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Applied and Environmental Microbiology, September 1999, p. 4171-4180, Vol. 65, No. 9
Department of Plant Quality, Bulb Research Centre, 2160 AB
Lisse, The Netherlands1; Division of
General Microbiology, Department of Biosciences, University of
Helsinki, SF-00014 University of Helsinki,
Finland2; Complex Carbohydrate
Research Center, University of Georgia, Athens, Georgia
306023; Department of Nematology,
Landbouw Universiteit Wageningen, 6700 ES Wageningen, The
Netherlands4; and Molecular
Microbiology, IMBW/BCA Faculty of Biology, Vrije Universiteit, 1081 HV Amsterdam, The Netherlands5
Received 11 February 1999/Accepted 1 July 1999
The objective of this study was to develop a specific immunological
diagnostic assay for yellow disease in hyacinths, using monoclonal
antibodies (MAbs). Mice were immunized with a crude cell wall
preparation (shear fraction) from Xanthomonas hyacinthi and
with purified type IV fimbriae. Hybridomas were screened for a positive
reaction with X. hyacinthi cells or fimbriae and for a
negative reaction with X. translucens pv. graminis or
Erwinia carotovora subsp. carotovora. Nine MAbs
recognized fimbrial epitopes, as shown by immunoblotting,
immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and
immunoelectron microscopy; however, three of these MAbs had weak
cross-reactions with two X. translucens pathovars in
immunoblotting experiments. Seven MAbs reacted with lipopolysaccharides
and yielded a low-mobility ladder pattern on immunoblots.
Subsequent analysis of MAb 2E5 showed that it specifically recognized
an epitope on the O antigen, which was found to consist of rhamnose and
fucose in a 2:1 molar ratio. The cross-reaction of MAb 2E5 with all
X. hyacinthi strains tested showed that this O antigen is
highly conserved within this species. MAb 1B10 also reacted with
lipopolysaccharides. MAbs 2E5 and 1B10 were further tested in ELISA and
immunoblotting experiments with cells and extracts from other
pathogens. No cross-reaction was found with 27 other
Xanthomonas pathovars tested or with 14 other bacterial
species from other genera, such as Erwinia and
Pseudomonas, indicating the high specificity of these
antibodies. MAbs 2E5 and 1B10 were shown to be useful in ELISA for the
detection of X. hyacinthi in infected hyacinths.
Xanthomonas hyacinthi (ex
Wakker 1883) sp. nov., nom. rev. from Xanthomonas campestris
pv. hyacinthi (36) infects hyacinths and some related
ornamental bulbous crops (14). The spread of an X. hyacinthi infection (yellow disease) can be a fast process (35) and can cause considerable economic loss for the
hyacinth growers; for example, 710,000 hyacinth bulbs were condemned
because of yellow disease in 1997. Consequently, a rapid diagnosis for the presence of X. hyacinthi in plant material is a
prerequisite for taking decisive actions to prevent further spread of
disease. Xanthomonas pathovars are difficult to distinguish,
as they are almost identical in bacteriological and biochemical traits
(7). Pathovars of Xanthomonas can be
differentiated by their ability to infect certain host plants, often
economically important plant crops. A reclassification study of the
genus Xanthomonas, using phenotypic, chemotaxonomic, and
genotypic approaches, showed that X. hyacinthi strains
constitute a homogenous group with a genotype distinct from that of
X. campestris (36).
Various reports describe the identification and detection of
Xanthomonas species and pathovars by serological (2, 4, 5) and DNA-based (17, 18, 30) methods. The detection of plant pathogens with antisera is still the method of choice for many
plant inspection services because of the relatively low costs and the
presence of technical infrastructure based on automated enzyme-linked
immunosorbent assays (ELISA). Therefore, we initiated a study of the
surface antigens of X. hyacinthi for the development of a
serological detection assay. There are several reports describing the
production of monoclonal antibodies (MAbs) specific for
Xanthomonas pathovars (2, 4, 5). Most of the
strategies used involved immunization of mice with whole-cell
preparations; however, raising antibodies to well-characterized
antigens would have obvious advantages.
Recently, the existence of type IV fimbriae among xanthomonads has been
reported (24, 34). These filamentous protein polymers have
been shown to have antigenic properties as good as those of type IV
fimbriae of other bacterial species, such as Neisseria gonorrhoeae and Pseudomonas aeruginosa (22,
31). Immunoblot experiments indicated that Xanthomonas
pathovars showed variation in molecular mass of the fimbrial subunit
(34). These findings suggested that Xanthomonas
type IV fimbriae may contain unique determinants, as found for other
type IV fimbriae, and that this multimeric surface antigen from
X. hyacinthi might be suitable for MAb production. Other
components of Xanthomonas known to have antigenic properties
include the outer membrane proteins and lipopolysaccharide (LPS)
(1-5), and it has been shown that variation in outer
membrane proteins and LPS is correlated with the pathovar grouping of
X. campestris (21, 23).
In this report, we obtained MAbs by immunizing mice with purified
fimbriae and shear fractions of X. hyacinthi and analyzed them for application to phytodiagnostic purposes. The antifimbria MAbs
reacted with different fimbrial epitopes. We found that the anti-LPS
MAbs recognized the O antigen of X. hyacinthi S148.
This antibody was found to be X. hyacinthi specific. The O
antigen of S148 was partially characterized and shown to consist of
rhamnose (Rha) and fucose (Fuc). The cross-reaction of these MAbs with all strains of X. hyacinthi tested showed that this Rha-Fuc
O antigen is conserved within the species.
Cultures and media.
The bacterial strains used in this study
are listed in Table 1. Bacterial cells
were cultured on nutrient yeast agar (NYA; Difco Laboratories, Detroit,
Mich.). Some Xanthomonas species and pathovars were grown on
different media as prescribed by the LMG Culture Collection, Ghent,
Belgium.
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Development and Application of Pathovar-Specific Monoclonal
Antibodies That Recognize the Lipopolysaccharide O Antigen and the
Type IV Fimbriae of Xanthomonas hyacinthi
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Bacterial strains used in this study
Cultivation and inoculation of hyacinths. The cultivars Anna Marie, Carnegie, Pink Pearl, and King of the Blues were maintained in a greenhouse with a day-night regimen of 12 h of light (25°C; relative humidity, 70%) and 12 h of darkness (10°C; relative humidity, 90%). Leaves were spray inoculated with bacterial suspensions (106 to 107 CFU/ml) or with phosphate-buffered saline (PBS) as a control. X. hyacinthi S148 and TV45 were used for these experiments. After 2 to 3 weeks, lesions were visible and leaf material was collected for experimental use.
Preparation of antigens and immunization of mice. Cell extracts were prepared by ultrasonication (Branson, Danbury, Conn.) of bacterial suspensions (109/ml), as previously described (34). Protein-free samples were prepared by incubation with 50 µg of proteinase K (Sigma, St. Louis, Mo.) per ml for 120 min at 60°C. A crude outer bacterial surface preparation was obtained by shearing cells of strain S148 as previously described (34) and concentrating to 0.5 mg of protein/ml in PBS. Two 6-week-old New Zealand mice were injected intraperitoneally with 0.25 ml of this preparation, mixed 1:1 with Freund's complete adjuvant (FCA), three times at 2-week intervals. Native fimbriae were isolated and purified from strain S148 (34) and concentrated to 0.5 and 0.1 mg/ml in PBS, respectively. The fimbrial preparations were mixed 1:1 with FCA, and from each preparation, 0.25 ml was injected intraperitoneally (three times with 2-week intervals) in two 6-week-old BALB/c mice. Two weeks after the third injection, the mice received a final booster injection with the same preparation but without FCA, and after 4 days, the spleens were collected.
Modification of protein antigens.
Differential epitope
recognition of the antifimbrial MAbs was visualized by immunoblotting
proteolytic fragments of the fimbrial 17-kDa protein subunit following
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
These fragments were obtained by partial proteolysis of the 17-kDa
protein with 
-chymotrypsin (Sigma) using 1 U/µg of protein for 10 min at 25°C.
Hybridoma production. Hybridoma cells were produced by using equal amounts of the myeloma cell line Sp2/0-Ag-14 and spleen cells from the immunized mice which showed the highest titer in ELISA against fimbrial preparations or X. hyacinthi whole-cell preparations. Fusion and culturing of hybridoma cell lines were performed by the method of Boonekamp et al. (6). To prevent growth of yeast, 5 µg of amphotericin B (Fungizone; Imperial Laboratories Ltd., Andover, United Kingdom) was added per ml of medium. Selected hybridoma cultures were subcloned by the limiting dilution technique.
MAb production and isotyping.
Hybridoma fluid samples were
centrifuged for 10 min at 1,000 × g (Sigma 2-15 centrifuge, 4×25 ml rotor, 4°C) to remove cellular debris. Culture
supernatants were used directly for immunological experiments or stored
at 
20°C. Immunoglobulin isotyping was performed with the Sigma
Mouse Monoclonal Antibody Isotyping kit.
ELISA.
The antigen-coated plate (ACP)-ELISA was used for the
screening of positively reacting hybridoma supernatants. Polyvinyl
chloride 96-well plates (Costar, Cambridge, Mass., and Greiner,
Frickenhausen, Germany) were coated with 100-µl portions of bacterial
suspension (approximately 108 bacterial cells/ml) in
coating buffer (0.05 M carbonate-bicarbonate buffer [pH 9.6]) by
drying in a 37°C ventilated incubator and stored at 20°C until
used. In some experiments, microtiter plates were coated with purified
fimbriae (3 µg/well). For direct antibody sandwich (DAS)-ELISA,
plates were coated with gamma globulin, isolated from polyclonal rabbit
antisera raised against whole cells of X. hyacinthi S148,
with 0.1 µg of gamma globulin/well. Subsequently, bacterial dilutions
or infected-hyacinth extracts were added and incubated at 20°C.
ACP-ELISA and DAS-ELISA plates were blocked with 0.5% skim milk powder
(Oxoid, Basingstoke, United Kingdom) in PBS with 0.5% Tween 20 (PBST),
washed three times with PBST, and hybridoma supernatant (100 µl/well)
was added. Following incubation for 2 h at 37°C, plates were
washed, and goat anti-mouse alkaline phosphatase (American Qualex, La
Miranda, Calif.) diluted 1:10,000 was added for 1 h at 37°C.
Plates were washed with PBST and developed by adding substrate,
consisting of p-nitrophenyl-phosphate in 10% (vol/vol)
diethanolamine buffer, pH 9.8. Absorbance at 405 nm was measured with
an Anthos Labtec ELISA reader after 1 and 2 h of incubation at
37°C. The positive-negative threshold of the ELISA was determined as
three times the mean of the absorbance of PBS control samples or
healthy hyacinth samples. In practice, this meant that absorbance
values above 0.08 were considered positive.
LPS isolation. For preparation of LPS, the cell pellet obtained from a 5-liter culture of strain S148 was extracted by a modified hot phenol-water method (10) and the aqueous-phase material was fractionated by size exclusion chromatography on a Sephadex G-150 superfine column (Pharmacia, Uppsala, Sweden), as previously described (26). The eluted fractions were assayed colorimetrically for 3-deoxy-D-manno-2-octulosonic acid (Kdo) by the thiobarbituric acid assay (38) and for hexose with phenol-sulfuric acid (40); LPS-containing fractions were identified by PAGE analysis.
PAGE and immunoblot analyses. LPS samples were analyzed by deoxycholic acid PAGE, using 18% acrylamide gels. The gels were either silver stained for LPS (32) or Alcian blue-silver stained for possible K antigens (26). For immunological analysis of LPS samples, polyacrylamide gels were blotted to Nytran+ membranes (Schleicher and Schuell, Keene, N.H.) with a Trans-Blot SD apparatus (Bio-Rad). Fimbriae or other protein samples were analyzed by SDS-PAGE, using 12% Laemmli gels (15). Immunoblotting of separated protein samples was performed as previously described (34). In some experiments, a slot blot apparatus (Miniblotter 16; Immunetics, Cambridge, Mass.) was used for simultaneous testing of several antisera for recognition of blotted antigens.
Processing of leaf tissue samples of hyacinths. Inoculated areas of hyacinth leaves were excised (1 cm2; average wet weight of 60 mg) and immersed in 1-ml portions of PBST in small plastic bags. Homogenization of the samples was performed with a hand homogenizer (Bioreba AG, Basel, Switzerland). Subsequently, the plant extracts were serially diluted in antibody-coated microtiter plates and processed as described above.
Immunofluorescence experiments. Samples of hyacinths infected with X. hyacinthi and samples of bacteria were incubated on microscope slides and examined as previously described (34). For fluorescence labeling, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse polyvalent immunoglobulins (immunoglobulin A [IgA], IgG, and IgM) and goat anti-rabbit IgG (Sigma) were used.
Glycosyl residue analysis. Sugar composition analysis was performed by gas chromatography-mass spectrometry of the trimethylsilyl methyl glycoside derivatives (40), using a 30-m DB1 fused silica column (J&W Scientific, Folsum, Calif.) on a 5890A GC-MSD (Hewlett-Packard, Palo Alto, Calif.). Inositol was used as an internal standard, and retention times were compared to those of authentic monosaccharide standards.
1H NMR analyses. Proton nuclear magnetic resonance (1H NMR) spectroscopy was performed with a Brüker AM 250 apparatus. The samples were dissolved in 2H2O, and the spectra were obtained at 308 K. Chemical shifts were established relative to acetone.
Immunoelectron microscopy. Incubation of X. hyacinthi cells on Formvar-carbon-coated nickel grids with antisera was performed as previously described (34). MAbs were used in dilutions of 1:5 or 1:10 in PBS. Goat anti-mouse IgG and IgM, conjugated with 10-nm-diameter gold particles (Biocell Laboratories International, Cardiff, United Kingdom), was used as the secondary antibody. After counterstaining with phosphotungstic acid and air drying, grids were examined with a Philips transmission electron microscope type 201 or CM100, each operated at 60 kV.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Selection of MAbs specific for X. hyacinthi.
Immunizations with LPS and fimbrial fractions yielded numerous
hybridoma cell lines. About 60 clones reacted positively with X. hyacinthi samples in ELISA or immunoblot analyses. Subsequent screening by ELISA and in immunoblotting experiments of the
hybridoma clones for negative reactions with Erwinia
carotovora subsp. carotovora 550 and X. campestris pv. graminis LMG 726 identified 15 clones which
recognized exclusively X. hyacinthi antigens (Table
2).
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Specificities of the MAbs. Two distinct groups of MAbs were identified: group 8 MAbs, resulting from immunization with a shear fraction of X. hyacinthi S148, reacted strongly in ACP-ELISA with X. hyacinthi isolates. Group 15 MAbs, resulting from immunization with fimbriae, reacted strongly with a protein band in immunoblotting but only weakly with whole-cell antigens in ACP-ELISA. The MAbs belonged to the immunoglobulin classes IgG1, IgG3, IgG2a, IgG2b, and IgM (Table 2).
To assess whether the MAbs reacted with a polysaccharide component or a protein epitope, X. hyacinthi cells were sonicated and incubated with or without proteinase K. Next, these extracts were separated by SDS-PAGE and blotted onto nitrocellulose membranes. Analyses of the immunoblots showed that the group 15 MAbs reacted with the 17-kDa fimbrial subunit protein (Fig. 1A). The group 8 MAbs yielded a ladder-like pattern, suggesting that the reaction was with a polysaccharide component of X. hyacinthi (Fig. 1B).
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Recognition of antigens by group 8 and group 15 MAbs. The group 15 MAbs were tested with different immunological techniques (Table 2). All nine MAbs reacted with the fimbrial subunit of X. hyacinthi isolates in immunoblotting experiments, but not with X. translucens pv. graminis. MAb 12G9 showed a relatively weak reaction, suggesting that it recognized a different subunit epitope than MAb 8C11. The group 15 MAbs reacted weakly in a DAS-ELISA with X. hyacinthi cells. When applied to immunofluorescence labeling, the group 15 MAbs 5D12, 1A7, 9A2, and especially MAb 6C9 showed polar labeling of individual cells; fluorescent fimbrial filaments could be visualized under the microscope (Fig. 3A). The group 15 MAbs were also used for immunogold localization studies using electron microscopy (Fig. 4B to D). A distinct tagging of the fimbriae from strain S148 with gold particles was found for MAbs 6C9, 5G8, and 9A2. Some unlabeled fimbriae were also visible in Fig. 4C. The fimbriae of the other strains of X. hyacinthi (Table 1) were labeled with gold as well (not shown). MAb 5D12 labeled only certain parts of the fimbriae but not the tips (not shown).
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-chymotrypsin
(Fig. 5). MAbs 12G9 and 1A7 recognized
only the intact 17-kDa fimbrial subunit, whereas MAbs 6C9, 8C11, and
5D12 recognized other, smaller peptide fragments as well. Polyclonal rabbit antiserum against the 17-kDa fimbrial subunit recognized multimers of the fimbrial subunit in immunoblotting experiments (Fig.
5, lane 9).
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Antigen characterization. Polysaccharides were extracted from S148 cells and analyzed by SDS-PAGE and Alcian blue-silver staining to characterize the antigen recognized by group 8 MAbs (Fig. 7A, lane 1). Two major components were found in the extract. The high-mobility banding component is probably rough LPS (R-LPS); the low-mobility component showing a ladder pattern (region I) is probably the result of a sequential degree of polymerization of structurally constant repeating units, which could be from capsular antigens or smooth LPS (S-LPS) (9, 25). The fact that both components were visible without the Alcian blue prestain (not shown) suggested that the ladder pattern was the result of the presence of S-LPS (26). A portion of the preparation was subjected to polymyxin chromatography, which specifically binds LPS. The ladder pattern material was bound by the polymyxin, which also indicated that region I is due to S-LPS (data not shown).
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Purification and structural analyses of the LPS component, recognized by MAb 2E5. A crude polysaccharide preparation was fractionated by size exclusion chromatography which resulted in two major pools that were analyzed by SDS-PAGE (Fig. 7A, lanes 2 and 3). This showed that the chromatography completely separated the R-LPS from the region I material. Both were analyzed by immunoblotting (Fig. 7B, lanes 2 and 3). As expected, a ladder pattern in region I was found. In contrast, there was no recognition of the highly mobile R-LPS. Both components were then analyzed for glycosyl residue composition and by 1H NMR spectroscopy (Fig. 8).
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-OH
fatty acids that are common components of LPS. Although a lack of
standards precludes a molar comparison to the glycosyl components, the
fatty acids were abundant and detected in a 1:1 ratio of 3-OH--C-12 and
3-OH--C-13, with minor amounts of 3-OH--C-14. 1H NMR
analysis of the R-LPS showed the signals that arise from the fatty
acids (Fig. 8A). The signals at 1.2 to 1.5 ppm are due to the various
CH2 protons, and the signal at 0.8 ppm is from the terminal
CH3. The resonances from the carbohydrate protons are found
between 3.4 and 4.4 ppm, and the two sharp resonances at 1.95 and 2.05 ppm indicate the presence of acetyl groups on some of the glycosyl
residues. Although poorly resolved (due to the poor solubility of the
sample), the resonances at 1.8 and 2.4 ppm indicate that there is, in
fact, some Kdo associated with the LPS core. In contrast to the R-LPS,
the low-mobility ladder pattern material contained predominantly Rha
and Fuc (>95% of total carbohydrate) in a 2:1 molar ratio. However,
the fatty acids and GlcNAc of lipid A, as well as the core glycosyl
components, were also present in the region I preparation. This
demonstrated that MAb 2E5 is specific for the O antigen of the LPS. The
NMR spectrum (Fig. 8B) showed that the signals associated with the C-6
methyl protons of the Rha and Fuc are found at 1.4 and 1.15 ppm,
respectively, and those of the anomeric region are found at 4.6 to 5.4 ppm. A minor resonance at 0.8 ppm is due to the terminal methyl protons
of the fatty acids, and the CH2 resonances are obscured by
the Rha and Fuc signals. The 2:1 molar ratio of Rha to Fuc is supported
by the relative areas of the C-6 methyl proton resonances; however, it
is not clear from the present data if the O antigen is comprised of
linear or branched trisaccharide repeats.
In conclusion, all data indicated that the ladder pattern is a result
of the sequential degree of polymerization of O antigen in the S-LPS.
Due to the fact that MAb 2E5 did not recognize the R-LPS, we can
conclude that the epitope is the O antigen, which consists of Rha and Fuc.
Sensitivity of MAbs and application in routine detection of yellow disease. To determine the lowest detection threshold for X. hyacinthi, microtiter plates were coated with serially diluted bacterial cells (ACP-ELISA). The range of healthy background was 0.00 to 0.02; which differed significantly (data not shown) from the lower limits of the ELISA. MAb 2E5 could detect as little as 10,000 CFU/100 µl; the detection limit of MAb 1B10 was about 85,000 CFU/100 µl (Fig. 6B). For routine detection of hyacinth plants suspected to be infected, leaf extracts were processed as described above and tested in DAS-ELISA with MAbs 2E5 and 1B10; the detection limits were about 20,000 CFU/100 µl (33 × 105 CFU per g of hyacinth leaf) and 100,000 CFU/100 µl (17 × 106 CFU per g of hyacinth leaf), respectively. When used in immunofluorescence labeling experiments, MAb 2E5 could detect approximately 1,000 cells/ml of hyacinth leaf extract.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In a previous study of X. hyacinthi fimbriae, polyclonal antisera raised against preparations of native or denatured fimbriae of X. hyacinthi recognized common epitopes of fimbriae and LPS from other xanthomonads (34). Although the signal in ELISA or immunoblots was considerably weaker than signal obtained with X. hyacinthi strains, these polyclonal sera cannot be used for routine detection purposes. The results of this study showed that two panels of anti-X. hyacinthi MAbs recognize two defined surface antigens: type IV fimbriae and LPS O antigen. The MAbs recognizing the O antigen of X. hyacinthi showed no cross-reaction with any other xanthomonads (Table 1). This was important, as we have occasionally isolated other pathovars from hyacinth plants, including X. translucens pv. graminis (33).
Pathovar- or species-specific MAbs have been made against several xanthomonads, including X. campestris pv. pelargonii, pv. begoniae, and pv. oryzicola. The antibodies recognizing these pathovars have been shown or suggested to react with LPS or other cell surface polysaccharides (4, 5). We found that the epitope recognized by anti-X. hyacinthi MAb 2E5 is the LPS O antigen. Although there was no reaction of MAb 2E5 with the R-LPS, composition analysis showed that the region I material contained typical lipid A fatty acid and the same sugars found in the R-LPS. We also found that the O antigen from X. hyacinthi S148 consists of rhamnose and fucose in a 2:1 molar ratio, which is different from the structurally characterized O antigen of X. campestris pv. campestris NCPPB 45. The latter has a complex hexasaccharide repeating structure consisting of rhamnose, galactose, and galacturonic acid in a molar ratio of 4:1:1 (8). There are other Xanthomonas pathovars (pv. pelargonii, malvacearum, and vasculorum) that produce fucose-containing LPSs (23, 37). However, these bacteria must have epitopes that are immunologically from those of X. hyacinthi, since no cross-reaction was found with O-antigen-specific MAbs or antifimbrial MAbs. All strains of X. hyacinthi were recognized by MAb 2E5, indicating that the O antigen is not strain specific. This is in contrast to what has been found for enteropathogens, such as Escherichia coli or Salmonella spp., which produce highly variable O antigens (39). Reports on the occurrence of X. hyacinthi in the field are (with a few exceptions) restricted to The Netherlands, where by far the greater part of hyacinth culture is situated (35). Recently, X. hyacnthi was reclassified as a new Xanthomonas species, based on biochemical characteristics and its high G+C content (69%) of DNA (36). The collection of X. hyacinthi isolates used in this study were isolated from sites separated in time, place, and host plant. The isolates showed differences in growth rate, production of extracellular polysaccharides, and rate of pathogenicity in hyacinths (33).
Analysis of X. hyacinthi S148 R-LPS core indicated the presence of Kdo and equimolar amounts of mannose, glucose, and galacturonic acid. The presence of low levels of Kdo is in agreement with the finding that the LPS of Xanthomonas sinensis contains one Kdo per LPS molecule (38). In addition, the core composition of X. hyacinthi S148 is generally similar to that of Xanthomonas sinensis (19) and, interestingly, the R-LPSs of Sinorhizobium spp. (27, 28). Future studies may determine whether the LPS core regions of these bacterial species are structurally related.
Fimbriae were only recently described for Xanthomonas (24, 34), and this is the first report of MAbs that recognize these filamentous protein structures. Most antifimbria (group 15) MAbs showed good reaction when applied in immunoblotting experiments, in contrast to their low titer in DAS-ELISA. Immunization of mice with purified type IV fimbriae from X. hyacinthi S148 resulted in nine MAbs. Three of these MAbs showed only weak cross-reactivity with X. translucens pv. hordei and pv. translucens, both of which were isolated from Hordeum vulgare (barley). The type IV fimbriae from X. hyacinthi and X. translucens might have common features and bind to compatible leaf surface receptors of these monocots. The type IV fimbriae are expressed by a number of bacterial genera, and many aspects of their structure and immunological properties have been described (29). The fimbrial subunit of type IV fimbriae has a conserved N-terminal amino acid sequence and an immunodominant variable central and C-terminal domain. The specificities of the antifimbrial MAbs described here suggest that these parts are recognized by the MAbs. Type IV fimbriae are found and expressed by a number of Xanthomonas species and their pathovars (24, 34). A strategy of raising MAbs against type IV fimbriae of other Xanthomonas species and their pathovars might be feasible. Another approach is the production of polyclonal sera against synthetic peptides, homologous to the variable and immunodominant domains of the fimbriae (12, 22), resulting in diagnostically applicable antibodies.
A drawback for using type IV fimbriae as the target antigen might be the existence of phase variation or antigenic variation as described for N. gonorrhoeae and Moraxella bovis (13, 20). However, these phenomena were not found for any of the isolates of X. hyacinthi under study. The presence of fimbria-like structures that are not recognized by the antifimbrial MAbs (Fig. 4C) indicates the existence of at least one other type of fimbriae. The expression of different fimbriae in the genus Xanthomonas has been detected with X. campestris pv. vesicatoria mutants (24). The attachment of X. hyacinthi and its type IV fimbriae to stomata of hyacinth leaves (34) suggests a role for these surface antigens in the first stages of yellow disease. Attachment could be inhibited by incubation with antifimbria Fab fragments (34). The adherence of Pseudomonas aeruginosa to receptors on human epithelial cells can be blocked by MAbs binding to the C-terminal region of the fimbrial subunit located at the tip (11, 16). As the X. hyacinthi antifimbria MAbs showed variation in epitope recognition (Fig. 5), we are currently investigating the application of these MAbs in inhibition experiments of fimbrial attachment.
Importantly, six of the antifimbrial MAbs and in particular the anti-LPS MAbs may be used for the specific detection of X. hyacinthi. MAbs 2E5 and 1B10 are currently being tested by the Bulb Inspection Service of The Netherlands. The sensitivity under laboratory conditions of MAb 2E5 is limited to 20,000 CFU/100 µl; under routine conditions, the detection limit in hyacinth leaf extract is close to 50,000 CFU/100 µl. In most cases, this is sufficient to detect the presence of X. hyacinthi in the infection sites; the application of these MAbs in immunofluorescence labeling experiments results in higher sensitivity of the detection of this bacterium. However, the use of the MAbs for routine detection with DAS-ELISA is preferred by most inspection services, due to the fast and automated logistics developed over the last decade.
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ACKNOWLEDGMENTS |
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T.O.-R. was supported by the Academy of Finland. B.L.R. was funded by grant MCB-9728564 from the National Science Foundation and by the U.S. Department of Energy-funded Center for Plant and Microbial Complex Carbohydrate Research under grant DE-FG02-93ER-20097.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Plant Quality, Bulb Research Centre, P. O. Box 85, 2160 AB Lisse, The Netherlands. Phone: 31(0)252462121. Fax: 31(0)252417762. E-mail: joop.van.doorn{at}lbo.agro.nl.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Applied and Environmental Microbiology, September 1999, p. 4171-4180, Vol. 65, No. 9
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