Hygiene-Institut, Universität Heidelberg, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany1
EMBL, Meyerhofstraße 1, 69123 Heidelberg, Germany2
Author for correspondence: Gerlinde Layh-Schmitt. Tel: +1 513 622 5509. Fax: +1 513 622 0085. e-mail: Layhschmitt.G{at}pg.com
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ABSTRACT |
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Keywords: Mycoplasma pneumoniae, P1 adhesin, paraformaldehyde cross-linking
Abbreviations: MALDI MS, matrix-assisted laser desorption/ionization mass spectrometry
a Present address: Procter & Gamble Pharmaceuticals, Health Care Research Center, Anti-Infective Research, PO Box 8006, 8700 Mason-Montgomery Road, Mason, OH 45040, USA.
b Present address: Protein Interaction Laboratory, The University of Southern Denmark, Campusvej 55, DK 5230, Denmark.
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INTRODUCTION |
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Adhesin P1 of M. pneumoniae, a transmembrane protein with a molecular mass of 170 kDa, as well as the adhesin-related 30 kDa protein and the two cytadherence-associated proteins of 40 and 90 kDa cluster densely in the membrane of the attachment organelle (Dallo et al., 1990 ; Feldner et al., 1982
; Franzoso et al., 1993
; Layh-Schmitt & Herrmann, 1992
, 1994
). The high density and close proximity of these proteins in the terminal structure seem to be prerequisite for effective cytadherence. The two accessory proteins of 40 and 90 kDa are post-translational cleavage fragments of the ORF6 gene product, which is encoded by an ORF of the P1 operon next to the P1 gene (Inamine et al., 1988
; Sperker et al., 1991
). Previous cross-linking studies with a non-permeant cross-linking reagent showed that the ORF6 gene products are located on the bacterial cell surface in close proximity to the P1 protein (Layh-Schmitt & Herrmann, 1994
).
Some of the ORF6 gene products and the P1 molecules are retained by the Triton-X-100-insoluble fraction of the M. pneumoniae cells, which apparently represents a filamentous structure composed of numerous proteins and resembling the eukaryotic cytoskeleton (Kahane et al., 1985 ). However, the lack of the ORF6 gene product in a spontaneous haemadsorption-negative mutant resulted in the release of all the P1 protein into the Triton-soluble phase (Layh-Schmitt & Harkenthal, 1999
). These findings suggest that the ORF6 gene product may be responsible for proper disposition of the P1 molecules in the mycoplasma membrane, which seems to be a prerequisite for interaction of the 40 and 90 kDa cytadherence-associated proteins and the P1 protein with the cytoskeleton. As suggested by Krause (1996
, 1998
) and Hahn et al. (1998)
, the cytoskeleton-forming proteins might play a scaffolding role in locating the P1 molecule in the membrane of the tip structure. Some components of the mycoplasma cytoskeleton have been well characterized: the cytoskeleton-forming, high-molecular-mass proteins HMW1, 2 and 3 (Krause, 1996
, 1998
; Krause et al., 1997
) and the P65 protein, which seems to be associated with the cytoskeleton (Proft et al., 1995
). All four proteins exhibit higher apparent molecular masses on SDS-polyacrylamide gels than expected due to their high proline content. However, the cytoskeleton-like protein complex consists of more than 25 proteins and, to date, there are no clear experimental data about which particular cytoskeleton proteins might interact directly with the P1 protein.
For this reason, in the present study, we focused on the determination of proteins which are located within the cell membrane or inside the cell in close proximity to the transmembrane protein P1. Hence, cross-linking studies using the permeant cross-linking reagent paraformaldehyde were carried out. The proteins linked to the P1 molecules were analysed by immunoblot analysis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS).
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METHODS |
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Antisera.
Rabbit antisera against the P1 protein (Proft & Herrmann, 1994 ), the 90 and 40 kDa proteins (Layh-Schmitt & Herrmann, 1992
), the 30 kDa protein (Layh-Schmitt et al., 1995
, 1997
) and the cytoskeleton-forming proteins (HMW1, HMW3 and P65) (Proft & Herrmann, 1994
; Proft et al., 1995
) were produced as described previously.
In vivo cross-linking by paraformaldehyde.
Cross-linking studies were performed according to the method described by Skare et al. (1992) . To evaluate the appropriate cross-linking conditions bacterial suspensions were incubated with 1% paraformaldehyde (Merck) in PBS, pH 6·5 [30 mg cells (wet weight) ml-1], at room temperature for different lengths of time (10, 20, 30, 40, 50, 60 and 75 min). Two hundred microlitre samples of the incubation mixture were transferred into microfuge tubes containing 500 µl PBS, pH 6·5. The cross-linking reaction was stopped by centrifuging the cells from the paraformaldehyde solution at 6000 g for 15 min. The bacteria were washed twice with PBS, and the pellets were finally resuspended in 50 µl 2x Laemmli buffer (Laemmli, 1970
). Prior to SDS-PAGE and immunoblot analysis, the cells were lysed by incubation at 60 °C for 10 min or heated to 95 °C for 15 min to break the cross-links.
To enrich cross-linked complexes, 200 mg (wet weight) M. pneumoniae cells was treated with 7 ml 1% paraformaldehyde in PBS for 60 min. Cross-linking was stopped by centrifugation of the cells at 6000 g for 10 min. The pellet was washed with PBS three times and frozen at -20 °C until needed for immunoaffinity chromatography.
Preparation of the immunoaffinity chromatography column.
A bed volume of 1 ml Sephacryl-protein A (Pierce) with a binding capacity of 15 mg human IgG was loaded in a 15 ml plastic column (Bio-Rad). The gel material was washed with 10 vols IgG-binding buffer (Pierce). One millilitre of rabbit antiserum against the P1 protein was added to the column. The gel was incubated overnight at 4 °C by gentle shaking in a rotating shaker. Unbound protein was released from the gel by washing with 10 vols IgG-binding buffer (Pierce). IgG was covalently bound to the protein A matrix by adding 2 ml of the cross-linking reagent dimethyl pimelimidate (Pierce) to a final concentration of 20 mM in 200 mM sodium borate buffer, pH 9·0. After 1 h incubation at room temperature the gel was washed with 5 ml 200 mM borate buffer, pH 9·0, and then the excess reactive groups of the cross-linking reagent were blocked with 2 ml ethanolamine (200 mM, pH 8·0) during incubation for 10 min. IgG molecules which were not covalently bound to protein A were released from the gel matrix by 5 ml 0·2 M glycine/HCl, pH 2·8. Thereafter the gel was washed twice with 10 ml IgG-binding buffer.
Immunoaffinity chromatography for isolating cross-linked protein complexes.
The paraformaldehyde-treated bacteria (200 mg wet weight) were lysed in 800 µl lysis buffer [2% SDS, 25 mM NaCl, 25 mM Tris/HCl (pH 7·2) and 10 U aprotinin (Sigma) ml-1], heated to 60 °C for 5 min, and then diluted in 4 vols (3·2 ml) dilution buffer [2·5% Triton X-100 (Pierce), 190 mM NaCl, 60 mM Tris/HCl, 10 U aprotinin ml-1]. The lysate was mixed with the antibody-bound gel material in a 15 ml column. The column was closed with a plastic cap and the gel was incubated with the lysate by gentle shaking with an overhead shaker overnight at 4 °C. The protein A matrix with the bound antibodyantigen complex was washed four times with 10 ml washing buffer I [0·1% Triton X-100, 0·02% SDS, 150 mM NaCl, 50 mM Tris/HCl (pH 7·5), 5 mM EDTA] and twice with washing buffer II [150 mM NaCl, 50 mM Tris/HCl (pH 7·5), 5 mM EDTA]. Cross-linked protein complexes containing the P1 protein were eluted from the gel with 2 ml 3·5 M KSCN. After dialysis against 3 mM Tris/HCl buffer (pH 7·4), isolated protein complexes were lyophilized by vacuum centrifugation. The lyophilized samples were dissolved in 50 µl 2x Laemmli buffer at 60 °C for 10 min. To break the cross-links the samples were incubated at 95 °C for 15 min.
SDS-PAGE and immunoblot analysis.
Cell pellets of 200 µl M. pneumoniae suspensions [30 mg cells (wet weight) ml-1] which were incubated with paraformaldehyde for different lengths of time were lysed in 50 µl 2x Laemmli buffer at 60 °C for 5 min. To break the cross-links the samples were incubated at 95 °C for 15 min. Five microlitres of each sample was separated by 10% SDS-PAGE (Laemmli, 1970 ) before and after breaking the cross-links. The same procedure was used for resolving cross-linked protein complexes eluted from immunoaffinity chromatography columns. Immunoblotting was carried out as described previously (Layh-Schmitt & Herrmann, 1992
, 1994
) according to the method of Towbin et al. (1979)
. Antibodies against the P1 protein, the 30, 40 and 90 kDa proteins, and P65 were diluted 1:1000. Antibodies against HMW1, HMW3 and DnaK were diluted 1:5000.
Identification of proteins by MALDI MS.
The determination of peptide masses by MALDI MS in combination with database searching is an excellent method of identifying proteins of an organism if the corresponding DNA or protein sequences are stored in the database. Since the complete genome sequence of M. pneumoniae is known (Himmelreich et al., 1996 ), we initiated analysis of the mass of tryptic fragments of proteins complexed to the P1 adhesin by MALDI MS.
After breaking the cross-links, individual proteins of the affinity-chromatography-purified protein complex were separated by 10% SDS-PAGE. The bands of interest were excised from the gel, washed, reduced in the gel, S-alkylated, and subjected to proteolytic digestion with an excess of sequence grade trypsin (Boehringer Mannheim) as described previously (Shevchenko et al., 1996 ). After 3 h digestion, 0·3 µl digestion supernatant was removed and deposited directly into an acidified water droplet placed on the top of the matrix surface. A mixture of a saturated solution of
-cyano-hydroxycinnamic acid (Sigma) and nitrocellulose in acetone (Bio-Rad) was used as a fast evaporation matrix. Peptide mass maps were recorded on a Bruker Reflex Matrix-Assisted Laser Desorption Ionization Time-of-Flight mass spectrometer (Bruker-Franzen). All of the spectra were obtained as 100-shot means. Tryptic autolysis peaks and some matrix-related peaks were used for internal calibration. Proteins were identified using Peptide Search software to search a set of tryptic peptide masses against a non-redundant protein sequence database (NRDB) (Mann & Wilm, 1994
). Mass accuracy was better then 50 p.p.m.
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RESULTS |
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Based on these results paraformaldehyde cross-linking of proteins associated with or located in close proximity to P1 was carried out for 60 min (Fig. 2).
Immunoblot analysis of proteins cross-linked to P1
Individual proteins of the protein complexes isolated by affinity chromatography were released by breaking paraformaldehyde cross-links at 95 °C for 15 min. The proteins were resolved by SDS-PAGE. Immunoblot analysis revealed the proteins complexed to the P1 protein. The P1 protein complex contained the P1 protein, the 90 and 40 kDa proteins, the adhesin-related 30 kDa protein, DnaK, the cytoskeleton-forming proteins HMW1 and HMW3 and the cytoskeleton-associated 65 kDa protein. The HMW proteins were detected in minute amounts, indicating that only a small proportion of the cytoskeleton-forming proteins was associated with the P1 protein complex (Fig. 3). When lysates of untreated M. pneumoniae cells were used for immunoaffinity chromatography only the P1 protein was purified (not shown). This indicated that proteins which were co-purified with the P1 protein were specifically cross-linked to each other by paraformaldehyde.
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DISCUSSION |
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To expand our knowledge of the interactions between cytadherence-associated membrane proteins (P1 adhesin, 90, 40 and 30 kDa proteins) and cytoplasmic proteins of the mycoplasma cell, we performed nearest-neighbour analyses using the permeant cross-linking reagent paraformaldehyde. In comparison to other chemical cross-linkers paraformaldehyde has the advantage of being non-specific with respect to its reaction partner and applicable for in vivo use. Cross-linking by paraformaldehyde is reversible by heat (95 °C). Therefore, individual proteins of paraformaldehyde cross-linked complexes can be analysed after breaking the cross-links. Furthermore, paraformaldehyde is a monomer in solutions, and therefore cross-linking of proteins by this reagent requires close associations. Since paraformaldehyde is able to permeate the cell membrane, cross-linking of proteins can occur inside the membrane or the bacterial cell as well as on the cell surface.
In this study we focused on the identification of individual proteins complexed to the P1 protein by paraformaldehyde by the use of a combination of two different methods: immunoblot analysis and MALDI MS.
We screened a series of defined antisera for their reactivity with particular proteins of the isolated protein complex. We were able to identify the membrane proteins of 90 kDa and 40 kDa (ORF6 gene product), the 30 kDa adhesin-related protein, the cytoskeleton-associated protein of 65 kDa, and minute amounts of the cytoskeleton-forming proteins HMW1 and HMW3, in addition to the P1 protein. The results achieved with nearest-neighbour analysis using the permeant cross-linker paraformaldehyde provided unequivocal proof of direct associations of the P1 protein and/or other cytadherence-associated membrane proteins (30, 40 and 90 kDa proteins) with defined cytoskeleton proteins. Since cross-linking of the P1 protein (and/or the 90 and 40 kDa proteins) with the 30 kDa protein, the HMW proteins and the 65 kDa protein was achieved with the permeant reagent paraformaldehyde but not with the impermeant cross-linking reagent DTSSP (Layh-Schmitt & Herrmann, 1994 ), we suggest that interactions between these proteins occur via their transmembrane or cytoplasmic regions. Furthermore, cross-linking with DTSSP is restricted to reactive amino groups whereas reactivity of paraformaldehyde is less specific and might result in a higher number of cross-linked proteins. Several of the proteins detected in the cross-linked complex (P1, the 30 and 90 kDa proteins, P65 and the HMW1 and HMW3 proteins) contain proline-rich repeat regions which might play a role in proteinprotein interactions. For a considerable number of eukaryotic membrane proteins homologous or heterologous interactions have been shown to be a common feature, with the transmembrane segments the principal sites of interaction (Brosig & Langosch, 1998
; Langosch & Herringa, 1998
).
The use of MS for the characterization of functional protein complexes has been described previously (Neubauer et al., 1997 ). Here we show that the same technology can be applied to determine which proteins of the mycoplasma membrane or cytoplasm are located in close proximity to each other to form complexes after cross-linking. By using MALDI MS we were able to confirm the results obtained by immunoblot analysis and to identify two additional proteins which were components of the P1 complex namely DnaK and the E1
subunit of pyruvate dehydrogenase.
DnaK may play a role in translocating membrane proteins from the cytoplasm into the cell membrane, resulting in an association of DnaK with the P1 protein or other membrane proteins. Such an association of DnaK with the membrane has been shown previously for Escherichia coli (Bukau et al., 1993 ). It is still unclear whether the truncated forms of DnaK and P1 detected in the cross-linked complex were an experimental artefact or whether these proteins are naturally expressed in variable forms. The pyruvate dehydrogenase complex has been shown to be located in the membrane of Acholeplasma laidlawii, which, like M. pneumoniae, is a member of the Mollicutes (Wallbrandt et al., 1992
). Therefore, in M. pneumoniae pyruvate dehydrogenase might also be associated with the membrane, leading to cross-linking with the isolated protein complex. Alternatively, the pyruvate dehydrogenase E1
subunit might function as a structural protein essential for the assembly and/or regulation of cytadherence-associated proteins which were detected in the cross-linked complex. This assumption is supported by the findings of Spellerberg et al. (1996)
, who proposed a role for a member of the pyruvate oxidase family in the regulation of adhesive properties and virulence of Streptococcus pneumoniae.
The data presented in this paper provide insights into the interactions between the membrane and cytoskeletal proteins of M. pneumoniae. Further biochemical studies are needed to define the regions of individual proteins essential for the mycoplasma proteinprotein interactions in the attachment organelle of M. pneumoniae. The mycoplasma cell might serve as a model prokaryotic organism for studying proteinprotein interactions as well as structurefunction relationships because its complete genome sequence is known, the number of genes and proteins (approx. 600) is relatively low, and its architecture (lack of cell wall) is simple.
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ACKNOWLEDGEMENTS |
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Received 22 July 1999;
revised 7 November 1999;
accepted 26 November 1999.