Inhibition of T. pallidum attachment to plasma and cellular fibronectin by recombinant TP0136 proteins.

<p>T. pallidum attachment to plasma and cellular fibronectin-coated slides is inhibited by slide pre-incubation with 800 pmol/well of recombinant proteins based on the TP0136 sequences form Nichols Houston and Nichols Seattle. T. pallidum σ70 and PBS were used as negative controls. Experiments were repeated twice using each time triplicate wells per condition. Bars represent the mean number of T. pallidum cells counted in 10 fields of triplicate experiments ± standard error. Significance was assessed by comparing wells coated with recombinant proteins to the PBS control wells with Student’s unpaired two-tailed t-test and significance set at p≤0.05. For comparison between different protein concentrations ANOVA test was used (*p<0.05; **p<0.001).</p

<i>Treponema pallidum</i> subsp. <i>pallidum</i> TP0136 Protein Is Heterogeneous among Isolates and Binds Cellular and Plasma Fibronectin via its NH₂-Terminal End

<div><p>Adherence-mediated colonization plays an important role in pathogenesis of microbial infections, particularly those caused by extracellular pathogens responsible for systemic diseases, such as <i>Treponema pallidum</i> subsp. <i>pallidum</i> (<i>T</i>. <i>pallidum</i>), the agent of syphilis. Among <i>T</i>. <i>pallidum</i> adhesins, TP0136 is known to bind fibronectin (Fn), an important constituent of the host extracellular matrix. To deepen our understanding of the TP0136-Fn interaction dynamics, we used two naturally-occurring sequence variants of the TP0136 protein to investigate which region of the protein is responsible for Fn binding, and whether TP0136 would adhere to human cellular Fn in addition to plasma Fn and super Fn as previously reported. Fn binding assays were performed with recombinant proteins representing the two full-length TP0136 variants and their discrete regions. As a complementary approach, we tested inhibition of <i>T</i>. <i>pallidum</i> binding to Fn by recombinant full-length TP0136 proteins and fragments, as well as by anti-TP0136 immune sera. Our results show that TP0136 adheres more efficiently to cellular Fn than to plasma Fn, that the TP0136 NH2-terminal conserved region of the protein is primarily responsible for binding to plasma Fn but that binding sites for cellular Fn are also present in the protein’s central and COOH-terminal regions. Additionally, message quantification studies show that <i>tp0136</i> is highly transcribed during experimental infection, and that its message level increases in parallel to the host immune pressure on the pathogen, which suggests a possible role for this protein in <i>T</i>. <i>pallidum</i> persistence. In a time where syphilis incidence is high, our data will help in the quest to identify suitable targets for development of a much needed vaccine against this important disease.</p></div

Primers used in this study.

<p>Primers used in this study.</p

TP0136 message quantification during experimental infection.

<p>For message quantification, a biopsy from the leading edge of a dermal lesion was obtained from each of the three infected rabbits every three days for 30 days. Each sample was amplified in triplicate. The data were reported as the mean values ± standard error (SE) for triplicate experiments. Left <i>y</i> axis shows real-time qPCR analysis of TP0136 message normalized to TP0547 mRNA (orange line) during progression of primary syphilitic lesions in the rabbit model. Although biopsies were obtained at day 0 and day 3 as well, no message quantification was possible from these samples. Newman-Keuls Multiple Comparison Test was used to assess significant differences in TP0136 message level between time points (*<i>p<</i>0.05) whenever a significant difference between sample means was found by ANOVA. Right <i>y</i> axis shows absolute quantification data for TP0574 message (black bars), reflecting absolute <i>T</i>. <i>pallidum</i> burden.</p

<i>T</i>. <i>pallidum</i> subsp. <i>pallidum</i> strains used in this study.

<p>¹The Nichols Seattle strain was provided by James N. Miller, University of California, Los Angeles, CA. The Nichols Houston strain was provided by Steven J. Norris, University of Texas Health Science Center, Houston, TX. The Nichols Dallas strain was provided by Michael Norgard, University of Texas Southwestern Medical Center, Dallas, TX.</p><p>²Year refers to the isolation of the parent Nichols strain by HJ Nichols and WH Hough [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003662#pntd.0003662.ref068" target="_blank">68</a>].</p><p>³ The Dal-1 strain [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003662#pntd.0003662.ref069" target="_blank">69</a>] was provided by Rob George, CDC, Atlanta, GA</p><p>⁴Strains provided by Paul Hardy and Ellen Nell, Johns Hopkins University, Baltimore, MD.</p><p>⁵Strain isolated in Seattle by Sheila A. Lukehart, University of Washington, Seattle, WA.</p><p>⁶Strain provided by Sandra A. Larsen, Center for Disease Control and Prevention, Atlanta, GA.</p><p><i>T</i>. <i>pallidum</i> subsp. <i>pallidum</i> strains used in this study.</p

Reactivity of anti-TP0136 immune sera against recombinant TP0136 proteins and Inhibition of <i>T</i>. <i>pallidum</i> attachment to plasma and cellular fibronectin by anti-TP0136 immune sera.

<p>(A) Sera obtained from TP0136-H and TP0136-S-immunized rabbits recognized both recombinant full-length proteins and Frag. 1 and Frag. 2. Anti-TP0136-H antiserum recognized Frag.3-H but reacted only weakly against Frag.3-S. Similarly, anti-TP0136-S antiserum recognized Frag.3-S but reacted less well against Frag F3-H. Bars represent the absorbance at 405 nm ± SEM for triplicate samples. Significance between reactivity to single peptides was assessed by Student’s unpaired two-tailed t test with significance set at * <i>p<</i>0.05. (B) Antisera directed against TP0136-H and TP0136-S inhibited treponemal attachment to slides coated with plasma and cellular Fn. Experiments were repeated twice using each time triplicate wells per condition. Bars represent the mean number of <i>T</i>. <i>pallidum</i> cells counted in 10 fields of triplicate experiments (± standard error) following incubation of <i>T</i>. <i>pallidum</i> with either anti-TP0136 immune sera, IRS (positive control) or NRS (negative control). Significance is calculated with respect to NRS using the Student two-tailed t test with significance set at * <i>p<</i>0.05.</p

Recombinant TP0136 binding to plasma and cellular fibronectin.

<p>Adhesion to plasma and cellular Fn of TP0136 variants was evaluated using full-length recombinant TP0136 (w/o signal peptide) from Nichols Houston and Nichols Seattle, as well as four additional recombinant proteins representing the NH₂-terminal (Frag.1), central (Frag.2), and COOH-terminal regions (Frag.3-H and Frag.3-S) of both TP0136 variants. <i>T</i>. <i>pallidum</i> transcription factor σ⁷⁰ served as negative control. Each experiment was repeated three times to ensure reproducibility of results. Each time, each sample was tested in triplicate. (A-F) Results of the binding assay to plasma and cellular Fn. Colors represent different recombinant proteins used to assess dose-dependent binding to plasma Fn. X axis data point correspond to 100 pmol of protein/well (log[protein] = -6.0), 250 pmol/well (-5.6), 500 pmol/well (-5.3), 750 pmol/well (-5.1), and 1000 pmol/well (-5.0). In all panels, data points represent the absorbance at 405 nm ± SEM for triplicate samples. Significance was assessed by ANOVA for data in panel A-F (*<i>p<</i>0.05; **<i>p<</i>0.001) (G) Comparison between binding of recombinant proteins to plasma and cellular Fn. Data in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003662#pntd.0003662.g003" target="_blank">Fig. 3G</a> represent binding to Fn variants of 1,000 pmol of protein from panels above. Data points represent the absorbance at 405 nm ± SEM for triplicate samples. Significance was assessed by Student’s unpaired two-tailed t test with significance set at <i>p<</i>0.05 (*).</p

Primers used for the preliminary analysis of a subset of PNG samples.

<p>Primers used for the preliminary analysis of a subset of PNG samples.</p

Phylogenetic analysis of <i>Tp</i> multilocus sequence types.

<p>Multilocus Sequence Types (MLSTs) were defined by sequencing regions of three genes: <i>tp0548</i>, <i>tp0136</i> and <i>tp0326</i>. Concatenated sequences were first aligned using the Muscle algorithm, using default parameters. The evolutionary history of the MLSTs was inferred using the Neighbor-Joining method. The optimal tree is shown, with branch lengths equivalent to the evolutionary distance as indicated by the scale. Evolutionary distance was measured using the number of differences per sequence, with pairwise deletion of gaps. The percentage of replicate trees in which the associated molecular types clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Analyses were conducted in MEGA version 7.0 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006113#pntd.0006113.ref032" target="_blank">32</a>].</p

Phylogenetic relationships of the <i>tp0136</i> typing region.

<p><i>tp0136</i> types are shown for <i>T</i>.<i>p</i>. <i>pertenue</i> and Fribourg Blanc isolates and PNG samples; typing designations are as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006113#pntd.0006113.g003" target="_blank">Fig 3</a>. Sequences were first aligned using the Muscle algorithm, using default parameters. The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree is shown, with branch lengths equivalent to the evolutionary distance as indicated by the scale. Evolutionary distance was measured using the number of differences per sequence, with pairwise deletion of gaps. The percentage of replicate trees in which the associated molecular types clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Analyses were conducted in MEGA version 7.0 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006113#pntd.0006113.ref032" target="_blank">32</a>].</p