Identification of a Treponema pallidum laminin-binding protein

Abstract

Host extracellular matrix (ECM) components represent ideal microbial adhesion targets that many pathogens use for colonization of tissues and initiation of infection. This study investigated the interaction of the spirochete Treponema pallidum with the ECM component laminin. To identify candidate laminin-binding adhesins, the T. pallidum genome was analyzed to predict open reading frames that encode putative outer membrane proteins, as these proteins interact directly with host ECM components. Subsequent recombinant expression of these proteins and analysis of their laminin-binding potential identified one protein, Tp0751, that demonstrated specific attachment to laminin. Tp0751 attached to laminin in a dose-dependent, saturable manner but did not attach to the ECM component collagen type I or IV or to the negative control proteins fetuin or bovine serum albumin. Sodium metaperiodate treatment of laminin reduced the Tp0751-laminin interaction in a concentration-dependent manner, suggesting that oligosaccharides play a role in this interaction. In addition, Tp0751-specific antibodies were detected in serum samples collected from both experimental and natural syphilis infections, indicating that Tp0751 is expressed in vivo during the course of infection. Collectively, these experiments identified Tp0751 as a laminin-binding protein that is expressed during infection and may be involved in attachment of T. pallidum to host tissues.

FIG. 1.

T. pallidum attachment to various ECM components. Each bar represents the average number of treponemes bound ± the standard deviation for six microscopic fields, and the results are representative of three independent experiments. For statistical analyses, attachment to each ECM component was compared to attachment to the negative control BSA by the Student two-tailed t test.

FIG. 2.

Binding of recombinant proteins to human laminin (A) and BSA (B). Each bar represents the mean absorbance (Abs.) value at 600 nm ± the standard deviation for three wells, and the results are representative of three independent experiments. For statistical analyses, the attachment of each of the recombinant proteins to laminin was compared to the attachment of the protein to BSA by the two-tailed t test. LBP, laminin-binding protein; F, full-length protein; N, N-terminal fragment; C, C-terminal fragment; I, internal fragment.

FIG. 3.

Specificity profile of attachment of recombinant Tp0751 to various coating reagents. Each bar represents the mean absorbance (Abs.) value at 600 nm ± the standard deviation for three wells, and the results are representative of three independent experiments. For statistical analyses, attachment to each component was compared to attachment to the negative control BSA by the Student two-tailed t test.

FIG. 4.

Binding of recombinant Tp0751 (□) to immobilized laminin as a function of varying laminin concentrations (A) and varying Tp0751 concentrations (B). Each datum point represents the mean absorbance (Abs.) value at 600 nm ± the standard deviation for three wells, and the results are representative of three independent experiments. Positive and negative control recombinant proteins tested in panel A were the S. aureus laminin-binding protein (▪) and T. pallidum ORF Tp0557 (▴), respectively.

FIG. 5.

Contribution of laminin carbohydrate moieties to recombinant Tp0751-laminin interaction. Shown is a bar graph representing Tp0751 attachment to untreated laminin (0 mM bar) and laminin treated with various concentrations of sodium metaperiodate (5 to 100 mM). Each bar represents the mean absorbance (Abs.) value at 600 nm ± the standard deviation for three wells, and the results are representative of three independent experiments. Shown above each bar is the percent reduction in recombinant Tp0751 attachment to sodium metaperiodate-treated laminin as a function of the sodium metaperiodate concentration. The data are expressed as percent reduction in attachment compared to the level of attachment to untreated laminin. In each case (*), the P value, as measured by the Student two-tailed t test, was ≤0.0009.

FIG. 6.

Reactivity of syphilitic serum samples to recombinant Tp0751. (A) Reactivity of sequential rabbit serum samples collected from experimentally infected rabbits. The serum samples on the x axis represent pools of serum samples collected from rabbits infected with T. pallidum for the indicated time periods. NRS, normal rabbit serum. (B) Reactivity of human serum samples from different stages of syphilis infection. The serum samples on the x axis are grouped by stages of syphilis: P, primary (n = 14); S, secondary (n = 13); L, latent (n = 8); N, neurosyphilis (n = 8). The overall mean absorbance (Abs.) of each group is represented by a horizontal line.