1 UMR Mycoplasmoses des Ruminants, AFSSA-Site de Lyon, 31 av Tony Garnier, FR-69364 Lyon Cedex 07, France
2 Department of Biotechnology, Royal Institute of Technology, AlbaNova University Center, SE-106 91 Stockholm, Sweden
3 UMR Mycoplasmoses des Ruminants, Pathologie du Bétail, Ecole Nationale Vétérinaire de Lyon, 1, Av Bourgelat, FR-69280 Marcy l'Etoile, France
4 Department of Bacteriology, National Veterinary Institute, SE-751 89 Uppsala and Division of Food Hygiene and Bacteriology DBS-VPH, PO Box 7009, Swedish University of Agricultural Sciences, SE-75007 Uppsala, Sweden
Correspondence
François Poumarat
f.poumarat{at}lyon.afssa.fr
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ABSTRACT |
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INTRODUCTION |
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Although Mmymy SC strains are homogeneous (based on protein analysis, Costas et al., 1987), they can be differentiated to some extent at the molecular level. African and European strains can be clearly distinguished by restriction analysis of whole DNA (Poumarat & Solsona, 1995
), by the chromosomal distribution of IS1296 (Cheng et al., 1995
; Frey et al., 1995
) and by multilocus sequence analysis (Lorenzon et al., 2003
). All the Mmymy SC strains isolated in Europe since 1980 have undergone a major chromosomal deletion of 8·84 kb, including certain genes in the ABC glycerol transporter operon and a putative lipoprotein lppB (Vilei et al., 2000
).
In 1990 it was first reported that mycoplasmas can undergo reversible high-frequency variation to alter their surface antigenic repertoire within a cell population (Rosengarten & Wise, 1990). New systems of surface protein variation in pathogenic mycoplasma species are regularly reported, and the observations strongly suggest that such variability confers the means of enhancing colonization and adapting to the host environment during the various stages of infection (Citti & Rosengarten, 1997
).
Protein variation not only enables mycoplasmas to escape the host immune defence system (Citti et al., 1997; Neyrolles et al., 1999
; Le Grand et al., 1996
), but is also involved in adhesion (Sachse et al., 2000
; Washburn et al., 1993
), haemadsorption (Markham et al., 1993
; Noormohammadi et al., 1997
, 2000
), membrane transport (Theiss & Wise, 1997
) and immunomodulation (Muhlradt et al., 1998
). The simplest form of surface variation is a reversible ON/OFF switch in protein expression, called phase variation. The wide range of mutations involved in such variation include promoter mutations (Yogev et al., 1991
; Persson et al., 2002
) or inversion (Horino et al., 2003
), changes in putative transcription activators (Glew et al., 1998
; Washburn et al., 1998
), mutations that lead to gene truncation (Theiss & Wise, 1997
), frameshift mutations (Boguslavsky et al., 2000
; Winner et al., 2003
; Zhang & Wise, 1997
) and DNA inversions or gene conversions that fuse the coding gene to an active promoter (Bhugra et al., 1995
; Glew et al., 2002
; Noormohammadi et al., 2000
; Lysnyansky et al., 2001b
).
Some surface proteins undergo antigenic variation by altering the size and/or epitope composition. Most of these proteins contain repetitive units of different structure. The repetitive sequences function like interchangeable cassettes that can be deleted, inserted or recombined to form a rich variety of protein variants (Zheng et al., 1995; Zhang & Wise, 1996
; Yogev et al., 1995
; Lysnyansky et al., 1999
, 2001a
; Boguslavsky et al., 2000
; Boesen et al., 1998
; Citti et al., 2000
). Epitope masking is a phenomenon in which the epitopes of a constitutively expressed surface protein are subject to variable surface exposure, either due to a secondary protein that sterically blocks accessibility of the surface epitopes or as a consequence of size variation (Citti et al., 1997
; Zhang & Wise, 2001
). Variable proteins are often subjected to both size and phase variation, and many are members of multigene families. The proteins of a multigene family are often differentially expressed, and chromosomal rearrangements occur to allow transcription of one gene instead of another (Lysnyansky et al., 1999
; Citti et al., 2000
; Markham et al., 1994
, 1999
; FlitmanTene et al., 2000
; Glew et al., 2000
; Roske et al., 2001
; Shen et al., 2000
; Horino et al., 2003
; Noormohammadi et al., 1998
).
Mmymy SC was originally thought not to exhibit intraclonal variability, but a variable surface protein Vmm (Persson et al., 2002) that undergoes reversible phase variation has recently been identified. The vmm gene has been demonstrated in all Mmymy SC strains as a single copy and is regulated at the transcriptional level by dinucleotide (AT) insertions or deletions in a repetitive region of the promoter spacer. Genome sequencing has shown that five additional genes encoding prolipoproteins that have promoters with variable TA-repeats are located upstream of the vmm gene and may belong to the same family (Westberg et al., 2004
).
Studies of the epitope recognized by mAb 3F3 started because the epitope is specific to Mmymy SC and because it is recognized in the host immune response (Brocchi et al., 1993). These features make 3F3 useful for diagnostic analysis. Unexpectedly, antigenic variation was observed with this mAb, indicating that Mmymy SC is undergoing a new phase variation.
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METHODS |
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mAb, oligopeptide and immunoassays.
mAb 3F3 was prepared from Mmymy SC type strain PG1 in collaboration with the AFSSA-Site de Lyon and the Istituto Zooprofilattico Sperimentale della Lombardia e dell' Emilia (Brocchi et al., 1993). Dot blotting was performed as described by Poumarat et al. (1991)
. Colony immunostaining was carried out as described by Rosengarten & Yogev (1996)
. Freshly grown mycoplasma colonies were transferred to nitrocellulose membranes by placing the membranes on the surface of agar plates. The membranes were gently removed and blocked in TBS-B buffer [TBS (50 mM Tris and 0·2 M NaCl) supplemented with 10 % horse serum], before they were incubated with mAb 3F3 (at a concentration of 9 µg ml1) diluted at 1/1000 in TBS-B, for 1·5 h at ambient temperature. Unbound antibody was removed by washing three times in TBS-T (TBS and 0·05 % Tween 20) and once in TBS. Thereafter, the membranes were incubated with peroxidase-conjugated rabbit anti-mouse antibodies (DAKO) in TBS-B (2·6 µg ml1) for 1·5 h, followed by three washes in TBS-T and one wash in TBS. Colonies expressing proteins that bind mAb 3F3 were specifically identified by an enzymic colour reaction with 4-chloro-1-naphthol, which gives a dark blue colour. The membranes were finally stained with ponceau red solution (Sigma Diagnostics), which non-specifically stains proteins red, to reveal colonies negative for mAb 3F3.
Western blotting was carried out as previously described (Le Grand et al., 1996) after SDS-PAGE in 816 % gradient polyacrylamide gels.
The synthetic peptide containing the target epitope for mAb 3F3, which covered the region that was shared by all phagemid clones, was obtained from Interactiva Biotechnologie. The peptide sequence was acetylNHNTQSEEVKKAFVDSYNKLHGTNHNNLKAICONH2.
Immunoelectron microscopy.
The ON-type and OFF-type variants of the Afadé strain were subjected to immunoelectron microscopy. Colonies of Mmymy SC were grown on agar plates and resuspended in 200 µl 0·05 M Tris/HCl buffer (pH 7·4) with 1 % BSA. One drop of cell suspension was placed on a 200 mesh nickel grid and coated with 1/1000 poly-L-lysine for 5 min, before being blocked for 5 min with Tris/HCl buffer (pH 7·4) containing 1 % BSA. Grids were transferred to a 50 µl drop of mAb 3F3 diluted 1 : 100 in Tris/HCl buffer (pH 7·4) with 1 % BSA and incubated for 15 min at room temperature. After rinsing twice for 5 min in Tris/HCl buffer (pH 7·4) and once for 5 min in Tris/HCl buffer (pH 8·2), the grids were pre-incubated for 5 min in Tris/HCl buffer (pH 8·2) with 1 % BSA. Grids were then incubated for 15 min at room temperature with the secondary antibody: gold conjugate anti-mouse immunoglobulins (10 nm) (Tebu, France) diluted 1 : 40 in Tris/HCl buffer (pH 8·2) with 1 % BSA. After rinsing twice for 5 min in Tris/HCl buffer (pH 8·2), once for 5 min in Tris/HCl buffer (pH 7·4) and once for 5 min in filtrated distilled water, the grids were negatively stained with 0·3 % (w/v) phosphotungstic acid (pH 7·0), blotted dry and observed with a JEOL 1200 EX transmission electron microscope equipped with numeric Megaview II camera and AnalySIS software.
Phage display.
A gene VIII-based whole-genome phage-display library of randomly fragmented chromosomal DNA from strain M223/90 of Mmymy SC (Persson et al., 2002) was used to identify the target epitope of mAb 3F3. The library was made from a mixture of phagemid vectors pG8PL0 and pG8SPA0 (Jacobsson & Frykberg, 1998
). It contained 107 independent clones and the titre of the phage stock was 1011 p.f.u. Both vectors were constructed so that the mycoplasma peptides were fused to a tag that binds human serum albumin (HSA), and permitted ligand-independent screening of clones that expressed mycoplasma peptides.
Affinity selection of phage that displayed mycoplasma peptides recognized by mAb 3F3 was then carried out by three consecutive pannings. Two Maxisorp microtitre wells (Nalge Nunc International) were coated with 125 µg protein G in 250 µl coating buffer (0·05 M Na2CO3, pH 9·7) for 1 h at room temperature. The wells were rinsed three times with PBS containing 0·05 % Tween 20, before addition of 250 µl mAb 3F3 diluted 1/3000 in PBS in one of the wells and BALB/c mouse serum in the other well as a negative control. The antibodies were immobilized for at least 1 h and the wells were then rinsed six times with PBS/Tween and blocked with PBS/Tween for 10 min. Meanwhile, the HSA-binding region of the phagemids was blocked by incubating the phage stock with 100 µg HSA ml1. After 1 h, 200 µl phage stock was transferred to each coated well, and the panning proceeded for 4 h at room temperature. The wells were rinsed 30 times in PBS/Tween, to remove unspecifically bound phage, then the captured phage was eluted with 200 µl sodium citrate buffer (50 mM sodium citrate, 140 mM NaCl, pH 2). The eluates were neutralized with 40 µl 2 M Tris/HCl buffer (pH 8·7), serially diluted in LuriaBertani (LB) medium and immediately used to infect a 150 µl overnight culture of E. coli CDJ64/14 (Rydén & Isaksson, 1984
). The infected cultures were incubated for 30 min at room temperature, spread on selective Luria agar (LA) plate (Sigma) and incubated overnight. The colonies were then counted, and 150 were transferred to a new selective LA plate for colony blot screening. New phage stocks were produced by resuspending the remaining colonies from the 3F3 panning in LB medium, infecting the bacteria with helper phage R408 and processing as above. A conjugate of HSA and horseradish peroxidase was used in the colony blot screening to detect the tag. Replica blots were screened with mAb 3F3 to identify the clones that expressed the 3F3 epitope.
DNA sequencing.
The mycoplasma inserts from phagemid clones that were positive for the tag and for mAb 3F3 by colony blot screening were sequenced. Phagemids were prepared with the Wizard Plus SV Miniprep DNA purification system (Promega), and the inserts first sequenced with the ALBP primer (Table 2), which is complementary to the HSA-binding region of the vector. Depending on the mycoplasmal DNA vector insertion, the phagemids were then sequenced with the Sasekv or Nypel primers (Table 2
). The complete sequence of the ptsG gene and flanking region was determined after searching the genome database of Mmymy SC strain PG1 with the consensus sequence (Westberg et al., 2004
).
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Southern blot hybridization.
The presence of the ptsG gene in 43 Mmymy SC strains (Table 1) was assessed by Southern blotting as described by Poumarat et al. (1999)
. Chromosomal DNA was digested to completion with the restriction enzyme HindIII and the fragments were separated by electrophoresis on 0·8 % agarose gel. Two probes were used: i) the 3F3-insert probe (Table 2
), corresponding to an inner part of the 3F3 epitope sequence (hybridization at 44 °C), and ii) the ptsG PCR probe (hybridization at 65 °C). The latter probe was produced by PCR with the 3F3-forward and 3F3-reverse primers (Table 2
) with DNA from strain PG1 as template. The reaction mixtures were prepared with the PCR DIG Probe Synthesis Kit (Roche Diagnostic).
Nucleotide sequence accession number and sequence analysis.
The nucleotide sequences for the two copies of the ptsG gene were assigned the ORF names MSC_0860 and MSC_0873 in the genome sequence database of the Mmymy SC strain PG1 (accession no. NC_005364). The ProDom database was used to study the protein domain arrangements (Corpet et al., 1999). Multiple sequence alignment was performed with the Multalin program (Corpet, 1988
).
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RESULTS |
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Identification and putative function of the gene encoding the protein that binds mAb 3F3
Serial affinity pannings of the phage-display library to mAb 3F3 resulted in an accumulation of phages that expressed peptides containing the target epitope of this mAb. The total amount of recovered phage determined from the viable count of infected E. coli was 2·5x104, 4·0x104 and 2·2x106 c.f.u. of the first, second and third pannings, respectively. Colony blot screening for expression of the tag in 150 of these colonies gave 5, 46 and 140 HSA-positive colonies. In the original phage stock, two colonies out of 150 were positive for HSA. Replica blots which were screened with mAb 3F3 showed that nearly all the tag-positive colonies were also positive for mAb 3F3. Control pannings with BALB/c serum performed in parallel with the mAb 3F3 pannings resulted in considerably lower amounts of recovered phagemids, namely 1·3x104, 5·9x103 and 6·0x102 c.f.u., respectively. Twenty colonies that were positive for both the tag and for mAb 3F3 were randomly selected from the third panning. The phagemid DNA was then isolated and the mycoplasmal inserts sequenced. An overlapping sequence covering a particular region was detected in all inserts, thus limiting the 3F3 epitope to an 85 bp gene region that was present in all clones. Several colonies turned out to be multiples of the same recombinant, but there were eight unique recombinants in which the gene segments differed in size. When the panning experiments were repeated, another five clones from the second panning were sequenced. The mycoplasmal inserts of these clones also contained the 85 bp gene region. The panning results were further evaluated by producing a synthetic peptide of 29 amino acids corresponding to the 85 bp region. Dot blotting of the synthetic peptide and detection with mAb 3F3 resulted in strong positive signals (data not shown). When the consensus of the mycoplasma inserts was compared to the genome database of Mmymy SC strain PG1 (Westberg et al., 2004), sequence data were retrieved for the full gene and its flanking regions. Two genes (MSC_0860 and MSC_0873) were found, both annotated ptsG. Using the BLAST program (Altschul et al., 1997
), we showed that MSC_0860 and MSC_0873 are identical, except in position 113, where the GCT (Ala) codon in MSC_0860 is replaced by the GTT (Val) codon in MSC_0873. Translation could theoretically occur from seven putative start codons, two of which are predicted to produce a signal sequence (Nielsen et al., 1997
). The first signal sequence, which starts at the first or second methionine, was predicted to be cleaved off between amino acid positions 49 and 50 in AIA-AN residues. A putative ribosome-binding site (AAAGAA in MSC_0860 and AAAGGA in MSC_0873) is located 12 bp upstream of the first start codon. Promoter regions TATTAA and TATAAT were identified 10880 bp before the same start codon. The longest ORF (2028 bp) would encode a native protein of 676 aa (about 75 kDa).
The ptsG gene encodes the permease of the phosphoenolpyruvate : glucose phosphotransferase system (glucose PTS). Bacterial sugar phosphotransferase catalyses the concomitant transport and phosphorylation of its sugar substrate.
Mmymy SC PG1 genome analysis (Westberg et al., 2004) revealed two other ORFs annotated ptsG, designated MSC_0054 and MSC_0161, but they do not harbour the protein sequence corresponding to the epitope recognized by mAb 3F3.
The protein sequence deduced from MSC_0860 and MSC_0873 showed 69 % similarity with PtsG of Spiroplasma citri (accession no. AAP55652), 56 % similarity with PtsG of Mycoplasma pulmonis (accession no. NP_325848) and 52 % similarity with PTS of Mycoplasma penetrans (accession no. NP_757560.1).
The protein sequence obtained from MSC_0054 shows 48 % similarity with PtsG of S. citri (accession no. AAP55652), 51 % similarity with PtsG of M. pulmonis (accession no. NP_325848), 64 % similarity with PtsG of M. penetrans (accession no. NP_757560.1) and 47 % similarity with MSC_0860 and MSC_0873.
MSC_0161 encodes a putative protein showing homology only with the N-terminal part of PtsG and which could be described as truncated. However, nucleic acid analysis showed that a complete PtsG protein could be obtained by removing three frameshifts (data not shown).
PtsG protein structures
PTS permeases consist of several distinct or fused polypeptide chains (Saier & Reizer, 1992). MSC_0860 and MSC_0873 code for a native protein of 676 aa (about 75 kDa) and MSC_0054 for a protein of 580 aa (about 65 kDa). Protein sequence analysis, deduced from MSC_0054, MSC_0860 and MSC_0873 by using ProDom (Corpet et al., 1999), indicates that these proteins consist of three domains linked to each other as follows: IIC domain, unidentified domain and IIB domain. Although the sequence signature of the IIB domain was found in all PtsG of Mmymy SC, it is not the consensus sequence described for most bacteria (i.e. N-[LIVMFY]-x(5)-C-x-T-R-[LIVMF]-x-[LIVMF]-x-[LIVM]-x-[DQ]; Reizer et al., 1994
). In MSC_0054, [DQ] is replaced by N and in MSC_0860 and MSC_0873, T is substituted by S. These substitutions are found in other bacteria, such as M. pulmonis and Pasteurella multocida. Immediately downstream of the IIC domain, a short sequence was observed containing two histidyl residues. It showed a low similarity to the sequence signature of the IIA domain and could be a degenerated IIA domain. However, a consensus sequence signature of the IIA domain was not found in the PtsG of Mmymy SC. Moreover, a multiple sequence alignment between the PtsG proteins of Mmymy SC and those of other mycoplasmas clearly confirmed the absence of a IIA domain in Mmymy SC. The observed structure of Mmymy SC differed from the IICBA organization described for the PTS mycoplasmas. However, an absence of IIAIICB fusion has also been observed for the phytopathogenic mollicute S. citri (André et al., 2003
).
The IIC domain (amino acids 42445 according to ProDom) deduced from MSC_0860 and MSC_0873 shows eight transmembrane regions as predicted by TopPred2 (Claros & von Heijne, 1994). The 85 bp gene region corresponding to the epitope recognized by mAb 3F3 is located between two conserved segments of the IIC domain at amino acid position 271299. This part of the gene is predicted to be in a loop of 87 aa at the outside of the cell surface between membrane helices 4 and 5. The sequence of the mAb 3F3 epitope did not show any similarity with any other genes or peptides in the NCBI database.
The ptsG gene in the Mmymy SC genome
MSC_0860 and MSC_0873 occur in a long chromosomal DNA direct repeat (Fig. 3). The observed organization of the regions containing MSC_0860 and MSC_0873 is similar. Differences are due to the insertion and orientation of IS1634 as well as to mutations creating frameshifts in the ORFs. Each repeat contains an IS1634 copy (IS1634AX and IS1634AZ according to Westberg et al., 2004
) inserted in different sequences. IS1634AX is inserted 86 bp after the start codon of the glk gene and, in consequence, glk is truncated at its 5' extremity. In the MSC_0873 region, the glk gene does not seem to be functional because of a frameshift resulting from the insertion of two guanosines 201 bp after the start codon of the glk gene. This gene encodes a putative protein with the sequence signature of the ROK (Repressor, ORF, Kinase) family. This family regroups transcriptional repressors, sugar kinases and uncharacterized ORFs (Titgemeyer et al., 1994
). Repressor proteins in this family possess an N-terminal region which contains an helixturnhelix DNA-binding motif. No such motif could be identified in the protein deduced from the glk gene, even when the frameshift was removed to restore the complete gene. This suggests that the protein in question is a sugar kinase. However, because of the frameshift or the IS1634 insertion, neither glk gene should be functional.
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Occurrence of the ptsG gene in field isolates of Mmymy SC
Forty-three Mmymy SC strains were subjected to Southern blot on HindIII-digested genomic DNA and hybridization with the ptsG PCR probe (Table 1). Both the ptsG-PCR probe and the specific 3F3 insert probe were tested beforehand on 10 strains, and both hybridizations gave the same profiles (Fig. 4
). Although all Mmymy SC strains contained the ptsG gene, three distinct patterns were obtained (Fig. 4
and Table 1
). All 26 strains isolated in Europe showed only a single band which was 4 kb in size in 23 strains and 2 kb in size in three strains (two from Italy and one from Spain). All strains from Africa, like the PG1 strain, had the same profile with two bands (7·8 kb and 4 kb) except one from Senegal, which showed a European profile. The profile of the Australian Gladysdale strain was of African type, whereas that of the V5 Australian vaccine strain was of European type. The two ptsG genes of the PG1 strain detected by the probes showed only three HindIII restriction sites that were in the 3' distal part of the gene outside the target sequences of the two probes. Thus, the HindIII fragments of Mmymy SC could not contain more than one copy of the ptsG gene, which could explain why European strains with a single band profile harbour only one copy of the ptsG gene. The HindIII restriction pattern, deduced from the DNA sequence of the MSC_0860 and MSC_0873 regions, indicates that the 4 kb fragment revealed by Southern blot hybridization corresponds to the MSC_0860 region, and that the 7·8 kb fragment corresponds to the MSC_0873 region.
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DISCUSSION |
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The phage-display library (Persson et al., 2002) and genome database (Westberg et al., 2004
) have already proved efficient in identifying a particular protein epitope and its corresponding gene. However, there is an obvious risk of creating false epitopes in fusion proteins (Fehrsen & du Plessis, 1999
; Murthy et al., 1999
). The specific recognition by mAb 3F3 of a synthetic oligopeptide deduced from a phage-display-selected DNA sequence abolished any remaining doubts about the reliability of the selection during pannings and confirmed identification of the true gene encoding the proteins which bind mAb 3F3. This gene was identified as ptsG, which encodes a putative glucose PTS permease. Phase variation in mycoplasma transporters has so far only been documented for one subunit in an ABC transporter of Mycoplasma fermentans (Theiss & Wise, 1997
). These authors speculated that variation might not only have consequences for immune evasion, but also provide alternative transport capacities through the use of different subsets of the same genes.
Genetics of glucose PTS
Two ptsG genes, MSC_0860 and MSC_0873, potentially code for the proteins recognized by 3F3 mAb and show homology with the glucose PTS permeases. The ptsG gene of Mmymy SC contained seven alternative initiation codons, and two of the theoretical ORFs were predicted to contain a signal sequence, suggesting the putative expression of at least two surface-exposed proteins of different sizes. Such a coordinate phase variation of two distinct surface antigens encoded by a single gene has also been reported in Mycoplasma synoviae (Noormohammadi et al., 1998). These genes are in a duplicated region containing several copies of IS1634. Sequence analysis showed that IS1634CE, which separates the two chromosomal repeats, is bordered by two different sequences, each corresponding to the direct repeats found for the insertion sequence bordering the chromosomal DNA repeat (Fig. 3
). Such chimerical organization leads to the conclusion that IS1634CE was involved in duplication of the ptsG region. The insertion of IS1634AX and IS1634AZ inside the chromosomal repeats occurred after the duplication of the ptsG region, as shown by different direct repeats bordering both insertion sequences.
The two regions could be distinguished by Southern blot hybridization. The copy containing MSC_0860 is carried by a 4 kb fragment and the 7·8 kb fragment contains the MSC_0873 gene. We showed that the European strains lack the 7·8 kb fragment, i.e. the MSC_0873 copy. As strains with only the MSC_0860 copy expressed the epitope recognized by 3F3 mAb, we concluded that the expression of MSC_0860 is sufficient to have the protein recognized by 3F3 mAb. The 3F3-OFF type does not express MSC_0860 and MSC_0873. Whether such bacteria are still able to use glucose or not remains unknown due to the presence of another ptsG gene, MSC_0054.
The three putative PtsG proteins show a structure atypical for mycoplasmas. Up till now, an IICBA organization has been described for mycoplasmas, but this association of the IIA domain with IICB was not found for Mmymy SC. Two genes, MSC_0274 and MSC_0394, encoding hypothetical IIA protein, were found in Mmymy SC (Westberg et al., 2004). The protein deduced from MSC_0274 is similar to the IIA of M. penetrans (accession no. NP_757834·1) and that deduced from MSC_0394 is similar to IIA protein of Mycoplasma capricolum (accession no. P45618) and to IIA of S. citri (accession no. AAP55649·1). Interestingly, this similarity pattern is the same as that described for IICB proteins. The organization observed for Mmymy SC was also described for a phytopathogenic mollicute, S. citri (André et al., 2003
). It was suggested that this structure (IIA+ IICB) is common to all bacteria that have arthropods as hosts, but Mmymy SC has no arthropods as hosts.
Preliminary results indicate that the genetic mechanism involved in ON-to-OFF transition is a base substitution (G to A) leading to the creation of a stop codon uspstream of the sequence encoding the region recognized by mAb 3F3. This substitution directly generates a stop codon, without a frameshift. This is an unusual mechanism of antigenic variation in Mmymy SC. However, a similar mechanism was observed for the gapA gene of Mycoplasma gallisepticum (Winner et al., 2003). Data collected in our study revealed that the 3F3-OFF variant of the Afadé strain spontaneously generates a 3F3-ON variant at high frequency (data not show), indicating that this mutation is reversible, as was observed for the gapA gene of M. gallisepticum.
Distribution of the ptsG gene within the Mmymy SC biotype
All the molecular analyses clearly distinguish the European strains from those of other geographical origins (Africa, Australia and Asia) (Cheng et al., 1995; Poumarat & Solsona, 1995
; Lorenzon et al., 2003
). The European strains lack a genomic segment of 8·84 kb that is present in all the other strains (Vilei et al., 2000
). As this 8·84 kb deleted segment does not include a copy of the ptsG gene, a new genetic specificity has been identified within the European cluster. European strains have a single copy of the ptsG gene, whereas all the other Mmymy SC strains have two copies. Within the African cluster, a large chromosomal segment including the ptsG gene has been duplicated at some stage in evolution. The European PO-67 strain showed a unique pattern intermediate between the two main Mmymy SC clusters, i.e. an IS1296 profile typical of the African cluster, but harbouring only one ptsG gene copy like the European cluster. The PO-67 strain is the oldest European strain kept in collection and was isolated from a French outbreak in 1967 (Table 1
). Seventeen severe CBPP outbreaks occurred during that year in the eastern part of the French Pyrenees and 500 bovines with clinical symptoms were slaughtered (Anonymous, 1967
). The PO-67 strain had never been included in previous studies of molecular epidemiology, except in a recent multilocus sequence analysis study (Lorenzon et al., 2003
) that also clearly classified the PO-67 strain in the European group, even though it did not exhibit the 8·84 kb deletion. This observation suggests that the deletion may be a recent feature of Mmymy SC evolution in Europe, and that the PO-67 strain may be reminiscent of the highly virulent form of CBPP that affected Europe in the nineteenth century. As the PO-67 strain did not show either the duplication or the deletion, it might be closely related to the unknown ancestor of all Mmymy SC strains.
Relation between PtsG and host
It was previously shown that mAb 3F3 targets a specific Mmymy SC epitope that is recognized by sera from CBPP-infected animals (Brocchi et al., 1993). The mAb 117/5 that is used in a diagnostic ELISA (Le Goff & Thiaucourt, 1998
) was tested on dot blots of the 3F3 synthetic peptide. Intense positive staining of the dot blots and confirmatory Western blottings with whole-cell lysates of the strain Afadé, in which mAbs 3F3 and 117/5 were compared, showed that these two antibodies target the same epitope (data not shown). Previous studies with mAb 117/5 can, therefore, give us a hint of the expression of PtsG in vivo. Amaro et al. (2000)
performed immunohistochemistry in tissues from CBPP-infected animals at different stages of infection with mAb 117/5 and a polyclonal reference serum raised against Mmymy SC. mAb 117/5 and the polyclonal serum targeted Mmymy SC cells in all infected tissues, but mAb 117/5 did not react with Mmymy SC cells localized in the follicular germinal centre of lymph nodes from animals with chronic lesions, although these were detected by the polyclonal. These results may indicate that this phase variation of PtsG proteins can occur in vivo, and might suggest a biological transformation that results in the establishment of a latent maintenance stage that circumvents the killing action of the host defence.
Colony immunostaining was used to compare the relative phenotypic proportions of the ON and OFF types for expanded cultures from i) fourth passage without cloning of the Afadé strain isolated from an acute form of CBPP, ii) a T1/44/2 strain vaccine vial and iii) a KH3J collection vial. The proportion of negative colonies varied greatly: 0·01 % for the Afadé strain, 50 % for the T1/44/2 strain and 99·999 % for the KH3J strain. These results suggest a potential relationship between the level of PtsG protein expression and the respective virulence (high, moderate and non-virulent) of these three strains. However, analysis of the phase variation of the PtsG protein did not reveal any difference between the Afadé and T1/44/2 strains other than a constant extremely low ON/OFF reversion rate in T1/44/2. Surprisingly, the vaccine was shown to consist of an equal mixture of ON-type and OFF-type 3F3 phenotypes. Antigenically distinct subpopulations in vaccine seed strains have already been reported (Rweyemamu et al., 1995). Further investigations would be useful to see if variations in the proportions of 3F3-ON and 3F3-OFF phenotypes in vaccine culture are correlated with virulence and the protective efficacy of the vaccine.
In conclusion, this report provides evidence for variation affecting a putative membrane transport system in mycoplasmas. This variation in a putative glucose-specific PTS of Mmymy SC involves a single base substitution leading to the truncation of the protein. Further investigations are required to see if such variation provides alternative transport capacities and also to see if it results in a regulation of substrate import.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 16 April 2004;
revised 3 September 2004;
accepted 7 September 2004.
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