Previous Article | Next Article 
Applied and Environmental Microbiology, December 1998, p. 5033-5038, Vol. 64, No. 12
Department of the Science of Food of Animal
Origin,1 and
Department of
Bacteriology,2
Institute of
Infectious Diseases and Immunology,
Received 7 April 1998/Accepted 25 August 1998
Salmonella typhimurium expresses two antigenically
distinct flagellins, each containing a different H antigen
(i and 1,2), the combination of which is highly
specific for this serotype. In this study, overlapping recombinant
flagellin fragments were constructed from the fliC
(H:i) and fljB (H:1,2) flagellin
genes, and the expression products were tested for binding to H
antigen-specific monoclonal and polyclonal antibodies. A minimal area,
86 amino acids for H:i and 102 amino acids for
H:1,2, located in the central variable domain of each
flagellin was required for the binding of serotype-specific antibodies,
providing further evidence for the presence of a discontinuous H
epitope. Two peptides comprising these areas were shown to be highly
suitable for application as antigens in an enzyme-linked immunosorbent
assay detecting S. typhimurium-specific antibody.
Worldwide, Salmonella
typhimurium is a major cause of human food poisoning. The
consumption of food products originating from infected animals is
a primary source of human infection. To efficiently detect and
ultimately eliminate the presence of Salmonella from animal reservoirs such as pigs and poultry, there is a need for a rapid
and sensitive assay. Flagella are immunodominant antigenic surface
structures of Salmonella that, upon infection, elicit an
early short-lived humoral response in the host, including very young
animals (19), making them ideal for the detection of
infections in livestock of all ages. Flagellin is the major structural
protein of flagella and carries the serotype-specific H-antigenic
determinants (5). These H antigens are located in the
central variable domain comprising flagellin regions IV, V, and VI
(11, 20) and probably induce the production of
serotype-specific antibodies in the infected host. On the other hand,
antibodies against the conserved N- and C-terminal flagellin domains or
regions I, II, and VIII give rise to cross-reactions between
Salmonella serotypes and other Enterobacteriaceae in serological tests based on whole purified flagellum antigen (3). Furthermore, flagellar proteins are easily produced
through heterologous expression systems and therefore can be obtained in large quantities that are relatively pure (7, 16). This is an advantage over the lipopolysaccharide (O antigens), the other
commonly used antigen in Salmonella-specific serological assays. In a previous study we determined the location of the serotype-specific H:gm antigen of Salmonella
enteritidis through the construction of overlapping recombinant
peptides from its flagellin (16). Testing of these peptides
with gm-specific antibodies resulted in the selection of a
peptide that contained the H:gm epitope specific for
S. enteritidis and allowed the development of an S. enteritidis-specific enzyme-linked immunosorbent assay (ELISA).
S. typhimurium (H:i:1,2) carries two distinct
flagellin genes (fliC and fljB), and through
phase variation it can express two antigenically different flagella on
its surface (9). In the present study we have isolated both
flagellin genes of S. typhimurium and cloned overlapping
fragments of these genes in an expression vector. We produced
monoclonal antibodies (MAbs) to both S. typhimurium
flagellins and used these to select two recombinant flagellin fragments
of 86 and 102 amino acids, specific to the H:i and
H:1,2 antigens, respectively. These peptides appeared to be
highly suitable antigens in an ELISA detecting S. typhimurium-specific antibodies.
Production and validation of H:i- and
H:1,2-specific MAbs.
Purified S. typhimurium flagellins enriched in H:i or
H:1,2 were prepared as described by van Zijderveld et al.
(19). MAb-producing hybridoma cell lines were obtained from
mice immunized with the purified flagellin. Three
H:i-specific MAbs (IH10, IC11, and VB5) and three
H:1,2-specific MAb-producing cell lines (XA1, XA9, and VIID6) were selected (19) and used for further analyses. The H:gm-specific MAb (gm3) from our previous studies (16,
19) was used as a negative control.
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Production of Monoclonal Antibodies Specific for the i
and 1,2 Flagellar Antigens of Salmonella
typhimurium and Characterization of Their Respective
Epitopes
ABSTRACT
Top
Abstract
Text
References
TEXT
Top
Abstract
Text
References
TABLE 1.
Validation of H antigen-specific MAbs compared to
commercial serotyping antisera (Murex) with whole-protein cell extracts
of various Salmonella serotypes in Western blots
View larger version (38K):
[in a new window]
FIG. 1.
Serotyping of Salmonella strains with MAb VB5
and commercial serotyping serum (batch 2) on Western blots. (A)
H:i flagellin-containing Salmonella stained with
VB5. (B) H:1,2 flagellin-containing Salmonella
stained with VB5. (C) H:i flagellin-containing
Salmonella stained with commercial H:i-specific
serotyping serum. The Salmonella strains used (with the H
serotype in parentheses) are as follows: AB, S. aberdeen (i:1,2); BA, S. bandia
(i:lw); BE, S. bergen (i:enz15);
BO, S. bonariensis (i:enx); EN,
S. enteritidis (gm:-); HE, S. heidelberg (r:1,2); JU, S. jukestown
(i:enz15); KE, S. kentucky
(i:z6); KD, S. kedougou (i:lw);
NE, S. newport (eh:1,2); MU, S. muenchen (d:1,2); TA, S. takoradi
(i:1,5); TY, S. typhimurium
(i:1,2); VI, S. virchow (r:1,2).
Molecular size markers in kilodaltons are shown at the right.
Construction of flagellin gene clones. The fliC gene (Fig. 2) was amplified from chromosomal DNA of S. typhimurium SL3261 (i,1,2 [14]) with the conserved flagellin domain primers E1 and E2 (Table 2 and Fig. 2). PCR was performed in a 50-µl reaction volume with 1 U of DNA polymerase (PrimeZyme kit; Biometra, Göttingen, Germany) and 25 pmol of each primer. For each reaction, 35 cycles, each consisting of three steps (94°C for 1 min, 50°C for 2 min, and 72°C for 3 min), were carried out, followed by a single step of 10 min at 72°C to complete elongation of the products. The amplified fliC gene was cloned into pEX11 (6) and electroporated into Escherichia coli POP2136 (8).
|
-galactosidase (cro-
|
Subcloning and expression of flagellin fragments. The fliC and fljB gene fragments were digested in several single and double digestions as outlined in Fig. 2, separated on an agarose gel, and isolated from the gel (QIAEX kit; Qiagen, London, United Kingdom). Smaller fragments were created by PCR amplification of either a PstI-PstI fliC fragment (PP-i [Fig. 2A]) or the 980-bp HindIII-EcoRI fljB fragment. All fragments were cloned into expression vector pEX11, pEX12, or pEX13 (6), and clones in E. coli POP2136 were selected by culturing at 30°C on agar supplemented with 100 µg of ampicillin per ml. Subclones containing the correct inserts were identified by restriction enzyme analysis of the plasmids. The expression and purification of fusion proteins were carried out as described previously (8). The expression products were separated by SDS-polyacrylamide gel electrophoresis and tested for recognition by specific MAbs and typing sera in Western blots. For each fragment, one representative subclone was selected for further analysis.
Epitope mapping of the H:i and H:1,2 flagellins. The purified recombinant flagellin fragments were screened for binding to H:i- and H:1,2-specific typing sera and MAbs in cross blots. For this procedure, a Hybond C nitrocellulose filter (Amersham) was soaked in 0.5 M Tris-HCl (pH 6.8) and then placed in a Miniblotter 16 (Immunetics, Cambridge, Mass.) according to the manufacturer's instructions. Purified fliC and fliB flagellin fragments (Fig. 2) were resuspended in 1 M Tris-HCl (pH 6.8)-6% SDS-30% glycerol-0.75% dithiothreitol and boiled for 5 min. Each of a maximum 10 channels of the Miniblotter was filled with 110 µl of a suspension containing 15 µg of protein and incubated for 1 h at room temperature. SDS was removed by electroelution for 10 min at 50 V in 25 mM Tris-HCl-192 mM glycine-20% methanol. Immunostaining was performed essentially as with Western blotting, except that incubation with typing antisera and MAbs was carried out with the filter placed crosswise in the immunoblotter.
The smallest H:i and H:1,2 flagellin fragments that were still strongly recognized by H antigen-specific MAbs were 86 (pcr15-i) and 102 (pcr2-1,2) amino acids, respectively (Fig. 2), and contained 67 to 70% of the variable residues identified by the above-mentioned sequence comparison of the H:i and H:1,2 flagellins. When 5 N-terminal (pcr2-i) or 11 C-terminal (pcr7-i) amino acids were deleted from pcr15-i, a significant decrease in antibody binding was observed (Fig. 2A). The simultaneous removal of both these ends (pcr3-i) or the deletion of 17 amino acids from the N terminus (RR-i) resulted in a complete loss of all detectable binding. When a larger fragment, PP-i (89-352), was cut into two parts at amino acid position 230, no binding by specific MAbs to fragment PS-i or SP-i could be detected (Fig. 2A). However, SP-i (data not shown) and RR-i (Fig. 2) were still weakly recognized by specific typing sera. Removing 18 N-terminal (AA-1,2) or 33 C-terminal (pcr3-1,2) amino acids from the minimal fragment pcr2-1,2 eliminated binding of all specific MAbs (Fig. 2B). However, pcr3-1,2 was still recognized weakly by commercial typing sera, but this binding disappeared after the removal of 34 additional C-terminal amino acids (pcr4-1,2). Obviously, the most critical parts of the i and 1,2 antigenic determinants of S. typhimurium are contained within the pcr15-i and pcr2-1,2 fragments and are located between positions 190 and 291 on the H:i and H:1,2 flagellins (Fig. 2), a site which is in the hypervariable flagellin region IV (20). The localization of the H:i- and H:1,2-specific antigenic determinants in this part of the flagellin is in accordance with the results of a study of Yoshioka et al. (22). These authors showed that a spontaneous deletion mutant, lacking a part of the flagellin that corresponded to our fragment pcr15-i, was motile in the presence of H:i-specific antisera, in contrast to wild-type S. typhimurium. A similar location for the H:d antigen was inferred for Salmonella muenchen by Newton et al. (11), who presented evidence that region IV codes for the major determinants. Flagellin fragments were recognized exclusively by homologous MAbs and not by heterologous MAbs (Fig. 2). The binding by H-specific typing antisera showed similar results. Apart from the overall pattern of strongly positive and clearly negative antibody binding to flagellin fragments, two H:i fragments (pcr2-i and pcr7-i) bound only weakly to both MAbs and serotyping sera. Since these two fragments were slightly smaller or shifted in position with respect to the smallest strongly positive fragment pcr15-i, we assume that they contain only part of the antigenic determinants. Furthermore, H:i and H:1,2 typing sera bound to some flagellin fragments (SP-i, RR-i, and pcr3-1,2), none of which was recognized by the corresponding MAbs (Fig. 2). Although we do not have conclusive evidence, we believe that due to incomplete absorption of the commercial typing sera, residual antibodies that recognize flagellin epitopes unrelated to the H antigen are still present and are probably the cause of this weak binding. All other flagellin fragments, however, showed similar binding of both MAbs and serotyping sera. It is evident that both MAbs and serotyping sera have one antibody binding site on the S. typhimurium flagellin which probably consists of a single epitope. Evidently, H:i (pcr15-i) and H:1,2 (pcr2-1,2) flagellin fragments, with minimum lengths of 86 and 102 amino acids, respectively, are required for the strong binding of H-specific MAbs. The lengths of these fragments correspond well to the length of our previously found flagellin fragment, 91 amino acids, the minimal fragment for the H:gm flagellar determinant of S. enteritidis (16). Since the binding site of an antibody cannot contain more than 15 amino acid residues (18), it is obvious from our data that the residues involved in the formation of the H epitope are dispersed over a flagellin area of approximately 100 amino acids and therefore must represent a discontinuous epitope. Support for this position comes from two other studies in which sequence comparisons of Salmonella flagellins could not identify a single stretch of amino acid residues as a possible epitope site for the H antigen (10, 17). Furthermore, Joys and Schödel (4) showed that synthetic octameric peptides recognized by specific antibodies originated from sites widely dispersed on the flagellin of S. muenchen. The presence of such a discontinuous epitope suggests that some conformation of the recombinant flagellin fragments is required for antibody binding. Apparently, this conformation is maintained during or recovered after exposure to the denaturing conditions of our assays. Indeed, in our cross blots, a significant reduction of antibody binding to the recombinant flagellin fragments was observed when the electroelution of the denaturing agent SDS was omitted (data not shown), indicating that regaining the conformation of the epitope is necessary for optimal binding. An important consequence of the discontinuity of the H-specific epitope is that we could determine only the flagellin region in which a significant portion of the H-antigenic determinants was dispersed. No conclusion could be drawn about the individual amino acid residues that interact with the antibody. Also, the outer termini of the smallest binding fragments do not necessarily represent amino acid residues that are part of the antibody binding site, since their sole function could well be to maintain the flagellin fragment in the conformation required for the formation of the three-dimensional antibody binding site. In an attempt to identify the amino acids that are involved in the binding of specific antibodies, alignments between flagellar amino acid sequences of various Salmonella serotypes present in the National Center for Biotechnology Information (NCBI) database were determined (data not shown). This analysis resulted in the identification of a considerable number of hypervariable residues, which may be important for the differences in antigenic properties of flagellins of different serotypes. Aside from the fact that the majority of these residues were located on the H:i- and H:1,2-containing flagellin fragments, no solid conclusions could be drawn from this alignment, and further studies are needed to establish which amino acid residues contribute to the formation of the H antigen-determining epitope.Use of H-specific flagellin fragments in ELISA for the detection of antibodies in chickens. To obtain a soluble protein antigen suitable for use in ELISA, two small flagellin fragments of fliC (pcr6-i) and fljB (pcr2-1,2) (Fig. 2) were cloned into pGEX4T-1 (12) (Pharmacia, Uppsala, Sweden). Expression in E. coli PC2495 (21) and purification of the fusion proteins were carried out as described before (12) (Pharmacia). The wells of a microtiter plate (Greiner, Alphen a/d Rijn, The Netherlands) were each coated with 100 µl of a solution containing 5 µg of either the pcr6-i or the pcr2-1,2 flagellin peptide per ml. ELISAs were performed as described previously (2). Sixteen-week-old SPF White Leghorn chickens were infected orally with S. typhimurium, S. enteritidis, or Salmonella enterica Pullorum and Gallinarum, and serum samples were obtained on day 14, 21, or 28 after infection. Only sera from S. typhimurium-infected chickens bound to both recombinant flagellin fragments by ELISA (Fig. 3).
|
Nucleotide sequence accession number. The amino acid sequences deduced from the fliC gene and the fliB gene were deposited in GenBank under accession no. AF045151.
| |
ACKNOWLEDGMENTS |
|---|
We thank the Veterinary Health Inspection (VHI) and Food Inspection (HIGB) of the Dutch Ministry of WVS for financial support for this study.
We thank H. Maassen of the National Institute of Public Health and Environment (RIVM), Bilthoven, The Netherlands, for supplying strains of various serotypes of Salmonella and D. Mekkes of the Animal Health Service, Deventer, The Netherlands, for supplying chicken sera for ELISA. N. Hendriks is acknowledged for her technical assistance.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Medical Microbiology, Vrije Universiteit, Van der Boechorststraat 7, 1081 BT, Amsterdam, The Netherlands. Phone: (31) 20 4448310. Fax: (31) 20 4448318. E-mail: jg.kusters.mm{at}med.vu.nl.
| |
REFERENCES |
|---|
|
Top
Abstract
Text
References
|
|---|
| 1. | Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.). 1997. Current protocols in molecular biology, vol. 1 to 3. John Wiley & Sons, Inc., Brooklyn, N.Y. |
| 2. | Baay, M. F. D., and J. H. J. Huis in't Veld. 1993. Alternative antigens reduce cross-reactions in an ELISA for the detection of Salmonella enteritidis in poultry. J. Appl. Bacteriol. 74:243-247[Medline]. |
| 3. | Ibrahim, G. F., G. H. Fleet, M. J. Lyons, and R. A. Walker. 1985. Immunological relationships between Salmonella flagellins and between these and flagellins from other species of Enterobacteriaceae. Med. Microbiol. Immunol. 174:101-113[Medline]. |
| 4. |
Joys, T. M., and F. Schödel.
1991.
Epitope mapping of the d flagellar antigen of Salmonella muenchen.
Infect. Immun.
59:3330-3332 |
| 5. | Kauffmann, F. 1964. Das Kauffmann-White schema, p. 21-66. In E. van Oye (ed.), The world problem of salmonellosis. Dr. W. Junk Publishers, The Hague, The Netherlands. |
| 6. |
Kusters, J. G.,
E. J. Jager, and B. A. M. van der Zeijst.
1989.
Improvement of the cloning linker of the bacterial expression vector pEX.
Nucleic Acids Res.
17:8007 |
| 7. | Kwang, J., and E. T. Littledike. 1995. Production and identification of recombinant proteins of Salmonella typhimurium and their use in detection of antibodies in experimentally challenged animals. FEMS Microbiol. Lett. 130:25-30[Medline]. |
| 8. | Lenstra, J. A., J. G. Kusters, G. Koch, and B. A. M. van der Zeijst. 1989. Antigenicity of the peplomer protein of infectious bronchitis virus. Mol. Immunol. 1:7-15. |
| 9. | Macnab, R. M. 1987. Flagella, p. 70-83. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology, vol. 1. American Society for Microbiology, Washington, D.C. |
| 10. |
Masten, B. J., and T. M. Joys.
1993.
Molecular analyses of the Salmonella g... flagellar antigen complex.
J. Bacteriol.
175:5359-5365 |
| 11. | Newton, S. M. C., R. D. Wasley, A. Wilson, L. T. Rosenberg, J. F. Miller, and B. A. D. Stocker. 1991. Segment IV of a Salmonella flagellin gene specifies flagellar antigen epitopes. Mol. Microbiol. 5:419-425[Medline]. |
| 12. | Smith, D. B., and K. S. Johnson. 1988. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67:31-40[Medline]. |
| 13. |
Smith, N. H., and R. K. Selander.
1990.
Sequence invariance of the antigen-coding central region of the phase 1 flagellar filament gene (fliC) among strains of Salmonella typhimurium.
J. Bacteriol.
172:603-609 |
| 14. | Stocker, B. A. 1990. Aromatic-dependent Salmonella as live vaccine presenters of foreign epitopes as inserts in flagellin. Res. Microbiol. 141:787-796[Medline]. |
| 15. |
Thompson, J. D.,
D. G. Higgins, and T. J. Gibson.
1994.
CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.
Nucleic Acids Res.
22:4673-4680 |
| 16. |
van Asten, A. J. A. M.,
K. A. Zwaagstra,
M. F. Baay,
J. G. Kusters,
J. H. J. Huis in't Veld, and B. A. M. van der Zeijst.
1995.
Identification of the domain which determines the g,m serotype of the flagellin of Salmonella enteritidis.
J. Bacteriol.
177:1610-1613 |
| 17. |
Vanegas, R. A., and T. M. Joys.
1995.
Molecular analyses of the phase-2 antigen complex 1,2, · · of Salmonella spp.
J. Bacteriol.
177:3863-3864 |
| 18. | van Regenmortel, M. H. V. 1989. Structural and functional approaches to the study of protein antigenicity. Immunol. Today 10:266-272[Medline]. |
| 19. |
van Zijderveld, F. G.,
A. M. van Zijderveld-van Bemmel, and J. Anakotta.
1992.
Comparison of four different enzyme-linked immunosorbent assays for serological diagnosis of Salmonella enteritidis infections in experimentally infected chickens.
J. Clin. Microbiol.
30:2560-2566 |
| 20. | Wei, L. N., and T. M. Joys. 1985. Covalent structure of three phase-1 flagellar filament proteins of Salmonella. J. Mol. Biol. 186:791-803[Medline]. |
| 21. | Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103-119[Medline]. |
| 22. |
Yoshioka, K.,
S. Aizawa, and S. Yamaguchi.
1995.
Flagellar filament structure and cell motility of Salmonella typhimurium mutants lacking part of the outer domain of flagellin.
J. Bacteriol.
177:1090-1093 |
Applied and Environmental Microbiology, December 1998, p. 5033-5038, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Perera, K., Murray, A.
(2009). Development of an indirect ELISA for the detection of serum IgG antibodies against region IV of phase 1 flagellin of Salmonella enterica serovar Brandenburg in sheep. J Med Microbiol
58: 1576-1581
[Abstract] [Full Text] -
Rementeria, A., Vivanco, A. B., Ramirez, A., Hernando, F. L., Bikandi, J., Herrera-Leon, S., Echeita, A., Garaizar, J.
(2009). Characterization of a Monoclonal Antibody Directed against Salmonella enterica Serovar Typhimurium and Serovar [4,5,12:i:-]. Appl. Environ. Microbiol.
75: 1345-1354
[Abstract] [Full Text] -
Tomoyasu, T., Ohkishi, T., Ukyo, Y., Tokumitsu, A., Takaya, A., Suzuki, M., Sekiya, K., Matsui, H., Kutsukake, K., Yamamoto, T.
(2002). The ClpXP ATP-Dependent Protease Regulates Flagellum Synthesis in Salmonella enterica Serovar Typhimurium. J. Bacteriol.
184: 645-653
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»