Defining the interaction of the Treponema pallidum adhesin Tp0751 with laminin

Abstract

Various invasive pathogens attach to host tissues via the extracellular matrix component laminin, the major glycoprotein found within basement membranes. Previous investigations identified the laminin-binding adhesin Tp0751 within the spirochete bacterium Treponema pallidum. In the current study, Tp0751 was shown to attach to a variety of laminin isoforms that are widely distributed throughout the host, including laminins 1, 2, 4, 8, and 10. Such universal attachment is conducive for an adhesin present within a highly invasive pathogen that encounters a variety of tissue sites during the course of infection. Additional studies systematically identified the amino acid residues within Tp0751 that contribute to laminin binding using synthetic peptides designed from the mature protein sequence. The minimum laminin-binding region of the adhesin was localized to 10 amino acids; peptides containing these residues inhibited attachment of Tp0751 and T. pallidum to laminin. Further, Tp0751-specific antibodies inhibited attachment of T. pallidum to laminin. This study furthers our knowledge of the interaction of T. pallidum with laminin, an association that is proposed to facilitate bacterial traversal of basement membranes and subsequent entry into the circulation and tissue invasion. As such, these investigations will reveal new targets for possible prevention of bacterial dissemination and establishment of chronic infection.

FIG. 1.

Overview of the synthetic peptide sequences used to delineate the minimum laminin-binding sequence of Tp0751. Thirteen synthetic peptides were prepared to cover the Tp0751 mature protein sequence (p1 to p13). Also depicted are the scrambled peptides (p4-scr, p6-scr, and p10-scr) and the mutated peptides that were created, including the peptides with each of the four amino acids unique to peptides 4, 6, and 10 mutated, either collectively (m4, m6, and m10) or individually (98P-A through 185S-A). The predicted signal sequence for Tp0751 is shown by a double underline, and the four amino acids unique to each of peptides 4, 6, and 10 are identified by a single underline.

FIG. 2.

Tp0751 Attachment to various laminin isoforms. The attachment potential of Tp0751-1 to laminin isoforms 1 (α1β1γ1), 2/4 (α2β1, 2γ2), 8 (α4β1γ1), and 10 (α5β1γ1) was determined. Each bar represents the mean absorbance value at 600 nm ± SEM for triplicate samples. For statistical analyses, attachment of Tp0751-1 to each of the laminin isoforms was compared with attachment of the negative control recombinant protein Tp0557 by the Student two-tailed t test (*, P < 0.0001).

FIG. 3.

Identification of the peptide sequences that mediate attachment of Tp0751 to laminin. (A) Attachment of Tp0751-2 and synthetic peptides 1 to 13 to laminin. Also shown is the attachment potential of scrambled versions of synthetic peptides 4, 6, and 10. For statistical analyses, attachment of each of the synthetic peptides was compared with attachment of the negative control recombinant protein Tp0557 by the Student two-tailed t test (*, P ≤ 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.

FIG. 4.

Delineation of the amino acid residues mediating attachment of Tp0751 to laminin. Shown is the laminin attachment potential of native and mutant peptides for the sequences covered by peptide 4 (A), peptide 6 (B), and peptide 10 (C). Data are reported as the mean absorbances at 600 nm ± SEM for triplicate wells. Statistical analyses compared the level of attachment of each mutant peptide to that of the native peptide by the Student two-tailed t test (*, P < 0.0001 [A and B] or P ≤ 0.0139 [C]).

FIG. 5.

Cellular attachment potential of recombinant Tp0751. Shown is attachment of the SW480 cell line to BSA (negative control), fibronectin (positive control), and Tp0751-1. Data are reported as the mean absorbance at 595 nm ± SEM for three wells. Statistical analyses compared the level of attachment to each cell line by the Student two-tailed t test (*, P < 0.001).

FIG. 6.

Peptide inhibition of T. pallidum attachment to laminin. (A) treponemes were incubated with laminin-coated slides in the presence of peptides 4, 6, and 10 (i), diluent alone with no peptide addition (negative control, ii), control peptides 1, 2, and 3 (negative control, iii), and mutagenized peptides 4, 6, and 10 (m4+m6+m10; iv). A representative spirochete is identified by an arrowhead in panel i. Spirochetes were visualized by dark-field microscopy using a Nikon Eclipse E600 microscope. (B) Quantitation of the number of treponemes attached per field under each reaction condition. Statistical analyses compared the level of attachment of each condition to that of the “no addition” sample by the Student two-tailed t test (*, P < 0.0001).

FIG. 7.

Antibody inhibition of T. pallidum attachment to laminin. (A) treponemes were incubated with laminin-coated slides in the presence of anti-Tp0751-1 serum (i), diluent alone with no antibody addition (negative control, ii), immune rabbit serum (positive control, iii), normal rabbit serum (negative control, iv), and the irrelevant control serum anti-Tp0155 (negative control, v). Representative spirochetes are identified by arrowheads in panels i, ii, iv, and v. Spirochetes were visualized by dark-field microscopy using a Nikon Eclipse E600 microscope. (B) Quantitation of the number of treponemes attached per field under each reaction condition. Statistical analyses compared the level of attachment of each serum condition to that of the “no antibody” sample by the Student two-tailed t test (*, P < 0.0001).