Abstract
The oral pathogen Tannerella forsythia is implicated in the development of periodontitis, a common inflammatory disease that leads to the destruction of the gum and tooth supporting tissues, often leading to tooth loss. T. forsythia is a unique Gram-negative organism endowed with an elaborate protein O-glycosylation system that allows the bacterium to express a glycosylated surface (S)-layer comprising two high molecular weight glycoproteins modified with O-linked oligosaccharides. The T. forsythia S-layer has been implicated in the modulation of cytokine responses of antigen presenting cells, such as macrophages, that play a significant role during inflammation associated with periodontitis. The macrophage-inducible C-type lectin receptor (Mincle) is an FcRγ-coupled pathogen recognition receptor that recognizes a wide variety of sugar containing ligands from fungal and bacterial pathogens. In this study, we aimed to determine if Mincle might be involved in the recognition of T. forsythia S-layer and modulation of cytokine response of macrophages against the bacterium. Binding studies using recombinant Mincle-Fc fusion protein indicated a specific Ca2+-dependent binding of Mincle to T. forsythia S-layer. Subsequent experiments with Mincle-expressing and Mincle-knockdown macrophages revealed a role for Mincle/S-layer interaction in the induction of both pro- and anti-inflammatory cytokine secretion in macrophages stimulated with T. forsythia as well as its S-layer. Together, these studies revealed Mincle as an important macrophage receptor involved in the modulation of cytokine responses of macrophages against T. forsythia, and thus may play a critical role in orchestrating the host immune response against the bacterium.
Highlights
Tannerella forsythia is a Gram-negative oral anaerobe and a member of the so called ‘red complex’ bacterial consortium of the subgingival cavity strongly implicated in periodontitis [1, 2]
Given the S-layer glycoproteins of T. forsythia are decorated with fucosylated sugar branches [11] (S1B Fig), we set out to determine whether the S-layer binds macrophage-inducible C-type lectin receptor (Mincle) and mediates T. forsythia-Mincle interactions
Human macrophage mannose receptor (MMR)-Fc construct used as control exhibited little to no binding to plate bound S-layer, while strongly binding mycobacterium mannose-capped lipoarabinomannan (ManLAM), a positive control for MMR (Fig 1A)
Summary
Tannerella forsythia is a Gram-negative oral anaerobe and a member of the so called ‘red complex’ bacterial consortium of the subgingival cavity strongly implicated in periodontitis [1, 2]. Inflammatory responses associated with chronic periodontitis potentially contribute to the development of systemic diseases such as atherosclerosis, PLOS ONE | DOI:10.1371/journal.pone.0173394. S-layers are found as the outermost cell envelope in many bacteria and archaea, formed by the self-assembly of proteins into 2-D crystalline arrays [8]. S-layers generally function in the maintenance of bacterial integrity, display of bacterial components and interaction with the host non-immune and immune cells [9]. T. forsythia is the only Gram-negative bacterium to possess a glycosylated S-layer, which is formed by two extensively glycosylated proteins, TfsA and TfsB [10]. We demonstrated that a terminal trisaccharide motif on S-layer proteins could play a role in blocking the recognition and processing of T. forsythia by dendritic cells [13]
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