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Clinical and Diagnostic Laboratory Immunology, July 1999, p. 627-629, Vol. 6, No. 4
1071-412X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Brucella Outer Membrane Lipoproteins
Share Antigenic Determinants with Bacteria of the Family
Rhizobiaceae
Axel
Cloeckaert,1,*
Anne
Tibor,2 and
Michel S.
Zygmunt1
Laboratoire de Pathologie Infectieuse et
Immunologie, Institut National de la Recherche Agronomique, 37380 Nouzilly, France,1 and Unité
d'Immunologie-Microbiologie, Facultés Universitaires Notre-Dame
de la Paix, Namur, Belgium2
Received 6 November 1998/Returned for modification 19 January
1999/Accepted 6 April 1999
 |
ABSTRACT |
Brucellae have been reported to be phylogenetically related to
bacteria of the family Rhizobiaceae. In the present study, we used a panel of monoclonal antibodies (MAbs) to Brucella
outer membrane proteins (OMPs) to determine the presence of common OMP epitopes in some representative bacteria of this family, i.e., Ochrobactrum anthropi, Phyllobacterium
rubiacearum, Rhizobium leguminosarum, and
Agrobacterium tumefaciens, and also in bacteria reported to
serologically cross-react with brucella, i.e., Yersinia enterocolitica O:9, Escherichia coli O:157, and
Salmonella urbana. In particular, most MAbs to the
Brucella outer membrane lipoproteins Omp10, Omp16, and
Omp19 cross-reacted with O. anthropi and P. rubiacearum, which are actually the closest relatives of
brucellae. Some of them also cross-reacted, but to a lower extent, with
R. leguminosarum and A. tumefaciens. The
putative Omp16 and Omp19 homologs in these bacteria showed the same
apparent molecular masses as their Brucella counterparts.
None of the antilipoprotein MAbs cross-reacted with Y. enterocolitica O:9, E. coli O:157, or S. urbana.
| |
TEXT |
Brucellae are gram-negative,
facultative, intracellular bacteria that can infect humans and many
species of animals. Six species are recognized within the genus
Brucella: B. abortus, B. melitensis, B. suis, B. ovis, B. canis, and
B. neotomae (7). These classifications are based
mainly on their differences in pathogenicity and host preference
(7). The Brucella species constitute a very
homogeneous group, as shown by their antigenic relatedness and by
DNA-DNA hybridization studies (>90% DNA homology for all species)
(8, 9, 25). On the basis of the 16S rRNA sequence, brucellae have been shown to belong to the family Rhizobiaceae
(27). This family includes plant and animal pathogens, such
as Agrobacterium, Bartonella, and
Brucella, that are characteristically associated pericellularly or intracellularly with eukaryotic cells; plant endosymbionts, such as Rhizobium and
Phyllobacterium; soil inhabitants, such as
Mycoplana; and isolates from soil and from human clinical specimens, such as Ochrobactrum (14, 18, 19).
Among all these bacteria, Ochrobactrum anthropi is the
closest known relative of brucellae (14, 24, 27). This
bacterium has gained interest in the past few years because of its
isolation from immunocompromised hosts (1, 11-13). Recent
reports have also described immunological cross-reactions between
Brucella spp. and O. anthropi (23,
24). The antigens containing common epitopes were described as
rough lipopolysaccharide and soluble and membrane proteins of unknown nature (23, 24). Since O. anthropi constitutes a
heterogeneous group of bacteria on the basis of classical phenotypical
characterization and DNA-DNA hybridization studies, further subdivision
of the genus into two species, O. anthropi and O. intermedium, has recently been proposed (24). The
latter, new species name has been suggested because of a closer genetic
and antigenic relationship with brucellae than with O. anthropi (24). Additionally, brucellae also share epitopes, mainly on the smooth lipopolysaccharide (S-LPS), with bacteria reported earlier to serologically cross-react with
Brucella, of which the most important is Yersinia
enterocolitica O:9 (7).
The Brucella outer membrane contains three major proteins
with molecular masses ranging from 25 to 27, 31 to 34, and 36 to 38 kDa
(2, 6). The largest protein has been identified and characterized as a porin (10, 17). The genes coding for
these proteins have been cloned and sequenced, and the current names for these outer membrane proteins (OMPs) are Omp25, Omp31, and Omp2b,
respectively (4, 5, 17). The other OMPs identified so far by
use of monoclonal antibodies (MAbs) are less abundant (minor) proteins
with molecular masses of 10, 16.5, 19, and 89 kDa (2). Gene
cloning, the predicted amino acid sequences, and the presence of
particular protein motifs have identified the 10-, 16.5-, and 19-kDa
OMPs as outer membrane lipoproteins (21, 22). The current
names for these OMPs are Omp10, Omp16, and Omp19, respectively
(21, 22). Omp16 actually belongs to the
peptidoglycan-associated lipoprotein family of proteins found in many
gram-negative bacteria (22). Homologs of Omp10 and Omp19 have not yet been reported for other bacteria. All of these proteins have been found as immunogenic proteins in infected cattle, sheep, and
goats (3, 15, 16, 21, 28).
In the present study, we used MAbs to analyze the occurrence of
epitopes common to Brucella OMPs in phylogenetically related bacteria of the family Rhizobiaceae and reported
S-LPS-cross-reacting bacteria as well. The importance of the epitopes
recognized by the MAbs in the antibody responses of
Brucella-infected cattle and sheep has been previously shown
by competitive enzyme-linked immunosorbent assay (ELISA) with these
MAbs (3, 28). The occurrence of common epitopes could
explain some of the serologic protein cross-reactivities reported
between Brucella and Ochrobactrum (23,
24). In addition, the present study also led to the
identification of new homologous proteins within the family
Rhizobiaceae.
The strains studied that belong to the family Rhizobiaceae
were O. anthropi 3301 (proposed as a reference strain for
O. intermedium), O. anthropi 3331, Phyllobacterium rubiacearum Pr1, Rhizobium
leguminosarum R11, and Agrobacterium tumefaciens At1
(26). The S-LPS-cross-reacting bacteria were Y. enterocolitica O:9 strain Ye8, Escherichia coli O:157
strain Ec2, and Salmonella urbana Su1 (26).
B. abortus 544 (biovar 1) was used as a reference. Strains
were grown on tryptic soy agar (Gibco BRL) supplemented with 0.1%
(wt/vol) yeast extract (Difco) at 37°C. R. leguminosarum
was cultured in tryptone-yeast medium at 30°C (20). MAbs
used were those of previous studies (2, 3, 6, 21, 22, 26,
28), and they were used as hybridoma culture supernatants
(twofold diluted in ELISA and immunoblotting).
The occurrence of cross-reacting epitopes was first screened by ELISA,
performed as described previously (2, 5, 28). Microtiter
plates were coated with bacterial suspensions in phosphate-buffered saline at an absorbance (600 nm) of 1.0. To improve accessibility of
OMPs, bacteria were sonicated prior to coating (5). MAbs were used at a dilution of 1/2. Positive control MAbs were 3D6, specific for peptidoglycan (6), and A53/09G03/D02, specific for DnaK, previously shown to cross-react with O. anthropi
and P. rubiacearum (26).
In particular, most MAbs to the outer membrane lipoproteins Omp10,
Omp16, and Omp19 cross-reacted in ELISA with both O. anthropi 3301 and 3331 and P. rubiacearum (Table
1). Fewer MAbs against the three OMPs
reacted with R. leguminosarum, and only one MAb, against
Omp16, reacted weakly with A. tumefaciens. None of these MAbs reacted with the S-LPS-cross-reacting bacteria Y. enterocolitica O:9, E. coli O:157, and S. urbana. The MAb bindings observed correlated with the genetic
closeness to brucellae. However, there was no significant difference in
MAb bindings between O. anthropi 3301 (proposed as O. intermedium) and O. anthropi 3331.
In immunoblotting after sodium dodecyl sulfate-polyacrylamide gel
electrophoresis, performed as described previously (2, 28),
the anti-Omp16 MAbs reacted strongly with O. anthropi 3301 and 3331, P. rubiacearum, and R. leguminosarum
and weakly with A. tumefaciens, thus confirming the ELISA
results (Fig. 1). The anti-Omp19 MAbs
reacted strongly only with O. anthropi and P. rubiacearum, which is also in accordance with the ELISA results. The putative Omp16 and Omp19 homologs detected by the MAbs in these
bacteria showed the same apparent molecular masses as their Brucella counterparts. The anti-Omp10 MAbs gave no positive
reactions in immunoblotting and reacted only weakly with B. abortus, which was used as the control (Fig. 1).
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FIG. 1.
Reactivity in immunoblotting of anti-Omp10 (lane 1),
anti-Omp16 (lanes 2 to 6), and anti-Omp19 MAbs (lanes 7 to 9) after
sodium dodecyl sulfate-polyacrylamide gel electrophoresis of B. abortus 544 (lanes 1, 2, and 7), O. anthropi (strains
3301 and 3331 gave the same result) (lanes 3 and 8), P. rubiacearum (lanes 4 and 9), A. tumefaciens (lane 5),
and R. leguminosarum (lane 6).
|
|
In conclusion, the present study showed the presence of epitopes
cross-reactive with Brucella outer membrane lipoproteins on
genetically related bacteria, of which the most important is O. anthropi. Of particular interest are the lipoproteins Omp10 and
Omp19, not yet reported for other bacteria. Thus, these proteins could
constitute a new family of OMPs specifically encountered in
Rhizobiaceae. As suggested by Velasco et al.
(23), the immunoresponse of Brucella-infected
hosts to protein antigens may not necessarily be specific for
brucellae, and the presence of O. anthropi or related
bacteria may explain previously described reactivities to OMPs in
healthy animals (16). The outer membrane lipoproteins Omp10,
Omp16, and Omp19 are the first identified among these OMPs.
| |
ACKNOWLEDGMENTS |
We thank J. M. Verger and M. Grayon for supplying the strains.
We also thank S. Baucheron for technical support.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Pathologie Infectieuse et Immunologie, Institut National de la
Recherche Agronomique, 37380 Nouzilly, France. Phone: 2 47 42 78 72. Fax: 2 47 42 77 79. E-mail: cloeckae{at}tours.inra.fr.
| |
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Clinical and Diagnostic Laboratory Immunology, July 1999, p. 627-629, Vol. 6, No. 4
1071-412X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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