Cloning and Characterization of a Gene (mspA) Encoding the Major Sheath Protein of Treponema maltophilum ATCC 51939^T

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 orRsaI, 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) ofT. 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).

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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).

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Fig. 2.

Restriction map of the T. maltophilum mspAregion. 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 themspA 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 ofT. 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. maltophilumchromosomal 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). AT. 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 completemspA gene), comigrates with the T. maltophilummajor surface protein (Fig. 1B, lanes 1 and 2). However, the level ofmspA expression in the recombinant system was low.

Heat modification experiments were done by mixing 20 μl of T. maltophilum cell suspension or 10 μg of protein of the OMF in a total volume of 40 μl with 10 μl of loading buffer and incubation at RT, 40, 45, 50, 60, 80, and 100°C, respectively, for 8 min. The analysis showed that the approximately 200-kDa protein in untreated fractions corresponded to the native MspA protein complex, which was converted into the 63-kDa monomeric form of MspA by heating (Fig. 1C, lanes 1 and 4). Temperatures below 50°C converted only small amounts of the MspA complex to the monomeric form, indicating that the transition occurs at 50 to 60°C (Fig. 1C, lanes 2 and 3). An additional heat-modifiable protein with a transition temperature of 80 to 100°C was identified at approximately 180 kDa. Heat treatment generated a new protein band at approximately 36 kDa, as seen in Fig.1C, lane 4. Other proteins (48, 52, and 85 kDa) visible in Fig. 1C were not heat modifiable (Fig. 1C, lanes 1 to 4). T. maltophilumOMF (10 μg) was treated with proteases in a total volume of 20 μl. Prior to proteinase treatment, two of the four samples were incubated at 100°C for 8 min. The final proteinase concentration was 50 μg ml⁻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 (Table2) 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 fromT. 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 andT. 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 ofTreponema 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 theT. 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 themsp gene (4, 5), we cloned a chromosomal DNA fragment of about 4.3 Mbp covering the mspA DNA region ofT. 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 themspA 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.