The Structure of <i>Treponema pallidum</i> Tp0624 Reveals a Modular Assembly of Divergently Functionalized and Previously Uncharacterized Domains

<div><p><i>Treponema pallidum</i> subspecies <i>pallidum</i> is the causative agent of syphilis, a chronic, multistage, systemic infection that remains a major global health concern. The molecular mechanisms underlying <i>T</i>. <i>pallidum</i> pathogenesis are incompletely understood, partially due to the phylogenetic divergence of <i>T</i>. <i>pallidum</i>. One aspect of <i>T</i>. <i>pallidum</i> that differentiates it from conventional Gram-negative bacteria, and is believed to play an important role in pathogenesis, is its unusual cell envelope ultrastructure; in particular, the <i>T</i>. <i>pallidum</i> peptidoglycan layer is chemically distinct, thinner and more distal to the outer membrane. Established functional roles for peptidoglycan include contributing to the structural integrity of the cell envelope and stabilization of the flagellar motor complex, which are typically mediated by the OmpA domain-containing family of proteins. To gain insight into the molecular mechanisms that govern peptidoglycan binding and cell envelope biogenesis in <i>T</i>. <i>pallidum</i> we report here the structural characterization of the putative OmpA-like domain-containing protein, Tp0624. Analysis of the 1.70 Å resolution Tp0624 crystal structure reveals a multi-modular architecture comprised of three distinct domains including a C-terminal divergent OmpA-like domain, which we show is unable to bind the conventional peptidoglycan component diaminopimelic acid, and a previously uncharacterized tandem domain unit. Intriguingly, bioinformatic analysis indicates that the three domains together are found in all orthologs from pathogenic treponemes, but are not observed together in genera outside <i>Treponema</i>. These findings provide the first structural insight into a multi-modular treponemal protein containing an OmpA-like domain and its potential role in peptidoglycan coordination and stabilization of the <i>T</i>. <i>pallidum</i> cell envelope.</p></div

Overall structure of Tp0624.

<p><b>(A)</b> Sequence features of Tp0624. SP—signal peptide (approximately residues 1 to 60). Green bar—construct used for structural studies (T61-D476). Dashed box—region with predicted secondary structure elements but no sequence similarity to any characterized protein. Dotted box—putative OmpA-like C-terminal domain. <b>(B)</b> Orthogonal views of Tp0624 tertiary structure shown as a cartoon with beta-strands in green, alpha helices in purple, and connecting coil in grey. Note the slightly flattened, triangle-like architecture with two distinct sides defined by either beta-strands (left) or alpha-helices (right).</p

Comparative Tp0624 domain analyses.

<p><b>(A)</b> Left: overlay of Tp0624 D3 (purple) on the OmpA-like domain of <i>Acinetobacter baumannii</i> (grey, PDB ID 3TD5) in complex with a peptidoglycan derivative (light grey ball-and-stick colored by element). Right: Structure-based sequence alignment of Tp0624 with the peptidoglycan binding residues of <i>A</i>. <i>baumanii</i> OmpA-like domain. Critical residues are bolded and displayed in the inset panels. Inset: Zoomed-in view of the peptidoglycan binding site on <i>A</i>. <i>baumanii</i> OmpA-like domain (grey, left), with hydrogen bonds to the diaminopimelic acid residue shown as dotted lines. Star indicates the loop that comprises the outer side of the pocket. Contrast with the same view of Tp0624 D3, which despite conservation of the essential Arg, shows the loss of a critical Asp to Ala, the displaced position of the outer loop (star), and the large residues (Leu/His) in the lower loop that project into the binding site. <b>(B)</b> Electrostatic surface of Tp0624 D3 in same orientation as (A) shows a clear basic patch comprising the putative ligand binding site. <b>(C)</b> Overlay of Tp0624 D1 (green) on the <i>E</i>. <i>coli</i> trigger factor (grey, PDB ID 1P9Y). Grey bracket, region of the trigger factor that has been functionalized for ribosome binding and is not conserved in Tp0624 D1. Green bracket, region of Tp0624 D1 that has been extended to form the interfaces with D2 and D3. <b>(D)</b> Tp0624 D2 is a previously uncharacterized domain showing no significant structural similarity to any domain in the PDB. Tp0624 consists of a four-stranded parallel top sheet (cyan), four-stranded mixed bottom sheet (orange), and flanking helices (yellow). Left: orthogonal views of Tp0624 D2 tertiary structure. Right: Topology diagram of Tp0624 D2.</p

Tp0624 amino acid sequence-based modularity analysis.

<p>Orthologs of Tp0624 were identified using BLASTp. Tp0624 signal peptide (SP), domains (D1, green; D2, cyan; D3, purple), and linkers (L1 and L2, yellow) are shown. Indicated on the right are the numbers of orthologs with different domain combinations from spirochetes (left number) or other bacteria (right number). Sequences homologous to D1 and D2 only occur together, while D3 homologs are ubiquitous, and all three domains only occur together in treponemes (shown by an asterisk).</p

Tp0624 is organized into two units consisting of D1-D2 and D3.

<p><b>(A)</b> Top: structure-based assignment of Tp0624 domains (D1, green; D2, cyan; D3, purple) and linkers (L1 and L2, yellow). Bottom: cartoon of Tp0624 tertiary structure colored by domain as in (top). <b>(B)</b> Table of inter-domain/linker interfaces of Tp0624 listed as “Interface Area in Ų (# Hydrogen Bonds, # Salt Bridges)” as defined by PISA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166274#pone.0166274.ref037" target="_blank">37</a>]. <b>(C)</b> Interface of D1-D2. Top: D2 (cyan) shown as B-factor defined coil with increased mobility indicated by thicker coil. D1 shown as green surface, with residues contacting D2 colored orange. Middle: Inverse display of (top). Inset: Sigma-A weighted electron density mesh (orange) contoured at 1.5 sigma for a loop of D2 (cyan) that protrudes into D1 (green).</p

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p

The Structure of <i>Treponema pallidum</i> Tp0751 (Pallilysin) Reveals a Non-canonical Lipocalin Fold That Mediates Adhesion to Extracellular Matrix Components and Interactions with Host Cells

<div><p>Syphilis is a chronic disease caused by the bacterium <i>Treponema pallidum</i> subsp. <i>pallidum</i>. <i>Treponema pallidum</i> disseminates widely throughout the host and extravasates from the vasculature, a process that is at least partially dependent upon the ability of <i>T</i>. <i>pallidum</i> to interact with host extracellular matrix (ECM) components. Defining the molecular basis for the interaction between <i>T</i>. <i>pallidum</i> and the host is complicated by the intractability of <i>T</i>. <i>pallidum</i> to <i>in vitro</i> culturing and genetic manipulation. Correspondingly, few <i>T</i>. <i>pallidum</i> proteins have been identified that interact directly with host components. Of these, Tp0751 (also known as pallilysin) displays a propensity to interact with the ECM, although the underlying mechanism of these interactions remains unknown. Towards establishing the molecular mechanism of Tp0751-host ECM attachment, we first determined the crystal structure of Tp0751 to a resolution of 2.15 Å using selenomethionine phasing. Structural analysis revealed an eight-stranded beta-barrel with a profile of short conserved regions consistent with a non-canonical lipocalin fold. Using a library of native and scrambled peptides representing the full Tp0751 sequence, we next identified a subset of peptides that showed statistically significant and dose-dependent interactions with the ECM components fibrinogen, fibronectin, collagen I, and collagen IV. Intriguingly, each ECM-interacting peptide mapped to the lipocalin domain. To assess the potential of these ECM-coordinating peptides to inhibit adhesion of bacteria to host cells, we engineered an adherence-deficient strain of the spirochete <i>Borrelia burgdorferi</i> to heterologously express Tp0751. This engineered strain displayed Tp0751 on its surface and exhibited a Tp0751-dependent gain-of-function in adhering to human umbilical vein endothelial cells that was inhibited in the presence of one of the ECM-interacting peptides (p10). Overall, these data provide the first structural insight into the mechanisms of Tp0751-host interactions, which are dependent on the protein’s lipocalin fold.</p></div

Expression and surface localization of Tp0751 in <i>B</i>. <i>burgdorferi</i>.

<p><b>(A)</b> Schematic depicting Tp0751 expression construct in <i>B</i>. <i>burgdorferi</i> including 1) constitutive <i>flaB</i> promoter (P_<i>flaB</i>), 2) Tp0751 lipoprotein localization signal sequence and Tp0751 coding sequence, and 3) a C-terminal 3X-FLAG tag. Inserts were cloned into XhoI and NotI sites of a pCE320 (<i>cp32</i>)-derived shuttle vector (resulting plasmid pCC_3–1), and transformed into a non-infectious, adhesion-attenuated B31-A-derived GFP-expressing parent strain (GCB706). Strain names and details are provided in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005919#ppat.1005919.s004" target="_blank">S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005919#ppat.1005919.s005" target="_blank">S2</a> Tables. (<b>B-D</b>) Fluorescence-activated cell sorting (FACS) analysis of Tp0751 localization on the surface of <i>B</i>. <i>burgdorferi</i>. Strains were mock-treated with phosphate based saline buffer or permeabilized with methanol before probing with antibodies to the FLAG tag (part of BBK32 and Tp0751 fusion proteins), or to constitutively expressed periplasmic (non-surface-localized) flagellin B protein (FlaB). (<b>B</b>) Representative histogram depicting fluorescence intensities of indicated mock-treated <i>B</i>. <i>burgdorferi</i> strains probed with anti-FLAG antibodies. The mean ± SEM percentages of bacteria that were FLAG-positive for each strain in all replicates are indicated. (<b>C-D</b>) Relative expression levels per bacterium of FLAG-tagged fusion proteins Tp0751 and BBK32 (<b>C</b>) and FlaB (<b>D</b>) in mock-treated and methanol-permeabilized <i>B</i>. <i>burgdorferi</i>. Mean ± SEM fluorescence intensities for each strain were expressed as a proportion of mean fluorescence intensity for <i>B</i>. <i>burgdorferi</i> expressing BBK32-FLAG. N = 8 independent cultures per strain analyzed in two FACS experiments. Statistical analysis: One-way Kruskal-Wallis ANOVA with Dunn’s post-test. * indicates p<0.05 vs. permeabilized BBK32-FLAG control within treatment (mock or permeabilized).</p

Tp0751 peptide 10 inhibits adhesion of Tp0751-expressing <i>B</i>. <i>burgdorferi</i> to HUVEC monolayers.

<p><b>(A)</b> Schematic showing the experimental design of the competitive inhibition assay. Confluent HUVEC monolayers seeded on artificial basement membrane were preincubated with synthetic Tp0751 peptides (p4, p6, p10, p11, p4scr, p6scr, and p10scr1) to allow for peptide-endothelial cell interactions. HUVECs were then co-incubated with parent (negative control) or Tp0751-expressing <i>B</i>. <i>burgdorferi</i> strains to assess the competitive inhibition capacity of the synthetic Tp0751 peptides. After washing to remove non-adherent bacteria, <i>B</i>. <i>burgdorferi</i> adhesion to HUVECs was quantified using fluorescence microscopy to count the number of adherent bacteria per field of view (FOV) at 400x magnification. <b>(B)</b> Bar graph illustrating the number of adherent <i>B</i>. <i>burgdorferi</i> per FOV. Mean counts ± SEM from ten FOV for each biological replicate are presented with standard error bars. For statistical analyses, attachment by strain <i>Bb</i>-Tp0751 to HUVEC monolayers preincubated with peptides p4, p6, and p10 was compared to the attachment of strain <i>Bb</i>-Tp0751 in the presence of corresponding scrambled peptides, using the Student’s two-tailed <i>t</i>-test. Strain <i>Bb</i>-Tp0751 exhibited statistically significant lower levels of binding to HUVEC monolayers preincubated with peptide p10 (*<i>p</i>≤0.005) when compared to the levels of binding when preincubated with the scrambled version of peptide 10 (p10scr1). <i>Bb</i>-Tp0751 adhered significantly more to endothelia than the parent under untreated conditions and when monolayers were pre-incubated with all other peptides (p˂0.05). The parent and <i>Bb</i>-Tp0751 strains are indicated by clear white bars and striped colored bars, respectively. <b>(C)</b> Line graph representing the effect of increasing concentrations (0 nM, 0.54 nM, 5.45 nM, 54.5 nM, and 545 nM) of p10 and negative control peptide p8 on adherence of Parent and <i>Bb</i>-Tp0751 to HUVECs. Mean counts ± SEM from ten FOV for each biological replicate are presented with standard error bars. Statistical analysis, was performed using the Student’s two-tailed <i>t</i>-test. Strain <i>Bb</i>-Tp0751 exhibited significantly lower levels of binding to HUVEC monolayers when preincubated with ≥ 5.45 nM p10 compared to the levels of binding when preincubated with ≥ 5.45 nM p8 (*<i>p</i>≤0.005). A non-linear regression curve was fitted and the IC50 for p10 inhibition of HUVEC binding by <i>Bb</i>-Tp0751 was calculated (IC50 = 17 nM) using GraphPad Prism data analysis software (San Diego, CA).</p