Identification of a unique TLR2-interacting peptide motif in a microbial leucine-rich repeat protein

https://doi.org/10.1016/j.bbrc.2012.06.008Get rights and content

Abstract

Pathogenesis of many bacterially-induced inflammatory diseases is driven by Toll-like receptor (TLR) mediated immune responses following recognition of bacterial factors by different TLRs. Periodontitis is a chronic inflammation of the tooth supporting apparatus often leading to tooth loss, and is caused by a Gram-negative bacterial consortium that includes Tannerella forsythia. This bacterium expresses a virulence factor, the BspA, which drives periodontal inflammation by activating TLR2. The N-terminal portion of the BspA protein comprises a leucine-rich repeat (LRR) domain previously shown to be involved in the binding and activation of TLR2. The objective of the current study was to identify specific epitopes in the LRR domain of BspA that interact with TLR2. Our results demonstrate that a sequence motif GC(S/T)GLXSIT is involved in mediating the interaction of BspA with TLR2. Thus, our study has identified a peptide motif that mediates the binding of a bacterial protein to TLR2 and highlights the promiscuous nature of TLR2 with respect to ligand binding. This work could provide a structural basis for designing peptidomimetics to modulate the activity of TLR2 in order to block bacterially-induced inflammation.

Highlights

► Leucine-rich repeat (LRR) domain of a bacterial protein activates TLR2. ► Peptide repeats in the LRR domain responsible for TLR2 activation were identified. ► A common peptide motif in these repeats interacts with TLR2. ► This peptide motif in a TLR2-dependent manner activates macrophages. ► This study identifies a novel non-acylated peptide ligand of TLR2.

Introduction

Pattern-recognition receptors (PPRs) of the Toll-like receptor (TLRs) family recognize distinct microbial patterns to transduce intracellular signaling for release of inflammatory cytokines involved in orchestrating innate and adaptive immune responses [1]. For example, TLR2 primarily binds acylated peptides, and in cooperation with TLR1 or TLR6 is able to differentially discriminate tri- or di-acylated peptides, respectively. However, TLR2 is also quite unrestricted with respect to its ligand specificity, evidenced from recent studies demonstrating its ability to bind non-acylated peptides and proteins of diverse origin with no structural similarity [2], [3], [4], [5], [6], [7], [8], [9], [10], [11]. In infectious diseases, such as periodontitis, a bacterially-induced chronic inflammation of the tooth supporting tissues often leading to tooth loss, TLRs modulate inflammatory responses of the host to oral bacteria during disease pathogenesis [12]. Tannerella forsythia, a pathogen strongly implicated in periodontitis, expresses a cell-surface associated and secreted protein BspA [13], which by activating TLR2 induces the secretion of inflammatory cytokines [14], [15]. Furthermore, TLR2 signaling leads to Th2 cell bias, which ultimately drives T. forsythia-induced periodontal inflammation and jaw associated bone loss in mice [16].

BspA comprises a horse-shoe shaped leucine-rich domain [17], which is formed by the 23 tandem repeats of a leucine-rich repeat (LRR) motif in the N-terminal portion, and four bacterial immunoglobulin-like domains in the C-terminal portion [13], [18]. The LRR motif in BspA belongs to a cysteine-containing subtype, which might allow the LRR domain to adapt a cysteine-ladder conformation reported in other cysteine containing LRR proteins [19]. We have previously shown that BspA activates TLR2/1 receptor heterodimer and that the LRR region comprising LRR repeats 1–16 is involved in direct binding to TLR2 [14].

The objective of the current study was to identify specific motif(s) within the LRR 1–16 region of BspA that bind and activate TLR2. Our approaches involved screening a series of overlapping synthetic peptides derived from the LRR 1–16 region for binding and activation of TLR2 by using reporter cell lines and macrophages from wild-type and TLR2 knockout mice.

Section snippets

Reagents

Toll-like receptor agonists Pam3CSK4 lipopeptide, highly purified Escherichia coli K-12 LPS (Ec. LPS), polymyxin B sulfate, human monocytic THP1-Blue cells and HEK293 (hTLR2/hTLR1) cells stably expressing human TLR2 and TLR1 and a reporter plasmid, pNiFty2, expressing a NF-κB-inducible secreted alkaline phosphatase (SEAP) were purchased from InvivoGen (San Diego, CA). Monoclonal anti-TLR2 (MAb) and isotype control (IgG2a) antibodies were obtained from eBioscience. Synthetic peptides were

Trypsin treated LRR1 domain retains the ability to activate TLR2

To ascertain whether smaller discrete peptide epitopes for TLR2 activation are present in the LRR1 domain of BspA (Fig. 1), we tested the activity of the trypsin digest of rBspLRR1. For this purpose, the fraction eluted in the exclusion volume of the G-25 column from the trypsin digest of BspLRR1 was tested for TLR2 activation using reporter cell lines HEK293 (hTLR2/hTLR1) and THP-Blue cells. The results showed that the tryptic digest of BspLRR1 induced NF-κB in HEK293 (hTLR2/hTLR1) (Fig. 2A)

Discussion

The current study was undertaken to identify the peptide motif(s) in the T. forsythia BspA virulence protein that mediate TLR2 binding and activation. Our study described here showed that a peptide motif GC(S/T)GLXSIT present in the LRR1 domain of the BspA protein is responsible for TLR2 binding and activation. Synthetic peptides derived from the LRR1 region of BspA containing this motif bound and activated reporter cell lines and mouse macrophages in a TLR2-dependent manner. Moreover, a

Acknowledgment

This study was supported by U.S. Public Health Research Service Grant DE014749.

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      T. forsythia does not possess any visible pili-like appendages in TEM or contain putative pilus or fimbrial genes in its genome sequence. However, it does possess the major surface antigen and adhesin BspA (Sharma et al., 1998) that is involved in coaggregation with F. nucleatum, T. denticola and P. gingivalis, attachment to oral epithelial cells (Mishima & Sharma, 2011), as well as immune system modulation (Myneni et al., 2012). BspA consists of four domains: The N-terminal, leucine-rich repeats (LRRs), bacterial Ig-like domains, and the CTD.

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