1 Department of Biology, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585, Japan
2 Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA
3 PRESTO, JST, Sumiyoshi-ku, Osaka 558-8585, Japan
Correspondence
Makoto Miyata
miyata{at}sci.osaka-cu.ac.jp
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ABSTRACT |
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Present address: Department of Oral Microbiology, Meikai University School of Dentistry, 1-1 Keyakidai, Sakado, Saitama, 350-0283, Japan.
Present address: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
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INTRODUCTION |
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M. mobile, isolated from the gill organ of a fish, has a flask-shaped cell structure (Kirchhoff & Rosengarten, 1984). It typically glides at a rate of 37 cell lengths per s (2·04·5 µm s1) (Kirchhoff, 1992
; Miyata et al., 2000
, 2002
; Rosengarten & Kirchhoff, 1987
) in the direction of the tapered end, which is designated the head-like structure, and exerts a maximum force of 27 pN (Miyata et al., 2001
, 2002
). The gliding behaviour of cells with beads attached to their surfaces and cells with elongated head-like structures has been examined (Miyata et al., 2002
; Miyata & Uenoyama, 2002
). Rapid-freezefracture electron microscopy of actively gliding cells showed 50 nm spikes that stick out from the cell membrane around the head-like structure and bind to the glass surface (Miyata & Petersen, 2004
). These observations suggested that the gliding machinery is localized at the head-like structure, which might be considered a specialized gliding organelle.
In this study, we isolated antibodies against the mycoplasma cell surface, identified the targets of these antibodies, localized the gliding machinery, and then showed that the cell surface is differentiated into at least three parts.
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METHODS |
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Mouse immunization and hybridoma production.
These procedures followed the methods previously described, with slight modifications (Harlow & Lane, 1999). Cultured cells of M. mobile 163K (ATCC 43663) were collected by centrifugation at 10 000 g for 3 min at room temperature and washed twice with PBS consisting of 75 mM sodium-phosphate buffer (pH 7·3) and 100 mM NaCl. Mycoplasma cells were suspended in PBS and emulsified in complete Freund's adjuvant. Female BALB/c mice were injected intraperitonially with the emulsion containing 1011 whole M. mobile cells. Three weeks after the first injection, a second injection was done with 5x1010 whole M. mobile cells emulsified with Freund's incomplete adjuvant. Two weeks after the second injection, the mice were injected with 5x1010 whole M. mobile cells without adjuvant. The mice were sacrificed 3 days after the last boost. The spleens were dissected, and dissociated spleen cells were fused to SP-1 myeloma cells. The hybridomas were cloned and screened three times using an ELISA assay. The class and subclass of the resulting antibodies were determined by the Mouse Immunoglobulin Typing Kit (Wako). mAb9 was typed as IgM and the others were typed as IgG1.
ELISA and immunoblotting.
For the ELISA assay, cultured mycoplasma cells were collected by centrifugation as before. After two washes with PBS, mycoplasma cells were suspended in 10 mM phosphate buffer (pH 7·0) containing 0·1 % SDS and adjusted to 10 µg ml1 mycoplasma protein solution. A 50 µl aliquot of mycoplasma protein solution was added to each well of a FALCON 96-well Microtitre Plate (Becton Dickinson) and incubated for 2 h. The remainder of the ELISA procedure was performed with a method described by Roggendorf et al. (1982).
For immunoblotting, cultured mycoplasma cells were collected by centrifugation, washed with PBS, resuspended in PBS and lysed by addition of sample loading buffer. The lysate containing 5 µg protein for each lane of 7 mm width was fractionated by 5 % and 7 % SDS-PAGE (Sambrook et al., 1989), and analysed by immunoblotting, using a hybridoma medium with appropriate dilutions.
Microscopy.
Cultured cells were collected by centrifugation and resuspended at a 30-fold higher concentration in Aluotto medium. Motility was assessed with a tunnel slide test (Uenoyama et al., 2004). Briefly, cells were adhered to a glass coverslip which was assembled into a rudimentary flow chamber constructed with a microscope slide and double-sided tape. Then, antibodies were applied by exchanging the medium in the chamber for medium containing a hybridoma supernatant. Immunofluorescence microscopy without permeabilization was performed as described previously (Seto et al., 2001
; Seto & Miyata, 2003
; Uenoyama et al., 2004
) by using monoclonal hybridoma supernatants at a dilution of 1 : 100 and polyclonal mouse serum at a dilution of 1 : 400 with PBS.
Identification of target protein of mAb9.
The target protein of mAb9 was purified by successive fractionation of cell lysate using immunoblotting against mAb9 and SDS-PAGE as indicators of specificity and purity. Cells from 3 l of culture were collected by centrifugation at 10 000 g for 10 min at 4 °C, washed twice with 5 ml of buffer consisting of 8 mM HEPES buffer (pH 7·0) and 0·28 M sucrose, and subjected to phase partitioning using 5 ml of a TritonX-114-containing solution (Bordier, 1981). The hydrophobic phase was mixed with 45 ml 50 mM HEPES buffer (pH 7·5) containing 0·1 % Triton X-100, and applied to a HiTrap CM Sepharose FF 1 ml column (Amersham). The flowthrough fraction was loaded onto a HiTrap DEAE Sepharose FF 1 ml column (Amersham). The target protein was retained by the column and eluted by 6 ml 50 mM NaCl. A total of 70 µg of protein was purified by SDS-PAGE following TCA-mediated precipitation, and subjected to in-gel partial digestion, using V8 protease and trypsin (Cleveland et al., 1977
). The peptide digests were transferred to a nylon membrane and an N-terminal amino acid sequence was determined by Edman degradation for each preparation. The amino acid sequences NTTTPLTIC and NSLITSSRGC determined for V8 and trypsin digests, respectively, were both found in an ORF in the genome sequence as described above.
Identification of target proteins of mAbs 13 and 14 by mass spectrometry.
To identify the target of mAb14, cells from 10 ml culture were washed with PBS, lysed and subjected to 5 % SDS-PAGE. Immunoblots and Coomassie-stained gels showed that the protein band was sufficiently separated from other bands so it was simply excised from the gel and subjected to trypsin digestion for mass spectrometry.
The target of mAb13 was isolated by using antibody-loaded Protein G-conjugated beads (Protein G Sepharose 4 Fast Flow; Amersham Biosciences). Cells from 50 ml of culture were washed and subjected to phase partitioning, whereby whole proteins were fractionated into hydrophobic, hydrophilic and insoluble fractions using Triton X-114 (Bordier, 1981). The hydrophilic fraction was mixed with 10 µl of a slurry of antibody-loaded beads saturated with mAb13 according to the manufacturer's instruction. After mixing at 4 °C for 2 h, the beads were washed three times with PBS and extracted by SDS-PAGE gel-loading buffer (Sambrook et al., 1989
). The target proteins were subjected to 10 % SDS-PAGE and excised from the gel after staining with Coomassie blue. A duplicate gel was subjected to immunoblotting to verify that the bands were reactive to the antibodies employed.
The excised bands of target proteins were reduced, alkylated, and digested by trypsin as described by Hanna et al. (2000). A nano-spray chromatography column (75 µm ID) was pulled in house and packed with 15 cm of Michrom MAGIC C18AQ media (Michrom). This column was directly interfaced to the mass spectrometer and separations were performed using a linear gradient of 540 % acetonitrile/0·1 % formic acid over 100 min at a flow rate of 250 nl min1. Tandem mass spectra were acquired on an LCQ Deca XP-plus ion-trap mass spectrometer (ThermoFinnigan) using a top five approach, where MS/MS spectra were obtained for the five most abundant peaks in the original MS full (parent) spectrum. Proteins were identified using SEQUEST to search against a database consisting of 6-frame-translated DNA sequences from the M. mobile genome (http://www-genome.wi.mit.edu/annotation/microbes/mycoplasma) (Eng et al., 1994
; Jaffe et al., 2004b
). SEQUEST scores >2·5 for charge state z=2 peptides and >3·75 for charge state z=3 peptides were considered valid peptide assignments. ORFs that corresponded to these peptide assignments were identified by comparison against the current M. mobile genome annotation (GenBank accession no. AE017308).
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RESULTS |
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Inhibition of gliding
Inhibitory effects of monoclonal antibodies on M. mobile attachment and gliding were examined by using the tunnel slide test (Fig. 1). An antibody, designated mAb7, displaced the gliding mycoplasmas from the glass surface within 3 min after addition, while control supernatants (including the other antibodies) did not show any effects. This suggests that the inhibitory effects were specific to mAb7.
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mAbs 9, 13 and 14 each recognize a surface structure; their target proteins were identified using a combination of biochemical purification, mass spectrometry and N-terminal Edman sequencing (Table 1). In addition, immunoblots were used to verify the number and molecular mass of the target proteins (Fig. 3
). The mAbs recognized various members of a family of related proteins. These proteins are annotated in the genome as the Mvsp proteins, for mobile variable surface protein. The Mvsp proteins were predicted, based on sequence analysis, to be surface exposed, and here we demonstrated (along with immunofluorescence data, Fig. 2
) that this prediction was indeed correct. mAb13 recognized two protein bands with estimated molecular masses of 48·0 and 57·5 kDa whose identities are MvspN and MvspO, respectively. mAb9 and mAb14 recognized proteins with estimated molecular masses of 74 and 191 kDa, respectively, whose identities are MvspK and MvspI, respectively.
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Sequence properties of target proteins
Three of the four surface-reactive antibodies raised for this study recognized proteins in the Mvsp family. This family is composed of 16 members in M. mobile and two in Mycoplasma pulmonis. The M. mobile members of the family share 4060 % similarity at the amino acid level, are tandemly coded in several clusters in the genome, can be aligned to each other in various registers using a multiple sequence alignment program such as CLUSTAL_W, and exhibit sequence features suggestive of transmembrane helices and/or lipid attachment sites (Jaffe et al., 2004b). Here we present a more detailed analysis of the sequence properties of the Mvsp family members detected in this study.
The amino acid sequence of MvspO (the larger target of mAb13) was found to have an internal repeat consisting of a duplication of amino acids 110209 at positions 210309. MvspO shares 87·5 % identity with the sequence of MvspN (the smaller target of mAb13) if they are compared excluding the second repeat. It is probable that mAb13 recognizes an epitope common to these two proteins. In addition, MvspN is encoded just downstream from MvspO in the genome with respect to the direction of transcription. The observed molecular masses of these proteins are significantly different from those predicted from ORF sequences (Table 1). This disagreement may suggest post-translational modification of these proteins. MvspK and MvspI (the target proteins of mAbs 9 and 14, respectively) also have internal repeats.
mAb7 recognized the Gli349 protein, which has been described elsewhere (Uenoyama et al., 2004). An orthologue of Gli349 was found in the genome of another gliding mycoplasma, M. pulmonis. M. pulmonis is phylogenetically the closest related mycoplasma to M. mobile to have a complete genome sequence (Chambaud et al., 2001
; Weisburg et al., 1989
).
The Gli349 protein and the Mvsp proteins detected in this study share some common sequence features, which are highlighted in Fig. 4. All of their sequences contain a transmembrane segment near the N-terminus (as predicted by SOSUI; Hirokawa et al., 1998
) preceded by a positively charged domain. These features suggest that these proteins may be anchored in the membrane via their N-terminal regions (Navarre & Schneewind, 1999
; Pugsley, 1993
). In addition, MvspK and MvspI are predicted to have a lipid attachment site by detection of a corresponding PROSITE motif (Hofmann et al., 1999
). These proteins might additionally or alternatively be anchored to the membrane by attached lipids.
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DISCUSSION |
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The four Mvsp proteins detected in this study are unlikely to be involved in motility or adhesion because antibodies against them did not affect gliding or glass binding. All of the Mvsp proteins localized to regions other than the neck region, as presented in Figs 2 and 5. Interestingly, MvspI localized to the head and body but specifically not to the neck region. These observations suggest that the neck region is specialized for glass binding and gliding. Further subcellular localization to the head could be seen for MvspN and MvspO, while MvspK localized to the body.
In the process of screening of hybridoma cell lines, we obtained around 20 hybridomas producing antibodies recognizing surface structures; however, only four of them could ultimately be established. In immunofluorescence microscopy all of the antibodies from the precursor hybridoma strains labelled the cells in one of the four patterns shown in Figs 2 and 5 (data not shown). These observations suggest that surface proteins other than those identified here may also be localized in the same patterns.
Genome sequencing revealed 16 highly related proteins that are encoded primarily by two gene clusters and share sequence features indicative of membrane localization. This evidence suggests that they might be involved in antigenic variation in M. mobile (Jaffe et al., 2004b). This assumption is supported by the fact that the Mvsp proteins are immunodominant, as Mvsp family members were recognized by three of the four hybridomas established by immunization of mice with whole mycoplasma cells. The vsa gene family involved in antigenic variation in M. pulmonis has been reported to express only one gene at one time (Bhugra et al., 1995
). However, the immunostaining of the Mvsp proteins in this study showed that multiple mvsp variants are co-expressed in a M. mobile cell, which is consistent with the result of proteomic analysis (Jaffe et al., 2004b
). The multiple and localized expression of mvsp genes in a M. mobile cell suggests that if they are involved in antigenic variation, the mechanism of variation may be different to that in M. pulmonis.
We previously quantified the number of Gli349 molecules on the M. mobile cell surface and concluded that around 450 molecules exist on a cell (Uenoyama et al., 2004). The 450 units of gliding machinery may form a large cluster, occupy the neck space and exclude other surface proteins from the region. Such a cluster might prevent free diffusion of proteins across this region of the cell membrane and thus provide a basis for differentiation of the cell surface into disparate regions. However, even if this was the case, specific mechanisms must be required for trafficking of newly synthesized proteins to their proper subcellular locations. Analysis of the amino acid sequences near the N-termini of the proteins did not identify obvious signals for protein localization (Fig. 4
), but this does not exclude the presence of such signals.
No surface structures specific for the head or body have been found by freezefracture or scanning electron microscopy of M. mobile (Kirchhoff et al., 1987; Miyata & Petersen, 2004
; Rosengarten et al., 1988
; Rosengarten & Kirchhoff, 1989
). However, sectioned images of chemically fixed cells showed increased electron densities under transmission electron microscopy at the front end of the head-like structure when it was stained by uranyl acetate (Kirchhoff et al., 1987
; Shimizu & Miyata, 2002
). The distribution of these increased electron densities may be consistent with the localization of the targets of mAb13, namely MvspN and MvspO.
While subcellular localization in a mycoplasma cell has been reported for several cytadherence proteins (Krause & Balish, 2004; Uenoyama et al., 2004
), localization has not been shown for other classes of proteins. In this study, we have shown subcellular localization of proteins apparently not related to cytadherence for the first time, and are able to reconstruct a detailed map of the surface differentiation of M. mobile. Such surface differentiation might be a feature found in other species of Mycoplasma as well.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 28 June 2004;
revised 1 September 2004;
accepted 2 September 2004.
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