<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

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

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

<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

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

Fine Analysis of Genetic Diversity of the <i>tpr</i> Gene Family among Treponemal Species, Subspecies and Strains

<div><p>Background</p><p>The pathogenic non-cultivable treponemes include three subspecies of <i>Treponema pallidum (pallidum, pertenue, endemicum)</i>, <i>T. carateum</i>, <i>T. paraluiscuniculi</i>, and the unclassified Fribourg-Blanc treponeme (Simian isolate). These treponemes are morphologically indistinguishable and antigenically and genetically highly similar, yet cross-immunity is variable or non-existent. Although all of these organisms cause chronic, multistage skin and systemic disease, they have historically been classified by mode of transmission, clinical presentations and host ranges. Whole genome studies underscore the high degree of sequence identity among species, subspecies and strains, pinpointing a limited number of genomic regions for variation. Many of these “hot spots” include members of the <i>tpr</i> gene family, composed of 12 paralogs encoding candidate virulence factors. We hypothesize that the distinct clinical presentations, host specificity, and variable cross-immunity might reside on virulence factors such as the <i>tpr</i> genes.</p><p>Methodology/Principal Findings</p><p>Sequence analysis of 11 <i>tpr</i> loci (excluding <i>tprK</i>) from 12 strains demonstrated an impressive heterogeneity, including SNPs, indels, chimeric genes, truncated gene products and large deletions. Comparative analyses of sequences and 3D models of predicted proteins in Subfamily I highlight the striking co-localization of discrete variable regions with predicted surface-exposed loops. A hallmark of Subfamily II is the presence of chimeric genes in the <i>tprG</i> and <i>J</i> loci. Diversity in Subfamily III is limited to <i>tprA</i> and <i>tprL</i>.</p><p>Conclusions/Significance</p><p>An impressive sequence variability was found in <i>tpr</i> sequences among the Treponema isolates examined in this study, with most of the variation being consistent within subspecies or species, or between syphilis vs. non-syphilis strains. Variability was seen in the <i>pallidum</i> subspecies, which can be divided into 5 genogroups. These findings support a genetic basis for the classification of these organisms into their respective subspecies and species. Future functional studies will determine whether the identified genetic differences relate to cross-immunity, clinical differences, or host ranges.</p></div

<i>tpr</i> alleles among treponemal species, subspecies and strains.

<p><i>tprD2</i>: A <i>tprD</i> allele which contains a 330-bp unique central region and three smaller heterogeneous regions at the 3′ end. <i>tprC</i>-like <i>and tprD</i>-like: similar to <i>tprC</i> or <i>tprD</i>, respectively, with small sequence differences in discrete variable regions (DVRs). <i>tprGJ</i>: A chimera where the 3′ end contains <i>tprJ</i> signatures. <i>tprGI</i>: A chimera where the 5′ end is homologous to <i>tprG</i>, and the central and 3′ regions are homologous to the corresponding regions of <i>tprI</i>. truncated: Predicted truncated proteins due to a frameshift. <i>tprA</i>-like, <i>tprI</i>-like and <i>tprL</i>-like contain small sequence differences. <i>tprE</i>-like and <i>tprH</i>-like contains small sequence differences that segregate syphilis from non-syphilis treponemes. <i>tprL1</i>: A unique <i>tprL</i> allele in <i>T. p. pertenue</i> and Fribourg-Blanc strain. * indicates that <i>tprC</i> and <i>tprD</i> in the Nichols strain and that <i>tprI</i>-like sequences in the <i>tprF</i> and <i>tprI</i> loci are also identical in the <i>pertenue</i> subspecies and the Fribourg-Blanc treponeme.</p

Encoded variants at the <i>tprL</i> (tp1031) locus.

<p><u>Coding sequences:</u> Three different coding sequences have been identified for treponemal species and subspecies: the proposed <i>tprL</i> ORFs in the Nichols and Street 14 genome sequences; an extended <i>tprL</i> for <i>pallidum</i>, <i>endemicum</i>, and <i>paraluiscuniculi</i> strains; and a fused <i>tprL</i> (called <i>tprL1</i>) for <i>pertenue</i> and the Fribourg-Blanc strains. The Nichols ORF was predicted to be 1542 bp, although lacks identifiable promoter elements upstream. In this study, an extended <i>tprL</i> of 1806 bp has been identified in the Nichols and other <i>pallidum</i> strains, as well as in <i>endemicum</i> and <i>paraluiscuniculi</i> strains. The initially shorter Nichols tprL was the result of sequencing errors in the reported Nichols genome sequence <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002222#pntd.0002222-Fraser1" target="_blank">[27]</a>. Typical promoter elements are shown for the extended <i>tprL</i> ORF (SC, start codon. RBS, ribosomal binding site. +1, transcriptional start site (TSS). −10 and −35, σ⁷⁰ signatures). A deletion of 278 bp (274 bp of the 5′ end of <i>tp1030</i>, whose coding sequence is located on the minus strand, and 4 bp of the 5′ end of the genome-derived <i>tprL</i>) creates an alternative start site in <i>tp1030</i> for <i>pertenue</i> and Fribourg-Blanc <i>tprL1</i>, resulting in a shorter ORF of 1668 base pairs. This ORF, however, lacks recognizable promoter elements. <u>Encoded proteins:</u> Differences in coding sequences result in two different proteins: 1) a shorter <i>pertenue</i>/Fribourg-Blanc variant with a 44 amino acid unique amino terminus and 2) a longer TprL in the remaining species/subspecies with a predicted signal peptide 25 amino acids long (green) in the longer product, but not identifiable in the <i>pertenue</i>/Fribourg-Blanc gene product. Blue color, region unique to <i>pertenue</i> and Fribourg-Blanc strains (132 nucleotides or 44 amino acids). Red color, region unique to the <i>pallidum</i>, <i>endemicum</i> and <i>paraluiscuniculi</i> species/subspecies (65 amino acids).</p