1 Zentrum für Molekulare Biologie, Universität Heidelberg (ZMBH), Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany
2 Institut für Medizinische Mikrobiologie und Hygiene, Technische Universität, Medizinische Fakultät Carl Gustav Carus, Fetscherstrasse 74, D-01307 Dresden, Germany
Correspondence
Richard Herrmann
r.herrmann{at}mail.zmbh.uni-heidelberg.de
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ABSTRACT |
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INTRODUCTION |
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The ORF6 gene is part of the P1 operon (Fig. 1), which consists, in the following order, of the genes ORF4 (MPN140), P1 (=ORF5 (MPN141)) and ORF6 (MPN142). Repetitive DNA sequences are located within both the P1 gene and the ORF6 gene. P1 contains RepMP2/3 and RepMP4 while ORF6 contains RepMP5 (Colman et al., 1990
; Ruland et al., 1990
, 1994
). Each of these repetitive sequences appears eight to ten times as similar copies dispersed on the genome of M. pneumoniae M129 (Himmelreich et al., 1996
; Ruland et al., 1990
). They vary in size and are about 1·51·8 kbp (RepMP2/3), 1·11·5 kbp (RepMP4) and 1·52 kbp (RepMP5) long. Most of the repetitive sequences consist of variable middle sections, which are bordered by two constant regions. Comparative sequence and PCR analyses of the P1 operon from 115 M. pneumoniae patient isolates had shown that there is a constant fixed subtype-specific combination of copies of RepMP2/3, RepMP4 and RepMP5. Although not proven, a switch from a M. pneumoniae M129-specific P1 or ORF6 gene to a M. pneumoniae FH-specific one could be explained by exchange of the subtype-specific RepMP copy by recombination between the constant regions (Ruland et al., 1994
). So far, M. pneumoniae patient isolates carrying the combination of a M. pneumoniae M129-specific P1 gene and a M. pneumoniae FH-specific ORF6 gene or vice versa have not been found (Dumke et al., 2003
), although a subtype switch would require such intermediates.
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For our studies we used the cytadherence-negative mutant M. pneumoniae B170 (ATCC 29343), a derivative of M. pneumoniae M129 (Lipman et al., 1969). It possesses M129-specific P1 and ORF6 genes but has lost the ability to synthesize P40 and P90 (Sperker et al., 1991
) due to the deletion of one nucleotide (G) at position 1040 of the ORF6 gene (genome position 186787, P. C. Hu, personal communication). We complemented this mutant by introducing into its genome, via transposon, different ORF6 genes and compared the growth characteristics, expression of selected proteins, adherence properties and survival in an immunocompetent host of the resulting transformants with those of M. pneumoniae M129 and M. pneumoniae B170.
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METHODS |
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Isolation of genomic DNA, restriction analysis and Southern blot.
These were done as described previously (Dumke et al., 2003).
DIG labelling of probes and hybridization.
The genomic DNAs from single clones were characterized by Southern blotting, using DIG-labelled probes. These were generated by incorporating Digoxigenin-11-dUTP (Roche) with Taq DNA polymerase (Promega) during polymerase chain reactions according to the manufacturer's recommendation. Two probes were synthesized, deriving either from IS256 or from the gentamicin-resistance gene of the transposon Tn4001mod (Hahn et al., 1996). The primer set 1 (Table 1
) was used for amplification of a part of the IS element and the primer set 2 for amplification of a subfragment of the gentamicin-resistance gene.
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The mutation in the ORF6 gene of M. pneumoniae B170 at gene position 1040 was confirmed by sequencing the relevant part of PCR products generated with primer set 3 or primer set 4. Primer set 11 was used for sequencing only.
The ORF6 gene from M. pneumoniae M129 was amplified with primer set 5 (primer o.7179 contains an additional ClaI restriction site). This PCR product was ligated to the expression unit of MPN531. The expression unit was amplified with primer set 6 (primer o.6531 contains an additional PstI restriction site). The PCR was done as described above except that pcosMPG12 (Wenzel & Herrmann, 1989) was used as template. The ligation product of the expression unit and ORF6 gene was amplified by PCR, using primers o.6531 and o.7179, and subcloned into the ClaI and PstI restriction sites of the vector pBC (Stratagene).
The ORF6 gene from M. pneumoniae FH was amplified with primer set 7.
The recombinant ORF6 gene was assembled by ligating three different PCR products, PCR1, PCR2 and PCR3. They were synthesized with primer set 8 (PCR1, primer o.7199 contains a BlpI restriction site), set 9 (PCR3, primer o.7204 contains a BstEII restriction site) and set 10 (PCR2) using the cosmids (Wenzel & Herrmann, 1989) pcosMPE07 (PCR1, PCR3) and pcosMPCO9 (PCR2) as templates. PCR2 and PCR3 were then ligated and the product used to amplify a PCR2/3 intermediate, which was ligated to PCR1. This new intermediate served as template for synthesizing, with primers o.7199 and o.7204, sufficient amounts of the final product. This DNA fragment, consisting in the 5'3' direction of PCR1, PCR2 and PCR3, contained a BlpI restriction site near the 5' end and a BstEII site near the 3' end.
To prove the presence of Tn4001 in reisolates of M. pneumoniae from bronchial alveolar lavage fluids a specific part of this transposon was amplified with primer set 2. The stable mutation in the ORF6 gene of the reisolated mutants at genome position 186787 was confirmed by amplifying the relevant part of the gene with primer set 3 and sequencing with primer set 11.
Sequencing was done using the BigDye terminator cycle sequencing kit and the DNA sequencer ABI PRISM 377 or 373A (Perkin-Elmer Applied Biosystems) or by using external sequencing facilities (GATC Biotech).
Cloning of ORF6 genes
(i) ORF6 of M. pneumoniae M129.
The purified PCR products of the expression unit of MPN531 and of the ORF6 gene were ligated with T4 ligase (New England Biolabs) and inserted into the unique SmaI site of the vector pBC (Stratagene), yielding the plasmid pIC1. The ORF6 gene together with the expression unit of MPN531 was transferred from pIC1 into the transposon Tn4001mod, which is part of the plasmid pKV74 (Hahn et al., 1996). For this cloning step, the ORF6 gene was excised from pIC1 with the restriction enzymes PstI and ClaI (New England Biolabs) and, after the sticky ends had been converted to blunt ends with the Klenow enzyme (AGS), ligated into the BamHI site of the vector pKV74. The sticky BamHI ends were also converted to blunt ends by the Klenow enzyme. The new plasmid was named pICT1.
(ii) ORF6 of M. pneumoniae FH.
Plasmid pICT1 was digested with nucleases BstEII and BlpI, yielding a 10·8 kbp and a 1·8 kbp fragment. The 10·8 kbp fragment was isolated from a 0·8 % agarose gel and ligated with a BstEII/BlpI DNA fragment carrying the FH-specific RepMP5 copy. This fragment was amplified with primer set 7 from the FH-specific ORF6 gene using genomic DNA of M. pneumoniae FH as substrate. The new plasmid was named pICT2.
(iii) Recombinant ORF6.
The PCR product containing RepMP5 833 was synthesized as described. It was directly cloned into the unique SmaI site of the vector pBC. The resulting plasmid was called pIC3. The new recombinant ORF6 was excised from pIC3 with BlpI and BstEII and ligated into the plasmid pICT2, also treated with BlpI and BstEII, such that it replaced the FH-specific ORF6 gene, generating the new plasmid pICT3.
Transformation of M. pneumoniae.
M. pneumoniae cells were transformed with the corresponding plasmids by electroporation as described previously (Hedreyda et al., 1993) and transformants were selected on PPLO agar plates containing gentamicin (80 µg ml1). Colonies were picked from PPLO agar plates and grown in 1 ml modified Hayflick medium also containing gentamicin (80 µg ml1). These cultures were then used for inoculation of new expanded cultures or stored at 70 °C.
Filtration technique for Mycoplasma.
To get single colonies from M. pneumoniae suspensions, the standard technique as described by Tully (1983) was used with the following modifications. The cells were passed sequentially through a 400 nm, a 220 nm and a 100 nm filter, plated on PPLO agar plates and incubated at 37 °C. Single colonies were picked after 1014 days from plates and cultivated in liquid broth culture.
Haemadsorption test.
The haemadsorption test was done as published (Krause et al., 1982) with the modification that concentrates of human erythrocytes were diluted in Dulbecco's modified Eagle medium (Gibco) until the OD600 was 0·8. Pictures were taken with an Olympus DP50 digital camera connected to a Zeiss Axiophot with the 20x objective.
Polyclonal antibodies and preparation of antisera.
The specific antisera against the P40 proteins of subtypes 1 and 2 were obtained by immunizing rabbits with the following peptides, which have been coupled to keyhole limpet haemocyanin (mcKLH, Pierce): pep 2528F CDESSWKNSEKTTAEND (amino acid residues 275290; specific for M. pneumoniae M129); pep 2527M CSDSSGSQGQGTTDNKFQKY (amino acid residues 330346; specific for M. pneumoniae FH see Fig. 5). For coupling the peptides to mcKLH, a cysteine residue was added at the amino-terminus of each peptide. The peptides were synthesized by M. Ellis and T. Ruppert (ZMBH). The antisera were delivered from Peptide Speciality Laboratories. The antisera against P1 (Dumke et al., 2003
), P65 (Proft & Herrmann, 1994
), P90 and P40 (Sperker et al., 1991
) have been described.
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Animal experiments.
Three male guinea pigs (450600 g, Dunkin-Hartley, Charles River) per group were intranasally infected with an identical inoculum of a specific M. pneumoniae strain on the same day (Dumke et al., 2004). The inoculum varied between groups from approximately 4x106 to 6x108 c.f.u. suspended in 300 µl PBS. The infected animals were kept separately in air-conditioned boxes with filter systems (Biozone) until they were sacrificed. The experiments with the different transformant groups were organized in such a way that the groups were infected one after the other to avoid any cross-contamination. Ten days after infection, the animals were killed by heart puncture under anaesthesia (Jacobs, 1991
). The trachea was prepared and a small catheter device was introduced into the trachea drainage hole. For bronchoalveolar washing we instilled approximately 10 ml PPLO broth. The regained solution (approx. 3 ml) was used to isolate M. pneumoniae from the lower respiratory tract of the animals.
Two hundred microlitres of the infection suspensions and of the bronchial alveolar lavage fluids were spread on PPLO agar and incubated for at least 3 weeks at 37 °C. In the case of negative results the remaining bronchial alveolar lavage fluid (stored at 20 °C) was concentrated by centrifugation (8000 g, 10 min), and the sediments were resuspended in 500 µl PPLO broth and incubated in the same way. In parallel, 100 µl concentrated bronchoalveolar washing fluid was added to 900 µl PPLO broth and incubated at 37 °C until the colour of the medium changed from red to orange, or for at least 3 weeks. For molecular characterization selected colonies were picked and incubated in 1·5 ml PPLO broth at 37 °C.
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RESULTS |
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Selection of RepMP5 copies for constructing new ORF6 genes
After proving that trans-complementation with the ORF6 gene products is in principle possible, we constructed two new P1ORF6 combinations composed of the M. pneumoniae M129-specific P1 gene complemented with either the M. pneumoniae FH-specific ORF6 gene (Ruland et al., 1994), or a RepMP5-specific copy occurring outside the P1 operon of M. pneumoniae. These combinations have, so far, not been detected in M. pneumoniae isolates (Dumke et al., 2003
).
To simulate possible recombination events between the ORF6 gene and the repetitive elements, a set of scripts was created in the Perl programming language to simulate in silico all possible recombination events between the ORF6 gene and the repetitive elements. Two DNA fragments one from ORF6, the other from a repetitive element were assumed to be possible recombination sites if (i) the recombination site was longer than 20 bp (Cohan, 1994), (ii) there was a perfect match of at least 10 bp at either end of one recombination site, (iii) there was a similarity of at least 90 % between the two fragments and (iv) the longest mismatch stretch was not longer than 3 bp. Next, all found recombination sites were permutated and the sequences were recombined at these sites, so that 5' and 3' regions came from the original ORF6 gene, and the central region from a repetitive sequence. Recombined sequences which contained a frame-shift or a stop mutation, or were more than 70 amino acids shorter than the original P1 protein, were rejected. Finally, a multiple alignment of all 601 resulting recombined sequences was done using the program CLUSTAL W. A recombined sequence contained in a cluster distant as far as possible from both M129- and FH-specific ORF6 gene sequences was chosen for further analysis. The central region of this sequence was derived from the repetitive element RepMP5 833, located on the genome between nucleotides 131796 and 133581 (Figs 1 and 5
; for more details see the supplementary files with the online version of this paper).
Construction of new ORF6 genes
The new ORF6 genes were constructed by replacing a 1824 bp inner DNA fragment of the ORF6 gene of M. pneumoniae M129 located between the two unique restriction sites for the endonucleases BlpI (genome position nucleotide 185989) and BstEII (genome position nucleotide 187815). These two restriction sites are located outside of the variable RepMP5 repeat region within the conserved part of the ORF6 gene M129, the ORF6 gene FH and the RepMP5 833 copy (Fig. 1). The DNA manipulations were done with either plasmid pIC1 or pICT1 (see above), both carrying the ORF6 gene M129, by exchanging their BlpI/BstEII fragment (1824 bp) and inserting either the BlpI/BstEII fragment (1623 bp) from the ORF6 gene FH or the BlpI/BstEII fragment (1680 bp) from Rep MP5 833 (=ORF6 gene R, for details see Methods). The plasmids carrying the ORF6 gene FH were called pIC2 and pICT2, and the plasmids carrying the ORF6 gene R were called pIC3 and pICT3 (Table 2
). The correctness of these gene constructions was confirmed by sequencing each of the newly constructed ORF6 genes. An amino acid sequence comparison of the variable regions of the three ORF6 gene products used in this study is shown in Fig. 5
.
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Intranasal infection of guinea pigs with selected M. pneumoniae strains
To determine whether the transformed bacteria were able to colonize the host respiratory tract, we infected guinea pigs with selected transformants and screened for surviving bacteria in the bronchial alveolar lavage fluids.
In five sets of separate experiments three guinea pigs each were infected with a specific M. pneumoniae strain. The infection doses varied between 3·9x106 and 6·3x108 c.f.u. The following strains were tested for their ability to survive in the respiratory tract of the infected animals: (i) M. pneumoniae M129 (positive control for host colonization), expressing the characteristic subtype 1-specific P1 and ORF6 proteins (P40/P90); (ii) M. pneumoniae B170 (negative control for host colonization), unable to synthesize P40 and P90; (iii) three transformants of M. pneumoniae B170 containing the described ORF6 genes (Table 3), namely M. pneumoniae B170/M31 (clone 31, subtype 1-specific ORF6 gene), M. pneumoniae B170/F3 (clone 3, subtype 2-specific ORF6 gene) and M. pneumoniae B170/R1 (clone 1, RepMP5 833-derived ORF6 gene). Ten days after infection the bronchial alveolar lavage fluids were tested for viable M. pneumoniae strains surviving in the host environment, on agar plates and, in the case of negative results, enriched in liquid medium (Table 4
).
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DISCUSSION |
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The number of recovered colonies after plating without prior enrichment in liquid medium is rather low (Table 4), but this is a general phenomenon of the fastidiously growing M. pneumoniae cells. For this reason it is recommended microbiological practice in diagnostics not only to spread infected samples directly on agar plates but also to enrich the bacteria in liquid medium. A positive result, the identification of an infectious M. pneumoniae is achieved if, with either procedure, bacteria were re-isolated. Applying these procedures we recovered the ORF6-complemented M. pneumoniae strains from almost all infected guinea pigs and confirmed by molecular methods that they were identical with the inoculum strains. In agreement with published data (Lipman et al., 1969
), the only strain that could not be recovered was the non-adhering M. pneumoniae B170. It was eliminated by the natural clearance of the bronchial tract within 10 days.
Due to the problems concerning the recovery of M. pneumoniae directly from samples of the bronchial washing fluids, plating efficiencies cannot be used to quantify differences in the ability of individual strains to adhere and to colonize the host. The re-isolation of M. pneumoniae from bronchial fluids on agar plates or in liquid medium is therefore only a qualitative parameter for colonization. Since colonization is only one step in the interaction of M. pneumoniae with its host, further studies are needed to explore whether these transformants are still pathogenic and elicit comparable cellular and humoral host responses as the infectious subtype 1 and 2 strains (Jacobs, 1991; Jacobs et al., 1988
). We conclude from the result of the described experiments that M. pneumoniae strains carrying different gene P1/ORF6 combinations are able to colonize and to persist in a host organism and therefore could also act as true intermediates in a switch from subtype 1 to subtype 2 and vice versa.
In contrast to these findings are the results from analyses of the composition of the P1 operons from 115 M. pneumoniae strains collected from patients in different countries over a period of 30 years. They showed that only two subtype 1- and 2-specific combinations of the genes P1 and ORF6 could be identified (Dumke et al., 2003). How can we explain the discrepancy between the results from the patient isolates and the in vitro generation of transformants with hitherto unknown combinations of P1 protein and ORF6 gene products? What is the function of the seven additional copies of RepMP5, which supply a potential reservoir at least in theory for generating many different ORF6 genes (Himmelreich et al., 1996
; Rocha & Blanchard, 2002
)?
If we exclude the possibility that the isolation procedure for M. pneumoniae strains from patients is selective, then we should find more new combinations of the P1 and ORF6 genes in the P1 operon carrying different repetitive DNA sequences, as we indeed observed in the described experiments (Dumke et al., 2003). These new combinations could be generated by homologous recombination leading, among others, to the same P1/ORF6 combination as in the transformants M. pneumoniae M129/F and M129/R. We explain the failure to detect such M. pneumoniae strains by the lack of an efficient system for homologous recombination.
We assume that the crucial enzyme for this reaction is RecA. M. pneumoniae contains the corresponding gene (MPN490), but in a transcriptome analysis of M. pneumoniae M129 (Weiner et al., 2003) only low signals were obtained for a RecA-specific mRNA. In addition, RecA has not been identified in a proteome analysis combining 2D gel electrophoresis and mass spectrometry (Ueberle et al., 2002
) although the features of RecA, pI 9·77 and no predicted transmembrane segment, should have allowed the identification, unless the amount is too low for detection by standard 2D gel electrophoresis and mass spectrometry. However, recently RecA was identified in a total protein extract of M. pneumoniae FH by a proteome analysis, which was done without separation of the total protein extract into individual proteins prior to tryptic cleavage (Jaffe et al., 2004
). This method is more sensitive, since it avoids the considerable losses of proteins occurring during 2D gel electrophoresis and allows the application of higher amounts of protein extracts. Therefore, the concentration of RecA in M. pneumoniae could be rather low. In agreement with these observations are the negative results from experiments aiming to construct mutants from M. pneumoniae by homologous recombination, which is in contrast to the results with the closely related species Mycoplasma genitalium (Dhandayuthapani et al., 1999
). The latter organism was successfully transformed by integration of foreign DNA via homologous recombination and its RecA was also identified on 2D gels of total M. genitalium cell extracts (Wasinger et al., 2000
).
Similar experiments as those carried out with the ORF6 gene should also be done with the P1 gene, although this will be more complicated, because this gene contains two repetitive DNA sequences, RepMP2/3 and RepMP4 (Fig. 1; Ruland et al., 1990
). In a first step, one of these sequences of a subtype 1-specific P1 gene could be exchanged with a subtype 2-specific one and the resulting M. pneumoniae strain tested for adherence, ability to infect hosts and its potential to serve as a true precursor for a complete subtype switch. Experiments along this line will help to explain whether the observed switch of the dominating subtype in epidemic outbreaks of M. pneumoniae appearing in intervals of 37 years is caused by conversion of one subtype to another one or by faster growth of one subtype in a mixture of both subtypes. Since the human immune defence would be primarily directed against the dominating infecting subtypes, the second subtype could grow uninhibited, at least for some time, and should become the dominating infectious subtype in the following years.
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ACKNOWLEDGEMENTS |
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Received 22 July 2004;
revised 22 September 2004;
accepted 23 September 2004.
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