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Applied and Environmental Microbiology, March 2009, p. 1345-1354, Vol. 75, No. 5
0099-2240/09/$08.00+0     doi:10.1128/AEM.01597-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Characterization of a Monoclonal Antibody Directed against Salmonella enterica Serovar Typhimurium and Serovar [4,5,12:i:–] {triangledown}

A. Rementeria,1* A. B. Vivanco,1 A. Ramirez,1 F. L. Hernando,1 J. Bikandi,1 S. Herrera-León,2 A. Echeita,2 and J. Garaizar1

Departamento de Inmunología, Microbiología y Parasitología, Universidad del País Vasco, Campus de Alava, Vitoria-Gasteiz, and Campus de Bizkaia, Leioa, Spain,1 Sección de Enterobacterias, Servicio de Bacteriología, Centro Nacional de Microbiología, ISCIII, Majadahonda, Spain2

Received 11 July 2008/ Accepted 23 December 2008


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ABSTRACT
 
Flagellar extracts of Salmonella enterica serovars expressing phase 2 H1 antigenic complex (H:1,2, H:1,5, H:1,6, and H:1,7) and a mutant flagellin obtained by site-directed mutagenesis of the fljB gene from serovar Typhimurium at codon 218, transforming threonine to alanine, expressed in Escherichia coli (fljB218A) were used to analyze the H1 antigenic complex. Cross-reactions were detected by Western blotting and dot blotting using commercial polyclonal antibodies against the different wild-type extracts and mutant FljB218A. Therefore, we produced a monoclonal antibody (MAb), 23D4, isotyped as immunoglobulin M, against H:1,2 S. enterica serovar Typhimurium flagellin. The mutant flagellin was not recognized by this MAb. When a large number of phase 1 and phase 2 flagellin antigens of different serovars were used to characterize the 23D4 MAb, only extracts of serovars Typhimurium and [4,5,12:i:–] reacted. The protein composition of phase 1 and phase 2 extracts and highly purified H:1,2 flagellin from serovar Typhimurium strain LT2 and extract of strain 286 (serovar [4,5,12:i:–]), which reacted with the MAb, was studied. Phase 2 flagellin (FljBH:1,2) was detected in phase 1 and phase 2 flagellar heat extracts of serovar Typhimurium and was the single protein identified in all spots of purified H:1,2 flagellin. FliC, FlgK, and other proteins were detected in some immunoreactive spots and in the flagellar extract of serovar [4,5,12:i:–]. Immunoelectron microscopy of complete bacteria with 23D4 showed MAb attachment at the base of flagella, although the MAb failed to recognize the filament of flagella. Nevertheless, the results obtained by the other immunological tests (enzyme-linked immunosorbent assay, Western blotting, and dot blotting) indicate a reaction against flagellins. The epitopes could also be shared by other proteins on spots where FljB is not present, such as aminopeptidase B, isocitrate lyase, InvE, EF-TuA, enolase, DnaK, and others. In conclusion, MAb 23D4 can be useful for detection and diagnostic purposes of S. enterica serovar Typhimurium and serovar [4,5,12:i:–] and could be also helpful for epitope characterization of flagellum-associated antigens.


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INTRODUCTION
 
Salmonella species are recognized as major zoonotic pathogens of animals and humans (16) and are the etiologic agents of different diseases collectively referred to as salmonellosis. Salmonella is classified into more than 2,500 serovars using the Kauffmann-White scheme. A serovar is determined on the basis of somatic (O), flagellar (H), and capsular (Vi) antigens present in the cell walls of Salmonella organisms. The bacterial flagellum consists of three distinct major substructures: the basal body, which contains a motor; the hook, working as a universal joint; and the filament, the helical propeller (21). Combinations of flagellin subunits shape the flagellum extracellular structure and form the H antigens. Salmonella can undergo phase variation to alternately express two different major flagellar antigens, phase 1 and phase 2, encoded by the fliC and fljB genes, respectively. These two genes are present at two different locations on the chromosome, but only one of them is expressed by the cell at one time due to a mechanism regulated by the operon fljBA. Although no biological function has been ascribed to this capacity, Ikeda et al. (20) have demonstrated that flagellar regulation is linked to virulence of Salmonella enterica serovar Typhimurium. Nevertheless, several monophasic exceptions of Salmonella serovars exist in nature.

For serological characterization of Salmonella isolates, many commercially available polyclonal and monoclonal antibodies (MAbs) may be used. The serotyping is mostly performed in reference laboratories through slide or tube agglutination techniques, and variable sensitivity and specificity values are obtained. Shrader et al. (33) obtained a good sensitivity (>92%) and specificity (100%) with Denka Seiken (Tokyo, Japan) somatic and flagellar antisera by tube agglutination assays. However, when Denka Seiken flagellar antisera were used in a slide agglutination assay, the sensitivity and accuracy dropped to 88.9% and the specificity decreased to 91%. Commercial antiflagellar antibodies are generally produced by immunizing animals with whole organism, and little or no adsorption of the antisera is performed. Therefore, the antisera could contain antibodies against the O antigens from the immunizing organisms, which could explain the drop in the sensitivity and accuracy of slide agglutinations. Moreover, multicentric serotyping studies performed in national reference laboratories found significant differences between participating laboratories to correctly serotype Salmonella strains (37). Cross-reactions of commercial antibodies in serotyping of Salmonella are well-known phenomena (11). MAbs, with their monoepitopic specificity, have many advantages over monospecific polyclonal sera (4). Several MAbs directed against H antigens of Salmonella have been described (7, 17, 29, 32).

The antigenic epitopes of the different flagellins produced are thought to be defined by internal variable regions (IVR) of Salmonella flagellar genes, although the exact definitions of their antigenic structures are still unknown. Using DNA sequencing of IVR of phase 2 H1 antigenic complex, allelic variation was denoted by Echeita et al. (8). A single nucleotide polymorphism was found between alleles, and consensus sequences were also defined. In order to confirm the relationship between the single nucleotide polymorphism observed by Echeita et al. (8) with a change at the flagellar epitope, we sought here to obtain a mutant of fljBH:1,2 using site-direct mutagenesis. Furthermore, the production of a MAb specific for the H:1,2 flagellin of the S. enterica serovar Typhimurium was carried out to delineate the epitopes of the phase 2 H1 antigenic complex. The molecular characterization of the MAbs and their bacterial targets detected by Western blotting, protein sequencing, and immunoelectron microscopy (IEM) are also presented.


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MATERIALS AND METHODS
 
Bacterial strains.
In the present study, 89 S. enterica strains belonging to different serovars were used, including two S. enterica serovar Typhimurium reference strains: strain LT2 (strain 722 from the Spanish Type Culture Collections [CECT]) and strain 4,300 (Salmonella Reference Laboratory, Majadahonda, Madrid). The complete list of Salmonella strains used in the present study and their antigenic formulas, according to the latest version of the Kauffmann-White scheme (26, 27), are shown in Table 1. The Salmonella strains were selected based on the antigenicity of serovar Typhimurium: phase 2 flagellar antigens (H:1,2 or similar H1 complexes H:1,5, H:1,6, and H:1,7), phase 1 flagellar antigen (H:i), or somatic antigens (O:1, O:4, O:5, and O:12), including several strains of a monophasic serovar [4,5,12:i:–] emergent in Spain, and other nonrelated serovars. Also, four strains of related genera were used.


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  		TABLE 1. Bacterial strains used in this study

Salmonella flagellin extract production.
In order to obtain the different phases of flagellar antigens, the Salmonella strains were grown three times for 16 h at 37°C on Sven Gard agar (Bio-Rad Laboratories, Hercules, CA) containing a suitable anti-phase 1 or anti-phase 2 antiserum mixture (Bio-Rad Laboratories). For each culture, the most motile microorganisms were selected.

Phase 2 flagellar antigens of S. enterica serovar Typhimurium strain no. 4,300 were used for experimental immunizations. The method of Ibrahim et al. (19) for acid extractions was applied at peripheral growth on Sven Gard agar with anti-i antibodies to produce the H:1,2 antigens. The purity of the flagellin protein was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

For immunological techniques, the phase 1 or phase 2 flagellar antigens were obtained as described by Chart et al. (5). Briefly, Salmonella serovars grown on Sven Gard agar were suspended in 1 ml of saline solution and then incubated at 60°C for 30 min. Then, suspensions were centrifuged at 10,000 rpm for 10 min, and 400 µl of supernatant was used in the assays. The protein concentration was determined by a Bio-Rad protein assay. When required, in order to concentrate the antigen extracts, 4 volumes of 10% trichloroacetic acid-acetone was added for protein precipitation for 1 h at –20°C, followed by centrifugation at 6,500 rpm for 10 min. The pellets were resuspended in appropriate buffer as indicated for the different methods. Flagellin extracts were stored at –80°C until use.

In some cases, H:1,2 antigens were highly purified from LT-2 strain according to the method of Yamashita et al. (38). Briefly, flagellar filaments were packaged with 2% polyethylene glycol 6000 and 0.5 M NaCl and were purified by ionic exchange chromatography loading onto a Mono-Q column (Pharmacia). The purified flagellin was eluted with a 30-ml gradient of 20 mM Tris-HCl buffer (pH 7.8) containing 80 mM to 130 mM NaCl, and eluted protein was monitored at 280 nm. The flagellin filaments were rebuilt by 1.0 M (NH4)2SO4, pelleted at 120,000 x g, and resuspended in 20 mM Tris-HCl buffer (pH 7.8) containing 150 mM NaCl. The purified flagellin was stored at –80°C until use.

Site-directed mutagenesis.
We amplified by PCR the fljBA operon of S. enterica serovar Typhimurium (H:1,2) of strain 4,300, using the primers P56 and PM2 (8, 35). The sequence was cloned in pCR 2.1 vector and Escherichia coli TOP10 cells were transformed by using a TOPO-TA cloning kit (Invitrogen, Barcelona, Spain). The IVR of the cloned operon was confirmed by sequencing (Sistemas Genómicos, Valencia, Spain). A site-directed mutagenesis experiment was carried out by using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA), with the primers sense-Mut-7 (5'-GGTGGTACGAATGGTGCAGCTTCTGTAACCG-3') and antisense-Mut-7 (5'-CGGTTACAGAAGCTGCACCATTCGTACCACC-3') (nucleotides replaced via mutagenesis are shown in boldface). Mutagenizing primers were designed to anneal with the polymorphic region inside IVR of the fljBH:1,2 gene. The two base modifications correspond to the consensus sequence of the previously determined fljBH:1,7 gene (8) and code for alanine (GCA) instead of threonine (ACG) in codon 218 of the flagellin sequence (fljB218A). For the mutagenizing PCR, 20 ng of DNA template, 2.5 U of proofreading Pfu Turbo DNA polymerase, and ready-to-make nucleotides were added in a final volume of 50 µl. Thermocycling conditions were as follows: 1 cycle of 95°C for 30 s and then 12 cycles of 95°C for 30 s, 55°C for 1 min, and 68°C for 12 min. Hybrid DNA was digested with DpnI enzyme for degradation of the nonmutated template strand. The mutagenized DNA was transformed in E. coli epicurean Coli XL1-Blue cells, and the mutant nucleotide sequence of the insert was confirmed by sequencing.

To express the mutant gene, a pET directional TOPO expression kit (Invitrogen) was used to express the mutant gene. The pCR 2.1 plasmid DNA carrying mutant fljB218A gene was used as a template in a PCR with the primers FEX2 (5'-CACCTAAAAGGAAAATTTTATGGCACAAGT-3') and REX1 (5'-TTAACGTAACAGAGACAGCACGTTC-3'). Thermocycling conditions were as follows: 1 cycle of 95°C for 2 min; 25 cycles of 95°C for 1 min, 47°C for 30 s, and 72°C for 2 min; and a final cycle of 72°C for 15 min. The FEX2-REX1 PCR product was cloned directly into pET100/D-TOPO expression vector, and the constructs were transformed into TOP10 One-Shot E. coli by the heat shock method. Plasmids of recombinant clones were sequenced as described above. Correctly oriented plasmid constructs were used to transform BL21 Star (DE3) One-Shot E. coli by the heat shock method. Expression of the mutant fljB218A gene was induced with IPTG (isopropyl-β-D-thiogalactopyranoside) as described by the supplier. Lysates of the E. coli cells expressing mutant FljB218A were analyzed by SDS-PAGE.

Mouse immunization.
Six- to seven-week-old female BALB/c mice were immunized with flagellar acid extracts. First immunization was performed with intraperitoneal injections of 30 µg of protein in 200 µl with complete Freund's adjuvant. Two additional immunizations were accomplished each 20 days with the same antigen concentration in 200 µl of incomplete Freund's adjuvant. Antibody titers of mice sera were determined by Western blotting against H:1,2 extracts, and the mice with the highest antibody titers were selected. The selected mice were inoculated intraperitoneally with the final dose of antigen in 200 µl of saline solution. Finally, immune sera were obtained, and mice were sacrificed. The present study was performed at the Animalarium Facility of the University of the Basque Country (Bilbao, Spain). Procedures involving animals and their care were conducted in conformity with national and international laws and policies.

MAb production.
MAbs were obtained, with minor modifications, by following the methods described by de StGroth and Scheidegger (6). Briefly, female BALB/c mice were immunized with H:1,2 flagellar acid extract of S. enterica serovar Typhimurium strain 4,300. The mice with the highest antiflagellar antibody titer in serum (enzyme-linked immunosorbent assay [ELISA], Western blot, and dot blot tested) were sacrificed by cervical dislocation, and the spleens were extracted aseptically. Fusion of splenocyte cells from the SP2/O murine myeloma cells was performed in RPMI medium at a 5:1 ratio, respectively. The cellular mixture was centrifuged (400 x g for 10 min), and the pellet was resuspended in 1 ml of fusion medium (0.5 g of polyethylene glycol 4000, 500 µl of RPMI, and 50 µl of dimethyl sulfoxide), gently drip added, for each 107 myeloma cells. After incubation for 1 min at 37°C, the cells were centrifuged for 90 s at 400 x g and then reincubated, without discarding the supernatant, for 90 s at 37°C. The pellet was then resuspended in RPMI, the cells were centrifuged at 400 x g for 10 min, and the pellet was gently resuspended in 5 ml of hypoxanthine-aminopterin-thymidine medium. The cells were adjusted at 5 x 105 cells per ml, and 100-µl portions were dispensed in each well of microtiter plates. After incubation for 5 days at 37°C in 5% CO2 and 95% humidity atmosphere, 50 µl of HT medium per well was added. At day 10, the culture supernatants were screened for antiflagellar antibodies by ELISA against H:1,2 antigens. Positive wells were expanded in 24-well plates, cloned twice by limiting dilution, and isotyped by ELISA. The anti-H:1,2 MAb production of individualized clones was confirmed by ELISA, Western blotting, and dot blotting. The selected cell line, identified as 23D4, was deposited (strain 05122301) in the European Collection of Cell Cultures (Salisbury, United Kingdom), stored at –80°C, and frozen in liquid nitrogen. The cell culture supernatants that contain the MAbs used in the immunological tests were harvested from 10- to 15-day-old cultures of the 23D4 cell line in RPMI plus penicillin-streptomycin supplied with 10% fetal calf serum. A patent describing MAb 23D4 was deposited in the Spanish Office of Patents (14).

ELISA.
The 96-well ELISA plates were coated overnight at 4°C with 50 ng of flagellar antigens in 100 µl of carbonate-bicarbonate buffer (pH 9.6) per well. After blocking the wells with 200 µl of bovine serum albumin (BSA; 1% in phosphate-buffered saline [PBS; pH 7.4]), 100 µl of 23D4 culture supernatant or a 1:100 dilution in BT-PBS (1% BSA-0.05% Tween 20 in PBS) was added to each well, followed by incubation at 37°C for 1 h, and then washed three times with BT-PBS. Then, 100 µl of a 1:2,000 anti-mouse immunoglobulin M (IgM)-horseradish peroxidase (Sigma, St. Louis, MO) dilution in BT-PBS was added per well, followed by incubation as described above. Afterward, the plates were washed three times with BT-PBS and incubated with 100 µl per well of ortho-phenylene diamine solution (Sigma) for 30 min at room temperature in darkness. The reaction was stopped with 50 µl per well of 0.5 M of H2SO4, and the absorbance was measured at 490 nm. The percent positive was calculated as follows: (optical density of sample/optical density of positive control) x 100. Samples were considered positive if the percent positive value was >40%.

Electrophoretic separation of flagellar extract.
Acid or heat protein extractions were separated by SDS-PAGE in one-dimensional (1D) or 2D electrophoresis. The 1D or 2D gels were stained with Coomassie blue stain or Bio-Safe (Bio-Rad, Madrid, Spain) stain or electrotransferred to suitable membranes. The 1D electrophoresis technique was performed in SDS-PAGE (12.5% polyacrylamide). In each track, 2.5-µg portions of antigen extracts were separated at 200 V for 45 min in MiniProtean II (Bio-Rad). Prestained SDS-PAGE low-range markers (Bio-Rad) were used as standards.

Isoelectric focusing was carried out in immobilized nonlinear pH 3 to 5.6 gradient 7-cm long Immobiline Drystrips (GE Healthcare, Uppsala, Sweden). The 2D electrophoresis technique was performed according to the methods described by Hernando et al. (15). For Western blot and Coomassie blue staining, 2D gels were loaded with 75 and 300 µg of protein, respectively.

Western blot analysis.
Proteins separated on 1D or 2D gels were blotted onto polyvinylidene difluoride membrane or Immobilon-P membrane (Millipore, Madrid, Spain) by using a semidry transfer system for 1 h at 5 mA/cm2 or 400 mA for 2 h, respectively. According to Hernando and coworkers (2, 15), these membranes were treated in each case with the blocking solution TBS-M (TBS [0.5% Tween 20 in 50 mM PBS] containing 8% nonfat skim milk powder) for 1 h at 37°C. After blocking, the samples were incubated with the MAbs diluted 1:50 in TBS-M for 1 h at 37°C. The membranes were then washed for 5 min three times with TBS-M. Antigen-antibody reactions were visualized by the addition of anti-mouse polyvalent or IgM-horseradish peroxidase (Sigma) diluted 1:2,000 in TBS-M. Finally, the membranes were extensively washed with TBS three times, and detected by Immobilon Western chemiluminescence (Millipore), according to the manufacturer's instructions. For band visualization, membrane was wrapped in plastic and exposed to Curix RP-2 X-ray film (Agfa-Gevaert NV, Mortsel, Belgium). Stained gels and X-ray films were analyzed by using Image Master 2D Platinum software (version 5.0; GE Healthcare, Alcobendas, Madrid).

Dot blot analysis.
Flagellar extract samples were diluted to 25 ng per µl in 0.9% saline solution, 2 µl was deposited on nitrocellulose membrane, allowed to absorb, and dried at room temperature. Dot blot membranes were blocked with the blocking solution TBS-M for 1 h at 37°C. After blocking, the samples were incubated with MAb 23D4 diluted 1:50 in TBS-M for 1 h at 37°C. The membranes were then washed three times for 5 min each time with TBS-M. To detect attached MAb 23D4, anti-mouse polyvalent or IgM-horseradish peroxidase (Sigma) diluted 1:2,000 in TBS-M was used. The membranes were extensively washed with TBS three times. Detection of reaction was performed by using a substrate solution containing 0.015% H2O2 and 0.05% 4-(chloro-1-naphthol) in TBS containing 16.7% methanol. The color was allowed to develop for 20 min and was stopped by soaking the blots in distilled water for 5 min.

IEM of bacterial flagellar antigens.
IEM was performed using fresh, nonformolized cultures of motile bacteria (3). Briefly, bacteria were adsorbed onto glow-discharge grids coated with Formvar resin, and these grids were preincubated for 30 min at room temperature with 0.1% BSA blocking buffer (Sigma). Specimens were incubated for 30 min at room temperature with droplets of unlabeled 23D4 culture supernatants. The grids were then washed twice for 10 min on blocking buffer to remove unbound antibody. Primary antibodies were detected using anti-mouse IgM-10-nm gold conjugates (Sigma) diluted 1:20 in blocking buffer for 30 min at room temperature. To remove the unbound conjugate, the grids were washed twice with blocking buffer and once with PBS. Before negative staining with 1% uranyl acetate (pH 4.0 to 4.5), the grids were washed rapidly with 4 droplets of double-distilled water, and the preparations were visualized and microphotographed with a Philips EM208S electron microscope at an accelerating voltage of 80 kV at the Analytical and High Resolution Microscopy Facility of the University of the Basque Country.

Protein identification in 2D gels.
Protein spots from immunoreactive bands were excised and identified by liquid chromatography-tandem mass spectrometry using a Q-Tof Micro mass spectrometer (Waters, Milford, MA) at the Proteomics Facility of the University of the Basque Country. Obtained spectra were processed by using ProteinLynx Global Server (Waters) and searched against the NCBI (http://www.ncbi.nlm.nih.gov/) and Swiss-Prot databases (http://www.expasy.org/) using MASCOT (Matrixscience, London, United Kingdom). For protein identification, the following parameters were adopted: carbamidomethylation of cysteines as fixed modification, oxidation of methionines as variable modification, 50 ppm of peptide mass tolerance, 0.1-Da fragment mass tolerance, and one missed cleavage. The score obtained by the different peptide combinations of identified protein in each spot was calculated, and a score of >22 indicated identity or extensive homology (P < 0.05). The scores are derived from ion scores as a nonprobabilistic basis for ranking protein hits. The ion score is –10 x log(P), where P is the probability that the observed match is a random event.


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RESULTS
 
Flagellar site-directed mutagenesis.
Sequencing of the mutagenized fljB218A gene confirmed the two-nucleotide substitution at codon 218 of flagellin sequence, so that alanine was codified instead of threonine. Lysates of the E. coli cells expressing flagellin mutant and flagellar extracts of Salmonella serovars showing complex H1 (H:1,2, strain 4,300; H:1,5, strain 589; H:1,6, strain 271; and H:1,7, strain 444) were analyzed by dot blotting and Western blotting with commercial polyclonal antisera (Fig. 1). When wild flagellar extracts of H1 antigenic complex were used, cross-reactions with commercial polyclonal antibodies (Sanofi Pasteur MSD, Madrid, Spain) were observed. The flagellin mutant was also strongly recognized by commercial polyclonal antisera.


Figure 1
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  		FIG. 1. (A and B) Detection of different extracts of phase 2 H1 antigenic complex (H:1,2, strain 4,300; H:1,5, strain 589; H:1,6, strain 271; and H:1,7, strain no. 444) and a mutant flagellin (FljB218A) expressed in E. coli by either dot blotting with commercial polyclonal antibodies (Sanofi) and MAb 23D4 (A) or Western blotting with MAb 23D4 (B).

Production of MAbs.
Hybridomes were obtained, and the clone 23D4C8F8 was selected due to the strong reactivity of culture supernatant against H:1,2 flagellar extracts as determined by ELISA and dot blot analyses. A unique band at 54 kDa in Western blotting was recognized by this MAb, so it was selected for further analysis (Fig. 1B). The 23D4C8F8 hybridome was renamed as 23D4, and the MAb was named MAb 23D4. The MAb 23D4 exhibited a specific reaction with H:1,2 antigen but not with other wild phase 2 flagellar antigens of H1 complex, as shown in Fig. 1. The mutant flagellin obtained by site-directed mutagenesis was not recognized by this MAb.

Characterization of MAb 23D4.
The MAb produced by 23D4 clone was isotyped as IgM by specific anti-isotype mouse antibodies (data not shown). By dot blot and Western blot analyses, MAb 23D4 did not react with non-Typhimurium flagellar extracts or with mutant flagellin (Fig. 1). MAb 23D4 was characterized by ELISA with a large number of phase 1 and phase 2 Salmonella flagellin antigens extracted by heat (Table 1). MAb 23D4 reacted only with phase 1 flagellar extracts of serovar Typhimurium and serovar [4,5,12:i:–]. These results were confirmed by Western blot and dot blot experiments (Fig. 2). Dot blotting showed that only the flagellar extract from serovar [4,5,12:i:–] strain 286 was not recognized by MAb 23D4. This negative result may be due to a low protein concentration in extract (data not shown). For phase 2 extracts, MAb 23D4 only recognized the serovar Typhimurium (Table 1). The MAb reacted with eight strains but failed to recognize two strains belonging to this serovar (strains 236 and 320).


Figure 2
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  		FIG. 2. Analysis of reactivity of phase 1 Salmonella flagellar extracts with MAb 23D4. (A and B) Western blotting against antigenic extracts separated by 1D SDS-PAGE (A) or dot blotting (B). The characteristics of Salmonella strains are given in Table 1.

The commercial anti-H:1,2 polyclonal monovalent antibodies (Sanofi) were used as a control in the dot blot analyses (Table 1). Positive reactions were obtained against almost all strains from serovars with an antigenic formula that included H:1,2 antigen (15 strains), except for strain 235 (serovar Lindenburg). Furthermore, this antibody showed false-positive results with the phase 2 extract (z6 antigens) of strains 531 and 534 (serovar Kentucky). Nevertheless, this commercial antibody reacted also with different phase 1 extracts. Unexpectedly, the commercial antibody reacted with phase 1 flagellar extracts of serovar [4,5,12:i:–] (except with the strain 286) that does not express H:1,2 antigens and also showed weak reaction with phase 1 extract of strain 534 strain (serovar Kentucky).

2D gel image analysis and protein identification by sequencing.
In order to detect which epitope reacts with MAb 23D4, the protein compositions of reactive strain extracts were studied by 2D SDS-PAGE and Western blotting. The results are shown in Fig. 3 and 4. In these studies, phase 1 and phase 2 extracts, highly purified H:1,2 flagellin from strain LT2 (serovar Typhimurium), and strain 286 (serovar [4,5,12:i:–]) were used. Spots containing the proteins that reacted with MAb 23D4 by Western blotting were excised manually from stained gels, and in-gel digestion with trypsin was performed. The peptides yielded by this digestion were detected and identified by using a Q-Tof Micro mass spectrometer. The proteins detected in the different analyzed spots are shown in Fig. 3 and 4. Phase 2 flagellin (FljB) of serovar Typhimurium was detected in phase 1 and phase 2 heat flagellar extracts and purified H:1,2 flagellin (spots 1, 2, 3, 4, 7, 8, and 9 in Fig. 3 and spots 12 and 14 in Fig. 4). When highly purified phase 2 extracts of serovar Typhimurium were studied, FljB was the only protein identified in all spots of the purified extract. In reactive spots 10 and 11 of these extracts, proteins were not detected (Fig. 3). In phase 1 extract of serovar Typhimurium, phase 2 flagellin was also detected (Fig. 4). Phase 1 flagellin (FliC) was detected on spots 12 and 14 of phase 1 heat extracts of serovar Typhimurium, but it was not detected on phase 2 extracts. The reactions of extract from serovar [4,5,12:i:–] were weaker and shared some spots with phase 1 extract of serovar Typhimurium. In this serovar, FliC was detected on spots 15, 18, and 19. At phase 2 flagellar extract of serovar Typhimurium, and in flagellar extracts of serovar [4,5,12:i:–], the FlgK protein was detected in spots 4 and 15 (Fig. 3 and 4). Other proteins were detected in immunoreactive spots. Among them, an invasion protein related to injectisome, InvE, was detected in spot 5 of phase 2 extracts. Two heat shock proteins or chaperones were detected: GRoEL was detected in spot 3 (phase 2 extract), which belongs to 60-kDa chaperone family, and DnaK (heat shock protein 70) was detected in spots 13 and 16 (phase 1 extract). Two ABC superfamily periplasmic proteins were identified in immunoreactive spots: a dipeptide transport protein detected in spot 1, and a spermidine-putrescine substrate binding component detected in spot 6, both in phase 2 extract. Several metabolic enzymes were also identified: phosphopyruvate hydratase (enolase), adenylosuccinate synthetase, aromatic amino acid aminotransferase, aminopeptidase B, and isocitrate lyase. Finally, two components of protein synthesis apparatus were identified: 30S ribosomal protein S1 and elongation factor Tu (EF-Tu).


Figure 3
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  		FIG. 3. Analysis of phase 2 heat extracts (A and B) and purified H:1,2 flagellin (C and D) of serovar Typhimurium by Coomassie blue staining of 2D electrophoretic separations (A and C) and by Western blotting with MAb 23D4 (B and D). Immunoreactive spots with MAb 23D4 are oval-shaped, marked, and numbered. The protein identifications of the spots are given in the center table, which indicates the identified protein by peptide combinations, the sequence access numbers of identified proteins in the NCBI protein database, and the score obtained by the different peptide combinations of identified protein in each spot. A score of >22 indicates identity or extensive homology (P < 0.05). The final column of the table indicates the number of peptides obtained that match the protein sequence as detected by a MASCOT search in the NCBI protein database.


Figure 4
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  		FIG. 4. Analysis of phase 1 heat extracts of serovar Typhimurium (A and B) and serovar [4,5,12:i:–] (C and D) by Coomassie blue staining of 2D electrophoretic separations (A and C) and by Western blotting with MAb 23D4 (B and D). Immunoreactive spots with 23D4 MAbs are oval-shaped, marked, and numbered. The protein identifications of the spots are given in the center table, which indicates the identified protein by peptide combinations, the sequence access numbers of identified proteins in the NCBI protein database, and the score obtained by the different peptide combinations of identified protein in each spot. A score of >22 indicates identity or extensive homology (P < 0.05). The final column of the table indicates the number of peptides obtained that match on the protein sequence as detected by a MASCOT search in the NCBI protein database.

IEM.
The antigenic target of MAb 23D4 was studied by IEM of complete bacteria in order to determine the structure where the MAb attaches. Phase 2-expressing cells of serovar Typhimurium LT2 strain were used in the present study. As shown in Fig. 5, the gold-marked antibody was detected clearly at the bottom of flagellar structure and in discrete locations on the cell surface. MAb 23D4 did not attach to filament flagellar structure in any assay, so the epitope recognized is not apparently the conformational flagellin (FliC or FljB) structure. In other Salmonella serovars sharing some antigenic factors with Typhimurium, serovar Muenchen strain 270 ([6,8:d:1,2]), serovar Kentucky strain 531([8,20:i:z6]), and the monophasic strain 448 ([4,12:–:1,2]), no reaction was detected with MAb 23D4 even though high MAb concentrations and overexposure were applied (data not shown).


Figure 5
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  		FIG. 5. IEM of phase 2 intact cells of LT2 strain of serovar Typhimurium (A) and scheme of flagellar protein composition (B). The union site of anti-mouse IgM-gold 10-nm conjugates is indicated by black arrows. Flagellar filaments are indicated by a white arrow.


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DISCUSSION
 
The second-phase flagellar antigens—H:1,2, H:1,5, H:1,6, and H:1,7—constitute the H1 antigenic complex. Echeita and Usera (8) sequenced the flagellin hypervariable domain region of H1 antigenic complex, where antigenic epitope is located, and only very short differences in the central nucleotide sequence (IVR) were detected. The way that these differences change the structural epitope of each antigen was not clear, but the antiflagellar antibodies used for serotyping in National References Laboratories recognized these differences in the flagellum structure. However, we found that these polyclonal antiflagellar antibodies showed cross-reactivity against flagellar extracts with unfolded structure when they were examined by Western blot and dot blot assays. Commercial antiflagellar antibodies could contain antibodies against the O antigens from the immunizing organisms, which could explain the cross-reactivity observed by us. The characterization of flagellin structure related to epitopes recognized on the H1 antigenic complex was hard to perform due to these cross-reactions. The specificity of MAbs could be useful for the characterization of flagellar epitope. Moreover, MAbs specific for flagellar antigens are used for detection of Salmonella flagellins of different serovars (7, 17, 18, 28, 30, 36), as well as for identification of a common enterobacterial flagellin epitope (12). However, MAbs could recognize structures and epitopes shared by different molecules from the same or different microorganisms (9, 23, 24, 31). The aim of our research group was to develop a MAb specific for the H:1,2 epitope of the S. enterica serovar Typhimurium able to delineate the epitopes of the phase 2 H1 antigenic complex. To do so, H:1,2 flagellar extracts of strain 4,300 were used to immunize mice, and different antibody-producing clones were extracted. Due to its strong reaction with H:1,2 extract, the selected hybridome was the 23D4 clone (Fig. 1). The antibodies produced by 23D4 did not react with other antigenic extracts of H1 antigenic complex, H:1,5, H:1,6, and H:1,7 (Fig. 1). In addition, the MAbs produced by 23D4 clone discriminated the H:1,2 native antigen from a mutant antigen with one amino acid changed in H:1,2 flagellin (FljB218A) and expressed in E. coli. Other authors have reported that a single amino acid substitution in bacterial FliC flagellin can alter the species-specific TLR5 response on hosts (22).

Culture supernatants of the selected 23D4 hybridome were assayed by ELISA against flagellar extracts of phase 1 and phase 2 from 89 Salmonella strains and 4 related species. The ELISA data were confirmed by Western blot and dot blot techniques. Only the phase 1 and phase 2 flagellar extracts of serovar Typhimurium, and flagellar extracts of serovar [4,5,12:i:–] reacted with MAb 23D4. The reaction observed with phase 1 extracts of serovar Typhimurium could be explained by the presence of phase 2 flagellum-expressing cells in the sample. The identification of FljB by protein sequencing on phase 1 extracts of serovars Typhimurium support this hypothesis. The selective pressure using antiflagellar-phase antibodies in culture media could be insufficient to change the flagella in all motile cells on cultures. However, phase 1 flagellin was not detected on phase 2 extracts of serovar Typhimurium. Higher selective pressure of anti-H:1,2 antibodies could explain this fact. Other serovars with H:1,2 antigen on phase 2 flagella, or with their phase 1 extracts, did not show reactivity with MAb 23D4 by ELISA, Western blot, and dot blot techniques. The epitope recognized by MAb 23D4 was not present on flagellar extracts of these serovars. Only flagellar extracts of serovar [4,5,12:i:–], a monophasic variant of serovar Typhimurium in which operon fljBA is deleted by mutation (13), reacted with MAb 23D4. In the other hand, the commercial antibody (antiflagellar H:1,2) used as a control reacted with serovar [4,5,12:i:–] and also with serovar Kentucky (phase 2 extracts and weakly on phase 1 of one strain). In both cases, the epitope recognized by our MAb and commercial antibody should be present in antigenic extracts of these serovars.

In order to determine which proteins were detected by MAb 23D4, flagellar extracts of the serovars Typhimurium and [4,5,12:i:–] were studied by 2D electrophoresis and Western blotting. The composition of heat flagellar extracts included mainly flagellins. Several spots were detected by protein-antibody reactions in each extract. Purified phase 2 flagellin and heat extracts of phase 2 of LT2 strain showed similar immunoreactive patterns, but the weak reactive spots 3, 5, and 6 were detected only in heat extracts of phase 2. Immunoreactive protein bands were excised manually, digested with trypsin, and sequenced. Combinations of identified peptides were searched against NCBI and Swiss-Prot databases. Different proteins were identified in each immunoreactive spot, and results indicating the obtained score and the numbers of peptides used for identification are presented in Fig. 3 and 4. As indicated above, FljB was detected in phase 1 and phase 2 extracts of serovar Typhimurium and in phase 2 purified flagellin. In purified phase 2 extracts, FljB was the unique protein identified in all immunoreactive spots. In heat phase 2 extracts, the flagellin of phase 1 was not detected. The pressure of anti-i antibody seems to eliminate FliC expression in Salmonella serovar Typhimurium cells. In extracts of phase 1 of LT2 strain, the flagellins FljB and FliC were jointly detected in spots. Heat extracts of serovar [4,5,12:i:–] did not contain the phase 2 flagellin as anticipated.

In order to detect the structural target of MAb 23D4, we applied IEM to complete bacteria with gold-marked antibodies. Using phase 2-expressing bacteria of serovar Typhimurium (LT2 strain), we observed that MAb was attached to the bottom of the flagellar structure but not to the filament of flagellin. Taking into consideration the possibility of lipopolysaccharide contamination of flagellum extracts, we analyzed by ELISA, Western blotting, and dot blotting several Salmonella strains that shared at least one O factor, but no reaction was observed (Table 1). Moreover, in IEM analyses of complete bacteria of different Salmonella serovars (strains 270, 448, and 531) with the same somatic, phase 1, and/or phase 2 flagellum antigens of serovar Typhimurium, no reaction was observed. The bacterial flagellum comprises a complex membrane-spanning organization encompassing numerous protein interactions. The bacterial flagellum assembles in a strict order, with structural subunits delivered to the growing flagellum by a type III export pathway (10, 25), and chaperone secretion is also involved in the regulation of flagellar assembly (1). Typical components in the assemblage of the extracellular structure of flagella are the hook (FlgE) and the filament (10). The latter comprises four types of filament-class subunits: FlgK, FlgL, and FliD, all named hook-associated proteins (HAPs), and flagellin (FliC or FljB). The major filament substructure comprises about 20,000 flagellin (FliC or FljB) subunits, which are polymerized under the distal filament cap, a FliD pentamer that is displaced farther from the cell as the filament grows. The filament is adapted to the flexible hook by a preformed hook filament junction made up of 11 subunits each of FlgK and FlgL. Bacteria can show multiple flagella at different stages of assembly (34). The proteins that could be related with our observed location of antibody attachment in the bottom of external flagellar structure can be viewed in Fig. 5. Some of the basal proteins—FlgE, FlgK, FlgL, and/or flagellin subunits at the bottom of the filaments—alone or jointly can form the epitope structure recognized by MAb 23D4. The IEM with MAb 23D4 failed to recognize the flagellum filament. Nevertheless, the results obtained by the other immunological tests indicate a reaction against flagellins. This fact could be explained either by acid or heat degradation treatments applied to obtain the flagellar extracts or by denaturing conditions of 1D and 2D SDS-PAGE. The degradations of 3D structure could be enough to expose the epitopes. The epitope exposition due to structural degradation did not explain why the other extracts from other serovars that express H:1,2 antigens were not recognized by MAb 23D4.

In addition to FliC and FljB, other proteins were detected in some of these immunoreactive spots. FlgK or HAP1 was weakly detected in spot 4 of phase 2 extracts of serovar Typhimurium and spot 15 of serovar [4,5,12:i:–]. This HAP (HAP1) is disposed in the bottom of the flagella and could explain the detected reaction. Thus, the structural epitope could be exposed on FlgK or jointly within the FljB/FlgK dimer or some other protein on the flagellar bottom. FlgK was weakly detected, probably due to its low representation in the flagellar structure. As mentioned before, each flagellum is composed of about 20,000 flagellin subunits and only 11 FlgK subunits (10). The molecular weight and pI of FlgK are similar to those of flagellin, and its low frequency could make it difficult to detect.

Other proteins, neither flagellins nor FlgK, were detected in other immunoreactive spots, including the following: InvE, present in Salmonella pathogenicity island 1 (SPI1); heat shock proteins GRoEL and DnaK, which function as molecular chaperones; the ABC-type superfamily of transporters, as dipeptide transport protein substrate binding domain (DppA) and spermidine/putrescine substrate binding component; several metabolic enzymes, such as as phosphopyruvate hydratase (enolase), adenylosuccinate synthetase, aromatic amino acid aminotransferase, aminopeptidase B, and isocitrate lyase; and two proteins related to protein synthesis as elongation factor Tu (EF-Tu) and 30S ribosomal protein S1. The detection using MAb 23D4 of several spots in flagellar extracts with only these proteins suggests that the recognized epitope could be shared by at least one protein in each spot, but the detection of this epitope was not addressed in this work. Additional research must be accomplished in order to determine the identity of this epitope.

Traditional serotyping is a time-consuming process and requires the use of 167 antisera and well-trained technicians (16). However, Salmonella serovars Enteritidis and Typhimurium constituted more than 80% of all the Salmonella serovars recorded in the Global Salm Surv database in 2000 to 2005 (http://www.who.int/salmsurv/en/). The implementation of the international regulations for Salmonella control (CE no. 584/2008 from the European Commission) requires more accurate and quicker methods than those based on immunological tests. The data obtained in the present study have demonstrated that the MAb 23D4 reacts with flagellar antigens of Salmonella serovars Typhimurium and [4,5,12:i:–] obtained by acid or heat treatment. A flagellar heat extract of a growing Salmonella strain could be used to detect and confirm these serovars through simple techniques such as dot blotting or ELISA. Phase reversal would be unnecessary for the identification of serovar Typhimurium strains. The MAb described here discerns the two serovars from other serovars of Salmonella that shared somatic, phase 1, and/or phase 2 flagellar antigens. No cross-reaction was observed with MAb 23D4 in the present study, except with the monophasic Salmonella serovar [4,5,12:i:–]. Punctual mutation of IVR that modified one amino acid on H:1,2 flagellin was not reactive with our MAb, which demonstrates its high specificity. In conclusion, the MAb 23D4 can be useful for the detection and diagnostic purposes for Salmonella serovar Typhimurium and serovar [4,5,12:i:–] and could also be helpful for the characterization of epitopes of the Salmonella H1 antigen complex.


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ACKNOWLEDGMENTS
 
We thank G. Lete for animal care in the Animalarium Facility; J. M. Arizmendi and K. Aloria for mass spectrometry analysis performed in the Proteomics Facility; and R. Andrade for the IEM analysis performed in the Biomedicine High Resolution and Analytic Microscopy Facility, SGIker, of the University of the Basque Country.

This study was supported in part by grant PI 1998/52 from the Basque Government, Subvención General a Grupos de Investigación grant 9/UPV 00093.125-13542/2001, and grant GIU05/42 from UPV/EHU. A. B. Vivanco was supported by a Beca de Investigación Predoctoral grant from the UPV/EHU of Spain.


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FOOTNOTES
 
* Corresponding author. Mailing address: Departamento de Inmunología, Microbiología y Parasitología, Facultad de Ciencia y Tecnología, UPV/EHU, Sarriena s/n, 48940-Leioa (Bizkaia), Spain. Phone: 34 946015964. Fax: 34 946013500. E-mail: aitor.rementeria{at}ehu.es Back

{triangledown} Published ahead of print on 5 January 2009. Back


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Applied and Environmental Microbiology, March 2009, p. 1345-1354, Vol. 75, No. 5
0099-2240/09/$08.00+0     doi:10.1128/AEM.01597-08
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