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Journal of Bacteriology, April 2008, p. 2565-2571, Vol. 190, No. 7
0021-9193/08/$08.00+0     doi:10.1128/JB.01537-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Heterologous Expression of the Treponema pallidum Laminin-Binding Adhesin Tp0751 in the Culturable Spirochete Treponema phagedenis

Caroline E. Cameron,^1,2* Janelle M. Y. Kuroiwa,² Mitsunori Yamada,³ Teresa Francescutti,¹ Bo Chi,³ and Howard K. Kuramitsu³

Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada V8W 3P6,¹ Department of Medicine, University of Washington, Seattle, Washington 98195,² Department of Oral Biology, SUNY at Buffalo, Buffalo, New York 14214³

Received 24 September 2007/ Accepted 25 January 2008

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Treponema pallidum subsp. pallidum, the causative agent of syphilis, is an unculturable, genetically intractable bacterium. Here we report the use of the shuttle vector pKMR4PEMCS for the expression of a previously identified T. pallidum laminin-binding adhesin, Tp0751, in the nonadherent, culturable spirochete Treponema phagedenis. Heterologous expression of Tp0751 in T. phagedenis was confirmed via reverse transcriptase PCR analysis with tp0751 gene-specific primers and immunofluorescence analysis with Tp0751-specific antibodies; the latter assay verified the expression of the laminin-binding adhesin on the treponemal surface. Expression of Tp0751 within T. phagedenis was functionally confirmed via laminin attachment assays, in which heterologous Tp0751 expression conferred upon T. phagedenis the capacity to attach to laminin. Further, specific inhibition of the attachment of T. phagedenis heterologously expressing Tp0751 to laminin was achieved by using purified antibodies raised against recombinant T. pallidum Tp0751. This is the first report of heterologous expression of a gene from an unculturable treponeme in T. phagedenis. This novel methodology will significantly advance the field of syphilis research by allowing targeted investigations of T. pallidum proteins purported to play a role in pathogenesis, and specifically host cell attachment, in the nonadherent spirochete T. phagedenis.

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Syphilis is a chronic sexually transmitted disease caused by infection with the spirochete bacterium Treponema pallidum subsp. pallidum. An estimated 12 million new syphilis cases occur globally each year, the majority of which are in developing nations (20). In recent years, there has been a resurgence of syphilis in many developed countries, particularly among populations of men who have sex with men, with notable outbreaks spreading rapidly across North America, Europe, and Australia (1, 8, 11, 25, 44). Existing public health measures are not successfully controlling the spread of this disease, and alternative means of syphilis prevention are needed. In particular, in order to design an effective syphilis vaccine it is necessary to gain a deeper understanding of the pathogenic mechanisms used by T. pallidum to establish and sustain infection.

At the molecular level, little is currently known about T. pallidum pathogenesis. The bacterium gains entry to the host through intact mucosal barriers or microscopic epidermal abrasions (42). The organism is highly invasive, with in vitro studies showing that T. pallidum is able to penetrate endothelial cell monolayers to enter the bloodstream (45, 50) within hours of infection (13, 43). Treponemal invasion results in widespread bacterial dissemination, which, in turn, sets the stage for establishment of chronic infection. One key factor that has severely hampered studies of the pathogenic mechanisms used by this important human pathogen is that it cannot be continuously cultured in vitro. Although limited multiplication has been achieved in an in vitro tissue culture system (12, 17, 32, 34-37), T. pallidum is a fastidious, obligate human pathogen for which intratesticular inoculation of rabbits is the only reliable method of bacterial propagation. This organism remains refractory to genetic manipulations, thus preventing direct investigation of the functions of individual gene products. To circumvent this issue, investigators in the field must use heterologous systems to express candidate genes that are hypothesized to be involved in T. pallidum pathogenesis or virulence. Successful expression of T. pallidum genes has been accomplished in Escherichia coli (9, 16, 22, 47, 49); however, the dissimilarity in outer membrane ultrastructure and physiology between the two bacteria has yielded limited functional data from these studies (23). Expression of T. pallidum genes has also been performed in the bacterium Treponema denticola, a cultivable spirochete found in subgingival plaques associated with periodontitis (10, 48). Although expression studies using this related treponeme are physiologically more relevant than those performed with E. coli, interpretation of functional studies is complicated by the pathogenic nature of T. denticola. Specifically, T. denticola attaches to host cells and cellular components (7, 14, 15, 21, 26, 40, 41), penetrates endothelial cell monolayers (41), and invades the gingival connective tissue in some forms of periodontal disease (18, 28, 31, 46). Therefore, expression within T. denticola of T. pallidum molecules that are predicted to be involved in adhesion or tissue invasion, and subsequent determination of the effects of these heterologously expressed virulence factors on the overall pathogenicity of T. denticola, is not straightforward.

Treponema phagedenis, another member of the Treponema genus, is a strict anaerobe that does not attach to host cells or invade cell monolayers (7, 41). We hypothesized that the nonadherent, noninvasive phenotype of T. phagedenis, combined with its ease of cultivation, would make this treponeme an ideal candidate for heterologous expression of T. pallidum virulence factors and establishment as a model treponeme. Further, T. phagedenis has a G+C ratio similar to that of T. denticola (39), and therefore it was predicted that a shuttle vector developed for use in T. denticola would also function in T. phagedenis.

Previously, we identified a T. pallidum adhesin, designated Tp0751, that attaches to the extracellular matrix protein laminin (3, 5), a major component of basement membranes that underlie endothelial cell layers (51). This adhesin is purported to be involved in treponeme dissemination and pathogenesis (3, 5), although direct evidence of the involvement of this adhesin in the treponemal invasion process is currently lacking due to the experimental limitations associated with research on T. pallidum. Here we report the expression of this T. pallidum laminin-binding protein on the surface of T. phagedenis. This methodology will advance the field of syphilis research by opening new possibilities for functional investigations of T. pallidum proteins involved in pathogenesis and will directly facilitate the investigation of T. pallidum adhesins within a nonadherent, related treponeme.

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Extracellular matrix proteins. Laminin isolated from the Engelbreth-Holm-Swarm murine sarcoma and the negative control protein fetuin were purchased from Sigma Chemical Co. (Oakville, Ontario, Canada).

Bacteria. T. pallidum subsp. pallidum (Nichols strain) was propagated in New Zealand White rabbits as described elsewhere (29). All animal studies were approved by the local Institutional Review Boards and conducted according to standard accepted principles. T. phagedenis biotype Kazan was grown in either TYGVS medium (38) or thioglycolate broth (Sigma) supplemented with 20% heat-inactivated rabbit serum (R-7136; Sigma) in the presence of 10 µg/ml rifampin and 40 µg/ml erythromycin (the latter antibiotic was added to all cultures except wild-type T. phagedenis) (Sigma) at 37°C in an anaerobic chamber (Oxoid, Hampshire, United Kingdom). The identity of T. phagedenis was confirmed by 16S rRNA gene sequencing with the primers 5'-CACACCGCCCGTCACACC and 3'-CTATTCTTTCGCTTGACC.

Recombinant protein expression and polyclonal antibody production. Recombinant T. pallidum Tp0751 was prepared as previously described (3). The open reading frame (ORF) encoding the T. pallidum periplasmic flagellar protein FlaA (Tp0249) was expressed in order to generate control polyclonal antibodies for use in the T. phagedenis immunofluorescence and attachment inhibition studies. Briefly, primers unique to the mature T. pallidum flaA ORF were used (5'-Tp0249, 5'-GGATCCATGAGTCAGTGCTCATCGACTTC; 3'-Tp0249, 5'-CTGCAGCTACTGCTGCTCTTCCTGCCG) under the following PCR conditions: 35 cycles of 1 min of denaturation at 95°C, 2 min of annealing at 63°C, and 2 min 30 s of extension at 74°C. After PCR, the resulting amplification product was digested with BamHI and PstI, ligated to a similarly digested pRSETc expression vector (Invitrogen, Carlsbad, CA), and transformed into E. coli expression strain BL21 Star(DE3) (Invitrogen). The reading frame and sequence of the expression construct were verified by DNA sequencing. Expression and purification of recombinant Tp0249 were performed as reported previously (6). Polyclonal Tp0751-specific and Tp0249-specific immunoglobulin Y (IgY) antibodies were produced in chickens by Aves Labs Inc. (Tigard, OR). Briefly, each of two hens was immunized with either recombinant Tp0751 or Tp0249 by a proprietary immunization regimen, and the IgY was purified from the egg yolks by a proprietary method (Aves Labs Inc.). For comparison, preimmune IgY was isolated from egg yolks of the same hens collected prior to immunization. The specificities and titers of the antibody preparations were verified by an enzyme-linked immunosorbent assay-based protocol. Briefly, 96-well NUNC-Immuno MaxiSorp plates (Sigma) were coated overnight at 4°C in triplicate with 100 µl of the recombinant proteins per well in phosphate-buffered saline (PBS), pH 7.4, with 0.1% sodium dodecyl sulfate at a concentration of 2 µg/ml. Negative control wells were coated with the dilution buffer only. Plates were blocked at room temperature for 2.5 h with 4% milk powder-PBS and washed four times with Tris-buffered saline containing 0.05% Tween 20. Chicken antibody dilutions in the range of 1:100 to 1:3,000 were prepared in 4% milk powder-PBS, and 100 µl was added and incubated for 1 h at room temperature. After washing, 50 µl of a 1:1,000 dilution of horseradish peroxidase-linked goat anti-chicken IgY (Aves Labs Inc.) prepared in 4% milk powder-PBS was applied and incubated at room temperature for 1 h. After washing with Tris-buffered saline-0.05% Tween 20, wells were developed with the tetramethylbenzidine peroxidase substrate system (Kirkegaard & Perry Laboratories, Gaithersburg, MD) and the absorbance at 600 nm was measured.

Heterologous expression of T. pallidum Tp0751 in T. phagedenis. The T. pallidum tp0751 ORF and upstream noncoding region (bp 815798 to 816534) were PCR amplified from T. pallidum subsp. pallidum (Nichols strain) genomic DNA with the primers 5'-GTCGACTACTGGCGTGCAAGGAAGCGTC (SalI site underlined) and 3'-AGATCTTCAGGGCGAAGGAGCACTAG (BglII site underlined). Following amplification, the PCR product was digested with SalI and BglII and ligated to a similarly digested pKMR4PEMCS shuttle vector. This vector is related to the previously reported pKMR4PE shuttle vector (10) but is more versatile with the introduction of a multiple cloning site (MCS). The map of the shuttle vector and resulting tp0751/pKMR4PEMCS construct is illustrated in Fig. 1. The construct was transformed into E. coli Top10 (Invitrogen), and the sequence of the tp0751 ORF was verified by DNA sequencing with pKMR4PEMCS vector-specific primers, as well as internal primers designed from the tp0751 ORF. A large-scale DNA isolation was performed with the Qiagen Plasmid Maxi kit (Qiagen, Valencia, CA), and the purified DNA was used to transform T. phagedenis by the electroporation protocol previously reported for the transformation of T. denticola (10, 27). Briefly, 4 x 10⁹ freshly prepared competent T. phagedenis cells were mixed with 2 µg of the plasmid preparations on ice in a 0.1-mm electroporation cuvette. Electroporation was accomplished with a Gene Pulser (Bio-Rad Laboratories, Melville, NY) set at 1.8 kV, 25 µF, and 200 , producing a time constant of 4 to 4.5 ms, after which 1 ml of TYGVS medium was added and the cells were transferred to a culture tube and incubated overnight. All culturing was performed at 37°C under anaerobic conditions. Transformants were selected on TYGVS plates supplemented with 0.8% SeaPlaque agarose (FMC BioProducts, Rockland, ME) and 40 µg/ml erythromycin. Transformants appeared within 7 to 10 days at an efficiency of approximately 1 colony/µg of plasmid DNA. Individual colonies were inoculated into TYGVS broth and grown to mid-logarithmic phase, and plasmid DNA was isolated with a Qiagen miniprep kit to verify plasmid presence; in all cases, the plasmid was observed. Transformants of T. phagedenis containing the pKMR4PEMCS plasmid alone or the tp0751/pKMR4PEMCS construct displayed growth profiles and generation times similar to those of wild-type T. phagedenis.

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FIG. 1. Construction of the tp0751/pKMR4PEMCS shuttle vector. Shown are the putative rep gene of pTS1 (REP), the T. denticola prtB promoter, the Emr cassette (ermF and ermAM), and relevant restriction sites.

RNA isolation and RT-PCR analysis. T. phagedenis transformants were freshly harvested by centrifugation. RNA was extracted with the TRIZOL reagent (Invitrogen) and the Fastprep system (Krackeler Scientific Inc., NY) and treated with DNase I (Sigma). T. phagedenis first-strand cDNA was synthesized from 1 µg of total RNA with Superscript III reverse transcriptase (RT; Invitrogen) with a specific primer complementary to the 3' end of the tp0751 gene (5'-TCAGGGCGAAGGAGCACTAG-3'). Specific oligonucleotide primer sets were designed to amplify a 700-bp internal region of the tp0751 gene (5'-TACTGCGTGCAAGGAAGGCT-3' and 5'-GTGATATCTGGTGTATTGACCG-3'), and RT-PCR was performed with 2 µl of the cDNA and a 0.5 µm concentration of the oligonucleotide primer sets by established techniques (30). The RT-PCR was accompanied by positive and negative control reactions that used the same set of primers and the tp0751/pKMR4PEMCS plasmid and cDNA synthesized from a control pKMR4PEMCS/T. phagedenis transformant, respectively. To confirm the absence of residual DNA in each RNA sample, additional negative control reactions were performed in which T. phagedenis transformant RNA samples were used as templates without the RT step. For analysis, 1 µg of each of the RT-PCR products, or total RNA for the control reactions, was separated on a 1.5% agarose gel and visualized by ethidium bromide staining.

Immunofluorescence studies. Twenty-five microliters of mid-logarithmic growth phase culture of either wild-type T. phagedenis or T. phagedenis transformed with pKMR4PEMCS or tp0751/pKMR4PEMCS (approximately 2 x 10⁷ bacteria) was used to coat, in duplicate, poly-L-lysine-coated 10-well, 5-mm microscope slides (Tekdon Incorporated, Myakka City, FL) for 1 h at room temperature. Wells were washed with saline three times for 5 min each by immersion of the slide in a Coplin jar (Fisher Scientific, Ottawa, Ontario, Canada) and gentle agitation at 50 rpm. To reduce background fluorescence levels, polyclonal Tp0751-specific IgY antibodies were preadsorbed with a T. phagedenis lysate as follows. Ten milliliters of mid-logarithmic growth phase wild-type T. phagedenis culture was harvested by centrifugation at 9,300 x g for 10 min; the pellet was resuspended in PBS containing 1 mg/ml lysozyme, 2 µg/ml aprotinin, 0.2 µg/ml pepstatin A, and 100 µg/ml phenylmethylsulfonyl fluoride; and the mixture was incubated for 30 min on ice, followed by sonication on ice (three 30-s bursts). A 1:12.5 dilution of the polyclonal Tp0751-specific IgY antibodies was adsorbed overnight at 4°C with 10% (vol/vol) of the T. phagedenis lysate, samples were centrifuged at 12,000 x g for 10 min at 4°C, and 25 µl of the preadsorbed antibodies was diluted to 1:8,000 and added to the wells. To rule out the possibility that detected fluorescence was due to a breach in the integrity of the T. phagedenis outer membrane, control slides were included that consisted of a 1:100 dilution of polyclonal Tp0249-specific antibodies (directed against the T. pallidum periplasmic flagellar protein FlaA) incubated with wells coated with either wild-type T. phagedenis (negative control) or wild-type T. phagedenis that had first been subjected to a membrane permeabilization step by incubation for 1 h with 25 µl of 2% Triton X-100 (positive control). All antibody incubation steps were carried out for 1 h at room temperature. Negative control wells consisting of each construct with no primary antibody were also included. After washing four times with saline, 25 µl of a 1:500 dilution of fluorescein-labeled goat-anti-chicken IgY (Aves Labs, Inc.) was added to the wells and incubated in the dark for 1 h at room temperature. After one wash with saline, 30 µl of 250 µg/ml 4',6'-diamidino-2-phenylindole (DAPI) dilactate (Invitrogen) was added to each well and incubated for 30 min in the dark at room temperature. After washing three times with saline, 5 µl of a 1:3 Vectashield (Vector Laboratories, Burlingame, CA)-saline mixture was added to each well and fluorescence was viewed with a Nikon 80i fluorescence microscope (Meridian Instrument Company, Inc., Kent, WA) fitted with a monochrome digital camera, a dark-field condenser, and fluorescein and DAPI filters. Photographs were captured with a 60x oil immersion objective (x600 total magnification, includes a 10x ocular). Three independent experiments were performed, the samples were blinded, and a total of 10 randomly selected fields that covered an entire well were viewed for each sample.

T. phagedenis attachment and inhibition assays. Lab-Tek II chamber slides (Nalge Nunc International, Rochester, NY) were coated overnight at 4°C with 250 µl of a 75-µg/ml dilution of either laminin or the negative control protein fetuin prepared in saline. Chamber slides were blocked with 3% bovine serum albumin-saline for 2 h at room temperature and washed with saline three times for 5 min each with gentle agitation at 50 rpm. Mid-logarithmic growth phase cultures of either wild-type T. phagedenis or T. phagedenis transformed with pKMR4PEMCS or tp0751/pKMR4PEMCS were harvested at 3,200 x g. For the attachment assays, treponemes were adjusted to 4 x 10¹⁰ bacteria/ml in saline, and 500 µl of each bacterium-saline mixture was added in duplicate to the fetuin- and laminin-coated chamber slides. For the attachment inhibition assays, treponemes were adjusted to 2.125 x 10⁹ bacteria/ml in saline and pretreated for 3 h at 37°C under anaerobic conditions with a 1:2 dilution (100 µl antibody-200 µl bacterium-saline mixture) of either preimmune or polyclonal Tp0249-specific IgY antibodies (negative controls) or with polyclonal Tp0751-specific IgY antibodies. Following the preincubation step, 300 µl of the bacterium-saline-antibody mixture was added in duplicate to laminin-coated chamber slides. Bacteria that underwent a similar pretreatment step in the absence of antibodies (saline control, no pretreatment) were included for comparison. Bacteria were incubated with the coated chamber slides at 37°C for 3 h under anaerobic conditions, and the wells were then gently washed five times with saline. To determine the quantitative attachment of the wild-type and transformed T. phagedenis cultures under each attachment condition, chambers were removed, 5 µl of saline was added to each well, and the attached spirochetes were visualized by dark-field microscopy. Quadruplicate assays were performed, the assays were blinded, and in each case the average number of attached treponemes per field was calculated by reading a total of eight fields for each attachment condition. Statistical analyses were performed with the two-tailed Student t test.

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Preparation of the tp0751/pKMR4PEMCS expression construct. Previous investigations used the E. coli-T. denticola shuttle vector pKMR4PE to successfully express the T. pallidum flagellar gene flaA in T. denticola (10). To extend the usefulness of this vector, an MCS was introduced to create the shuttle vector pKMR4PEMCS, as shown in Fig. 1. The MCS was placed downstream of the erythromycin resistance (Em^r) gene cassette, which was, in turn, located downstream of the constitutively expressed T. denticola prtB promoter (2). In previous studies, this genetic organization resulted in the favorable expression of flaA in T. denticola (10) with no major polar effects observed downstream of the Em^r gene cassette (24). The entire tp0751 ORF, including the putative signal sequence, and upstream ribosome-binding site were PCR amplified and cloned into the MCS of the pKMR4PEMCS shuttle vector by using the SalI and BglII sites, as illustrated in Fig. 1, to create the construct tp0751/pKMR4PEMCS.

Expression of T. pallidum tp0751 in T. phagedenis. T. phagedenis was transformed with either the tp0751/pKMR4PEMCS construct or the shuttle vector alone by the electroporation protocol developed for T. denticola (10). Transformants harboring the correct chimeric plasmid were verified by antibiotic selection and DNA sequencing. RT-PCR analysis of total RNA extracted from T. phagedenis transformed with the tp0751/pKMR4PEMCS construct revealed the presence of an mRNA transcript corresponding to the tp0751 gene (Fig. 2), thus demonstrating that the heterologous T. pallidum tp0751 gene was successfully transcribed in T. phagedenis. To confirm the expression of Tp0751 on the T. phagedenis surface, immunofluorescence assays were performed. In these assays, polyclonal antibodies raised against recombinant T. pallidum Tp0751 were reacted with intact wild-type T. phagedenis or T. phagedenis transformed with either the tp0751/PKMR4PEMCS construct or the pKMR4PEMCS shuttle vector alone. Control antibodies included preimmune IgY antibodies, as well as antibodies raised against the periplasmic T. pallidum FlaA sheath protein (designated Tp0249) (19) which exhibit cross-reactivity with T. phagedenis biotype Kazan (33). As shown in Fig. 3, immunofluorescence was observed with antibodies against the periplasmic flagellar protein Tp0249 incubated with wild-type T. phagedenis whose outer membrane had been permeabilized by treatment with the detergent Triton X-100 (Fig. 3B), while no fluorescence was observed with Tp0249-specific antibodies on nonpermeabilized wild-type T. phagedenis (Fig. 3D), thus confirming the maintenance of treponemal structural integrity during experimentation. Importantly, Tp0751-specific antibodies demonstrated fluorescence with nonpermeabilized, intact T. phagedenis transformed with tp0751/pKMR4PEMCS (Fig. 3F), while no fluorescence was observed with Tp0751-specific antibodies incubated with either T. phagedenis transformed with the shuttle vector alone (Fig. 3H) or with wild-type T. phagedenis (Fig. 3J). Further, analysis of fluorescent T. phagedenis expressing Tp0751 by DAPI staining confirmed that all of the treponemes present displayed fluorescence (compare Fig. 3E and F). These results definitively demonstrate successful heterologous expression of the T. pallidum Tp0751 protein on the surface of T. phagedenis.

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FIG. 2. RT-PCR analysis of T. phagedenis transformants. Lane 1, PCR analysis of plasmid tp0751/pKMR4PEMCS (positive control); lane 2, RT-PCR analysis of RNA purified from the tp0751/pKMR4PEMCS/T. phagedenis transformant; lane 3, RT-PCR analysis of RNA purified from the pKMR4PEMCS/T. phagedenis transformant (negative control); lane 4, PCR analysis of RNA purified from the tp0751/pKMR4PEMCS/T. phagedenis transformant (no RT step, negative control); lane 5, PCR analysis of RNA purified from the pKMR4PEMCS/T. phagedenis transformant (no RT step, negative control). Size standards (1 kb) are shown at the left.

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FIG. 3. Heterologous expression of T. pallidum Tp0751 on the surface of T. phagedenis with the pKMR4PEMCS shuttle vector. Panels A, C, E, G, and I show DAPI-stained images of the T. phagedenis constructs, while panels B, D, F, H, and J show the corresponding immunofluorescence (IF) images. Panels A and B, Tp0249-specific antibodies, Triton X-100-treated wild-type T. phagedenis (positive control); panels C and D, Tp0249-specific antibodies, non-detergent-treated wild-type T. phagedenis (negative control); panels E and F, Tp0751-specific antibodies, tp0751/pKMR4PEMCS/T. phagedenis; panels G and H, Tp0751-specific antibodies, pKMR4PEMCS/T. phagedenis; panels I and J, Tp0751-specific antibodies, wild-type T. phagedenis.

Laminin attachment assays. To investigate the laminin attachment capacity of T. phagedenis transformed with the tp0751/pKMR4PEMCS construct, laminin attachment assays were performed with laminin-coated or fetuin-coated (negative control) chamber slides. As shown in Fig. 4A, T. phagedenis transformed with tp0751/pKMR4PEMCS demonstrated attachment to laminin (frame vi), while wild-type T. phagedenis (frame ii) and T. phagedenis transformed with the shuttle plasmid alone (frame iv) exhibited only minimal levels of background attachment to laminin. Further, neither wild-type T. phagedenis (frame i), T. phagedenis transformed with the shuttle vector alone (frame iii), nor T. phagedenis transformed with the tp0751/pKMR4PEMCS construct (frame v) attached to the highly glycosylated negative control protein fetuin. Figure 4B reports the quantitation of the number of treponemes attached per field to each of the coated surfaces. T. phagedenis transformed with tp0751/pKMR4PEMCS demonstrated an 8-fold increase in attachment to laminin over the attachment of wild-type T. phagedenis, an 8-fold increase in attachment to laminin over that of T. phagedenis transformed with the shuttle vector alone, and a 52-fold increase in attachment to laminin compared to the attachment to fetuin. To further define the laminin attachment potential of T. phagedenis transformed with the tp0751/pKMR4PEMCS construct, and to verify that this observed attachment capacity was conferred by heterologous expression of Tp0751, attachment inhibition experiments were performed. As shown in Fig. 4C, a 7-fold inhibition of the attachment of T. phagedenis expressing Tp0751 to laminin was observed upon bacterial pretreatment with Tp0751-specific polyclonal antibodies, compared to the 1.8-fold and 2.3-fold inhibitions observed for bacteria pretreated with the control preimmune and Tp0249-specific IgY antibodies, respectively. The minimal level of inhibition observed with the control antibodies likely represents cross-reactive chicken antibodies that bind to T. phagedenis, thereby providing a small degree of steric hindrance and thus a low level of inhibition of attachment.

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FIG. 4. Attachment of wild-type and transformed T. phagedenis to laminin and fetuin (negative control). (A) Wild-type (WT) T. phagedenis (i and ii), T. phagedenis transformed with the pKMR4PEMCS shuttle vector (iii and iv), and T. phagedenis transformed with the tp0751/pKMR4PEMCS shuttle vector (v and vi) were incubated with either fetuin-coated negative control slides (i, iii, and v) or laminin-coated slides (ii, iv, and vi), and spirochetes were visualized by dark-field microscopy with a Nikon Eclipse E600 microscope. (B) Quantitation of T. phagedenis attachment. Statistical analyses compared the level of attachment of each T. phagedenis construct to laminin with that of attachment to fetuin by the two-tailed Student t test (*, P < 0.0001). (C) Quantitation of attachment of T. phagedenis transformed with the tp0751/pKMR4PEMCS shuttle vector to laminin-coated slides following no pretreatment or pretreatment of bacteria with either preimmune IgY antibodies or polyclonal Tp0249-specific IgY antibodies (negative controls) or with polyclonal Tp0751-specific IgY antibodies. Statistical analyses compared the level of attachment of nonpretreated bacteria to that of bacteria pretreated with Tp0751-specific IgY antibodies by the two-tailed Student t test (*, P < 0.0001).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The results presented herein extend the usefulness of the pKMR4PEMCS shuttle vector, previously used for the expression of heterologous genes in T. denticola (10), to the expression of such genes in the nonadherent treponeme T. phagedenis, a significant advancement in the field of heterologous spirochete gene expression. Specifically, we describe the novel expression of the T. pallidum tp0751 gene encoding the laminin-binding adhesin within T. phagedenis. Expression of the tp0751 gene within T. phagedenis was verified at the mRNA level via RT-PCR analysis and at the protein level by immunofluorescence assays performed on the heterologous T. phagedenis construct with Tp0751-specific polyclonal antibodies. The latter assay verified the expression of the laminin-binding adhesin on the T. phagedenis surface. Expression of Tp0751 within T. phagedenis was functionally confirmed by performing treponemal laminin attachment assays. In these studies, Tp0751 expression within T. phagedenis conferred an adherent phenotype upon this nonadherent treponeme, with an eightfold enhancement of attachment to laminin being observed compared to controls. This laminin attachment capability could be specifically inhibited with polyclonal antibodies directed against Tp0751, with a threefold inhibition of attachment to laminin being observed compared to control, nonspecific antibodies.

Two important inferences can be made from the successful heterologous expression of Tp0751 within T. phagedenis. First, this result suggests proper recognition of the ribosome-binding site upstream of the tp0751 gene sequence and proper processing of the heterologously expressed protein within T. phagedenis, as evidenced by the laminin-binding capability conferred upon the heterologous T. phagedenis construct. Second, the observed attachment of viable T. phagedenis expressing Tp0751 to laminin-coated slides, combined with the immunofluorescence analyses showing surface immunofluorescence in non-detergent-treated, viable treponemes, verifies the expression of Tp0751 on the treponemal surface. The definitive identification of proteins that reside on the T. pallidum surface has been elusive due to the experimental limitations associated with research on T. pallidum (4), and evaluation of the surface exposure of a particular protein via direct extrapolation from experiments performed with T. phagedenis is useful in this regard.

Similarly, the nonculturable nature and genetic intractability of T. pallidum preclude the direct study of the function of Tp0751 within its native organism. The generation of a T. phagedenis construct heterologously expressing Tp0751 provides an important tool for investigating the hypothesized role of the Tp0751-laminin interaction in treponemal attachment and dissemination. Specifically, the creation of this nonadherent "model" treponeme will allow determination of the role of Tp0751, both alone and in combination with other T. pallidum and host proteins, in treponemal dissemination.

To our knowledge, these studies represent the first report of the successful transformation of T. phagedenis. This will advance our understanding of the pathogenesis of unculturable spirochetes, including T. pallidum, by allowing functional and ultrastructural investigations to be performed on putative outer membrane proteins, adhesins, and virulence factors within the context of a nonadherent treponeme. Although both the knowledge of treponemal molecules involved in pathogenesis and the process of treponemal genetic manipulation are still in their infancy, results presented here lay the foundation for future studies in these important, interrelated areas of research.

 
We thank Nathan Brouwer, Lisa Tisch, Scott Scholz, and Paul Cullen for expert technical assistance and Barbara Molini and Sheila Lukehart for providing T. pallidum.

This work was supported by Public Health Service grants AI-51334 (C.E.C.) and DE09821 (H.K.K.) from the National Institutes of Health, the Canada Foundation for Innovation (C.E.C.), and the British Columbia Knowledge Development Fund (C.E.C.). C.E.C. is a CIHR Canada Research Chair in Molecular Pathogenesis.

 
* Corresponding author. Mailing address: Department of Biochemistry and Microbiology, University of Victoria, 3800 Finnerty Road, Victoria, BC, Canada V8W 3P6. Phone: (250) 853-3189. Fax: (250) 721-8855. E-mail: caroc{at}uvic.ca

Published ahead of print on 8 February 2008.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Journal of Bacteriology, April 2008, p. 2565-2571, Vol. 190, No. 7
0021-9193/08/$08.00+0     doi:10.1128/JB.01537-07
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