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Department of Medicine, University of Washington, Seattle, Washington
Received 9 May 2005/ Returned for modification 17 June 2005/ Accepted 12 August 2005
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ABSTRACT |
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INTRODUCTION |
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Despite community outreach programs, the rates of primary and secondary syphilis have been steadily increasing in the United States over recent years, with a 12.4% increase in reported cases observed between the years 2001 and 2002 and an overall syphilis rate of 2.4 cases per population of 100,000 (9). The limited effectiveness of both local and global public health programs to control syphilis emphasizes the need for implementation of alternative means of syphilis prevention and specifically highlights the need for a greater understanding of the pathogenic mechanisms used by T. pallidum to establish and sustain infection.
Treponema pallidum gains entry to the host through intact mucosal barriers or microscopic epidermal abrasions (57). The pathogen has limited toxigenic properties, and tissue destruction associated with the disease appears to be due to the strong inflammatory response mounted by the host following infection (64). The organism is highly invasive; treponemes disseminate widely within hours of infection in experimental animals (11, 59), and in vitro studies have shown that T. pallidum is able to penetrate intact membranes and endothelial cell monolayers (62, 69). Treponemal invasion results in widespread bacterial dissemination, which in turn sets the stage for establishment of chronic infection.
Little is known about the virulence mechanisms utilized by invasive pathogens. No universal proteins contributing to bacterial invasion and dissemination have been identified, and proteins involved in these processes generally exhibit little or no sequence conservation. Several proteins involved in adhesion, invasion, and/or dissemination have been characterized in pathogens that maintain an intracellular lifestyle at some point during the course of infection. Substantially less information is currently available about extracellular pathogens and the virulence factors facilitating initiation of infection and spread to distant tissue sites. One factor which appears to be shared among many invasive pathogens, both intracellular and extracellular, is the capacity to attach to extracellular matrix (ECM) components. In particular, interaction with ECM components has been associated with the invasive ability of various pathogens (54), and the ECM component laminin is specifically targeted by a number of disseminating pathogens, including Paracoccidioides brasiliensis (76), Histoplasma capsulatum (47), Toxoplasma gondii (23), virulent mycobacteria (56, 66), Candida albicans (27), Sporothrix schenckii (40), and Leishmania donovani (25). In addition, tumor cells specifically interact with laminin during metastasis and dissemination, and a recent study has indicated that a unique tumor cell line with increased metastatic potential shows enhanced laminin attachment (51).
Laminins are a growing family of large multidomain heterotrimeric glycoproteins that comprise the most abundant component of the basement membrane, a specialized dense sheet of ECM that separates the epithelium from the connective tissue and surrounds the vascular endothelium, muscle cells, lipocytes and nerve cells (10, 71, 72). T. pallidum must cross several basement membranes throughout the course of infection, such as during both intravasation and extravasation, and during invasion of other tissues, including muscles or nerves. Previous literature has documented the capacity of T. pallidum to attach to laminin-coated surfaces (22), and prior investigations have identified a T. pallidum laminin-binding adhesin, Tp0751, via bioinformatic analyses performed on the T. pallidum genome and subsequent ECM attachment assays (7). Tp0751 is expressed during infection and exhibits a strong affinity for laminin (7). In this report, we further investigate the laminin attachment capability of the Tp0751 adhesin in order to identify potential targets for inhibition of bacterial dissemination and prevention of chronic infection.
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MATERIALS AND METHODS |
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Laminin sources. Laminins isolated from Engelbreth-Holm-Swarm (EHS) murine sarcoma (laminin 1) and human placenta (laminin 2/4) were purchased from Sigma (St. Louis, Mo.). Recombinant human laminin 8 and laminin 10 were a generous gift from Masayuki Doi (Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan).
Synthetic peptides. Peptides were synthesized by Bio-WORLD (Dublin, OH) using Fmoc (9-fluorenylmethoxycarbonyl)-based solid phase chemistry. Peptides were purified using high-performance liquid chromatography, and the purity and identity of the synthetic peptides were confirmed by high-performance liquid chromatography and mass spectroscopy, respectively. Peptides were reconstituted in dimethyl sulfoxide to a concentration of 80 mg/ml, followed by dilution to the appropriate working concentration with phosphate-buffered saline (PBS), pH 7.4. Thirteen overlapping peptides were synthesized that spanned the Tp0751 amino acid sequence from residue 46 through 237 (Fig. 1). This encompassed the entire mature Tp0751 sequence, including eight amino acids of the predicted signal sequence (7). Each peptide was 24 amino acids in length and shared 10 overlapping residues with the preceding and subsequent peptides. Scrambled peptides were prepared that used the exact amino acid composition of the original peptide but in a scrambled organization. Mutagenized peptides, corresponding to peptides 4, 6, and 10, were prepared that incorporated alanine or glycine residues in place of the four amino acids that are unique to each peptide (refer to Fig. 1). Except for the synthetic peptides used in the peptide inhibition experiment, all Tp0751 peptides incorporated six histidine residues at the amino terminus to allow for detection.
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Laminin-binding adherence assays. Laminin-binding adherence assays were performed as described previously (7). To determine the specificity of Tp0751 for various laminin isoforms, wells were coated with laminin isolated from EHS murine sarcoma (laminin 1) or human placenta (laminin 2/4) or with recombinant human laminin 8 or laminin 10. For all other laminin-binding adherence assays, wells were coated with EHS laminin. Wells were washed three times with PBS-0.05% Tween 20 (PBST) and blocked for 30 min with 1% bovine serum albumin (BSA). Recombinant Tp0751 proteins or synthetic Tp0751 peptides were incubated for 1.5 h at a concentration of 2 µg per well diluted in PBS. For the peptide inhibition assays, Tp0751-2 (2 µg per well) was added in the presence of non-histidine-tagged Tp0751 synthetic peptides p1, p4, p6, and p10, either individually or in combination, at concentrations ranging from 0 to 30 µM. Wells were washed six times with PBST, and adherent recombinant proteins and histidine-tagged synthetic peptides were detected via nickel-labeled horseradish peroxidase and the 3,3',5,5'-tetramethylbenzidine peroxidase substrate (both from Kirkegaard & Perry Laboratories, Gaithersburg, MD) as previously described (7). All incubations were performed at 37°C. Plates were read at 600 nm with an enzyme-linked immunosorbent assay plate reader (Bio-Tek Instruments, Winooski, VT), and statistical analyses were performed with the Student two-tailed t test.
Cell adhesion assay. The colon carcinoma cell line SW480 was obtained from ATCC (Rockville, MD), and cells were maintained in Dulbecco's modified Eagle medium (BioWhittaker, Walkersville, MD) with 10% fetal bovine serum (HyClone). To determine the capacity of Tp0751 to mediate cell attachment, assays were performed as described previously (63). Briefly, 96-well non-tissue-culture-treated enzyme-linked immunosorbent assay plates (Fisher Scientific, Pittsburgh, PA) were coated for 24 h at 4°C with 100 µl of either the recombinant T. pallidum protein Tp0751-1, the negative control protein BSA, or the positive control protein fibronectin, all at a concentration of 20 µg/ml in PBS. Wells were blocked with 1% BSA prepared in serum-free Dulbecco's modified Eagle medium. Confluent SW480 cells were detached by treatment with 2 mM EDTA for 10 min at 37°C. Detached cells were collected by centrifugation for 5 min at 200 x g and washed once by centrifugation with serum-free medium. Fifty thousand cells were added to each of the wells, and plates were centrifuged for 1 min at 30 x g to ensure uniform settling of cells and incubated for 3 h at 37°C. Following the incubation, nonadherent cells were removed by centrifugation (top side down) at 30 x g for 1 min. The attached cells were fixed and stained with 1% formaldehyde, 0.5% crystal violet. After washing with PBS, adherence was determined by measuring absorption at 595 nm in a microplate reader (Bio-Rad, Hercules, CA). The data are reported as the mean absorbance of triplicate wells ± standard error of the mean (SEM).
Antiserum. Anti-Tp0751 and anti-Tp0155 (negative control) polyclonal sera were raised in New Zealand White rabbits by immunizing them five times at 3-week intervals with 125 µg of either Tp0751-1 or Tp0155 recombinant protein emulsified in the Ribi adjuvant system (Sigma). Normal rabbit serum was collected prior to the initial immunization, and immune rabbit serum was collected from rabbits infected with T. pallidum for >90 days.
T. pallidum inhibition assays. For the treponemal adherence peptide inhibition assays, Lab-Tek II chamber slides (Nunc, Rochester, NY) were coated for 1.5 h at room temperature with 4 µg of laminin diluted in saline. The slides were washed once with saline and then incubated overnight at 4°C with either saline (negative control) or 250 µg of the following peptides, either individually or in combination: 1, 2, and 3; 4, 6 and 10; or m4, m6, and m10. Slides were washed three times with saline, followed by the addition of 1.5 x 108 treponemes per well. Slides were incubated for 3 h at 34°C under anaerobic conditions and washed gently with saline (eight times for 5 min each). For the treponemal adherence antibody inhibition assay, chamber slides were coated for 16 h at 4°C with 6 µg of laminin diluted in saline. Wells were washed two times with saline and blocked with 3% BSA-saline for 4 h at room temperature. During the blocking step, 4.5 x 107 treponemes were preincubated at 34°C under anaerobic conditions with a 1:2 dilution of either immune rabbit serum, normal rabbit serum, or anti-Tp0155- or anti-Tp0751-specific polyclonal serum. Control wells were incubated under the same conditions with no antibody addition. Following blocking, wells were washed two times with saline, and the treponeme-antibody mixtures or control treponeme sample were introduced and incubated for 2 h at 34°C under anaerobic conditions. After gentle washing with saline (six times for 5 min each), treponemes were fixed with 4% paraformaldehyde and washed an additional five times with saline. For each assay, attached spirochetes were visualized by dark-field microscopy and quantitative attachment was determined by calculating the number of attached treponemes per field. The assays were blinded, and a total of six fields were read for each attachment condition.
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RESULTS |
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1ß1
1), 2/4 (2ß1,22), 8 (4ß11), and 10 (5ß11) and the Tp0751-1 recombinant preparation previously shown to mediate attachment to laminin (7). The results of these attachment studies are shown in Fig. 2. Recombinant Tp0751-1 demonstrated a significant level of attachment to each of the selected laminin isoforms (P < 0.0001), whereas minimal binding of the negative control recombinant protein Tp0557 to each of the laminin isoforms was observed.
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The observation that peptides 3, 5, 7, 9, and 11 did not exhibit any detectable binding to laminin suggests that the linear configuration of amino acids contained within these peptides does not support laminin attachment. Since each peptide includes 10 overlapping residues from the preceding and succeeding peptides (refer to the peptide design in Fig. 1), this implicates the four amino acids unique to each peptide as being part of the minimum sequence within Tp0751 that mediates laminin attachment. To verify this finding, mutagenized peptides were prepared that incorporated alanine or glycine residues in place of the four amino acids that are unique to each of peptides 4, 6, and 10 (refer to Fig. 1). As shown in Fig. 4A to C, replacement of all four unique amino acids abolished the capacity of each peptide to attach to laminin. Subsequent investigations revealed that 10 amino acids are critical to the laminin attachment potential of the Tp0751-derived peptides, since mutagenesis of each of these residues resulted in elimination of the laminin attachment potential for the particular peptide. These residues included the four unique amino acids P98, V99, Q100, T101 within peptide 4 (Fig. 4A), amino acids W127 and I128 within peptide 6 (Fig. 4B), and the four unique amino acids T182, A183, I184, and S185 within peptide 10 (Fig. 4C). Mutagenesis of the other amino acids unique to peptide 6 had no effect on the laminin-binding capacity of the peptide (Fig. 4B), suggesting that these amino acid residues do not contribute to the ability of peptide 6 to bind to laminin.
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DISCUSSION |
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Previous investigations identified Tp0751, a T. pallidum adhesin expressed during infection that exhibits specific attachment to laminin (7). In this report we expand upon these studies and further characterize the interaction of Tp0751 with laminin. Tp0751 was demonstrated to mediate attachment of cells expressing laminin, and further, antibodies specific for Tp0751 inhibited attachment of T. pallidum to laminin, thus confirming the identity of this T. pallidum protein as an adhesin. Through the use of synthetic peptides, we have identified 10 amino acids that are critical to the laminin attachment potential of Tp0751, including amino acids 98 to 101, 127 to 128, and 182 to 185, with the major laminin-binding epitope comprising residues 127 to 128. Peptides containing these sequences inhibited attachment of T. pallidum to laminin, while mutagenized versions of these peptides did not inhibit treponemal attachment. The interaction of residues 98 to 101 and 127 to 128 with laminin appears to be dependent upon conservation of the linear peptide sequence; supporting evidence includes the observations that these peptides inhibited attachment of Tp0751 to laminin, and scrambled versions of these peptides did not exhibit an affinity for laminin. Scrambled versions of the peptide encompassing residues 182 to 185 retained the ability to attach to laminin; this observation suggests that this region of Tp0751 requires merely the presence of those amino acids, and not the contiguous linear sequence, to interact efficiently with laminin.
The identification of the residues mediating Tp0751 attachment to laminin represents the first step in the process of devising an antiadhesive therapy to block bacterial attachment and dissemination. Multiple examples exist of bacterial adhesins that function as effective vaccine candidates, including the Hap adhesin from Haemophilus influenzae (42) and the collagen adhesin from Staphylococcus aureus (50). Peptide inhibitors have also been used to prevent adherence of selected bacteria to their attachment ligands, including the bacterial pathogens Streptococcus mutans (35) and Porphyromonas gingivalis (38) and the noscomial pathogen Pseudomonas aeruginosa (6). These studies have successfully prevented infection by targeting adhesins via multiple routes, including vaccination with recombinant protein antigens, therapeutic administration of selected peptides to competitively inhibit pathogen attachment, and passive administration of antibodies raised against peptide sequences mediating host attachment. As a result, these studies demonstrate the feasibility of developing antiadhesin vaccines and therapies.
Laminins are large heterotrimeric glycoproteins consisting of an -, a ß-, and a -type chain. To date, 5 , 3 ß, and 3 chains have been identified that give rise to at least 15 different laminin isoforms (16, 48, 70, 74). Although certain isoforms, such as laminin 10, show widespread distribution, other isoforms are functionally distinct and expressed in a tissue-specific and developmentally regulated manner, thus creating marked heterogeneity among basement membranes found throughout the host (3, 12, 77). For example, laminin 1 is expressed in tissues containing epithelial basement membranes (13), laminin 2 and 4 are components of skin, skeletal muscle, heart muscle, and nerve cells (29, 39), and laminin 8 is localized in adipocytes, muscle cells and tissues containing epithelial and endothelial basement membranes (33, 55). The widespread distribution of laminin makes it an appropriate target for a disseminating pathogen such as T. pallidum, which has been shown to localize to a diverse range of these and other tissue sites. As shown in this report, Tp0751 efficiently attached to each of the laminin isoforms tested, including laminins 1, 2, 4, 8, and 10, thus strengthening the concept that laminin located in anatomically distinct tissue sites could function as a common target to allow attachment of T. pallidum to the host via Tp0751.
The intimate association of T. pallidum with the vascular endothelium is exemplified by the perivasculitis and endothelial cell abnormalities that are characteristically observed upon histopathologic analysis of infected tissues (45). Studies have shown that T. pallidum specifically activates endothelial cells (60, 61), and in turn, endothelial cell activation leads to increased vascular permeability (4, 73, 79). Laminin plays an important role in regulating endothelial cell morphogenesis (28), and we hypothesize that the specific interaction of the T. pallidum adhesin Tp0751 with laminin in the endothelial cell layer may facilitate treponemally induced endothelial cell activation and subsequent transendothelial treponemal passage to promote tissue invasion. Other situations which cause endothelial cell activation and in turn increased vascular permeability include tumor growth and chronic inflammation (46, 52) and infection with the pathogens Escherichia coli (67) and Trypanosoma brucei (26).
Significant insight can be gleamed from the study of mechanisms of dissemination of tumor cells and interaction of such cells with laminin. Tumor cells specifically attach to basement membranes, and laminin-binding proteins on tumor cell surfaces are crucial for metastasis (41). Tumor cells intravasate by penetrating the basement membrane, and although this step is incompletely understood, degradation of basement membrane components by proteolytic enzymes is proposed to be involved (80). Similarly, various invasive bacterial pathogens, including Haemophilus influenzae, Salmonella enterica, E. coli, and Yersinia pestis, adhere to laminin and initiate a proteolytic cascade that facilitates basement membrane degradation and bacterial invasion (36). Along these lines, the related spirochete Treponema denticola binds to extracellular matrix components (18, 19) and expresses a chymotrypsin-like protease that degrades basement membrane components and promotes treponemal invasion (14, 17, 30). Whether a similar situation contributes to the remarkable invasive capability of T. pallidum remains to be determined.
In summary, in this report we have determined that Tp0751 mediates attachment of mammalian cells expressing laminin and that this adhesin exhibits an attachment profile similar to those of multiple laminin isoforms that are widespread throughout the host. We have delineated the amino acid residues involved in attachment of the T. pallidum adhesin Tp0751 to laminin and have identified the minimum laminin-binding epitope for attachment. Future investigations will target these identified laminin-binding regions of Tp0751 to determine if in vivo inhibition of treponemal attachment to laminin alters the course of T. pallidum infection, dissemination, and disease progression.
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ACKNOWLEDGMENTS |
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This work was supported by the Public Health Service grant AI-51334 from the National Institutes of Health and faculty awards from the University of Washington (Royalty Research Fund and STD New Investigator Award AI-31448).
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FOOTNOTES |
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0.0002). (B) Peptide inhibition of Tp0751-2 attachment to laminin. The capacities of peptides 1, 4, 6, and 10 to inhibit attachment of Tp0751-2 to laminin were investigated.
