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Conservation of seven genes involved in the biosynthesis of the lipopolysaccharide O-side chain in Brucella spp.
Res. Microbiol. 151 (2000) 209–216 © 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S092325080000141-8/FLA
Conservation of seven genes involved in the biosynthesis of the lipopolysaccharide O-side chain in Brucella spp. Axel Cloeckaerta*, Maggy Grayona, Jean-Michel Vergera, Jean-Jacques Letessonb, Fabrice Godfroidb a
b
Laboratoire de pathologie infectieuse et immunologie, Institut national de la recherche agronomique, 37380 Nouzilly, France Unité de recherche en biologie moléculaire (URBM), laboratoire d’immunologie-microbiologie, facultés universitaires Notre-Dame de la Paix, Namur, Belgium (Submitted 6 September 1999; accepted 8 December 1999)
Abstract — Seven genes of the wb locus of Brucella melitensis 16M involved in the biosynthesis of the lipopolysaccharide O-side chain have been recently identified, i.e. wbkA, gmd, per, wzm, wzt, wbkB, and wbkC, coding, respectively, for proteins homologous to mannosyltransferase, GDP-mannose 4,6 dehydratase, perosamine synthetase, ABC-type transporter (integral membrane protein), ABC-type transporter (ATPase domain), a hypothetical protein of unknown function, and a putative formyl transferase. The seven genes have a G + C content lower (around 48%) than that typical of Brucella spp. (58%) and thus may have been acquired from a species other than Brucella. In the present study, we analyzed by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) the seven O-chain biosynthetic genes for polymorphism among Brucella spp. PCR-RFLP showed that the seven genes are highly conserved and occur even in the naturally rough species B. ovis and B. canis and also in rough strains of B. abortus and B. melitensis. Nevertheless, the few polymorphisms that were observed consisted of absence of additional restriction sites sometimes allowing differentiation at the species level (e.g. B. ovis) or at the biovar or strain level. There were no apparent deletions or insertions in the PCR-amplified genes in any of the Brucella strains studied. In conclusion, the seven O-chain biosynthetic genes studied appear to be highly conserved among Brucella spp. and thus may have been acquired before species differentiation. Some of the species- or biovar-specific markers detected could be used for molecular typing of brucellae in addition to those previously described. © 2000 Éditions scientifiques et médicales Elsevier SAS Brucella / DNA polymorphism / PCR-RFLP / O-chain gene
1. Introduction Brucellae are Gram-negative, facultative intracellular bacteria that can infect many species of animals and occasionally man. Six species are at present recognized within the genus Brucella: B. abortus, B. melitensis, B. suis, B. ovis, B. canis, and B. neotomae [10]. They can be distin-
* Correspondence and reprints Tel.: +33 (2)2 47 42 77 50; fax: +33 (0)2 47 42 77 79; [email protected] Abbreviations: PCR-RFLP, polymerase chain reaction– restriction fragment length polymorphism.
guished by differential phenotypical tests [2]. This classification is mainly based on differences in pathogenicity and host preference [2]. Nevertheless, it has been shown, on the basis of DNA–DNA hybridization studies, that the genus Brucella constitutes a highly homogeneous group of species (> 90% DNA homology for all species) and it was proposed in the mid-1980s that this genus should comprise only one genomic species [20]. That proposal, however, has not yet been agreed upon by the Subcommittee on the Taxonomy of Brucella of the International Committee on Systematic Bacteriology and the classification into six species is still preferred.
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Figure 1. Organization of the O-chain biosynthetic genes in the 14-kb region of B. melitensis 16M. Insertion sequence (IS) regions are indicated by black boxes. For more detail see Godfroid et al. (unpublished data) and EMBL/GenBank accession number AF047478.
The main pathogenic species worldwide are B. abortus (responsible for bovine brucellosis), B. melitensis (the main etiologic agent of ovine and caprine brucellosis), and B. suis (responsible for swine brucellosis). These three Brucella species may cause abortion in their hosts, which results in huge economic losses. B. abortus, B. melitensis, and B. suis strains may occur as either smooth (S) or occasionally rough (R) strains, expressing smooth lipopolysaccharide (S-LPS) or rough lipopolysaccharide (R-LPS) as the major surface antigen, respectively, while B. ovis and B. canis are two naturally R species, expressing R-LPS as the major surface antigen. The latter species are responsible for ram epididymitis and canine brucellosis, respectively [3, 5]. For B. neotomae, only S strains isolated from desert rats have been reported [2, 16]. Smooth B. abortus, B. melitensis, and B. suis strains are further classified into biovars based on differential tests such as serotyping, phage typing, CO2 requirement, H2S production, and metabolic properties [2]. The antigenic determinants involved in serotyping of S strains with polyclonal sera are borne by the O-polysaccharide (O-PS) or O-chain moiety of the S-LPS lacking in the R-LPS. At present, S Brucella strains are classified into three serotypes, i.e. A+ M–, A– M+, and A+ M+, according to slide agglutination with A and M monospecific polyclonal sera [2]. These agglutination results correspond to strains expressing mainly the A (A dominant) or M (M dominant) epitope or both epitopes in nearly equivalent amounts. Additionally, S Brucella strains, as shown by use of monoclonal antibodies, share common epitopes on their O-PS crossreacting or not with Yersinia enterocolitica O:9 [8, 11, 17, 22]. These epitopes have been named
C/Y and C, respectively. O-PS epitopic heterogeneity at the surface of Brucella spp. has been shown by use of the monoclonal antibodies [8]. Some biovars such as B. suis biovar 2 (A dominant) are lacking in C epitopes and could constitute a different serotype not detected by the A or M monospecific polyclonal sera [8]. Recently, a chromosomal fragment of 13.8 kb from B. melitensis 16M-containing genes involved in the biosynthesis of the LPS O-side chain has been cloned and sequenced ([13], Godfroid et al., unpublished data). Seven genes have been identified on this fragment, i.e. wbkA, gmd, per, wzm, wzt, wbkB, and wbkC coding, respectively, for proteins homologous to mannosyl transferase, GDP-mannose 4,6 dehydratase, perosamine synthetase, ABC-type transporter (integral membrane protein), ABCtype transporter (ATPase domain), a hypothetical protein of unknown function, and a putative formyl transferase. The organization of these genes is shown in figure 1. The first is separated from the others by insertion sequences. The latter six are probably organized in an operon and are also delimited by insertion sequences (Godfroid et al., unpublished data). The essential roles of per and wbkC in the synthesis of the O-chain have been demonstrated and also the role of both ABC-type transporters in export of the O-chain ([13], Godfroid et al., unpublished data). The seven genes have a G + C content lower (around 48%) than that typical of Brucella spp. (58%) and thus may have been acquired from a species other than Brucella. In the present study, we analyzed by PCRRFLP the seven O-chain biosynthetic genes for polymorphism among Brucella spp.
Biosynthesis of LPS O-side chain in Brucella
2. Materials and methods 2.1. Bacterial strains
The Brucella strains used in this study are listed in table I. All strains were maintained freeze-dried in the Inra Brucella Culture Collection, Nouzilly (BCCN), France. Cultures were grown on tryptic soy agar (Difco) supplemented with 0.1% (w/v) yeast extract (Difco) (TSAYE). For fastidious strains (B. abortus biovar 2 and B. ovis), 5% sterile horse serum (Gibco BRL) was added to TSAYE (TSAYES medium). The strains were checked for purity and species and biovar characterization by standard procedures [2]. 2.2. DNA preparation
The strains were cultured for 24 h at 37 °C on a TSAYE or TSAYES slope and harvested, in 3 mL sterile distilled water, by centrifugation at 2 000 g for 10 min. The pellet was suspended in 567 µL TE/sodium buffer (50 mM Tris, 50 mM EDTA, 100 mM NaCl, pH 8.0). Then, 30 µL of a 10% (w/v) SDS solution and 3 µL of a 2% (w/v) proteinase K solution were added and the mixture was held at 37 °C for 1 h. The lysed cell suspension was extracted twice with phenolchloroform and nucleic acids were precipitated by gently mixing the aqueous phase with two volumes of cold ethanol. The precipitate was dissolved in 100 µL of TE (10 mM Tris, 1 mM EDTA, pH 8.0). The amount of DNA was estimated by electrophoresis of an aliquot of each sample through 0.8% agarose gels and comparison with standard DNA solutions. 2.3. Primers and amplification conditions
The 20-mer primers (Isoprim) were selected to amplify each O-chain biosynthetic gene separately according to the reported B. melitensis 16M wb nucleotide sequence (EMBL/Genbank accession number AF047478, Godfroid et al., unpublished data). These primers and their position on the wb nucleotide sequence are indicated in table II. Amplification reaction mixtures were prepared in volumes of 100 µL containing 10 mM
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Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 0.2 mg mL–1 gelatin (1X PCR buffer; Appligene), 200 µM each deoxynucleoside triphosphate, 1 µM each primer, 100 ng of genomic DNA, and 2.5 U of Taq DNA polymerase (Appligene). The temperature cycling for the amplification was performed in a GeneAmp PCR System 9600 thermocycler (Perkin Elmer) as follows: cycle 1 was 94 °C for 5 min (denaturation); the next 30 cycles were 58 °C for 30 s (annealing), 70 °C for 30 s (extension) and 94 °C for 30 s (denaturation); the last cycle was 58 °C for 30 s (annealing) and 70 °C for 10 min (extension). 2.4. Restriction digestion
For polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP), the following restriction enzymes were used: AluI, AvaII, BglI, BglII, ClaI, EcoRI, HindIII, KpnI, MluI, NheI, Sau3AI, SmaI, TaqI, and XbaI (Appligene), BanI and HaeII (Promega), HaeIII, HinfI, and PvuII (New England Biolabs), SalI (Boehringer), and StyI (Eurogentec). Restriction enzymes were chosen according to the B. melitensis 16M determined wbkA, gmd, per, wzm, wzt, wbkB, and wbkC nucleotide sequences (Godfroid et al., unpublished data). The restriction map of each gene is shown in figure 2. Five microliters of each PCR product were cleaved with 1–5 U of each restriction enzyme in a 20-µL volume. Buffer and temperature conditions recommended by the manufacturers were used. Restriction fragments from each reaction were separated in gels with 1.5% (w/v) agarose (Eurobio) and 0.5 µg mL–1 ethidium bromide (Sigma). The 100-bp DNA ladder (Gibco) was used as molecular size marker.
3. Results and discussion PCR-RFLP study of the seven O-chain biosynthetic genes was conducted on representative strains of each Brucella species and their different biovars with particular attention to their reported lipopolysaccharide (LPS) charac-
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Table I. Brucella strains and restriction patterns of their O-chain biosynthetic genes. O-chain biosynthetic gene restriction patterns: Species B. melitensis
a
Biovar Serotype 1
M
1 2
S-R Mb A
3
AM
R 1
A
2
A
3
A
4
M
5
M
6
A
9
M
R R 1
– – A
2
A
3
A
4
AM
5
M
–
–
–
–
B. canis
– –
– –
B. neotomae
– – –
– – A
B. abortus
B. suis
B. ovis
Strain
Host/ source
Geographic wbkA gmd origin
16M (ATCC 23456; goat USA BCCN R1) EP (BCCN 87.92) human USA 63/9 (ATCC 23457; goat Turkey BCCN R2) Ether (ATCC 23458; goat Italy BCCN R3) B115 (BCCN R19) goat Malta 544 (ATCC 23448; cattle England BCCN R4) 86/8/59 (ATCC cattle England 23449; BCCN R5) Tulya (ATCC human Uganda 23450; BCCN R6) 292 (ATCC 23451; cattle England BCCN R7) B3196 (ATCC cattle England 23452; BCCN R8) 870 (ATCC 23453; cattle Africa BCCN R9) C68 (ATCC 23455; cattle England BCCN R11) 45/20 (BCCN V2) cattle England RB51 (BCCN V5) cattle USA 1330 (ATCC 23444; swine USA BCCN R12) Thomsen (ATCC swine Denmark 23445; BCCN R13) 686 (ATCC 23446; swine USA BCCN R14) 40 (ATCC 23447; reindeer Former BCCN R15) Soviet Union 513 (BCCN R21) wild Former rodent Soviet Union 63/290 (ATCC sheep Africa 25840; BCCN R17) Reo 198 (BCCN sheep USA R22) BCCN 76.250 sheep France RM6/66 (ATCC dog USA 23365; BCCN R18) D519 (BCCN C1) dog Madagascar BCCN 87.65 dog Canada 5K33 (ATCC 23459; desert USA BCCN R16) rat
per
wzm wzt wbkB wbkC
Ac
A
A
A
A
A
A
A A
A A
A A
A A
A A
A A
A A
A
A
A
A
A
A
A
A A
A A
A A
A A
A B
A A
A A
A
A
A
A
B
A
A
A
A
A
A
A
B
A
A
A
A
A
B
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A A A
A A A
A A B
A A A
B B A
A A A
A A A
A
A
B
A
A
A
A
A
A
B
A
A
Cd
A
A
A
B
A
A
A
A
A
A
A
A
A
A
A
Bd
A
A
A
A
A
A
B
A
A
A
A
A
A
B A
A A
A B
A A
A A
A A
A A
A A A
A A A
B B A
A A A
A A A
A A A
A A B
a
O-PS related serotype, A and M for A dominant or M dominant, – for R strains. b S-R M strain EP has a lack in O-PS expression and expresses a reduced amount of M-dominant O-PS at its surface [4]. c Restriction pattern A (see figure 3 for the corresponding restriction maps) corresponds to that expected from the nucleotide sequence of B. melitensis 16M (Godfroid et al., unpublished data; EMBL/GenBank accession number AF047478). d Restriction patterns B and C: see corresponding restriction maps in figure 3.
teristics, i.e. S (A dominant, M dominant, or A+ M+) and R (table I). Besides the naturally R species, B. ovis and B. canis, some well known R
derivatives of the other Brucella species were included such as B. melitensis strain B115 shown to produce the O-chain intracellularly [9] and B.
Biosynthesis of LPS O-side chain in Brucella
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Table II Primers used for PCR. Target DNA wbkA gmd per wzm wzt wbkB wbkC
Primer name
Sequence (5’-3’)
Position in the wbk region*
wbkAA wbkAB gmdA gmdB perA perB wzmA wzmB wztA wztB wbkBA wbkBB wbkCA wbkCB
TGCGAATTGGTGTCGACGCT AGAAGCCTCCTTGCTATCCG CATGGCAAAAACGGGAATGA GGAGAGTAAACTGGTATATC CGGCGGCTCGTGAGAATTTG GCGTACCTTCCAGACATTAG CAACCACATTTAGTGAGACG CAGGGTAATCGATGGCTGGA CCTATGATCCAGCCATCGAT GGAGCGCCTGAATCTCTTAT CTCGGGAATGGGAGCTATAG GCCTCTTTTTCGTCAGTTAC TACGAATTGCAGCGCGAACT GCCAGAAGCCTTTATCATCA
2060–2078 907–926 5312–5331 6437–6456 6365–6384 7568–7587 7525–7544 8335–8354 8328–8347 9089–9108 9065–9084 9940–9959 9868–9887 10799–10818
* EMBL\Genbank\DDBJ databank accession number AF047478.
abortus vaccine strain RB51 [18]. B. melitensis S-R strain EP shown to express a reduced amount of O-PS at its surface [4] was also of interest in the present study. The seven O-chain biosynthetic genes were successfully PCR-amplified in all Brucella species. There were no apparent deletions or insertions in the PCR-amplified genes in any of the Brucella strains studied. The few polymorphisms detected by RFLP (figures 2 and 3) consisted of absent or additional restriction sites sometimes allowing the differentiation at the species level or at the biovar or strain level (table I). The differences in restriction patterns observed, however, did not correlate with the LPS phenotype (S or R) nor with the dominant antigenic profile (A or M) of the S strains. The restriction maps of the seven O-chain biosynthetic genes with the restriction enzymes used are shown in figure 3. No polymorphism was detected in the gmd and wzm genes. B. abortus biovars 1, 2, and 4 showed a distinct restriction pattern of their wzt gene consisting of absence of the SalI site (figure 2). These biovars are typically thioninesensitive [2] and were also previously shown to have a specific molecular marker consisting of a 120-bp deletion in their porin omp2a gene [6, 12]. The R B. abortus strains showing the same wzt SalI pattern (table I) are actually derived from S B. abortus biovar 1 strains. B. abortus biovar 3
strain Tulya could be differentiated from the other Brucella strains by AluI restriction of its wbkB gene. B. ovis strains had a distinct wbkA HindIII restriction pattern (figures 2 and 3). B. suis biovars 1, 2, 3, and 4 and B. canis strains also formed a separate group on the basis of AluI and HindIII restriction patterns of their per genes. B. suis and B. canis are in fact closely related on the basis of phenotypic characteristics [16]. Finally, B. neotomae strain 5K33 lacked the SmaI site in its wbkC gene. Probably mutations resulting in the R phenotype of the Brucella strains studied are located elsewhere on the chromosome. Complementing R B. melitensis strain B115 with the functional wzm and wzt genes shown to be involved in export of the O-chain (Godfroid et al., unpublished data) did not result in restoration of the S phenotype. By Tn5 transposon mutagenesis other genes distinct from those reported in our study have been identified as being involved in the biosynthesis of the O-chain [1, 14]. Moreover, in the case of R B. abortus strain RB51 it has been reported that a wbkA-related gene named wboA (not identical to that from our study) is disrupted by an IS711 insertion sequence [19]. No correlation could be found between the antigenic profiles (A or M dominant) of the S Brucella strains and the PCR-RFLP patterns observed. Genes determining the S serotypes are probably also located elsewhere on the
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chromosome. The Brucella O-PS structure has been described as being constituted by homopolymers of 4,6-dideoxy-4-formamido-aD-mannopyranose residues. O-PS from A-dominant strains is a linear a-1,2-linked polymer with about 2% a-1,3 linkages, while O-PS from M-dominant strains is a linear polymer of pentasaccharide repeating units containing one a-1,3-linked and four a-1,2-linked monosaccharide residues [15]. The presence of a-1,2 and a-1,3 linkages in the O-PS of Brucella suggests the existence of at least two mannosyltransferases (Godfroid et al., unpublished data). Here we studied polymorphism of the a-mannosyltransferase gene wbkA. A gene encoding a second mannosyltransferase (WboA) was
Figure 2. Restriction maps of the O-chain biosynthetic genes with the restriction enzymes used. For each gene, restriction map A corresponds to that deduced from the nucleotide sequence of B. melitensis 16M (Godfroid et al., unpublished data). Only differences compared to the nucleotide sequences of B. melitensis 16M are indicated in restriction maps B and C. The restriction patterns A, B, or C are further indicated in table I for each gene and for each Brucella strain studied.
Figure 3. PCR-RFLP of the the O-chain biosynthetic genes. A. Lanes: 1, molecular size markers; 2, wbkA from B. melitensis 16M uncut; 3, wbkA from B. melitensis 16M cut by HindIII; 4, wbkA from B. ovis 63/290 cut by HindIII; 5, per from B. melitensis 16M uncut; 6, per from B. melitensis 16M cut by AluI; 7, per from B. suis 1330 cut by AluI; 8, per from B. melitensis 16M cut by HindIII; 9, per from B. suis 1330 cut by HindIII; 10, wzt from B. melitensis 16M uncut; 11, wzt from B. melitensis 16M cut by SalI; 12, wzt from B. abortus 544 cut by SalI. B. Lanes: 1, molecular size markers; 2, wbkB from B. melitensis 16M uncut; 3, wbkB from B. melitensis 16M cut by AluI; 4, wbkB from B. abortus Tulya cut by AluI; 5, wbkB from B. melitensis 16M cut by TaqI; 6, wbkB from B. suis 686 cut by TaqI; 7, wbkB from B. melitensis 16M cut by Sau3A; 8, wbkB from B. suis 686 cut by Sau3A; 9, wbkC from B. melitensis 16M uncut; 10, wbkC from B. melitensis 16M cut by SmaI; 11, wbkC from B. neotomae 5K33 cut by SmaI.
Biosynthesis of LPS O-side chain in Brucella
recently identified in Brucella abortus strain 2308 [14]. WbkA could interact with WboA to elongate the Brucella LPS O chain by a-1,2 and a-1,3 links. In conclusion, the seven O-chain biosynthetic genes studied appear to be highly conserved among Brucella spp. and thus may have been acquired before species differentiation. Some of the species- or biovar-specific markers detected could be used for molecular typing of brucellae in addition to those previously described [6, 7, 12, 21]. Résumé — Conservation de sept gènes impliqués dans la biosynthèse de la chaîne O du lipopolyoside chez les Brucella. Sept gènes du locus wb de Brucella melitensis 16M impliqués dans la biosynthèse de la chaîne O du lipopolyoside ont été récemment identifiés : les gènes wbkA, gmd, per, wzm, wzt, wbkB et wbkC codant respectivement pour des protéines homologues à une mannosyltransférase, une GDPmannose 4,6 déshydratase, une perosamine synthétase, un transporteur de type ABC (protéine membranaire intégrale), un autre transporteur de type ABC (domaine ATPasique), une protéine hypothétique de fonction inconnue et une formyl transférase putative. Les sept gènes ont un pourcentage GC plus faible (autour de 48 %) que celui typique de Brucella (58 %) et auraient pu être ainsi acquis d’une espèce autre que Brucella. Nous avons étudié le polymorphisme de ces sept gènes par PCR-RFLP chez les Brucella. Par cette technique nous montrons que les sept gènes sont très conservés et sont même présents dans les espèces naturellement rugueuses B. ovis et B. canis et également chez des souches rugueuses de B. abortus et B. melitensis. Néanmoins le peu de polymorphisme détecté consistant en l’absence de sites de restriction ou en des sites supplémentaires permet parfois la différentiation au niveau de l’espèce (ex. B. ovis), du biovar ou de la souche. Nous n’avons pas détecté, par PCR-RFLP, la présence de délétions ou d’insertions dans aucun des gènes et aucune souche de Brucella étudiée. En conclusion, les sept gènes de synthèse de la chaîne O apparaissent très conservés parmi les espèces et souches de Brucella et pourraient donc avoir été acquis avant la différentiation en espèces des Brucella. Certains marqueurs d’espèce ou de biovar détectés pourraient être utilisés pour le typage moléculaire des Brucella en complément de
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ceux décrits auparavant. © 2000 Éditions scientifiques et médicales Elsevier SAS Brucella / polymorphisme de l’ADN / PCR-RFLP / gène de la chaîne O
References [1] Allen C.A., Adams L.G., Ficht T.A., Transposon-derived Brucella abortus rough mutants are attenuated and exhibit reduced intracellular survival, Infect. Immun. 66 (1998) 1008–1016. [2] Alton G.G., Jones L.M., Angus R.D., Verger J.M., Techniques for the Brucellosis Laboratory, Institut National de la Recherche Agronomique, Paris, 1988. [3] Blasco J.M., Brucella ovis, in: Nielsen K., Duncan J.R. (Eds.), Animal Brucellosis, CRC Press, Inc., Boca Raton, 1990, pp. 351–378. [4] Bowden R.A., Verger J.M., Grayon M., Limet J.N., Dubray G., Simultaneous expression of smooth and rough phase properties related to lipopolysaccharide in a Brucella melitensis strain, J. Med. Microbiol. 39 (1993) 363–370. [5] Carmichael L.E., Brucella canis, in: Nielsen K., Duncan J.R. (Eds.), Animal Brucellosis, CRC Press, Inc., Boca Raton, 1990, pp. 335–350. [6] Cloeckaert A., Verger J.M., Grayon M., Grépinet O., Restriction site polymorphism of the genes encoding the major 25 kDa and 36 kDa outer-membrane proteins of Brucella, Microbiology 141 (1995) 2111–2121. [7] Cloeckaert A., Verger J.M., Grayon M., Grépinet, O., Polymorphism at the dnaK locus of Brucella species and identification of a Brucella melitensis species-specific marker, J. Med. Microbiol. 45 (1996) 200–205. [8] Cloeckaert A., Weynants V., Godfroid J., Verger J.M., Grayon M., Zygmunt M.S., O-polysaccharide epitopic heterogeneity at the surface of Brucella spp. studied by enzyme-linked immunosorbent assay and flow cytometry, Clin. Diagn. Lab. Immunol. 5 (1998) 862–870. [9] Cloeckaert A., Zygmunt M.S., Nicolle J.C., Dubray G., Limet J.N., O-chain expression in the rough Brucella melitensis strain B115: induction of O-polysaccharide-specific monoclonal antibodies and intracellular localization demonstrated by immunoelectron microscopy, J. Gen. Microbiol. 138 (1992) 1211–1219. [10] Corbel M.J., Brinley-Morgan W.J., Genus Brucella Meyer and Shaw (1920), in: Krieg N.R., Holt J.G. (Eds.), Bergey’s Manual of Systematic Bacteriology, vol. 1, Williams Wilkins, Baltimore, 1984, pp. 377–388. [11] Douglas J.T., Palmer D.A., Use of monoclonal antibodies to identify the distribution of A and M epitopes on smooth Brucella species, J. Clin. Microbiol. 26 (1988) 1353–1356. [12] Ficht T.A., Bearden S.W., Sowa B.A., Marquis H., Genetic variation at the omp2 porin locus of the brucellae: species-specific markers, Mol. Microbiol. 4 (1990) 1135–1142. [13] Godfroid F., Taminiau B., Danese I., Denoel P., Tibor A., Weynants V., Cloeckaert A., Godfroid J., Letesson J.J., Identification of the perosamine synthetase gene of Brucella melitensis 16M and involvement of the lipopolysaccharide O side chain in Brucella survival in mice and in macrophages, Infect. Immun. 66 (1998) 5485–5493. [14] McQuiston J.R., Vemulapalli R., Inzana T.J., Schurig G.G., Sriranganathan N., Fritzinger D., Hadfield T.L., Warren R.A., Snellings N., Hoover D., Halling S.M., Boyle S.M., Genetic characterization of a Tn5-disrupted glycosyltransferase gene homolog in Brucella abortus and its effect on lipopolysaccharide composition and virulence, Infect. Immun. 67 (1999) 3830–3835. [15] Meikle P.J., Perry M.B., Cherwonogrodzky J.W., Bundle D.R., Fine structure of A and M antigens from Brucella biovars, Infect. Immun. 57 (1989) 2820–2828. [16] Meyer M.E., Current concepts in the taxonomy of the genus Brucella, in: Nielsen K., Duncan J.R. (Eds.), Animal Brucellosis, CRC Press, Inc., Boca Raton, 1990, pp. 1–17.
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[17] Palmer D.A., Douglas J.T., Analysis of Brucella lipopolysaccharide with specific and cross-reacting monoclonal antibodies, J. Clin. Microbiol. 27 (1989) 2331–2337. [18] Schurig G.G., Roopii R.M., Bagchi T., Boyle S., Buhrman D., Sriranganathan N., Biological properties of RB51; a stable rough strain of Brucella abortus, Vet. Microbiol. 28 (1991) 171–188. [19] Vemulapalli R., McQuiston J.R., Schurig G.G., Sriranganathan N., Halling S.M., Boyle S.M., Identification of an IS711 element interrupting the wboA gene of Brucella abortus vaccine strain RB51 and a PCR assay to distinguish strain RB51 from other Brucella species and strains, Clin. Diagn. Lab. Immunol. 6 (1999) 760–764.
[20] Verger J.M., Grimont F., Grimont P.A.D., Grayon M., Brucella, a monospecific genus as shown by deoxyribonucleic acid hybridization, Int. J. Syst. Bacteriol. 35 (1985) 292–295. [21] Vizcaino N., Verger J.M., Grayon M., Zygmunt M.S., Cloeckaert A., DNA polymorphism at the omp-31 locus of Brucella spp.: evidence for a large deletion in Brucella abortus, and other species-specific markers, Microbiology 143 (1997) 2913–2921. [22] Weynants V., Gilson D., Cloeckaert A., Tibor A., Denoel P.A., Godfroid F., Limet J.N., Letesson J.J., Characterization of smooth lipopolysaccharides and O polysaccharides of Brucella species by competition binding assays with monoclonal antibodies, Infect. Immun. 65 (1997) 1939–1943.
Conservation of seven genes involved in the biosynthesis of the lipopolysaccharide O-side chain in Brucella spp. Axel Cloeckaerta*, Maggy Grayona, Jean-Michel Vergera, Jean-Jacques Letessonb, Fabrice Godfroidb a
b
Laboratoire de pathologie infectieuse et immunologie, Institut national de la recherche agronomique, 37380 Nouzilly, France Unité de recherche en biologie moléculaire (URBM), laboratoire d’immunologie-microbiologie, facultés universitaires Notre-Dame de la Paix, Namur, Belgium (Submitted 6 September 1999; accepted 8 December 1999)
Abstract — Seven genes of the wb locus of Brucella melitensis 16M involved in the biosynthesis of the lipopolysaccharide O-side chain have been recently identified, i.e. wbkA, gmd, per, wzm, wzt, wbkB, and wbkC, coding, respectively, for proteins homologous to mannosyltransferase, GDP-mannose 4,6 dehydratase, perosamine synthetase, ABC-type transporter (integral membrane protein), ABC-type transporter (ATPase domain), a hypothetical protein of unknown function, and a putative formyl transferase. The seven genes have a G + C content lower (around 48%) than that typical of Brucella spp. (58%) and thus may have been acquired from a species other than Brucella. In the present study, we analyzed by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) the seven O-chain biosynthetic genes for polymorphism among Brucella spp. PCR-RFLP showed that the seven genes are highly conserved and occur even in the naturally rough species B. ovis and B. canis and also in rough strains of B. abortus and B. melitensis. Nevertheless, the few polymorphisms that were observed consisted of absence of additional restriction sites sometimes allowing differentiation at the species level (e.g. B. ovis) or at the biovar or strain level. There were no apparent deletions or insertions in the PCR-amplified genes in any of the Brucella strains studied. In conclusion, the seven O-chain biosynthetic genes studied appear to be highly conserved among Brucella spp. and thus may have been acquired before species differentiation. Some of the species- or biovar-specific markers detected could be used for molecular typing of brucellae in addition to those previously described. © 2000 Éditions scientifiques et médicales Elsevier SAS Brucella / DNA polymorphism / PCR-RFLP / O-chain gene
1. Introduction Brucellae are Gram-negative, facultative intracellular bacteria that can infect many species of animals and occasionally man. Six species are at present recognized within the genus Brucella: B. abortus, B. melitensis, B. suis, B. ovis, B. canis, and B. neotomae [10]. They can be distin-
* Correspondence and reprints Tel.: +33 (2)2 47 42 77 50; fax: +33 (0)2 47 42 77 79; [email protected] Abbreviations: PCR-RFLP, polymerase chain reaction– restriction fragment length polymorphism.
guished by differential phenotypical tests [2]. This classification is mainly based on differences in pathogenicity and host preference [2]. Nevertheless, it has been shown, on the basis of DNA–DNA hybridization studies, that the genus Brucella constitutes a highly homogeneous group of species (> 90% DNA homology for all species) and it was proposed in the mid-1980s that this genus should comprise only one genomic species [20]. That proposal, however, has not yet been agreed upon by the Subcommittee on the Taxonomy of Brucella of the International Committee on Systematic Bacteriology and the classification into six species is still preferred.
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Figure 1. Organization of the O-chain biosynthetic genes in the 14-kb region of B. melitensis 16M. Insertion sequence (IS) regions are indicated by black boxes. For more detail see Godfroid et al. (unpublished data) and EMBL/GenBank accession number AF047478.
The main pathogenic species worldwide are B. abortus (responsible for bovine brucellosis), B. melitensis (the main etiologic agent of ovine and caprine brucellosis), and B. suis (responsible for swine brucellosis). These three Brucella species may cause abortion in their hosts, which results in huge economic losses. B. abortus, B. melitensis, and B. suis strains may occur as either smooth (S) or occasionally rough (R) strains, expressing smooth lipopolysaccharide (S-LPS) or rough lipopolysaccharide (R-LPS) as the major surface antigen, respectively, while B. ovis and B. canis are two naturally R species, expressing R-LPS as the major surface antigen. The latter species are responsible for ram epididymitis and canine brucellosis, respectively [3, 5]. For B. neotomae, only S strains isolated from desert rats have been reported [2, 16]. Smooth B. abortus, B. melitensis, and B. suis strains are further classified into biovars based on differential tests such as serotyping, phage typing, CO2 requirement, H2S production, and metabolic properties [2]. The antigenic determinants involved in serotyping of S strains with polyclonal sera are borne by the O-polysaccharide (O-PS) or O-chain moiety of the S-LPS lacking in the R-LPS. At present, S Brucella strains are classified into three serotypes, i.e. A+ M–, A– M+, and A+ M+, according to slide agglutination with A and M monospecific polyclonal sera [2]. These agglutination results correspond to strains expressing mainly the A (A dominant) or M (M dominant) epitope or both epitopes in nearly equivalent amounts. Additionally, S Brucella strains, as shown by use of monoclonal antibodies, share common epitopes on their O-PS crossreacting or not with Yersinia enterocolitica O:9 [8, 11, 17, 22]. These epitopes have been named
C/Y and C, respectively. O-PS epitopic heterogeneity at the surface of Brucella spp. has been shown by use of the monoclonal antibodies [8]. Some biovars such as B. suis biovar 2 (A dominant) are lacking in C epitopes and could constitute a different serotype not detected by the A or M monospecific polyclonal sera [8]. Recently, a chromosomal fragment of 13.8 kb from B. melitensis 16M-containing genes involved in the biosynthesis of the LPS O-side chain has been cloned and sequenced ([13], Godfroid et al., unpublished data). Seven genes have been identified on this fragment, i.e. wbkA, gmd, per, wzm, wzt, wbkB, and wbkC coding, respectively, for proteins homologous to mannosyl transferase, GDP-mannose 4,6 dehydratase, perosamine synthetase, ABC-type transporter (integral membrane protein), ABCtype transporter (ATPase domain), a hypothetical protein of unknown function, and a putative formyl transferase. The organization of these genes is shown in figure 1. The first is separated from the others by insertion sequences. The latter six are probably organized in an operon and are also delimited by insertion sequences (Godfroid et al., unpublished data). The essential roles of per and wbkC in the synthesis of the O-chain have been demonstrated and also the role of both ABC-type transporters in export of the O-chain ([13], Godfroid et al., unpublished data). The seven genes have a G + C content lower (around 48%) than that typical of Brucella spp. (58%) and thus may have been acquired from a species other than Brucella. In the present study, we analyzed by PCRRFLP the seven O-chain biosynthetic genes for polymorphism among Brucella spp.
Biosynthesis of LPS O-side chain in Brucella
2. Materials and methods 2.1. Bacterial strains
The Brucella strains used in this study are listed in table I. All strains were maintained freeze-dried in the Inra Brucella Culture Collection, Nouzilly (BCCN), France. Cultures were grown on tryptic soy agar (Difco) supplemented with 0.1% (w/v) yeast extract (Difco) (TSAYE). For fastidious strains (B. abortus biovar 2 and B. ovis), 5% sterile horse serum (Gibco BRL) was added to TSAYE (TSAYES medium). The strains were checked for purity and species and biovar characterization by standard procedures [2]. 2.2. DNA preparation
The strains were cultured for 24 h at 37 °C on a TSAYE or TSAYES slope and harvested, in 3 mL sterile distilled water, by centrifugation at 2 000 g for 10 min. The pellet was suspended in 567 µL TE/sodium buffer (50 mM Tris, 50 mM EDTA, 100 mM NaCl, pH 8.0). Then, 30 µL of a 10% (w/v) SDS solution and 3 µL of a 2% (w/v) proteinase K solution were added and the mixture was held at 37 °C for 1 h. The lysed cell suspension was extracted twice with phenolchloroform and nucleic acids were precipitated by gently mixing the aqueous phase with two volumes of cold ethanol. The precipitate was dissolved in 100 µL of TE (10 mM Tris, 1 mM EDTA, pH 8.0). The amount of DNA was estimated by electrophoresis of an aliquot of each sample through 0.8% agarose gels and comparison with standard DNA solutions. 2.3. Primers and amplification conditions
The 20-mer primers (Isoprim) were selected to amplify each O-chain biosynthetic gene separately according to the reported B. melitensis 16M wb nucleotide sequence (EMBL/Genbank accession number AF047478, Godfroid et al., unpublished data). These primers and their position on the wb nucleotide sequence are indicated in table II. Amplification reaction mixtures were prepared in volumes of 100 µL containing 10 mM
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Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 0.2 mg mL–1 gelatin (1X PCR buffer; Appligene), 200 µM each deoxynucleoside triphosphate, 1 µM each primer, 100 ng of genomic DNA, and 2.5 U of Taq DNA polymerase (Appligene). The temperature cycling for the amplification was performed in a GeneAmp PCR System 9600 thermocycler (Perkin Elmer) as follows: cycle 1 was 94 °C for 5 min (denaturation); the next 30 cycles were 58 °C for 30 s (annealing), 70 °C for 30 s (extension) and 94 °C for 30 s (denaturation); the last cycle was 58 °C for 30 s (annealing) and 70 °C for 10 min (extension). 2.4. Restriction digestion
For polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP), the following restriction enzymes were used: AluI, AvaII, BglI, BglII, ClaI, EcoRI, HindIII, KpnI, MluI, NheI, Sau3AI, SmaI, TaqI, and XbaI (Appligene), BanI and HaeII (Promega), HaeIII, HinfI, and PvuII (New England Biolabs), SalI (Boehringer), and StyI (Eurogentec). Restriction enzymes were chosen according to the B. melitensis 16M determined wbkA, gmd, per, wzm, wzt, wbkB, and wbkC nucleotide sequences (Godfroid et al., unpublished data). The restriction map of each gene is shown in figure 2. Five microliters of each PCR product were cleaved with 1–5 U of each restriction enzyme in a 20-µL volume. Buffer and temperature conditions recommended by the manufacturers were used. Restriction fragments from each reaction were separated in gels with 1.5% (w/v) agarose (Eurobio) and 0.5 µg mL–1 ethidium bromide (Sigma). The 100-bp DNA ladder (Gibco) was used as molecular size marker.
3. Results and discussion PCR-RFLP study of the seven O-chain biosynthetic genes was conducted on representative strains of each Brucella species and their different biovars with particular attention to their reported lipopolysaccharide (LPS) charac-
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Table I. Brucella strains and restriction patterns of their O-chain biosynthetic genes. O-chain biosynthetic gene restriction patterns: Species B. melitensis
a
Biovar Serotype 1
M
1 2
S-R Mb A
3
AM
R 1
A
2
A
3
A
4
M
5
M
6
A
9
M
R R 1
– – A
2
A
3
A
4
AM
5
M
–
–
–
–
B. canis
– –
– –
B. neotomae
– – –
– – A
B. abortus
B. suis
B. ovis
Strain
Host/ source
Geographic wbkA gmd origin
16M (ATCC 23456; goat USA BCCN R1) EP (BCCN 87.92) human USA 63/9 (ATCC 23457; goat Turkey BCCN R2) Ether (ATCC 23458; goat Italy BCCN R3) B115 (BCCN R19) goat Malta 544 (ATCC 23448; cattle England BCCN R4) 86/8/59 (ATCC cattle England 23449; BCCN R5) Tulya (ATCC human Uganda 23450; BCCN R6) 292 (ATCC 23451; cattle England BCCN R7) B3196 (ATCC cattle England 23452; BCCN R8) 870 (ATCC 23453; cattle Africa BCCN R9) C68 (ATCC 23455; cattle England BCCN R11) 45/20 (BCCN V2) cattle England RB51 (BCCN V5) cattle USA 1330 (ATCC 23444; swine USA BCCN R12) Thomsen (ATCC swine Denmark 23445; BCCN R13) 686 (ATCC 23446; swine USA BCCN R14) 40 (ATCC 23447; reindeer Former BCCN R15) Soviet Union 513 (BCCN R21) wild Former rodent Soviet Union 63/290 (ATCC sheep Africa 25840; BCCN R17) Reo 198 (BCCN sheep USA R22) BCCN 76.250 sheep France RM6/66 (ATCC dog USA 23365; BCCN R18) D519 (BCCN C1) dog Madagascar BCCN 87.65 dog Canada 5K33 (ATCC 23459; desert USA BCCN R16) rat
per
wzm wzt wbkB wbkC
Ac
A
A
A
A
A
A
A A
A A
A A
A A
A A
A A
A A
A
A
A
A
A
A
A
A A
A A
A A
A A
A B
A A
A A
A
A
A
A
B
A
A
A
A
A
A
A
B
A
A
A
A
A
B
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A A A
A A A
A A B
A A A
B B A
A A A
A A A
A
A
B
A
A
A
A
A
A
B
A
A
Cd
A
A
A
B
A
A
A
A
A
A
A
A
A
A
A
Bd
A
A
A
A
A
A
B
A
A
A
A
A
A
B A
A A
A B
A A
A A
A A
A A
A A A
A A A
B B A
A A A
A A A
A A A
A A B
a
O-PS related serotype, A and M for A dominant or M dominant, – for R strains. b S-R M strain EP has a lack in O-PS expression and expresses a reduced amount of M-dominant O-PS at its surface [4]. c Restriction pattern A (see figure 3 for the corresponding restriction maps) corresponds to that expected from the nucleotide sequence of B. melitensis 16M (Godfroid et al., unpublished data; EMBL/GenBank accession number AF047478). d Restriction patterns B and C: see corresponding restriction maps in figure 3.
teristics, i.e. S (A dominant, M dominant, or A+ M+) and R (table I). Besides the naturally R species, B. ovis and B. canis, some well known R
derivatives of the other Brucella species were included such as B. melitensis strain B115 shown to produce the O-chain intracellularly [9] and B.
Biosynthesis of LPS O-side chain in Brucella
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Table II Primers used for PCR. Target DNA wbkA gmd per wzm wzt wbkB wbkC
Primer name
Sequence (5’-3’)
Position in the wbk region*
wbkAA wbkAB gmdA gmdB perA perB wzmA wzmB wztA wztB wbkBA wbkBB wbkCA wbkCB
TGCGAATTGGTGTCGACGCT AGAAGCCTCCTTGCTATCCG CATGGCAAAAACGGGAATGA GGAGAGTAAACTGGTATATC CGGCGGCTCGTGAGAATTTG GCGTACCTTCCAGACATTAG CAACCACATTTAGTGAGACG CAGGGTAATCGATGGCTGGA CCTATGATCCAGCCATCGAT GGAGCGCCTGAATCTCTTAT CTCGGGAATGGGAGCTATAG GCCTCTTTTTCGTCAGTTAC TACGAATTGCAGCGCGAACT GCCAGAAGCCTTTATCATCA
2060–2078 907–926 5312–5331 6437–6456 6365–6384 7568–7587 7525–7544 8335–8354 8328–8347 9089–9108 9065–9084 9940–9959 9868–9887 10799–10818
* EMBL\Genbank\DDBJ databank accession number AF047478.
abortus vaccine strain RB51 [18]. B. melitensis S-R strain EP shown to express a reduced amount of O-PS at its surface [4] was also of interest in the present study. The seven O-chain biosynthetic genes were successfully PCR-amplified in all Brucella species. There were no apparent deletions or insertions in the PCR-amplified genes in any of the Brucella strains studied. The few polymorphisms detected by RFLP (figures 2 and 3) consisted of absent or additional restriction sites sometimes allowing the differentiation at the species level or at the biovar or strain level (table I). The differences in restriction patterns observed, however, did not correlate with the LPS phenotype (S or R) nor with the dominant antigenic profile (A or M) of the S strains. The restriction maps of the seven O-chain biosynthetic genes with the restriction enzymes used are shown in figure 3. No polymorphism was detected in the gmd and wzm genes. B. abortus biovars 1, 2, and 4 showed a distinct restriction pattern of their wzt gene consisting of absence of the SalI site (figure 2). These biovars are typically thioninesensitive [2] and were also previously shown to have a specific molecular marker consisting of a 120-bp deletion in their porin omp2a gene [6, 12]. The R B. abortus strains showing the same wzt SalI pattern (table I) are actually derived from S B. abortus biovar 1 strains. B. abortus biovar 3
strain Tulya could be differentiated from the other Brucella strains by AluI restriction of its wbkB gene. B. ovis strains had a distinct wbkA HindIII restriction pattern (figures 2 and 3). B. suis biovars 1, 2, 3, and 4 and B. canis strains also formed a separate group on the basis of AluI and HindIII restriction patterns of their per genes. B. suis and B. canis are in fact closely related on the basis of phenotypic characteristics [16]. Finally, B. neotomae strain 5K33 lacked the SmaI site in its wbkC gene. Probably mutations resulting in the R phenotype of the Brucella strains studied are located elsewhere on the chromosome. Complementing R B. melitensis strain B115 with the functional wzm and wzt genes shown to be involved in export of the O-chain (Godfroid et al., unpublished data) did not result in restoration of the S phenotype. By Tn5 transposon mutagenesis other genes distinct from those reported in our study have been identified as being involved in the biosynthesis of the O-chain [1, 14]. Moreover, in the case of R B. abortus strain RB51 it has been reported that a wbkA-related gene named wboA (not identical to that from our study) is disrupted by an IS711 insertion sequence [19]. No correlation could be found between the antigenic profiles (A or M dominant) of the S Brucella strains and the PCR-RFLP patterns observed. Genes determining the S serotypes are probably also located elsewhere on the
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chromosome. The Brucella O-PS structure has been described as being constituted by homopolymers of 4,6-dideoxy-4-formamido-aD-mannopyranose residues. O-PS from A-dominant strains is a linear a-1,2-linked polymer with about 2% a-1,3 linkages, while O-PS from M-dominant strains is a linear polymer of pentasaccharide repeating units containing one a-1,3-linked and four a-1,2-linked monosaccharide residues [15]. The presence of a-1,2 and a-1,3 linkages in the O-PS of Brucella suggests the existence of at least two mannosyltransferases (Godfroid et al., unpublished data). Here we studied polymorphism of the a-mannosyltransferase gene wbkA. A gene encoding a second mannosyltransferase (WboA) was
Figure 2. Restriction maps of the O-chain biosynthetic genes with the restriction enzymes used. For each gene, restriction map A corresponds to that deduced from the nucleotide sequence of B. melitensis 16M (Godfroid et al., unpublished data). Only differences compared to the nucleotide sequences of B. melitensis 16M are indicated in restriction maps B and C. The restriction patterns A, B, or C are further indicated in table I for each gene and for each Brucella strain studied.
Figure 3. PCR-RFLP of the the O-chain biosynthetic genes. A. Lanes: 1, molecular size markers; 2, wbkA from B. melitensis 16M uncut; 3, wbkA from B. melitensis 16M cut by HindIII; 4, wbkA from B. ovis 63/290 cut by HindIII; 5, per from B. melitensis 16M uncut; 6, per from B. melitensis 16M cut by AluI; 7, per from B. suis 1330 cut by AluI; 8, per from B. melitensis 16M cut by HindIII; 9, per from B. suis 1330 cut by HindIII; 10, wzt from B. melitensis 16M uncut; 11, wzt from B. melitensis 16M cut by SalI; 12, wzt from B. abortus 544 cut by SalI. B. Lanes: 1, molecular size markers; 2, wbkB from B. melitensis 16M uncut; 3, wbkB from B. melitensis 16M cut by AluI; 4, wbkB from B. abortus Tulya cut by AluI; 5, wbkB from B. melitensis 16M cut by TaqI; 6, wbkB from B. suis 686 cut by TaqI; 7, wbkB from B. melitensis 16M cut by Sau3A; 8, wbkB from B. suis 686 cut by Sau3A; 9, wbkC from B. melitensis 16M uncut; 10, wbkC from B. melitensis 16M cut by SmaI; 11, wbkC from B. neotomae 5K33 cut by SmaI.
Biosynthesis of LPS O-side chain in Brucella
recently identified in Brucella abortus strain 2308 [14]. WbkA could interact with WboA to elongate the Brucella LPS O chain by a-1,2 and a-1,3 links. In conclusion, the seven O-chain biosynthetic genes studied appear to be highly conserved among Brucella spp. and thus may have been acquired before species differentiation. Some of the species- or biovar-specific markers detected could be used for molecular typing of brucellae in addition to those previously described [6, 7, 12, 21]. Résumé — Conservation de sept gènes impliqués dans la biosynthèse de la chaîne O du lipopolyoside chez les Brucella. Sept gènes du locus wb de Brucella melitensis 16M impliqués dans la biosynthèse de la chaîne O du lipopolyoside ont été récemment identifiés : les gènes wbkA, gmd, per, wzm, wzt, wbkB et wbkC codant respectivement pour des protéines homologues à une mannosyltransférase, une GDPmannose 4,6 déshydratase, une perosamine synthétase, un transporteur de type ABC (protéine membranaire intégrale), un autre transporteur de type ABC (domaine ATPasique), une protéine hypothétique de fonction inconnue et une formyl transférase putative. Les sept gènes ont un pourcentage GC plus faible (autour de 48 %) que celui typique de Brucella (58 %) et auraient pu être ainsi acquis d’une espèce autre que Brucella. Nous avons étudié le polymorphisme de ces sept gènes par PCR-RFLP chez les Brucella. Par cette technique nous montrons que les sept gènes sont très conservés et sont même présents dans les espèces naturellement rugueuses B. ovis et B. canis et également chez des souches rugueuses de B. abortus et B. melitensis. Néanmoins le peu de polymorphisme détecté consistant en l’absence de sites de restriction ou en des sites supplémentaires permet parfois la différentiation au niveau de l’espèce (ex. B. ovis), du biovar ou de la souche. Nous n’avons pas détecté, par PCR-RFLP, la présence de délétions ou d’insertions dans aucun des gènes et aucune souche de Brucella étudiée. En conclusion, les sept gènes de synthèse de la chaîne O apparaissent très conservés parmi les espèces et souches de Brucella et pourraient donc avoir été acquis avant la différentiation en espèces des Brucella. Certains marqueurs d’espèce ou de biovar détectés pourraient être utilisés pour le typage moléculaire des Brucella en complément de
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ceux décrits auparavant. © 2000 Éditions scientifiques et médicales Elsevier SAS Brucella / polymorphisme de l’ADN / PCR-RFLP / gène de la chaîne O
References [1] Allen C.A., Adams L.G., Ficht T.A., Transposon-derived Brucella abortus rough mutants are attenuated and exhibit reduced intracellular survival, Infect. Immun. 66 (1998) 1008–1016. [2] Alton G.G., Jones L.M., Angus R.D., Verger J.M., Techniques for the Brucellosis Laboratory, Institut National de la Recherche Agronomique, Paris, 1988. [3] Blasco J.M., Brucella ovis, in: Nielsen K., Duncan J.R. (Eds.), Animal Brucellosis, CRC Press, Inc., Boca Raton, 1990, pp. 351–378. [4] Bowden R.A., Verger J.M., Grayon M., Limet J.N., Dubray G., Simultaneous expression of smooth and rough phase properties related to lipopolysaccharide in a Brucella melitensis strain, J. Med. Microbiol. 39 (1993) 363–370. [5] Carmichael L.E., Brucella canis, in: Nielsen K., Duncan J.R. (Eds.), Animal Brucellosis, CRC Press, Inc., Boca Raton, 1990, pp. 335–350. [6] Cloeckaert A., Verger J.M., Grayon M., Grépinet O., Restriction site polymorphism of the genes encoding the major 25 kDa and 36 kDa outer-membrane proteins of Brucella, Microbiology 141 (1995) 2111–2121. [7] Cloeckaert A., Verger J.M., Grayon M., Grépinet, O., Polymorphism at the dnaK locus of Brucella species and identification of a Brucella melitensis species-specific marker, J. Med. Microbiol. 45 (1996) 200–205. [8] Cloeckaert A., Weynants V., Godfroid J., Verger J.M., Grayon M., Zygmunt M.S., O-polysaccharide epitopic heterogeneity at the surface of Brucella spp. studied by enzyme-linked immunosorbent assay and flow cytometry, Clin. Diagn. Lab. Immunol. 5 (1998) 862–870. [9] Cloeckaert A., Zygmunt M.S., Nicolle J.C., Dubray G., Limet J.N., O-chain expression in the rough Brucella melitensis strain B115: induction of O-polysaccharide-specific monoclonal antibodies and intracellular localization demonstrated by immunoelectron microscopy, J. Gen. Microbiol. 138 (1992) 1211–1219. [10] Corbel M.J., Brinley-Morgan W.J., Genus Brucella Meyer and Shaw (1920), in: Krieg N.R., Holt J.G. (Eds.), Bergey’s Manual of Systematic Bacteriology, vol. 1, Williams Wilkins, Baltimore, 1984, pp. 377–388. [11] Douglas J.T., Palmer D.A., Use of monoclonal antibodies to identify the distribution of A and M epitopes on smooth Brucella species, J. Clin. Microbiol. 26 (1988) 1353–1356. [12] Ficht T.A., Bearden S.W., Sowa B.A., Marquis H., Genetic variation at the omp2 porin locus of the brucellae: species-specific markers, Mol. Microbiol. 4 (1990) 1135–1142. [13] Godfroid F., Taminiau B., Danese I., Denoel P., Tibor A., Weynants V., Cloeckaert A., Godfroid J., Letesson J.J., Identification of the perosamine synthetase gene of Brucella melitensis 16M and involvement of the lipopolysaccharide O side chain in Brucella survival in mice and in macrophages, Infect. Immun. 66 (1998) 5485–5493. [14] McQuiston J.R., Vemulapalli R., Inzana T.J., Schurig G.G., Sriranganathan N., Fritzinger D., Hadfield T.L., Warren R.A., Snellings N., Hoover D., Halling S.M., Boyle S.M., Genetic characterization of a Tn5-disrupted glycosyltransferase gene homolog in Brucella abortus and its effect on lipopolysaccharide composition and virulence, Infect. Immun. 67 (1999) 3830–3835. [15] Meikle P.J., Perry M.B., Cherwonogrodzky J.W., Bundle D.R., Fine structure of A and M antigens from Brucella biovars, Infect. Immun. 57 (1989) 2820–2828. [16] Meyer M.E., Current concepts in the taxonomy of the genus Brucella, in: Nielsen K., Duncan J.R. (Eds.), Animal Brucellosis, CRC Press, Inc., Boca Raton, 1990, pp. 1–17.
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Cloeckaert et al.
[17] Palmer D.A., Douglas J.T., Analysis of Brucella lipopolysaccharide with specific and cross-reacting monoclonal antibodies, J. Clin. Microbiol. 27 (1989) 2331–2337. [18] Schurig G.G., Roopii R.M., Bagchi T., Boyle S., Buhrman D., Sriranganathan N., Biological properties of RB51; a stable rough strain of Brucella abortus, Vet. Microbiol. 28 (1991) 171–188. [19] Vemulapalli R., McQuiston J.R., Schurig G.G., Sriranganathan N., Halling S.M., Boyle S.M., Identification of an IS711 element interrupting the wboA gene of Brucella abortus vaccine strain RB51 and a PCR assay to distinguish strain RB51 from other Brucella species and strains, Clin. Diagn. Lab. Immunol. 6 (1999) 760–764.
[20] Verger J.M., Grimont F., Grimont P.A.D., Grayon M., Brucella, a monospecific genus as shown by deoxyribonucleic acid hybridization, Int. J. Syst. Bacteriol. 35 (1985) 292–295. [21] Vizcaino N., Verger J.M., Grayon M., Zygmunt M.S., Cloeckaert A., DNA polymorphism at the omp-31 locus of Brucella spp.: evidence for a large deletion in Brucella abortus, and other species-specific markers, Microbiology 143 (1997) 2913–2921. [22] Weynants V., Gilson D., Cloeckaert A., Tibor A., Denoel P.A., Godfroid F., Limet J.N., Letesson J.J., Characterization of smooth lipopolysaccharides and O polysaccharides of Brucella species by competition binding assays with monoclonal antibodies, Infect. Immun. 65 (1997) 1939–1943.