Previous Article | Next Article 
Journal of Bacteriology, February 1999, p. 1025-1029, Vol. 181, No. 3
Institut für Mikrobiologie und
Hygiene1 and
Institut für
Pharmakologie und Toxikologie,
Received 17 August 1998/Accepted 21 November 1998
The major sheath protein-encoding gene (mspA) of the
oral spirochete Treponema maltophilum ATCC
51939T was cloned by screening a genomic library with an
anti-outer membrane fraction antibody. The mspA gene
encodes a precursor protein of 575 amino acids with a predicted
molecular mass of 62.3 kDa, including a signal peptide of 19 amino
acids. The native MspA formed a heat-modifiable, detergent- and
trypsin-stable complex which is associated with the outer membrane.
Hybridization with an mspA-specific probe showed no
cross-reactivity with the msp gene from Treponema
denticola.
The postulated etiologic role of
oral treponemes in human periodontitis is based on the presence of
elevated numbers of these organisms in periodontal lesions (6, 11,
12, 19, 20). The interaction of the periodontal bacteria with
host tissue is complex, involving motility, mechanisms of adherence,
and modulation of the immune response (12, 13, 20). Adhesion
and the interaction with other subgingival plaque organisms may be
important steps in colonization (9). Several surface
antigens have been found in oral treponemes (1, 3, 5, 7, 16, 18,
23, 27, 28), some of which elicit a humoral response in
periodontitis patients (25). Recently it has been reported
that group IV treponemes (2) had the highest prevalence in
periodontal lesions (15). A representative novel species of
this phylogroup, Treponema maltophilum (30), was
selected to study the role of these bacteria in the pathogenesis of
periodontal infections. First we began to analyze outer membrane
antigens of T. maltophilum because they were considered relevant in the pathogen-host interaction.
Bacterial strains.
All bacterial strains used in this study
are listed in Table 1 and were maintained
as described previously (30).
Cloning the major sheath protein of T. maltophilum.
An
expression gene library of T. maltophilum was generated by
partially digesting chromosomal DNA with HaeIII or
RsaI, followed by packaging with a Gigapack II Gold cloning
kit (Stratagene, Heidelberg, Germany). The screening for T. maltophilum-specific antigens was done by immuno-colony dot assay
(26) using a chicken polyclonal antibody (immunoglobulin Y)
raised against the purified outer membrane protein fraction (OMF) of
T. maltophilum. Recombinant pBK-CMV phagemids from strongly
reacting clones were isolated. Whole-cell extracts of the corresponding
recombinant Escherichia coli XLOR strains were subjected to
sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE and
Western blot analysis with the anti-OMF antibody, as described
elsewhere (10, 24). Two clones (pKH30 and pKH33) showed a
positive band at approximately 63 kDa (Fig.
1B, lanes 3 and 4), and one clone (pKH32)
showed a positive band at 58 kDa (lane 5). The 63-kDa protein
comigrated with the T. maltophilum major surface protein
(designated MspA). DNA sequence analysis showed that all three inserts
contained the same genomic region (Fig.
2). The missing 5' region was obtained by
reverse PCR with internal mspA primers (msprR1 and msprR2
[Fig. 2]) and SacII-digested, religated chromosomal DNA.
The resulting fragment was cloned and sequenced (Fig. 2 [pKH58]).
Sequencing was done as described earlier (30). The primer
pair mspU3 and mspR2 (Fig. 2) was then used to amplify and clone the
entire chromosomal mspA gene of T. maltophilum,
resulting in plasmid pKH55 (Fig. 2).
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Cloning and Characterization of a Gene (mspA) Encoding
the Major Sheath Protein of Treponema maltophilum ATCC
51939T
ABSTRACT
Top
Abstract
Text
References
TEXT
Top
Abstract
Text
References
TABLE 1.
Distribution of mspA homologs in various
Treponema strains
View larger version (77K):
[in a new window]
FIG. 1.
SDS-PAGE and Western blot analysis of the MspA protein.
(A) SDS-PAGE and the corresponding Western blot, obtained with the
anti-OMF antibody (2-h incubation), of different T. maltophilum protein samples. M, molecular mass standards
(Bio-Rad); TM, sonicated T. maltophilum cells; P3, pellet of
the OMF; P3s and P4, soluble fractions of OMF; P13, extracellular
proteins. (B) Western blot analysis of cloned recombinant MspA. Equal
amounts of extracts of E. coli K-12 strains harboring
plasmids pKH55 (lane 2), pKH30 (lane 3), pKH33 (lane 4), pKH32 (lane
5), pKH34 (lane 6), pKH42 (lane 7), pKH51 (lane 8), pCAL-n (lane 9),
and pBK-CMV (lane 10) and sonicated T. maltophilum cells
(lane 1) were applied to each lane. (C) Western blot analysis with the
anti-OMF antibody (overnight incubation), showing the effects of heat
modification (lanes 1 to 4) and proteinase treatment (lanes 5 to 8) of
the OMF of T. maltophilum. Try, trypsin; PK, proteinase K. (D) SDS-PAGE illustrating the results of detergent and chemical
treatment of the native MspA complex (OMF). P, pellet; S, soluble
fraction; Tris, 20 mM Tris (pH 7.2) (lanes 1 and 2 [control]);
TX-114, 1% Triton X-114 (lanes 3 and 4); SDS, 1% SDS (lanes 5 and 6);
Na2CO3, 0.1 M Na2CO3
(pH 11) (lanes 7 and 8); NaCl, 0.6 M NaCl (lanes 9 and 10); Urea, 1.6 M
urea (lanes 11 and 12).
View larger version (20K):
[in a new window]
FIG. 2.
Restriction map of the T. maltophilum mspA
region. Coding regions corresponding to mspA, ORF232,
ORF262, and ORFU are indicated by large arrows. Small arrows represent
the primers used for PCR. E, EcoRI; H,
HindIII; Ha, HaeIII; K, KpnI; P,
PstI; S, SalI, Sc, SacII. SD,
Shine-Dalgarno ribosome binding site.
,
signal peptide; 
, putative
lipoprotein attachment site.
Nucleotide sequence analysis of the DNA region containing the mspA locus. The nucleotide sequence of the entire insert of plasmids pKH30, pKH32, pKH55, and pKH58 was determined (Fig. 2). An open reading frame (ORF) of 1,728 bp encoding a protein of 575 amino acids was identified. The predicted molecular mass of 62.3 kDa is in agreement with the size of the major outer membrane protein of T. maltophilum reacting with the anti-OMF antibody. A putative Shine-Dalgarno (SD) consensus sequence (AAGGAGG) is located 11 bp upstream of the ATG start codon. Downstream of the TAA stop codon of mspA, the sequence shows a GC-rich stem-loop representing a potential rho-independent transcriptional termination signal. Upstream of the mspA gene, we identified an ORF of 699 bp (named ORF232) encoding a putative protein of 232 amino acids with a theoretical molecular mass of 25.4 kDa (Fig. 2). A putative prokaryotic membrane lipoprotein lipid attachment site characteristic of bacterial lipoproteins (29) was found in the N-terminal part of the protein by computer analysis (Motifs program, Genetics Computer Group package, Husar 4.0). Downstream of mspA, on the complementary DNA strand, an ORF of 789 bp (ORF262) was located that encoded a putative protein of 262 amino acids (30.1 kDa) (Fig. 2). Computer analysis ("Signalseq" program, Husar 4.0) revealed a putative signal peptidase I cleavage site at the N-terminal part of the protein. A search through current protein databases revealed no homologous proteins. Downstream of the cloned T. maltophilum chromosomal DNA fragment, we identified the start of a putative ORF (named ORFU) (Fig. 2). The encoded first 182 N-terminal amino acids exhibited 73 and 72% similarity to the conserved hypothetical proteins YebB of Bacillus subtilis and MJ0326 of Methanococcus jannaschii, respectively.
Analysis of the MspA peptide.
Fractionation experiments were
done by a modified SDS method as described earlier (22). A
T. maltophilum culture (200 ml) was harvested by
centrifugation (5,000 × g, 15 min, 4°C).
Extracellular proteins were denatured with 15% trichloroacetic acid
(TCA) and pelleted (pellet P13) by centrifugation. The cell pellet was
washed with phosphate-buffered saline (PBS) (pH 7.2) and resuspended in
10 ml of PBS containing 0.01% SDS. The suspension was gently shaken
for 30 min at room temperature (RT). The cells were pelleted (P2) by
centrifugation (5,000 × g, 15 min, 4°C). The
supernatant was again centrifuged (25,000 × g, 30 min,
4°C). Pellet P3 (OMF fraction) was resuspended in 100 µl of 0.05 M
Tris-HCl (pH 7.2) and stored at 
20°C. Proteins of the supernatant
were precipitated with TCA, pelleted by centrifugation (P4),
resuspended in 100 µl of 0.05 M Tris-HCl (pH 7.2), and stored at
20°C. SDS-PAGE and Western blotting revealed that the 63-kDa MspA
protein was the major component of the outer membrane (Fig. 1A, lane 3 [fraction P3]) recognized by the anti-T. maltophilum OMF
antibody (Fig. 1A, lane 8). Only small amounts of MspA are found in the
P3s (soluble), P4, and P13 fractions (Fig. 1A, lanes 9, 10, and 11, respectively). Additional bands were found at approximately 48, 52, 85, 180, and 200 kDa (Fig. 1A, lane 3 and 3C, lane 1). Western blot
analysis showed that the recombinant MspA protein of the E. coli clone, harboring plasmid pKH55 (containing the complete
mspA gene), comigrates with the T. maltophilum
major surface protein (Fig. 1B, lanes 1 and 2). However, the level of
mspA expression in the recombinant system was low.
1 for proteinase K, and the final trypsin concentration
was 5% (wt/vol). Samples were incubated at 37°C for 30 min, mixed
with 5 µl of loading buffer, boiled for 8 min, and then analyzed by SDS-PAGE (Fig. 1C). Whereas the native form of MspA was trypsin resistant, the monomeric form was not (Fig. 1C, lanes 5 and 6). Both
forms of the MspA protein were sensitive to proteinase K treatment
(Fig. 1C, lane 7 and 8). Detergent and chemical treatment studies were
done with T. maltophilum in a whole-cell suspension (10 µl) or with purified OMF protein (5 to 10 µg). The pelleted protein
was suspended in 20 µl of detergent in 20 mM Tris-HCl (pH 7.2) and
incubated at room temperature for 30 min. To separate the soluble and
insoluble fractions, samples were centrifuged at 14,000 rpm for 10 min
in a Labofuge 400 R (Hereus). Supernatant (soluble [S]) and pellet
(insoluble [P]) fractions were mixed with SDS-PAGE loading buffer,
incubated at 100°C for 8 min, and analyzed by SDS-PAGE. Except for 8 M urea, the native MspA complex was resistant to treatment with
detergents disrupting noncovalent protein bonds (25 mM EDTA, 25 mM
MgCl2, 25 mM CaCl2, 100 mM NaCl) (data not
shown). After treatment of the OMF with 1% Triton X-114 or 1% SDS,
MspA was found in the supernatant (Fig. 1D, lanes 3 and 4 and 5 and 6).
Treatment of the OMF with buffer (control [Fig. 1, lane 2]) and
chemicals known to remove peripheral proteins from the membrane (0.1 M
Na2CO3 [pH 11], 0.6 M NaCl, 1.6 M urea) did
not extract MspA from the outer membrane of T. maltophilum (Fig. 1D, lanes 7 to 12).
The MspA protein of T. maltophilum has a typical prokaryotic
signal peptidase I consensus sequence and a leader peptide of 19 amino
acids. Kyte-Doolittle plot analysis indicated a typical strong
hydrophobic peak for the signal peptide (data not shown). The mature
protein contains 556 amino acids and a theoretical molecular mass of
60.2 kDa. To determine the N-terminal amino acid sequence of the 63-kDa
OMF protein, the proteins of fraction P3 (OMF) were separated by
SDS-PAGE, transferred electrophoretically to polyvinylidene difluoride
membranes (PVDF-Westran; Schleicher & Schuell), and subjected to Edman
degradation with a Procise (Applied Biosystems) gas phase sequencer.
The sequence AEPAAEAKVAEFSGN was identical to the
deduced amino acid sequence of the mature MspA protein, indicating that
the MspA protein is cleaved at the predicted peptidase I cleavage site
(data not shown). Comparison of the amino acid sequences of the Msp
peptides of T. denticola (strains OTK, ATCC 35405, and ATCC
32520) and the MspA protein of T. maltophilum showed an
overall similarity in the total amino acid composition (Table
2) and in the hydropathy plot (data not shown). Like the Msp proteins of T. denticola ATCC 35405 and
ATCC 32520, the MspA of T. maltophilum contains no cysteine
residue, and the numbers of small, small hydrophobic, and aromatic
amino acids were nearly identical for all four proteins (Table 2). MspA
exhibited similarities of 49, 45, and 43% to the Msp proteins from
T. denticola OTK, ATCC 32520, and ATCC 35405, respectively.
|
Occurrence of the mspA gene-containing region in other treponeme species. Chromosomal DNAs of various treponeme strains were analyzed by Southern blotting with the 1.2-kb internal mspA HindIII restriction fragment (Fig. 2) and the approximately 1.8-kb amplicon of the msp gene (primers KX14 and KX04; see reference 9) as T. maltophilum mspA and T. denticola ATCC 33521 msp-specific probes. Labeling of the probes and hybridization were performed as described earlier (8). Hybridization with the T. maltophilum-specific probe has shown a positive signal in all group IV treponemes looked at so far (Table 1). However, DNA of Treponema brennaborense (21) and Treponema socranskii subsp. socranskii showed only a weak signal. All other treponemal chromosomal DNAs investigated did not show any cross-hybridization. Hybridization of the same blot with the T. denticola msp probe gave positive signals with the T. denticola strains and Treponema vincentii (Table 1). This was corroborated by Western blot analysis of total cell protein from various treponemal strains by using the T. maltophilum-specific anti-OMF antibody (Table 1). All available isolates of phylogroup IV and T. socranskii subsp. socranskii, but no other treponemal strain investigated so far, have reacted with the antibody (data not shown). However, these data have to be verified with an MspA-specific antibody.
We conclude that MspA forms a heat-modifiable, detergent- and trypsin-stable, high-molecular-mass complex within the outer membrane. No strong hydrophobic regions other than the signal sequence were found, as predicted by the Kyte-Doolittle algorithm. These properties are common to porin proteins (17) and also described for the outer sheath protein (Msp) of T. denticola (3, 4, 7) and the MompA protein of Treponema pectinovorum (27). While T. denticola Msp protein formed porins within membranes (3, 14), this characteristic remains to be shown for the T. maltophilum MspA complex. However, the physical characteristics described in this study suggest that the T. maltophilum MspA protein represents another member of the so-called "msp-like" protein group (5). Whereas no information has been given so far on regions flanking the msp gene (4, 5), we cloned a chromosomal DNA fragment of about 4.3 Mbp covering the mspA DNA region of T. maltophilum encoding about four proteins, possibly associated with or integrated within the outer membrane. Currently, we are characterizing these putative proteins to show whether they are associated with the MspA protein complex. Preliminary PCR analysis suggests that T. maltophilum reference strain ATCC 51940 and the clinical isolate I exhibit a similar gene arrangement of the mspA DNA region, suggesting that this arrangement may be conserved among oral treponemes (data not shown).Nucleotide sequence accession number. The mspA gene DNA sequence of T. maltophilum has been submitted to the EMBL and GenBank databases under accession no. Y17800.
| |
ACKNOWLEDGMENTS |
|---|
We thank M. Kachler for excellent technical assistance and Bettina Brand for careful review of the manuscript.
This study was supported by a grant (01KI9318) from the Bundesministerium für Bildung und Forschung to U.B.G.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Institut für Mikrobiologie und Hygiene, Universitätsklinikum Charité, Humboldt-Universität zu Berlin, Berlin, Germany. Phone: 49-3020934715. Fax: 49-3020934703. E-mail: ulf.goebel{at}charite.de.
| |
REFERENCES |
|---|
|
Top
Abstract
Text
References
|
|---|
| 1. |
Blanco, D. R.,
K. Reimann,
J. Skare,
C. I. Champion,
D. Foley,
M. M. Exner,
R. E. W. Hancock,
J. N. Miller, and M. A. Lovett.
1994.
Isolation of the outer membranes from Treponema pallidum and Treponema vincentii.
J. Bacteriol.
176:6088-6099 |
| 2. |
Choi, B. K.,
B. J. Paster,
F. E. Dewhirst, and U. B. Göbel.
1994.
Diversity of cultivable and uncultivable oral spirochetes from a patient with severe destructive periodontitis.
Infect. Immun.
62:1889-1895 |
| 3. |
Egli, C. E.,
W. K. Leung,
K.-H. Müller,
R. E. W. Hancock, and B. C. McBride.
1993.
Pore-forming properties of the major 53-kilodalton surface antigen from the outer sheath of Treponema denticola.
Infect. Immun.
61:1694-1699 |
| 4. |
Fenno, J. C.,
K.-H. Müller, and B. C. McBride.
1996.
Sequence analysis, expression, and binding activity of recombinant major outer sheath protein (Msp) of Treponema denticola.
J. Bacteriol.
178:2489-2497 |
| 5. |
Fenno, J. C.,
G. W. K. Wong,
P. M. Hannam,
K.-H. Müller,
W. K. Leung, and B. C. McBride.
1997.
Conservation of msp, the gene encoding the major outer membrane protein of oral Treponema spp.
J. Bacteriol.
179:1082-1089 |
| 6. | Fiehn, N. K. 1989. Small-sized oral spirochetes and periodontal disease. Acta Pathol. Microbiol. Scand. 97(Suppl. 7):1-31. |
| 7. |
Haapasalo, M.,
K.-H. Müller,
V.-J. Uitto,
W. K. Leung, and B. C. McBride.
1992.
Characterization, cloning, and binding properties of the major 53-kilodalton Treponema denticola surface antigen.
Infect. Immun.
60:2058-2065 |
| 8. | Heuner, K., L. Bender-Beck, B. C. Brand, P. C. Lück, K.-H. Mann, R. Marre, M. Ott, and J. Hacker. 1995. Cloning and genetic characterization of the flagellum subunit gene (flaA) of Legionella pneumophila serogroup 1. Infect. Immun. 63:2499-2507[Abstract]. |
| 9. |
Kolenbrander, P. E., and J. London.
1993.
Adhere today, here tomorrow: oral bacterial adherence.
J. Bacteriol.
175:3247-3252 |
| 10. | Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685[Medline]. |
| 11. | Listgarten, M. A., and S. Levin. 1981. Positive correlation between the proportions of subgingival spirochetes and motile bacteria and susceptibility of human subjects to periodontal deterioration. J. Clin. Periodontol. 8:122-138[Medline]. |
| 12. | Listgarten, M. A. 1987. Nature of periodontal diseases: pathogenic mechanisms. J. Periodontal Res. 22:172-178[Medline]. |
| 13. | Loesche, W. J. 1993. Bacterial mediators in periodontal disease. Clin. Infect. Dis. 16:S203-S210. |
| 14. | Mathers, D. A., W. K. Leung, C. Fenno, Y. Hong, and B. C. McBride. 1996. Major surface protein complex of Treponema denticola depolarizes and induces ion channels in HeLa cell membranes. Infect. Immun. 64:2904-2910[Abstract]. |
| 15. |
Moter, A.,
C. Hoenig,
B.-K. Choi,
B. Riep, and U. B. Göbel.
1998.
Molecular epidemiology of oral treponemes associated with periodontal disease.
J. Clin. Microbiol.
36:1399-1403 |
| 16. | Nakamura, Y., T. Umemoto, Y. Nakatani, I. Namikawa, and A. Wadood. 1993. Common and specific antigens of several treponemes detected by polyclonal antisera against major cellular proteins. Oral Microbiol. Immunol. 8:288-294[Medline]. |
| 17. | Nikaido, H. 1992. Porins and specific channels of bacterial outer membranes. Mol. Microbiol. 6:435-442[Medline]. |
| 18. |
Norris, S. J., and the Treponema pallidum Polypeptide Research Group.
1993.
Polypeptides of Treponema pallidum: progress toward understanding their structural, functional, and immunologic roles.
Microbiol. Rev.
57:750-779 |
| 19. | Penn, C.-W. 1991. Pathogenicity and molecular biology of treponemes. Rev. Med. Microbiol. 2:68-75. |
| 20. | Saglie, R., M. G. Newman, F. A. Carranza, Jr., and G. L. Pattison. 1982. Bacterial invasion of gingiva in advanced periodontitis in humans. J. Periodontol. 53:217-222[Medline]. |
| 21. |
Schrank, K.,
B.-K. Choi,
S. Grund,
A. Moter,
K. Heuner,
H. Nattermann, and U. B. Göbel.
1999.
Treponema brennaborense sp. nov., a novel spirochete isolated from a dairy cow suffering from digital dermatitis.
Int. J. Syst. Bacteriol.
49:43-50 |
| 22. |
Stamm, L. V.,
R. L. Hodinka,
P. B. Wyrick, and P. J. Bassford, Jr.
1987.
Changes in the cell surface properties of Treponema pallidum that occur during in vitro incubation of freshly extracted organisms.
Infect. Immun.
55:2255-2261 |
| 23. |
Tall, B. D., and R. K. Nauman.
1994.
Outer membrane antigens of oral Treponema species.
J. Med. Microbiol.
40:62-69 |
| 24. |
Towbin, H.,
T. Staehelin, and J. Gordon.
1979.
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Proc. Natl. Acad. Sci. USA
76:4350-4354 |
| 25. |
Umemoto, T.,
J. J. Zambon,
R. J. Genco, and I. Namikawa.
1988.
Major antigens of human oral spirochetes associated with periodontal disease.
Adv. Dent. Res.
2:292-296 |
| 26. | van Die, I., B. van Geffen, W. Hoekstra, and H. Bergmans. 1984. Type 1C fimbriae of an uropathogenic Escherichia coli strain. Cloning and characterization of the genes involved in the expression of the 1C antigen and nucleotide sequence of the subunit gene. Gene 34:187-196. |
| 27. |
Walker, S. G.,
J. L. Ebersole, and S. C. Holt.
1997.
Identification, isolation, and characterization of the 42-kilodalton major outer membrane protein (MompA) from Treponema pectinovorum ATCC 33768.
J. Bacteriol.
179:6441-6447 |
| 28. |
Weinberg, A., and S. C. Holt.
1991.
Chemical and biological activities of a 64-kilodalton outer sheath protein from Treponema denticola strains.
J. Bacteriol.
173:6935-6947 |
| 29. | Wu, H. C., and M. Tokunaga. 1986. Biogenesis of lipoproteins in bacteria. Curr. Top. Microbiol. Immunol. 125:127-157[Medline]. |
| 30. |
Wyss, C.,
B.-K. Choi,
P. Schüpbach,
B. Guggenheim, and U. B. Göbel.
1996.
Treponema maltophilum sp. nov., a small oral spirochete isolated from human periodontal lesions.
Int. J. Syst. Bacteriol.
46:745-752 |
Journal of Bacteriology, February 1999, p. 1025-1029, Vol. 181, No. 3
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Edwards, A. M., Jenkinson, H. F., Woodward, M. J., Dymock, D.
(2005). Binding Properties and Adhesion-Mediating Regions of the Major Sheath Protein of Treponema denticola ATCC 35405. Infect. Immun.
73: 2891-2898
[Abstract] [Full Text] -
Lee, S.-H., Kim, K.-K., Choi, B.-K.
(2005). Upregulation of Intercellular Adhesion Molecule 1 and Proinflammatory Cytokines by the Major Surface Proteins of Treponema maltophilum and Treponema lecithinolyticum, the Phylogenetic Group IV Oral Spirochetes Associated with Periodontitis and Endodontic Infections. Infect. Immun.
73: 268-276
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»