TEXT

Top Abstract Text References

Spirochetes have unique morphology and motility. Their periplasmic flagella, located between the outer membrane and the cytoplasmic membrane, play an important role in cellular morphology and motility (5, 15). The Treponema genus contains several important pathogens, and many of these pathogenic spirochetes cannot be cultured in vitro. One of the most important spirochete pathogens is Treponema pallidum, the causative agent of syphilis, which can be grown experimentally only in rabbit testes, but no gene transfer system for the organism is available. For identifying the virulence factors of these pathogens, potential virulence genes must be expressed in heterologous systems. Although some T. pallidum genes can be expressed in Escherichia coli (6, 15), the distinct physiological differences between spirochetes and E. coli limits the use of the E. coli system for functional investigations.

One of the oral spirochetes, Treponema denticola, which has been shown to be associated with periodontitis (11, 12, 16), can be cultured in the laboratory. In addition, a gene transfer system for T. denticola was recently developed in our laboratory (8, 9). These advantages together with the similarity of T. denticola with other spirochetes suggest that T. denticola may serve as a suitable host for expressing heterologous spirochete genes.

Previously, the broad-host-range plasmid pKT210 was shown to serve as a shuttle vector in a variety of bacterial hosts, including T. denticola (8). However, this plasmid proved to be unstable in several host systems. Therefore, in the present study we constructed a novel E. coli-T. denticola shuttle vector based on the naturally occurring spirochete plasmid pTS1 (3) and demonstrated the expression of the heterologous T. pallidum flaA gene from the plasmid.

Construction of a novel shuttle vector and transformation of T. denticola. The cryptic plasmid pTS1 of T. denticola ATCC u9b (3) was used for shuttle vector construction. The sequence of pTS1 (3a) revealed an open reading frame homologous to a gene on plasmid pJDB23, a cryptic plasmid of Selenomonas ruminantium subsp. lactilytica (2). The fact that the gene on pJDB23 is responsible for the plasmid replication in E. coli (2) suggested that the open reading frame on pTS1 encodes a Rep protein. BamHI digestion of the pTS1 plasmid generated two fragments, and the larger, 2.8-kb fragment, which contains the potential Rep-encoding gene, was ligated into the BamHI site of an E. coli plasmid, pKMOZ19 (14), yielding the chimeric plasmid pKMRep4, which should replicate in both T. denticola and E. coli (Fig. 1A). The erythromycin resistance gene cassette (4), which has been shown to be expressed in T. denticola (9), was chosen as the selective marker for the shuttle vector. To ensure the transcription of the Em^r cassette in T. denticola, the promoter of a T. denticola proteinase gene, prtB (1), was placed upstream of the Em^r cassette. Both the Em^r cassette and the prtB promoter were PCR amplified and cloned into the E. coli plasmid pBK-CMV (Stratagene, La Jolla, Calif.). The fragment which contained the promoter and the Em^r cassette was removed from pBK-CMV, blunt ended, and ligated into the HincII site of plasmid pKMRep4 to generate the 7.7-kb pKMR4PE (Fig. 1A).

View larger version (35K): [in this window] [in a new window]   FIG. 1.   Construction of shuttle vectors pKMR4PE (A) and pKMflaA (B). The position and orientation of the putative Rep-encoding gene of pTS1 (Rep), the T. denticola prtB promoter (prtBp), and the Emr cassette (ermF and ermAM) are shown. Relevant restriction sites are indicated.

View larger version (113K): [in this window] [in a new window]   FIG. 2.   Electrophoretic analysis of plasmids isolated from T. denticola pKMR4PE and pKMflaA transformants. Lane 2, plasmid from wild-type T. denticola 33520; lane 3, plasmid from pKMR4PE transformants; lane 4, plasmid from pKMflaA transformants; lanes 5 to 9, SmaI digestions of plasmids from lane 2, lane 3, original pKMR4PE, lane 4, and original pKMflaA, respectively; lanes 1 and 10, 1-kb DNA ladder.

Expression of the T. pallidum flaA gene in T. denticola. Our next step was to use the new shuttle vector to express heterologous spirochete genes. The gene of T. pallidum endoflagellum protein FlaA was chosen as a suitable gene because its sequence is known (5) and a monoclonal antibody, H9-2 (13), is available (gift from Sheila Lukehart, Harborview Medical Center, Seattle, Wash.). PCR primers were designed according to the T. pallidum flaA gene sequence (5), and the flaA gene was amplified from T. pallidum genomic DNA (gift from Kayla Hagman, University of Texas, Dallas). Our first attempt to clone the flaA gene together with its native promoter onto pKMR4PE in E. coli was not successful. This is consistent with previous reports that the strong expression of this flaA gene cannot be tolerated by E. coli (5). It was also known that the Em^r cassette does not have transcriptional termination signals (10). The flaA gene was then placed downstream of the Em^r cassette to be expressed from the prtB promoter. By using the XbaI restriction sites (underlined) which were incorporated into the forward and reverse PCR primers (5'-TTTTTTCTAGAGAGTGGTTATCTTATTGTGCG-3' and 5'-TTTTTTCTAGATAGCCATCCTACCACGCATCC-3', respectively) the amplified 1.25-kb flaA gene, which begins 24 bp upstream of its ribosome-binding site, was inserted into the unique XbaI site of pKMR4PE (Fig. 1B). The E. coli XL1-Blue colonies were screened by restriction endonuclease mapping for the flaA gene inserted in the same orientation as the Em^r cassette. The resulting plasmid, pKMflaA, was transformed into T. denticola, and erythromycin-resistant T. denticola colonies were analyzed for plasmids and flaA gene expression. As shown in Fig. 2, pKMflaA-transformed T. denticola contained an additional band larger than that in pKMR4PE (Fig. 2, lane 4). Linearization of the plasmid with SmaI indicated that the plasmid had the same size as the original pKMflaA plasmid (Fig. 2, lanes 8 and 9). The pKMflaA plasmid from T. denticola was next retransformed into E. coli XL1-Blue cells. The rescued plasmids were further analyzed by restriction endonuclease mapping and proved to be identical to the original pKMflaA plasmid (data not shown).

View larger version (62K): [in this window] [in a new window]   FIG. 3.   Western blot analysis of T. denticola pKMflaA transformants. Cell extracts were separated on a sodium dodecyl sulfate-12.5% polyacrylamide gel and transferred to a nitrocellulose membrane. Monoclonal antibody H9-2 of T. pallidum FlaA protein (1:10 dilution) was used as the primary antibody. Lanes 1 and 5, prestained sodium dodecyl sulfate-polyacrylamide gel electrophoresis standards (Bio-Rad, Hercules, Calif.); lane 2, cell extract of T. denticola pKMflaA transformants; lane 3, cell extract of T. denticola pKMR4PE transformants; lane 4, cell extract of T. pallidum.