Institute for Veterinary Bacteriology, University of Berne, Länggass-Strasse 122, CH-3012 Berne, Switzerland1
Laboratório Nacional de Investigação Veterinária, Estrada de Benfica 701, 1500 Lisbon, Portugal2
Author for correspondence: Joachim Frey. Tel: +41 31 631 2484. Fax: +41 31 631 2634. e-mail: jfrey{at}vbi.unibe.ch
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ABSTRACT |
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Keywords: contagious bovine pleuropneumonia, virulence, epidemiology, lipoprotein, genomic deletion
Abbreviations: ABC, ATP-binding cassette; CBPP, contagious bovine pleuropneumonia; DIG, digoxigenin; SC, small-colony type
The GenBank accession numbers for the nucleotide sequences determined in this work are: AF165134 for the 3·4 kb HindIII fragment from M. mycoides subsp. mycoides SC strain L2; AF165135 for the analogous locus in strain Afadé (containing lppB and IS1634); and AF1651136 for the DNA segment containing lppB[MmymyLC] and ORF6[MmymyLC] from M. mycoides subsp. mycoides LC strain Y-goat.
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INTRODUCTION |
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In a broad molecular epidemiological study of M. mycoides subsp. mycoides SC strains from various continents and countries using fingerprinting with the insertion element IS1296, two different clusters of strains were identified: an African cluster, including strains from endemic African countries and historical Australian strains; and a European cluster containing strains isolated since 1980 from outbreaks in four different European countries (Cheng et al., 1995 ). Characteristically, strains of the European cluster have a HindIII fragment of 3·4 kb detected by the IS1296 probe, which is absent in African strains. African-cluster strains, in contrast, had a 4·4 kb HindIII fragment which was not found in the European strains (Cheng et al., 1995
) (see also Fig. 1
). This clustering was confirmed by typing the strains with the most recently discovered insertion element, IS1634 (Vilei et al., 1999
), and this also confirmed that the outbreaks in Europe since 1980 are of different origin than those in Africa. Examination of the antigenic profiles of M. mycoides subsp. mycoides SC strains using immunoblot analysis of total cell proteins also revealed differences between European and African strains (Poumarat & Solsona, 1994
). In particular, European strains were found to lack a dominant antigen with an apparent molecular mass of 7071·5 kDa which was present in all African and Australian strains (Gonçalves et al., 1998
).
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METHODS |
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Cloning and sequencing strategies.
A partial genomic library of M. mycoides subsp. mycoides SC strain L2 was made by cloning total DNA digested with HindIII into the HindIII site of vector pBluescriptII SK(-) (Stratagene). Clones were selected for the presence of IS1296 by colony screening with the DIG-labelled IS1296 probe. Plasmid DNA of the positive colonies was isolated using the QIAprep Spin Plasmid kit (Qiagen). Double-stranded nested deletion, using exonuclease III (Pharmacia Biotech), was carried out following the manufacturers protocol to sequence cloned DNA fragments. Sequencing was performed with a DNA Sequenator AB310 and the Taq Dye Deoxy Terminator Cycle Sequencing Kit (Perkin Elmer) using primers complementary to the T3 and T7 promoters of the vector. Comparisons of DNA sequences and their deduced amino acid sequences with the EMBL/GenBank and NBRF databases were performed using the programs BLASTN, BLASTX and BLASTP (Altschul et al., 1990 ). Analysis of protein sequences for characteristic motifs was done using the programs PROSITE (Bairoch et al., 1995
) and SignalIP (Nielsen et al., 1997
).
PCR reactions.
Oligonucleotide primer sequences used for PCR amplifications and their corresponding annealing temperatures are given in Table 2. The PCR reactions were carried out in a DNA thermal cycler Gene Amp 9600 (Perkin Elmer) in 20 µl reaction mix (50 mM Tris/HCl, pH 9·2, 1·75 mM MgCl2, 16 mM (NH4)2SO4, 350 µM of each dNTP, and 300 nM forward and reverse primers) using as template 210 ng purified mycoplasma DNA. To each reaction, 2·5 units Taq DNA polymerase or 1·75 units of a mixture of Taq DNA and Pwo DNA polymerase (Expand Long Template PCR System kit, Boehringer Mannheim) were added. The latter was used for amplification of long DNA fragments (>3 kb) or for DNA segments that were used for cloning and for sequencing. The samples were subjected to 30 cycles of amplification consisting of 30 s at 94 °C, 30 s at 48 °C and elongation at 68 °C according to the length of the amplicon desired. The PCR amplification products were analysed by electrophoresis through 0·7% agarose gels and visualized after staining with ethidium bromide on a UV transilluminator (Ausubel et al., 1990
).
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Immunoblot analysis was carried out using standard protocols (Ausubel et al., 1990 ) and phosphatase-conjugated goat anti-mouse IgG+IgM (H+L) (Kirkegaard & Perry Laboratories) at a dilution of 1:2000, or phosphatase-conjugated goat anti-rabbit IgG (H+L) (Kirkegaard & Perry Laboratories) at a dilution of 1:5000, respectively.
Triton X-114 phase partitioning.
M. mycoides subsp. mycoides SC strain Afadé components were separated into hydrophobic and hydrophilic fractions by the Triton X-114 (Fluka) partitioning method as previously described (Cheng et al., 1996 ). A 50 ml culture of mycoplasmas was grown to the stationary phase and then harvested by centrifugation. The cells were washed three times in TS buffer (10 mM Tris/HCl, pH 7·5 and 150 mM NaCl) and resuspended in 1 ml TS buffer. Prewashed, condensed Triton X-114 was added to the 1 ml sample to give a final concentration of 1% (w/v) and the mixture was incubated for 30 min at 4 °C with gentle rocking. Insoluble components were then removed by centrifugation at 4 °C for 5 min at 13000 g. The Triton X-114-solubilized material was incubated for 15 min at 37 °C to allow condensation of the detergent phase, which was then separated by centrifugation at 37 °C for 5 min at 13000 g. The lower, detergent phase was adjusted to its original volume with TS buffer without the addition of Triton X-114. The upper, aqueous phase was transferred to a new tube and chilled to 4 °C, then Triton X-114 was added to a final concentration of 1%. This mixture was rocked at 4 °C for 5 min, incubated for 15 min at 37 °C and then centrifuged at 37 °C for 5 min at 13000 g. This cycle was repeated three times to ensure complete removal of hydrophobic fractions from the aqueous phase. Both phases were finally adjusted to the same volume. Samples from the detergent phase, the aqueous phase and whole mycoplasma cells were mixed with protein sample buffer, run on SDS/10% acrylamide gels and blotted onto nitrocellulose. The filter was subsequently used for immunoblotting with the monospecific, polyclonal antibodies directed against LppB.
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RESULTS |
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Analysis of the analogous genomic locus in the African M. mycoides subsp. mycoides SC strain Afadé
In order to clone and subsequently analyse the locus analogous to the above-characterized 3·4 kb HindIII fragment from strains of the African cluster, PCR amplification using genomic DNA of strain Afadé as template and oligonucleotide primers 3480bp-L and 3480bp-R, complementary to sequences in the 3·4 kb HindIII fragment of strain L2, was performed. This PCR amplification resulted in a fragment of about 11·3 kb from strain Afadé and from all other strains of the African cluster, except strain 91130, which gave a 9·0 kb fragment (Table 1). PCR with these primers using DNA from European strains as template resulted in a 2·5 kb fragment, indicating that European strains were lacking approximately 8·8 kb at this locus. The DNA fragment of 11·3 kb obtained by PCR amplification from strain Afadé was sequenced directly using oligonucleotide primers 3480bp-L and 3480bp-R, and further primers which were subsequently derived from sequence data. Sequence analysis revealed the presence of an 8841 bp DNA fragment in the genome of Afadé which was not present in the clone from the L2 genomic library, while the remaining 2·5 kb of the sequence was the same for both strains. This explained the difference in the above-described PCR reactions, which amplified an 11·3 kb fragment from African-cluster strains and a 2·5 kb fragment from European strains. During sequence analysis we observed that certain sequencing primers gave two superimposed sequences. This was due to the presence of two directly repeated segments of 478 bp flanking a copy of IS1634 (Fig. 2
). These repeats are the longest direct repeats created by IS1634 found thus far in M. mycoides subsp. mycoides SC (Vilei et al., 1999
).
The 11·3 kb fragment of strain Afadé contained, in addition to a full copy of IS1634, five different open reading frames. One of them had the structure of a gene encoding a peptide with a typical signal sequence for lipoproteins, but with no homology to any other product deposited in the EMBL/GenBank database. It was designated lppB (Fig. 2), following the proposal for nomenclature of lipoproteins in M. mycoides (Monnerat et al., 1999
). The lppB gene encoded a potential lipoprotein precursor of 622 aa. The N-terminal signal sequence had a signal peptidase II cleavage site after amino acid 23. Hence the mature LppB was predicted to contain 599 aa and to have a molecular mass of 67·4 kDa. There were 6 UGATrp codons in the lppB gene. A further open reading frame, ORF6, was found to encode a putative membrane protein of 516 aa with 23% identity in a 320 aa portion to the surface-located membrane protein Lmp3 of Mycoplasma hominis (accession number JC6009). Neither lppB nor ORF6 was present in European-cluster strains of M. mycoides subsp. mycoides SC. In addition, the 11·3 kb amplicon from strain Afadé contained a complete ORF5 encoding the analogue of the proline-rich membrane protein of E. faecalis on one side of the splice site corresponding to the 8·84 kb deletion found in the European strains, and a complete ORF2 encoding the previously mentioned putative ABC transporter protein on the other side of the splice site (Fig. 2
). Moreover, ORF2 was followed by a partially overlapping ORF3 encoding another putative ABC transporter (Fig. 2
).
Presence of the lppB gene in M. mycoides subsp. mycoides SC strains
A DIG-labelled probe prepared by PCR with primers DIG-4L and 7500bp4R (Table 2) was used in Southern blot analysis to detect the presence of lppB in the genomic DNA of a selection of strains of M. mycoides subsp. mycoides SC digested with HindIII. None of the European strains reacted with the probe, while the strains belonging to the African cluster showed three predicted DNA fragments (Fig. 3
). This result suggested that there are no additional allelic, silent copies of lppB present other than the one identified in the African strains. The presence of the three reacting bands of 3·9, 1·0 and 0·4 kb on the Southern blot was explained by the fact that the lppB gene has two HindIII restriction sites, as shown in Fig. 2
. Hence, lppB occurs as a single copy in the African strains.
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DISCUSSION |
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The aim of the present study was to identify major genetic differences between strains of the African and European clusters and to find a correlation with the characteristic antigenic difference between the two clusters, represented by the lack of an antigen at 7071·5 kDa in European strains, as observed previously (Gonçalves et al., 1998 ). Sequence comparison of an analogous DNA locus in two representative strains of both clusters showed that the genome of the European-cluster strains lacked a segment of 8·84 kb, compared to strains from the African cluster. A copy of IS1296 was found approximately 600 bp from the locus involving this deletion event. This single difference resulted in two distinct changes in the IS1296-profile between African- and European-cluster strains (a HindIII band of 3·4 kb for European strains and a HindIII band of 4·4 kb for African strains, as shown in Fig. 1
). Characterization of both ends of the deletion site in the African-cluster strains revealed the presence of open reading frames at each side. Both encoded full-length genes, one a putative proline-rich membrane protein and the other a putative ABC transporter protein, the latter belonging to a class of transporters which is abundant in mycoplasmas (Fraser et al., 1995
; Himmelreich et al., 1996
). In contrast, in strains of the European cluster only fragments of each of these open reading frames were found (Fig. 2
), indicating that the observed genetic difference was caused by a deletion event which gave rise to the cluster of European strains, rather than an insertion event in the African-cluster strains. Hence, our genetic data suggest that the European strains descended from an ancestral strain belonging to the African cluster of M. mycoides subsp. mycoides SC.
While all European strains analysed by PCR were similar in the vicinity of the deletion, one strain of the African cluster, strain 91130, had a 2·3 kb smaller fragment between the splicing sites compared to the other African strains. This difference corresponds to the absence in strain 91130 of one copy of IS1634 plus the corresponding 478 bp direct repeat, found in all other African strains studied. This reflects previous findings that showed that IS1634 patterns among African field strains varied and suggested that there might be a higher frequency of transposition of IS1634 compared to IS1296.
Among the different open reading frames found on the DNA segment that is specific for African-cluster strains, we identified the gene lppB, encoding a potential membrane-associated lipoprotein, as deduced from sequence comparisons and analysis. The lppB gene was found as a single copy in the African cluster, while European strains were devoid of this gene. This further supports the hypothesis that the European cluster was derived from the African cluster by genomic deletion. Thus, expression of LppB seems to be a significant phenotypic difference distinguishing the African-cluster strains from the European strains and explains the previously observed difference in the antigen profile at 7071·5 kDa between different strains of M. mycoides subsp. mycoides SC (Poumarat & Solsona, 1994 ; Gonçalves et al., 1998
). LppB seems to have homologues in M. mycoides subsp. mycoides LC and Mycoplasma sp. bovine group 7, as revealed by immunoblot analysis using monospecific antisera directed against recombinant LppB.
It has to be noted that beside African field strains, also strains from Australia, and the African and Australian vaccine strains of M. mycoides subsp. mycoides SC, possess the lppB gene. Historical isolates from Australia were previously shown by IS1296 typing to belong to the same cluster as strains isolated from Africa (Cheng et al., 1995 ). The presence of the lppB gene in vaccine strains and the fact that the LppB protein is expressed in these strains indicates that their virulence is attenuated at another genetic locus.
Membrane lipoproteins of several pathogenic mycoplasmas have been shown to induce blastogenesis and secretion of proinflammatory cytokines by a mechanism distinct from that of lipopolysaccharides, and hence may be important mycoplasma virulence factors (Brenner et al., 1997 ; Herbelin et al., 1994
; Mühlradt & Frisch, 1994
; Rawadi & Roman-Roman, 1996
). In addition they play an important role in the stability and integrity of the fragile mycoplasma membrane (Razin et al., 1998
). LppB therefore might contribute indirectly to the virulence of M. mycoides subsp. mycoides SC. However, it remains to be determined exactly how the lack of LppB might be responsible for the lower virulence and, in particular, the lower infectivity of European strains, compared to African cluster strains, as observed in comparative experimental infections (Abdo et al., 1998
).
In addition to the lppB gene, the strains of the European cluster lack several other genes, including putative ABC transporter genes, which might also be involved in the reduced pathogenicity of these strains. Such an effect of putative ABC transporters on virulence was postulated for M. hyopneumoniae (Blanchard et al., 1996 ).
In summary, our results show that the current European strains of M. mycoides subsp. mycoides SC lack a substantial segment of genetic information, which must have occurred by a deletion event. We therefore conclude that the strains found in the reemerging outbreaks of CBPP in Europe during the last 15 years are derived from an ancestral strain which belonged to the African cluster and which seems to represent the original type of M. mycoides subsp. mycoides SC.
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ACKNOWLEDGEMENTS |
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This study is part of the European COST Action 826 Ruminants Mycoplasmoses, and was supported by grant no. C96.0073 of the Swiss Ministry of Education and Science and by the Swiss Federal Veterinary Office.
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Received 9 July 1999;
revised 28 October 1999;
accepted 15 November 1999.