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>