co.) twice at 16 000 psi (1 psi = 6.895 kPa) (Nikaido 1983). The extract was centrifuged at 1000 x g for 10 min to pellet the unbroken cells, and the supernatant was centrifuged at 100 000 x g for 1 h at 4°C in a Beckman model L8-70 M ultracentrifuge, using the Ti 70.1 rotor. The nonporin proteins were extracted with 2% sodium dodecyl sulfate (SDS) in 10 mM Tris-HC1, pH 7.7, followed by extraction of the porin proteins with NaCl buffer (1% SDS, 50 mM Tris-HC1, pH 7.7,0.4 M NaC1,5 mM EDTA, 0.05 % 2-mercaptoethanol, and 3 mM sodium azide) (Nikaido 1983). The NaCl extract was applied to a Sephacryl S-200 column (1.6 x 90 cm), eluted with NaCl buffer, precipitated with acetone, and suspended in 0.01 M Tris-HC1, p H 7.5. Protein concentration of the porin suspension was determined on the Gilford Response spectrophotometer equipped with a single cell holder at 280 nm, using the Bio-Rad protein standard. The monomer was prepared by heating the native trimer in boiling water for 10 min at 100°C (Fig. 1) (Bentley and Klebba 1988). Preparation of the outer membrane The cell pellet as obtained above was suspended in 10 mM Hepes buffer, p H 7.4. Each pellet was treated with 0.05 mg of DNAse and RNAse, French pressed at 14 000 psi twice, and centrifuged at 3000 x g for 5 min. The supernatant was centrifuged at 150 000 x g for 1 h at 4°C. The OM, separated and purified by sucrose density gradient centrifugation, was suspended in Dulbecco's phosphate-buffered saline (DPBS) (Gibco) and stored at 4°C until use (Nikaido 1983). Isolation of lipopolysaccharide Lipopolysaccharide of S. typhimurium LT2 was isolated according to the method of Galanos et al. (1969). Polyacrylamide gel electrophoresis The porin and OM preparations were solubilized with SDS mix (0.0625 M Tris, p H 6.8, 2% SDS, 5% 2-mercaptoethanol (BME), and 10% glycerol) and loaded directly for trimeric forms, boiled (5 min) for monomeric forms, and analyzed using separating gels containing 11.5% acrylamide and 0.2% SDS. Electrophoresis was carried out at 25 mA, increasing to 30 mA when the sample reached the separating gel and until the dye front reached the bottom. Coomassie Brilliant Blue was used to detect protein bands (Laemmli 1970). Anti-OmpD MAbs BALB/c mice were immunized with either the T or the M OmpD antigen, depending on the monoclonal antibody to be produced. The 0.05-mL volume of the antigen (100 pg) was inoculated on day 1, in complete Freund's adjuvant, subcutaneously into both foot pads. On days 6, 10, 14, 26, 32, and 42 the antigen was suspended in DPBS, and injections were given in alternating foot pads. Half the concentration of the antigen (50 pg/O.O5 mL) was
FIG. 1. Detection of trimeric (top arrowhead) and monomeric, after boiling the antigen for 5 min (bottom arrow), Omp by SDSPAGE. The proteins were stained with Coomassie Brilliant Blue. Marker proteins had molecular sizes of 92.5, 66.2, 45, 31, 21.5, and 14.4 kDa, as shown on left. used for the last two inoculations. The animal was sacrificed on day 45. Lymph-node cells were fused with P3-x63-Ag8.653, using polyethylene glycol (PEG-4000) (Accurate Chemical and Scientific Corp., New York), and the fusion products were diluted in 96-well plates containing hypoxanthine-aminopterin-thymidine medium (Goding 1983; Kearney et al. 1979). Hybridomas were screened by ELISA either with the T or the M Omp antigen. Those producing anti-porin MAbs were cloned twice by limiting dilution and injected intraperitoneally into pristane-primed BALB/c mice for ascitic tumors. Iso types Heavy- and light-chain isotypes were identified either by double diffusions of hybridoma culture supernatants against class-specific antisera (Miles Laboratories, Inc.) or by ELISA using clonotyping kit system I (Fisher Scientific, Orangeburg). EL ISA ELISA
was performed according t o the method described by Kenneth et al. (1980). The OmpC, OmpD, OmpF, or OM antigen trimer or monomer was suspended in a buffer (0.01 M ammonium acetate, 0.02 M ammonium carbonate, pH 8.2), 0.1-mL aliquots containing 50 pg of the above antigen were dispensed in polystyrene microtiter wells (Immunolon 11; Dynatech), and the plate was incubated overnight at 4°C. The wells were blocked with 0.1% bovine serum albumin (BSA) in borate-buffered saline (BS) (0.1 M boric acid, 0.025 M sodium borate, 0.075 M sodium chloride, 0.015 mM sodium azide, p H 8.2) for 1 h. Supernatant (0.1 mL) from hybridoma-containing wells or ascites was added to each well and incubated overnight in a humidified chamber. After the BS-BSA block, between each stage, the plate was washed with (BS) on a Titertek Microplate washer 120. The substrate, 0.1 mL of
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A
B
C
D
E
F
G
H
I
TABLE1. Anti-Salmonella OmpD MAbs and their isotypes
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MAb
FIG. 2. Western immunoblots of anti-T MAbs against the denatured whole-cell lysate of S. typhimurium SH7454. After Western transfer, the nitrocellulose paper was cut into strips and incubated with various MAbs as follows: lane A, 3C6; lane B, 5D4; lane C, 8D4; lane D, 10D1; lane E, 14A2; lane F, 15A4; lane G, Fll-2. All the selected monoclonals were positive on Western blots and are indicated by an arrowhead at 34 000 Da. The last two strips were probed with ascites fluid from plasmacytoma cells, P3-x63-Ag8.653, and normal mouse serum, respectively.
Isotype
Trimeric 3C6 5D4 8D4 1OD 1 14A2 15A4 Fll-2 Monomeric C12 91D8 4.1G5 1B4 1A5 2All 3 1B9 NOTE: The light chain of all the MAbs was found to be of the k type.
FIG. 3. Western immunoblots of anti-M MAbs against the denatured whole-cell lysate of S. typhimurium SH7454 as described in Materials and methods. After Western transfer, the nitrocellulose paper was cut into strips and incubated with various MAbs as follows: lane A, C12; lane B, 91D8; lane C, 41G5; lane D, 1B4; lane E, 1A5; lane F, 2A11; and lane G, 3 1B9. Positive reaction is indicated at 34 000 Da (indicated by arrowhead). The last two strips were probed with ascites fluid from plasmacytoma cells, P3-x63Ag8.653, and normal mouse serum, respectively.
p-nitrophenylphosphate diluted 1:1000 in a buffer (0.5 1 mM MgCl,, 9.6% diethanolamine, pH 9.8), was added for an enzymatic color change in 15-30 min. The reaction was stopped by adding 3 M NaOH (50 pL), and the absorbance at 405 nm was read in the BioTek model EL 310 reader. Western blot analysis The antigens separated by PAGE were electrophoretically transferred onto a nitrocellulose (NC) paper (BA 85 0.45 pM; Schleicer and Schuell, Inc.) at 15 V for 21 h in a Hoefer model TE 52 transbolt cell (Bentley and Klebba 1988; Burnette 1981; Towbin et al. 1979). The NC paper was sliced into 5 mm wide strips on an Accutran strip cutter (Schleicher and Schuell) and incubated with MAbs (130 dilution) overnight. The immunodetection of OmpD and its cross-reaction with OmpC, OmpF, and OM was carried out by incubation of the strips in goat antimouse immunoglobin (Fisher Scientific) overnight. The strips were then incubated with alkaline phosphatase conjugate anti-goat IgG in rabbit for 90 min. The blots were developed with 5-bromo-4chloro-3-indolyl phosphate - nitro blue tetrazolium. The reaction was stopped after 15-20 min by washing the strips in distilled water. Anti-porin immunoblot reactions were evaluated by comparing these immunoblots with those using normal mouse serum and ascitic fluid from P3-x63-Ag8.653, the cell fusion partner, which is a nonsecretor of immunoglobulins (Kearney et al. 1979).
Results and discussion Hybridomas were observed in more than 60% of .the wells in all fusion experiments. They were cloned by limiting dilution following the Poisson distribution and until only a single clone in one-third of .the wells was evident. A total of 28 anti-T and 21 anti-M OmpD specific clones were observed and were found to be positive by ELISA with their respective antigen. Eight anti-T and four anti-M ascites that were anti OmpD porin specific also showed reactivity with LPS by ELISA and Western blots at a concentration of 10, 1, and 0.01 pg/mL. There is a possibility that the porins become associated with LPS during electrophoretic separation in Western blots since whole cell lysates were used, which may give a false-positive LPS reaction (Poxton et al. 1985). These antibodies were excluded from the present study. The remaining MAbs did not bind to LPS, as indicated by ELISA and Western blots. Twelve anti-T and 10 anti-M MAbs were positive on the ELISA but negative on Western blots. This may be attributed to a change in configuration of the epitope as a result of the SDS treatment prior to electrophoresis, which causes unfolding and denaturation. Alternatively some epitopes may have been buried too deep in the cell lysate used in the Western Blots, and these were not accessible to its homologous antibody. Two MAbs (T), F11-1 and F11-2, obtained from the same master well from the initial plating indicated the probable identification of a common epitope. Of these two MAbs, F11-2 was used in the present study. Seven anti-T and seven anti-M MAbs were subsequently judged to produce monospecific antibody on the basis of their specific affinity to pure antigen when assayed by ELISA and Western blots (Figs. 2 and 3). The T antibodies were observed to be IgG, except for antibody 15A4, which was an IgM. The monomeric antibodies belonged to isotype IgM, except for 31B9, which was IgG (Table 1). The ELISA on the T and M MAbs with the homologous antigen was highly positive as a result of the initial selection of the dilution for a strong positive titre of r 0.5. The same concentration was used for normalization of the conditions for all cross-reactivity experiments (Tables 2 and 3).
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TABLE2. Anti-Salmonella Omp T MAbs and their cross-reactivity with Salmonella porins in ELISA Cross-reacting porina
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OmpD
OmpC
OmpF
OM
TMAb
Antibody dilution
T
M
T
M
T
M
T
M
3C6 5D4 8D4 lODl 14A2 15A4 Fll-2
1:1000 1 :2000 1:1000 1 : 25 000 1:25000 1:1000 1:300
1.25 1.28 1.07 1.60 1.56 1.44 0.50
0.39 0.28 1.56 0.61 1.43 1.86 0.49
0.32 0.34 1.83 0.24 0.19 1.63 0.59
0.30 0.24 1.96 0.45 0.17 1.86 0.57
0.19 0.16 0.46 0.14 1.85 0.98 0.38
0.17 0.15 0.45 0.17 1.67 1.09 0.33
0.74 0.24 2.51 2.18 2.41 1.90 0.83
0.43 0.26 2.29 2.23 1.18 2.02 0.56
"Antibody reading of 0.5 or higher at A405in homologous reaction at the dilution shown in Table 2. 'b~ntibodiesreading 0.5 or higher were considered strongly cross-reactive, and their numbers are indicated outside the parentheses. The numbers inside the parentheses represent weakly positive antibodies, which produce reading of 0.3 to 0.49 in heterologous reaction. Values below 0.3 were considered negative.
TABLE3. Anti-Salmonella OmpD M MAbs and their cross-reactivity with Salmonella porins in ELISA
OmpD MMAb
Antibody dilution
T
M
OmpC T
M
OmpF
T
M
OM T
M
"See footnote a in Table 2. b ~ e footnote e b in Table 2.
The T MAbs 3C6, 5D4, and lODl showed less binding to the M than to the T form of OmpD antigen. This nonrecognition could be due to .the loss of symmetry or denaturation as a result of boiling the antigen. The trimeric MAbs 8D4 and 15A4 showed greater specificity for the monomer compared with the trimer. The T Omp antigen was initially subjected to SDS-PAGE to determine its molecular weight before inoculation of mice for the T MAbs. It is possible that owing to steric hindrance the large trimeric antibody molecule was unable to recognize the epitope on the trimeric antigen and produce a strong reaction. However, when the antigen was monomeric, the antigenic determinants were exposed to produce a strong reaction. It is also possible that anti-M antibodies were isolated as well as anti-T antibodies when trimers were the immunogens as a result of antigen processing in the mouse. This would require confirmation of specificity of the MAb on a nondenaturing gel with porin trimer as antigen. Cross-reaction of OmpD MAbs to OmpC antigen was greater than to OmpF antigen, with the exception of T MAb 14A2. Both the T and M MAbs 3C6, 8D4, 15A4, and F11-2 reacted with greater binding affinity to the
T and M OmpC antigen compared with the T and M OmpF antigens. The T MAb 5D4 showed greater binding to the T OmpC antigen, and the T MAb lODl showed greater binding to the M OmpC antigen. Only MAb 5D4 crossreacted with OmpC and OmpF antigens, whereas F l l - 2 antibody cross-reacted with OmpC antigen and 15A4 antibody cross-reacted with OmpF antigen on Western blot. Cross-reaction of monoclonals with OmpF and OmpC in ELISA suggests the possibility of some common epitopes in all three OM proteins. An earlier study (Tokunaga et al. 1979) on the OM proteins of S. typhimurium indicated that the three proteins were coded by separate genes. Structural homologies between the porins as well as reproducible differences, showing lack of homology, were expected on the basis of amino acid analysis. Tryptic fingerprints showed that a small fraction of the primary structure of the three porins shared a common sequence. Alanine and phenylalanine are observed to be common by the amino terminal and the carboxyl terminal analysis, respectively. Isoelectric points have suggested some resemblance as well as reproducible differences. Ultraviolet absorption spectra give
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TABLE 4. Western blots of anti-Salmonella OmpD MAbs and their cross-reactions with OmpC, OmpF, and OM
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MAb Trimeric 3C6 5D4 8D4 1OD1 14A2 15A4 Fll-2 Monomeric C12 91D8 4 1G5 1B4 1A5 2All 3IB9
SH7454 SH7457 SH7455 SH5014 ( O ~ P D ) ( O ~ P C ) ( O ~ P F ) (OM)
+ + + + + + + + + + +. + + +
-
+ -
+ + + + -
-
+
+ +
-
-
-
-
+ -
-
-
+
-
-
-
+
-
+ -
essentially similar profiles. The common sequences show signs that the Omp may have evolved by domain shuffling from a single ancestral gene that duplicated in the course of evolution to give rise to other genes to produce a related protein with new functions. Using SDS-PAGE, it is possible that some of the antigens of SH5014 lost their native conformation as a result of denaturation by SDS, BME, or heat and thus were not detected on immunoblots, whereas in the ELISA,the antigens retained their native conformation. Also, owing to its sensitivity, it is possible that ELISA was able to detect even weak antigen presence, except in the case of T MAb 5D4, where both the T and M OM were negative, and M MAb 1A5, where only M OM was positive (Klebba et al. 1990). Monoclonal antibody 5D4 was able to identify a common epitope in all the three Omp porins on Western blots (Table 4). However, all ELISA titres to M Omp were negative, but reaction with T OmpD was strongly positive, with T OmpC was weak, and with T OmpF was negative. It is difficult to determine the causes for a positive immunoblot and negative ELISA with antibodies against 5D4 and when similar results were observed with 1A5 against M OmpF. However, the positive reaction may be due to the difference in the dilution of the antibody for Western blots, 1:50 as compared with the dilutions 1:2000 and 1:500 for MAbs 5D4 and 1A5, respectively, for ELISA. Additional methodologies such as immunoprecipitation and peptide mapping may also be helpful. Cross-reaction of anti-OmpD M antibodies was higher with T and M OmpC antigen than with T and M OmpF antigen. The M OmpD antibodies were observed to bind more strongly with their homologous M OmpD antigen, except for antibody 31B9, indicating the presence of large number of common exposed epitopes. The binding of the M OmpD antibodies was also stronger with OmpC, OmpF, and OM M than T antigens, except for C12, where a lower binding was seen for unknown reasons. Binding of the monomeric antibodies was stronger with OmpC than with OmpF antigen, indicating higher homology and greater structural similarity of OmpD to OmpC than OmpD t o
OmpF. Only C 12,41G5, and 1B4 M antibodies cross-reacted with OmpC antigen on Western blot. M antibody 1A5 crossreacted with OmpF but escaped detection on ELISA. Reactions with OM on ELISA were as expected, as it carries all the three OM proteins. The high probability of strong homologous and heterologous cross-reaction between antiOmpD M MAbs and the respective trimeric and monomeric OM proteins suggests the Omp receptors are covered by reactive epitopes. OmpC shares a larger percentage of common epitopes with OmpD than OmpF with OmpD. A high percentage of common epitopes with structural similarity suggests a similar evolutionary origin. Our conclusion that OmpD is more closely related to OmpC than to OmpF is based solely on the overall performance of the MAbs and the total number of positive cross-reactions. It is unlikely that all the MAbs are similar and react with the same antigenic determinants, as they were produced from different experiments and belong to different antibody types and subtypes. In conclusion, we have raised an extensive panel of MAbs against OmpD T and M forms of S. typhimurium and studied their cross-reactions with ELISA and Western blots. The evidence supports the conclusion that the structural epitopes of OmpD, OmpC, and OmpF porins recognized by these MAbs are broadly overlapping but not identical. The OmpC appears to be more closely related to OmpD than OmpF antigenically. The feasibility of these monoclonals for epidemiological typing of serotype strains of S. typhimurium along with monoclonals against OmpC and OmpF (Pai et al. 1992) also needs to be investigated.
Acknowledgements The authors thank Dr. Richard Curtis Bird, Professor Gerald Wilt, and Dr. Victor Panangala for critical reading of the manuscript. The work was supported by Public Health Service grants GM 08167 and GM 08219. Bentley, A.T., and Klebba, P.E. 1988. Effect of lipopolysaccharide structure on reactivity of antiporin monoclonal antibodies with the bacterial cell surface J . Bacteriol. 170: 1063-1068. Benz, R. 1988. Structure and function of porins from gram-negative bacteria. Annu. Rev. Microbiol. 42: 359-393. Blasband, A.J., Marcotte, W.R., Jr., and Schnaitman, C.A. 1986. Structure of the lc and nmpC outer membrane porin protein genes of lambdoid bacteriophage. J. Biol. Chem. 261: 12 723 - 12 732. Burnette, W.N. 1981. Western blotting: electrophoretic transfer of proteins from sodium dodecyl sulfate - polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112: 195-203. Chai, T. J., and Foulds, J. 1979. Isolation and partial characterization of protein E, a major protein found in certain Escherichia coli K-12 mutant strains: relationship to other membrane proteins. J. Bacteriol. 139: 418-423. Gabay, J., Benson, S., and Schwartz, M. 1983. Genetic mapping of antigenic determinants on a membrane protein. J. Biol. Chem. 258: 2410-2414. Galanos, C., Luderitz, O., and Westphal, 0 . 1969. A new method for the extraction of R lipopolysaccharides. Eur. J . Biochem. 9: 245-249. Garavito, R.M., and Rosenbusch, J .P. 1986. Isolation and crystallization of bacterial porin. Methods Enzymol. 125: 309-328.
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Goding, J . W. 1983. Monoclonal antibodies: principles and practice. Academic Press Inc., London. Hancock, R.E.W. 1987. Role of porins in outer membrane permeability. J . Bacteriol. 169: 929-933. Hofstra, H., and Dankert, J . 1980. Major outer membrane proteins: common antigens in Enterobacteriaceae species. J. Gen. Microbiol. 117: 437-447. Hofstra, H., and Dankert, J . 1981. Porin from the outer membrane of Escherichia coli: immunological characterization of native and heat-dissociated forms. J. Gen. Microbiol. 125:
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/15/14 For personal use only.
285-292.
Inouye, M. 1979. What is the outer membrane? In Bacterial outer membranes. Edited by M. Inouye. John Wiley & Sons, Inc., New York. pp. 1-12. Ishii, J., and Nakae, T. 1980. Subunit constituent of the porin trimers that form the permeability channels in the outer membrane of Salmonella typhimurium. J. Bacteriol. 142: 27-3 1. Kearney, J.F., Radbruch, A., Liesgang, B., and Rajewsky, K. 1979. A new mouse myeloma line that has lost immunoglobulin expression but permits construction of antibody-secreting hybrid cell lines. J. Immunol. 123: 1548-1550. Kenneth, R.H., McKearn, T. J., and Bechtol, K.B. (Editors).1980. Monoclonal antibodies: a new dimension in biological analysis. Plenum Press, New York. p. 423. Klebba, P.E., Benson, S.A., Bala, S., Abdullah, T., Reid, J., Singh, S.P., and Nikaido, H. 1990. Determinants of ompF porin antigenicity and structure. J . Biol. Chem. 265: 6800-6810. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227: 680-685. Lee, D.R., and Schnaitman, C.A. 1980. Comparison of outer membrane porin proteins produced by Escherichia coli and Salmonella typhimurium. J . Bacteriol. 142: 1019- 1022. Lugtenberg, B., and Van Alphen, L. 1983. Molecular architecture and functioning of the outer membrane of Eschericha coli and other gram-negative bacteria. Biochim. Biophys. Acta, 737: 51-1 15.
Mizuno, T., Chou, M.Y., and Inouye, M. 1983. A comparative study on the genes for three porins of the Escherichia coli outer membrane. J. Biol. Chem. 258: 6932-6940. Nakae, T. 1986. Outer-membrane permeability in bacteria. CRC Crit. Rev. Microbiol. 13: 1-62. Nikaido, H. 1983. Proteins forming large channels from bacterial and mitochondria1 outer membranes: porins and phage lambda receptor protein. Methods Enzymol. 97: 85-100. Nikaido, H., and Vaara, M. 1985. Molecular basis of bacterial outer membrane permeability. Microbiol. Rev. 49: 1-32.
1107
Nurminen, M., Lounatmaa, K., Sarvas, M., Makela, P.H., and Nakae, T. 1976. Bacteriophage-resistant mutants of Salmonella typhimurium deficient in two major outer membrane proteins. J . Bacteriol. 127: 941-955. Overbeeke, N., van Scharrenburg, G., and Lugtenberg, B. 1980. Antigenic relationships between pore proteins of Escherichia coli K12. Eur. J . Biochem. 110: 247-254. Overbeeke, N., Bergmans, F., van Mansfield, F., and Lugtenberg, B. 1983. Complete nucleotide sequence of phoE, the structural gene for the phosphate limitation inducible outer membrane pore protein of Escherichia coli K12. J. Mol. Biol. 163: 513-532. Pai, S.R., Upshaw, Y., and Singh, S.P. 1992. (Alabama State University.) Unpublished data. Poole, K., and Hancock, R.E. W. 1986. Phosphate-starvation induced outer membrane proteins of members of the families Enterobacteriaceae and Pseudomonodaceae: demonstration of immunological cross reactivity with an antiserum specific for porin protein P of Pseudomonas aeruginosa. J . Bacteriol. 165: 987-993.
Poxton, I.R., Bell, G.T., and Barclay, G.R. 1985. The association of SDS-polyacrylamide gels of lipopolysaccharide and outer membrane proteins of Pseudomonas aeruginosa as revealed by monoclonal antibodies and western blotting. FEMS Microbiol. Lett. 27: 247-251. Rosenbusch, J.P. 1974. Characterization of the major envelope protein from Escherichia coli. Regular arrangement on the peptidoglycan and unusual dodecyl sulfate binding. J . Biol. Chem. 249: 8019-8029. Rosenbusch, J.P. 1987. Three-dimensional structure of membrane proteins. In Bacterial outer membranes as model systems. Edited by M. Inouye. John Wiley & Sons, Inc., New York. pp. 141-162. Schindler, M, and Rosenbusch, J.P. 1984. Structural transitions of porin, a transmembrane protein. FEBS Lett. 173: 85-89. Tokunaga, H., Tokunaga, M., and Nakae, T. 1979. Characterization of porins from the outer membrane of Salmonella typhimurium. 1. Chemical analysis. Eur. J. Biochem. 95: 434-439.
Towbin, M., Staehelin, T., and Gordon, J . 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76: 4350-4354. Van der Ley, P., Amesz, H., Tommassen, J., and Lugtenberg, B. 1985. Monoclonal antibodies directed against the cell surface exposed part of PhoE pore protein of the Escherichia coli K-12 outer membrane. Eur. J . Biochem. 147: 401-407.
